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THE USE OF TRANSFORMAWONAL MUTAGENESIS TO STüDY THE GENETICS OF DIMORPHISM IN
OPHIOSTOM ULM
A thesis suknitted in confomity with the requirements for the degree of Master of Science
Graduate Department of Botany University of Toronto
O Copyright by Karl Fiederid< Kduirewicr 1997
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Tho uw d tnnsf01llllUonil r n u t i ~ ~ s l a to rtudy tho grntlcr of dlmorphlrm In OphIoatomr ulml. W r e e al' Master d Science, 1997. Karl F. Kokurewicz, Graduate Department of Batany, University of Toronto.
This thesis reports the generation of Ophbstoma uhni mutants dispîaying an altered
dirnorphic distribution ushg the tedinique of transformational mutagenesis, and
presents a partial genetic analysis of the recwered mutants. Using the methoâology
developed by Royer et el (1991,1997) with 0. ulmistrain MH75, the
nonhomologous integretive vector. pPS57, was used as a mutagenic tool to
transform the aggressive VA30 strain of O. ulmi. Selection of transformants on a
hygrornycin wntaining solM medium induced ôudding-yeast growth. Reevaluation of
the meaiodology with VA30, founâ linearited vector and selection with 200pg/rnl
hygromycin to be optimal for the recovery of translormants. An estirnated û6ûû
transformants were screened. Six of the fourteen dirnorphic mutants obtained were
transformants, as detertnined by Southern analysis. One mutant, 1 ", had a
predominantly budding-yeast cell phenotype. Yml cultures producd sectord
growth composed predominantly of hyphae. Meiotic analysis of yml and a p l
sWor isdate, found bath to be inheritabie mutations which were not taggeâ by
pPS57, and sugg8Steâ that the two mutations were cloeely linked.
Table ot Contenta
Abstract
TaMe of Contents
List of TaMes
List of Figures
Introduction
An ovemiew of dimorphisrn
Ce11 wall formation in yeast and hy@d f m s
Significance of fungaI dimorphism to medicine and agn'wIture
Medically important dimorphic fungi
Ophiostoma ulmi and the agriculturally important dimorphic fungi
The genetics and molecular biology of fungal dimorpMsm
Pseudohyphal grawth in ÇaCCI)aromyc8s cefev&iae
Control of dimorphism in: UslYîago maydis
Candùh alk'cans
Mucol spp.
Ophidstom ulmi
Fungal trans fo~matim
Transfomational mutagenesis
ii
iii
vil
viii
xii
Évaluation of aansformational mutagenaokr
iii
(table d contents continued) Page
lmplementing transfomational mutagenesis to stuûy
fungal dimorphism in Ophiostana ulmi
OpMaPrna ulmi as a "modei dimorphic f ungusa
Charactenirabun and anelysis of the mutants
Classification of mutants
Analysis for reanangements and chromomal aiterations
Analysis of ONA rnethylation
Materials and methods
Materials
Strains and cultural conditions
Plasmid pPS57
Protoplasting of Ophiostuma ulmi cells
Transformation
Screening strategy for recovery of dimorphic mutants on solid media
DNA isolations
Restriction digestions, gel electrophotesis and Southern analysis
Meidc analysis
CHEF electrophoresis and Southern analysis
ONA methylation measurements
Resultr
T~ansfom,atimal mutag8nesi.S; definhg dhe systm îbf VA30
Hygromycin contakiing seledon media altm VA30 phenotype
Determinhg transformation statu8 of cdonh
iv
(taMe d contents continu&)
VA30 versus MH75 transformation; transformation with circular and
linear vector, and seledion with varying concentrations of hygromycin
Screening strategy for recovery of dimorphic mutants
Number of transformation experiments performed and putative
trarrsformarits screeried
Chamcterùadion of remveret# mutants
Stability of mutants
Sectoring of ym 1 mutant
Meiotic enalysis of mutants
Mating reactions
Meiotic analysis of: ym l
yml secîor
other mutants
CHEF electrophoresis and Southem analysis
DNA methykitjon
Analysis of pPS57 methylation with Hpa II and Msp I isoschizomen
Analysis of rDNA methylation with HpP II and Msp I isoschizomers
Analysis of DNA methylation with Sazacytidine
Dl8cussion
Defining the systetn with VA30
Induction of budding-yeast growth by hygromycin
Transformation status of colonies appearing on transformation plates
Transformation parameters for strain VA30
Seledion of dimarphic mutants
(table d contents continued)
The ym 1 kiddingyeast mutant
ûther mutants
Fudher anelysis of ym 1 and ym l sector
CHEF analysis
DNA methylation
Citerature clted
List of tables
Page
Table 1. Phenotypes of colonies on transformation plates, and upon 52
transfer to CM plates with and without hygrornycin, obsenred
at vaiious time periods.
Table 2. Number and morphology of colonies growing up on MH75 and 56
VA30 transformation plates, utilizing both circular and
linearized pPSS7 vector, and with varying levels of hygromycin
selection.
Table 3. Summary of mutants, indicating transformation conditions 61
utilized, results of Southern analysis using J2P-labelled pPS57
as a probe, and general characteristics on complet8 medium
(CM) plates.
TaMe 4. Radial growth extension of strain VA30 and generated mutants 64
after 9 days of growth on complete medium (CM), complete medium
wntaining hygrom ycin at 200pg/ml, and minimal medium (MM)
plates.
Table 5. Summary of meiodic analysis for yml X W2-TOL progeny. 90
Table 6. Summary of meiotic analysis for yml sectlor X W2-TOC 92
P r o g r n Y a
vii
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Rgun 8.
Figun 7.
Cotnponenîs of aie Saccharomyces cwevisiae pheromone
response pathway, th& utilkation in other S. oerevisiae
signal transduction pathways, and homologs in
Ustilago meydis.
Comparison of mutant and wild type colony growth.
Southern hyôridization of undigested DNAs from colonies
isolated from transformation plates, probed with
aP-labelled pPS57.
Growth oi wild type strain VA30 and budding-yeast mutants
ym 1, ym2, ym3 and ym4 on complete medium (CM), CM
containing hygromycin at 2Wpg/ml, and minimal medium
(MM) plates.
Comparison of Ophiostma ulmi wild type strain VA30 and
yml budding-yeast mutant cdony morphologies on cornpiete
medium (CM) plates, after 15 days of growth at 25°C.
Liquid cuture growth of OpMarlome ulmi straki VA30 and
mutants ym l(a, ym f(0) sector, ym2, ym3 and yml.
Microphotogr- of wild type strain VA3û and mutant ml. viii
(list of figures continued)
Figure 8. Southern hykidization of undigestecl and 801 II digested DNA
of Ophiostoma ulmi, buddlng-yeasî mutants ym1. ym2, ym3
and yml, probed with 32P-laôeiled pPS57.
Figure 9. Colony morphologies and radial growth rates d O. ulmi
nontransformed mutants.
Figure 10. Colony morphologies and radial growth rates of O. u/mi
transformed mutants.
Figure 1 1. Liquid culture growth of Ophiostoma ulmi strain VA30 and
mutants 26, 26 maintained on hygromycin. 94-7-27-1,
94-7-27-2, 94-7-27-3, 94-8-4-3. 94-8-44, 918-4-6,
94-8-8-20, and SM-8-8-30.
Figure 12a. Southern hyôridization of Bgl II and Hind Ill digested DNA
of Ophiostoma ulmi mutants probed with 32P-labelled pPS57.
Figure 12b. Southern hykidization of Bgl II end #id III digestd D M of 74
Ophiostoma ulmi mutants probed with 32P-labelid pPS57.
Figure 13. Convoluted giowth Os mutant 158.
Figure 14. Southern analpis of mutant 158.
(list of figures continued) Page
Figure 15. Sectord growth of budding-yeas? mutant yml on complete 80
medium (CM) plates.
Figure 16. Colony morphdogies and radial growth rates of VA30,
yml and yml sector.
Figure 17. Microphotographs of wild type strain VA30 and mutant 83
ym 1 (O) sector.
Figure 18. Southern analysis of pPSS7 insertion in ym 1 and ym l sectors. 84
Figure 19a. Southern analysis of Bgl II digested ymi X W2-TOL progeny 88
probed with 32P-labelled pPS57.
Figure 19b. Southern analysis of Bgl II digested yml X W2-TOL progeny 89
probed with 32P-labelled pPS57.
Figure 20. CHEF gel analysb of yml and yml sector. 95
Figure 21. Analysis of pPS57 methylatian in yml and yml sectm. 100
Figure 22. Anaîysis of pPS57 methylation in ophiostlome ulmi 1 O1
strain VA3 transformants.
(Iist of figures continuecl) Page
Figure 23. Analysis of rDNA methylation in ~ i o s t o m a ukni strain VA30 102
and VA30 transformants.
Figure 24. Effect of the methylation inhibitor 5-azacyodkie on radial IO4
growth rates of VA30, ym 1 and ym 7 sector.
Figure 25. Effect of agar concentration on the growîh of VA30 on variws 105
solid media.
I would like to thank my supervisor, Dr. Paul Horgen, for hi8 support and for the
opportunity to sing during aie past few years. I would also like to thank my
supewisory cornmittee rnembers, Dis. J.B. Anderson and M. Hubbes for their
guidance and counsel. I am especially appredative of the encouragement,
mentorship and friendship offered by Dr. John Rayer, and for assistance given, I am
also indeôted to Ors. Paul de la Bastide and Barry Saville. I would like to
acknowledge the generosity of Dr. Arabinda Guha and family. F inally, and m a t of
ail, I would like to thank my parents for support of al1 kinds through some difficult
times.
This research was supported in part by the Guha Graduate Scholarship and
graduate scholarships from the University of Toronto.
xii
Introduction
Fungal dimorphisrn, as defined by Romano (1966), is an envimnmentally
controlled reversiMe interconversion between the yeast and hyphal gmwth foim.
Fungi capable of dimorphic growth are found in the Ascomycetes,
Basidiomycetes, Deuteromycetes and Zygornycetes (Griffin, 1 994). The
mechanism undetîying dirnorphic switching hm attracted considerable interest as
a mode1 for cellular moiphogenesis (Sypheid et el. 1978), partly due to its
perceived role in the pathogenesis of several dimorphic fungi th& cause
afflictions in humans and in plants (Stewart and Rogers, 1978). Dirnorphic
switching is initiated by environmental stimuli, such as temperature or nutritionai
changes, which are sensed and transâuced by the ceIl. Some dimorphic fungi
require ôoth temperature and nutritional factors, whle others require a change in
pH (Griffin. 1994).
The p m s s of dirnorphic switching involves reception of the
environmental signal by the d l , and transduction of the signal into a cellular
response, induding activation of the cellular controls which detemine cell wall
formation. The cellular contrds determining fom may indude control over the
p m s s of cell division, polarity, location and rate of deposition of newly
synthesized cell wall cornponents, positioning of the vesiâe supply center (VSC)
and the rate of delivery of vesides to the gmwing cell wail, as well as alterations
to the cell wall composition itaelf (Bartnicki-Garda and Gien, 1993; Gliflin, 1994).
Dirnoiphic switching may play an integrel part in deterrnining th8
pathogenicity of Ophiastom ukni (8uism.) Nannf. (Kulkarni and NMecson,
2
1981), the causal agent of Dutch elm disease (DED). The molecular end genetic
processes controlling dimorphic switching in O. ulmi have not been well
characterized. The genetic mechanisms which contrd pseudohyphal growth
(PHG) in Sechammyces c8nMsiae have been shown to be partially consenred
amongst several fungi capable of tnie dimorphic growth, induding Ustiktgo
maydis and CandiMa albicans. In addition to PHG. the genetic cantrols governing
cellular morphogenesis, growth and metabolism, and cellular stress responses
have been well characteiized in S. cerevisiae, and together may provide a
framewoili for the investigation and evaluation of dirorphisrn in 0. ulmi.
Ml ml1 formation in ymst and hyph.l ldm
The fungal cell wall irnparts structural integnty to the cell and determines
the shape of the cell. The composition of the cell wall varies between species,
but in geneial the cell walls of ADcomycetes consist of chitin and
8-glucans, to fom microfibrils which impart strength and fom, (see Griffin. 1994).
The presence of chitin or cellulose is usually considerd to be mutually exdusive.
However, the cell wall of O. ulmi has been shown to contain both chitin and
cellulose polymers (Smith et al. 1967). Ascornycetes typically contain gcilacto-
manno-proteins and pglucans (see Griffin, 1994). A general mode1 for cell wall
biosynthesis in the Ascomycete C. albicans hes been describeci by Shepard and
Gopal (1 993). The cell wdl of C. 8Ibims is composed of mannoproteins, 81 ,3-
linked glucans, pl ,ô-linked gluoans, chitin, protein and lipM (Shepard and Gopal.
1993). Vesides from the Gdgi Iendoplasmic mticulum contain materials foi
construction of the plasma membrane, membrane bound enzymes, soluble
enzymes, mannoproteins and poseiôly wdl polymer primem. Vesides are
directed dong adin fibrils to the pîasma membrerio at the @nt of cell growth.
Chitin and glucan polymeis are synthesized by transmembrane enzymes and
products are extnided into the outside environment to add to the growing cell
wall. The mechanism by which pl ,6-glucan is synthesized is still unknown.
Constituents are inseited into the growing dl wall, with glucan and chitin
polymers becoming cross-linked with existing constituents of the cell wall. Cross-
linking is believed to be accomplished by the activity of lodized hydrolases and
a branching enzyme, thus the dynamics of cell wall growth are believed to
depend on ôoth cell wall synthesizing and degrading enzymes (Shepard and
Gopal, 1993).
The site of deposition of new cell wall constituents may be the major
difference between yeast and hyphal forms of growth (Kerridge, 1993). In the
hypha of C. elbicans, 990% of the wall extension occun near the hyphai tip, and
1 0% occurs over the rest of the cell (Shepard and Gopal, 1 993). In budding
yeasts, wall extension is divided 70%:30% between the tip and the rest of the cell
respectively, until a certain sire is reached, then 90% occun ove? the entire
sutface. What detemines lacation of cell wall deposition in fungi? It appean
that the spatial organization of cytoskeletal components determines the location
of cell wall synthesis, and that actin granules and microfilaments may be
particulerly important (Yokayama et al. 1 990). Adin granules, essodateci
microfilaments and calmodulin are found at the apex when hyphd growth occuis,
while in budding-yeasts, th8 grmules are dispersed throughout the cell.
Bartnicki-Garda and Gien (1 993), have suggested that the key moleailar events
regulating dimorphisrn rnay be those iesponsiôie for moving and deteminhg the
rate of ôisplacemenVadvanoe of the veside supply centei (VSC), and
detemininu the number of wall-kriiding veskles pmdUCBd per unit time. It is not
known how these processes or indeed the organization of cytobJ<eIetal dements
are affeded by environmental changes (Kertiôue, 1983).
4
The extent to which differences in cell wall composition are important to
the formation of yeast and hyphal cells is not known, howevei, little variation has
been found between the yeast and hyphal forms of several species of dimorphic
fungi (Bartnicki-Garda. 1 968; Bartnicki-Garcia and Gierz, 1 991 ).
There has been an increased interest in fungal pathogens afflicting
humans, largely because of the growing numben of immunologically
compromised patients who are at risk of opportunistic fungal infection. This
indudes cancer patients undergoing chemotherapy and radiation therapy, AIDS
(acquired immune deficiency syndrome) patients, and patients being treated with
anti-rejedion dnigs used for organ transplantation (Wamodc and Campbell,
1996). Under these circumstances, fungal species which normally do not cause
pathology in the human host may becorne pathogenic and cause life-threatening
systemic infections.
The human fungal pathogens, Blestomyces diematitidis, CCoccidioides
immitis, Hisroplesma capsulatum, P a ~ i o ~ e s bmsiSensis and Spomthnx
schenkii a n ail dimorphic, growing saprophytically in the environment as hyphae
and switching to a yeast forrn of gmwth at the elevated temperature of 37OC in
the human host. With the exception of @omlhrix schmkii, dl of these
dimorphic fungi are primary pathogens of humans; they are capable of infecting
via the respiratory tract and causing serious systemic infections in healthy
individuals who are free of trauma and immune system suppression (Koôayashi
and Mamsca, 1993; Kenidge, 1993). Spomthfix sdimkii is different in that il
requires traumatic implantation into the tissue for infection to ocair, and under
normal cimmstances remains subcutaneowr (M~vhy, 1 99 1 ). The morphogenic
changes that these dimorphic fungi undergo are viewed as adaptations which
allow an infection to establish itself, and then enable growth to ocair in the host
environment where the temperature is elevated to 37OC, where oxidation-
reduction potentiels are reduced, and where host defense mechanisms are
piesent (Kobayashi and Maresca, 1993).
The important opportunistic fungi capable of causing systemic infections in
humans include Asperpiilus furnigaius, Candida albicans and Pneumocystis
can'nii. Within this gmup of opportunistic fungi, only C. aibimns displays
dimorphism (Kerridge. 1 993). Candida a/&ians, an Ascomycete which causes
canddiosis, is responsible for the most common fungal infection of humans
(Murphy. 1 991 ). Candida albicans grows principall y as a saprophytic yeast. It
colonizes mucosal surfaces including those of the intestinal tract and vagina. It
becornes parasitic in a debilitated host and may become systemic thrwgh the
passage of yeast cells into the circulatory system (Kerridge, 1 993). The role that
dimorphic switching plays in the development of systemic infections by C.
albicms is not dear. However, hyphal outgrowth from the yeast fonn allows the
fungus to evade phagocytosis by leukocytes (Borgem, 1993). The hyphal form of
gmwth may also allow for greater adhesion because of a hig her surlace to
volume ratio, and greater penetration eitttar due to a tip fodng its way through
the plasma membrane or possibly by the secretion of lytic enzymes at the hyphal
tip. Since the hyphal phase is thought to be lese effectively dealt with by the
immune system, there has b e n interest by medical reseaichers to inhibit the
hyphal phase (Bergers, 1993).
The four piimary âimorphic pathogens, BlaSIOmycies demiatidids,
bmsiliensis, as well asSjwrnthd'x schenkii, have been less extensively studied
than C. a l h n s (Kerridge, 1993). Sparothriw sch8ndIii has recently been
placed within the genus Ophiosforna baseâ on 18s ribosomal RNA sequeme
data (Berbee and Taylor, 1 991 ).
Genes that play a mle in dimorphic switching may be targets for ami-
fungal dnigs (Gimeno et al. 1 993). Antifungal compounds such as azoles, for
example, have k e n shown to inhibit the yeast-hyphel transition in C. e ~ s ,
and allow leukocytes to eliminate Candi& infections. The major antifungais in
use appear to interfere with cell wall formation (Yamaguchi et al. 1993). These
are corn prised of the azoles, allylami ne derivatives and morpholine derivatives,
al1 of which target the biosynthetic pathway for ergosterol, a lipid component of
the fungai cell membrane (Yamaguchi et al. 1993). Disruption of ergosterol
synthesis may cause degeneration of the cell membrane, which impairs ceIl wall
formation by affecting the cooidinated synthesis aWor assembly of glucans,
chitin and other cell wall constituents (Yamaguchi et al. 1993). Several new
classes of antihingal compounds which target the cell wall are currently under
study: the nikkornycins which target chitin synthase, papulacandins and
echinocandins which inhibit glucan synthase, and the benanomicins and
pradimicins which act on cell wall mannoproteins (Wamod< and Campbell, 1996).
The Basidiomycete Smuts (Usfi&go spedes) inlecl a variety of
monocotyledonous and d'iledonous plants and cause severe economic
losses (Banuett, 1995). The corn pathogen. UsIr'- mavas, is a well-studied
mernbei of this group and demonstrates a case where dimwphism is rdeled to
pathogenicity. This fungus is pathagenk in its hyphaî km but non-pathogenic
(Le. incapable of infecting the host plant) in the yeast phase (Bamiett, 1991).
The Ascomycete Ophioslame species infect a numbei of trees which are
valuable to the lumber and landscaping industries. The best stuâied of this group
is O. ulmi. Dutch dm disease has killed a large proportion of the highly
susceptible Amencan elrn (Ulmus amen'cana L.) population in North America,
with a signifiant economic and aesthetic impact upon the uiban landscape.
Ophiostoma u/mi exists as two distinct subgroups, known as "non-aggressive"
(NAG) and "aggressive"; the latter is divided into the two races North American
aggressive (NAN) and European aggressive (EAN). Braiser (1 991 ) renameâ the
aggressive subgroup (NAN and EAN) as the separate species 0. nom-u/mi
Brasier, while the non-aggressive subgroup remained 0. ulmi. Because of the
controversial nature of this taxonornic change, the aggiessive VA30 strain used
in this study will still be referred to as 0. ulmi.
Within an infected elrn tree, O. ulmigrows in hyphal and budding-yeast
phases, and produces both Spor~thtix (Cepha110sporium) and Graphiumtypes of
spores (Harris and Taber, 1 970; Holmes, 1 978). The process of dimorphic
switching is thougM to be integral to the pathogenicity of O. uimi (Kulkarni and
Nickeison, 1981). @hiostom ukniis transpolted from intected to healthy trees
on and within the Wies of the native elrn bah beetle (Hylurpopinus mfips) or
the European elm bark beetle (Scdyfus mullistnatus) each of which is attracfed
by (-)~rcubebene, which is relessed by the elrn tree (see Sticklen, 1 991 ).
Infection may also be spiead through mot grafts. Grrqphiumtype spores are
produced on stalked, spore-beaiing coremia and mycelia are produced in the
beetle galleires of infected trees (Agiios, 1 988). Them cipores are canied by
aduit beetles, to the ôaik and wood of uninfected trees where they geminate;
the resutting hyphal growth allows the fungus to penetrate and spread between
vessels (Campana, 1978), eventually reaching the large xylem vessels of the
spring wooô. f he invasiveness of the hyphaî fom may make O. ulmi similar to
8
C. albims (Kulkami and Nidterson, 1981 ). While in the sap Stream, hyphae
pioduce Sporothrix-type spores, which repmûuce in a budding- yeast fashion.
Conversion of hyphae to a ôudding-yeast cell growth allows the fungus to be
passively carried up the sap stream within individual xylem vessels and to be
spmad to new locations, where budding-yeast cells genninate and initiate new
sites of infection (Agrios, 1988).
Ophiostome ulmi exists vegetatively as a haploid, and sexual repmduction
is rarely observed in nature since single mating types are found over large areas
(Agrios, 1988). Ophiostoma uhni has two mating types, designated by two alleles
'Aa and 'Ba at a single locus (Brasier, 1984). When mating does occur between
compatible strains, spherical, Nad< perithecia containing crescent shaped
ascospores are produced (Agtios, 1 988).
Ophiostoma ulmi produces several metabdites thoug ht to play a role in
pathogenesis. One is cerato-ulmin, which has been shown to be a hydrophobin
(Stringer and firnôerlake, 1 993), and another is a high moleailar weight
peptidorhamnomannan (see Stiûûen 1 al. 1 991 ). Ophiostome u/mi also
produces elidtocs, a group of glycoproteins known to stimulate mansonone
production in host elm cells (Yang et al. 1989). Mansonones aie phytoalexin-like
sesquiteipenes, and their production represents one molecular defense
mechanisrn utilized by elms when faced with infection by O. uhni (Stiden et al.
1991). Resistance to DED occuis mainly through a damage contrd and
containment mechanisrn to prevent spreading of the pathogen.
Compartmentalization and containment of the pathogen ocairs thmgh the
formation of tyîoses - outgmwths of xylem parenchymai ce#s which ocdude
vessels. The tyloses dog the vesse1 mity and plevent rnovement of the fungus
to other vascular ekments (StMen et al, 1 991 ).
Strategies for the control of DED have invohred manipulations of the elrn
tree host, the beetle vector, or the 0. ulmi pathogen itself, none of which have
been succ8ssful in preventing the continued destruction of the elrn tree
population. These results have led to a belief that control of DE0 shoukl utilize
an integrated approach involving: i) the use of chemicals for disease protection.
ii) the induction, identification and genetic manipulation of the elrn tree's defense
system and. iii) control of 0. ulmivinilence (Hubbes, 1988). Manipulations of the
host elrn tree have included treatrnent with fungicides (Smalley, 1978), breeding
strategies to increase resistance to DED, pre-inoculation of trees with non-
aggreasive strains of O. u/mi in an attempt to elicit a pmtective response, and
pending the availability of appropriate resistance genes, the use of gene-transfer
methods to create resistant elms (see Brasier, 1991 ). The recent successful
production of fungus-tolerant transgenic oilseed rape (Bmssica napus), which
expresses the chitin degrading enzyme chitinase (Grison et al. 1996), suggests a
possible strategy for the production of resistant elms. The resistance obtained
wit h oilseed rape, may have resulted from both the dired degradation of chitin in
growing hyphae, and from stimulation of plant defense mechanisms in response
to the release of chitin oligorners, which may act as elicitors (Grison et al. 1996).
Strategies focusing on control of the bah beetle, either pioposed or attempted,
have induded the use of insecticides, ettractant-baited beetle traps, the use of
insect pathogens such as bacteria, fungi, viruses or nematodes specific to the
beetles, and the release of sterile male beetles into the environment (see StMen
et a/. 1 991 ). Becterial biocontrol agents such as Pseudomnas fluomsoens and
Baailiks subtiNs have also been used to inoculate d m s e d i n p (see StWen et
al. 1991 ). A proposed strategy invoives the aiteration of the pathogen itself; the
mycwiius-like btactom, which decrease pathogenicity of infectai 0. u l d strains,
may be used as a natural control rneasuie (Brader, 1983,1988, Webbei, 1987)-
10
Another proposed strategy would devekp dominant, nonpathogenic, mating-
type isolates of O. ulmi to be released into nature to mate with aggressive
isolates (Stidden et al. 1991). This would require a greater understanding of the
determinants of pat hogenidty and aggressiveness (StiMen el a/. 1 991 ), wit h
dimorphism Ming one of the possible determinants.
Pseudohyphal grauth in Saccharomyces cerevisàe
01 al1 dirnorphic fungi studied, it has been the budding yeast,
Saccharomyces cerevisiae , which has proviâed a 'rnodel systema for the study
of fungal dimorphism. This is despite the fact that yeast genetidsts had
"forgottena and subsequently "rediscovereb dimorphism in S. oerevisiae
(Gimeno et al. 1992). Since its rediscovery, eluddation of the genetic controls
goveming pseudohyphal growth (PHG) in S. cereMsiae has ben rapid.
Gimeno et al. (1992) showed that S. œrevisiae undergoes a yeast to
hyphal conversion in response to nitrogen stawation; this is brought about by
either the use of ptoline as the sole nitrogen source or by a low ammonia
medium in the presence of glucose (Blacketer et al. 1995). Pseudohyphal growth
occuis only in heteiothallic diploid yeasts which are heterdogous at the mating
type locus (MATWa). However, MATa and MATa haploids are capable of a
sirnilar 'invasive gmwtha form that exhiMts siightly dongated cells and kids with
a bipolar pattern, ocairring under the sudace of colonies growing on rlch agar
plates that are a few days dd (Roberts and Fink, 1994). A gmwth f o n descilbed
as pseudohyphal, which is both rapidly reversible and oxygen-âependent, has
also been reportecl to exist in hapîoid cells (Wright et al. 1993). Furthetmore, an
apparently irreversible pseudohyphal form in haploM cells grown in liquid media
with 2% ethanol and 2% leucine has also b e n describeci (Dickinson, 1994).
Although a Rlamentous form had been reported to ocair in S. œrevisiae
(Guillienond, 1920; Schen and Weaver, 1953; etc., in Gimeno et al. 1992).
there were no descriptions for the conditions required for its induction.
Pseudohyphal cells have a unipdar pattern of growth. aie elongated, display
incomplete cell separation to fom attachecl chains of cells, and grow invasively
into the surface of solid media (Gimeno et a/. 1992). The resulting colonies
resemble those produced by filamentous fungi, half of whose cells typically can
be found to be foraging below the surface of the solid medium (Gimeno et al.
1992). Pseudohyphae rnay allow S. cerevisiae to forage for nutrients and reach
substrates at a distance frorn the initial site of colonization, when nitrogen stanred
(Gimeno et al. 1992). Pseudohyphae may also uniquely secrete glycohydrolases
which are nat found in the yeast phase (Gimeno et al. 1992).
qe~ie
PHG requires a unipdar pattern of W i n g , and is controlled by the
rnating type locus (Gimeno et al. 1992). MATWa diploids divide prirnarily with a
unipolar pattern of giowth, where the daughter cells krd and rebud at the free
end, which pemits PHG to ocarr. In contrest, haploids, and MATda or MATda
diploids primarily divide axiaily, whereby the mther and daughter cells bud
adjacent to their cell pole (Gimeno et al. 1992). Oenes which contml budding
patterns indude RSRfBUD1, BUD2, BUD3, BUM end B U M (Bendei and
Pringle. 1989; Chant and HerSkowitz, 1991 ; see Chant and Herskowitz, 1993).
When none of the BU0 genes are fundkning, a iandom W i n g pattern results,
however, when BUOl end BUD2 genes are fundioning, a unipolar pattern
results. When BU03 and BU04 genes also function, an axial pattern of
budding results (Chant and Herskowitz, 1991 ). Chant and Herskowitz (1991 )
suggested a mechanism by which MATala diploids kid with a unipdar pattern;
M A W a diploids produce a mating type encoded repiessor al a2 that represses
the function of either or both BUD 3 and BUb4 genes, allowing a unipdar pattern
of budding to occur.
PHG was only found in MATa/a diploids and not in a or a haploids, or da
or da diploids (Gimeno et al. 1992). 1 his implicated the mating response in the
control of dimorphic switching in S. cerevisiae. The mating or pheromone
response pathway, is one of the rnost thoroughly investigated signal transduction
pathways; however, the discovery of new genes continues to increase this
pathway's complexity (reviewed by Watson et al. 1992; Kurjan, 1 993;
Herskowitz, 1995).
Haploid cells with a and a mating types each secrete a small polypeptide
pheromone. The a mating type haploid releases &factor and a mating type
hapkiû cells secrete a-factor. Recepüon of these pheiomones by cells of the
opposite rnating type triggers a G-protein linked protein kinase signal
transduction cascade t hat cul minates in the activation or inactivation of a nurnber
of pnes which allow mating to occw (Kurjan, 1 993). Celh respond by
undergoing a change in shape to form elongated "schmoo ceIlsa, and the cell
cyde anests in 0 1 so that m l n g can ocarr (Kurjan, 1993). Cell cyde anest is
accomplished by the tmnscirptional inactivation of y d i n genes required foi
Cdc28p protein kinase activity (Kuifan, 1 QQ3). Cydin proteins inte- with
Cddûp protein kinase to fonn a iunctionaî protein eerlneithreonine kinase
cornplex, allowing the cells to proceed through 01 (Watson et al. 1992).
Inactivation of cyclin transcription in response to phemmone binding prevents the
formation of the functional Cln-Cdc28p protein Wnsse cornplex, and arrests the
cell cycle in G1 (Watson et al. 1992). Several genes encoding components of
the pathway itself, for example FUS1 and STE2, are transciiptionally induced by
this pathway. Other genes regulated by the pheromone response signal
transduction pathway indude the gene encdng agglutinin which allows th8 two
mating cells to adhere, genes required for cellular and nuclear fusion, and genes
needed for recovery from cell cycle arrest (Kurjan. 1 993).
Figure 1 illustrates the mating response pathway of S. arevisiae in
schematic fonn. The mting pheromones are glycosylated peptides, 1 2 or 1 3
amino acids in length, and are encoded by the MATa and MATa mating type
alleles, respectively (Watson et al. 1992). Upon reception of the pheromones by
receptors on the opposite mating type cells, the pheromone binds to the receptor
on the extracellular surfam of the yeast ceil membrane (see Fig. 1). This
receptor has seven hydrophobie helioes which span the lipid bilayer, with an
extracellular amino terminal and a cytoplasmic carboxyl terminal. The ieceptor is
linked to a O-protein (a trimeric guanine nudeotide-binding protein), a three
subunit protein with a, pl and y subunits, which coupies the membrane-spanning
receptor molecule to a cytoplasmic effedor moleaile, a protein kinase encoded
by the SE20 gene (Watson 1992; Kurjan, 1993). O-pioteins are activatd
when they release GDP and bind GTP, and have an intrinsic enzymatic Wvity
that hydrdyzes bound GTP back to GDP (Stryei, 1986). All receptors that are
couplecl to their effectois by O-proteins have k e n fwnd to have this seven-helix
structure (Watson, 1992). A number of the genes encding proteins involved in
Flgun 1. Components of the Saochammyces œoerevisiae pheromone response pathway, their utilization in other S. cetwisiae signal transduction pathways, and homologs in UsfMgo maydis. Input for the pheromone response pathway involves reception of pheromones by G protein-linked pheromone receptors in hapîoid cells. -20 protein kinase and a MAP kinase module transduce the signai to the 5181 2 transcription factor, which Wulates the activity of several genes involved in the mating response. Two other pathways, the invasive gmwth pathway of haploid cells and the pseudohyphal gmwth pathway of diploid cells, utilize several of the sarne cornponents; however, pheromone receptors and G- proteins are not required, and other signals pmide the input (likely nitrogen starvation). Neither pathway requires the acüvity of Fus3 or Kssl , but may utilize other undiûcovered MAP kinases. The pheromone response pathway of Ustilago maydis is required for fusion of haploid cells and filamentous growth in the resuhing dikaryon. The acHvity of pheromones and pheromone mceptors and unknown signais provided by the host plant, are required for continued filamentous growth. F uz7, an Ste? MAPKK horndog, and an Ste20-iike protein kinase have ben identifid in U. maydis, while the existence of other components in this pathway an speculated. Involvement of this pathway in the filamentous arowth of U. maydis hadoid ceils has not been evaluated. Csndida aI6ians mai also utilize a simila? signal transduction cascade for the formation of hyphae (not illustrated). An S T E ~ ~ homolog (CPHI, or C H & pseudohyphal regulator) hm been found, and can conect both PHG and mating ôefects in S. cemvisiae ste12 mutants. Adapted from Heiskowitz (1995) and ûanuett (1995).
Ustilago maydis
Haploid Diploid Ir-
Stol 1 [Fj Skl t [y] transcription C F ) Factor
Pheromone Pheromone Invasive Growth
16
the pathway are STE genes. 'STE" stands for sterile; one of the mating
responses elicited by the mating pheromone is cell cyde anest. To select foi
mutants containing mutations in this pathway, hapîoid yeast cells were grown up
on media containing the a factor pheromone. Cells that continued to grow (and
therefore were unable to mate) and fom colonies were those mutants that dd
not respond to the pheromone, and wem therefore non-functional in some part of
the pathway (Watson et el. 1 992). In the absence of the phemmone. the G-
protein exits as a trimer with GDP bound to the a subunit homolog. Binding of
pheromone to the receptor results in an exchange of GD? for GTP in the a
subunit homolog. This causes dissociation of a-GTP from &y, allowing free By to
ad downstream upon the effector molecule (Kurjan, 1993). In the case of the
yeast mating response pathway, the effector appears to be a protein kinase
encoded by STE30 (Herskowitz, 1995). Protein kinases function to
phosphorylate other proteins to activate or inactivate thern, and are the
mdecules that really do the woik in signal transduction pathways. Protein
kinases are phosphotransferases that celalyze the transfer of the y phosphoryl
gmup from ATP to an amino acid side chain of the iedpient protein in the
presence of magnesium. Accepton for the phosphoryl groups are the alcohol
gmups of serine and threonine, and the phenol group of tyrosine (protein-serine,
t hreonine kinase, and piotei n-tyrosine kinase respeaively) (se0 Giiault, 1 993).
The protein kinase effector, STE20, links the G-protein to a MAPK
(mitogen-acthrated protein kinase) module in which a cascede of sequential
phosphorylations occurs (see Fig. 1 ). In S. œrevishe the MAPK module consists
of the MAP kinase kinase kinase, the MAP kinase kinase, encoded by Si€ l 1
and S E 7 respectively, and two MAP kinases encoded by FUS3 and KSSI
(Heiskowitz, 1995). The kinase casde poRlon of the pathway is paiHy
conservecl when compared with other known signal transduction pathways;
Fus3p and Kssl p proteins of the pheromne response pathway have sequeme
similarity to the mammalian MA? kinase gene which responds to extracellular
signais (i.9. marnmalian growth factor) and stimulates growth and diflemntiation
(Costigan et al. 1 994).
The MAP kinase cascade induces the phosphorylation of Ste12p, a
transcription factor, which is then activata and able to bind to a pheromone
response element in the upstream activation sequences of a number of genes
transcriptionaiiy regulated by t his pathway (Kurjan, 1 993). As discussed eailier,
this would include some of the genes encoding components of the pathway itself,
those that confer the ability to mate, and those that arrest the cell cycle at Start in
01. To induce cell cyde arrest, the genes encoding cydins are transcriptionally
inactivated so that an active Clnp-Cdc28p cornplex no longer foms (Kurjan,
1 993).
Genes of the S. cerevisiae pheromone response pathway fall into two
groups with respect to their role in PHG; those which are only utilized in the
pheiornone response pathway in haploid cells, with no known function in dploids,
and those genes wMch play a second role in PHG of diploids and invasive gmwth
of hapîoids (see Fig. 1 ). Lui et al. (1 993), found that the S T E ' , STE 1 1, S E 7
and STEllgenes which encode the putative protein kinase effedor, MAP protein
kinase kinase kinase, MA? kinase kinase homobgs, and the pheromone
rmponse transcription factor, respectivdy, were requiied for ?HG, but not for
nomai yeast growlh in dipldds, suggesting a role for thme genes in PHG. The
same genes, SfE20, S E 1 1, STE7and STE 12 were also found to be required
for the invasive giowlh of haploids (Rokfts and Fink, 1994). The portion of the
18
pheromone respnse pathway encoding the phemmone recepton, and the three
G-protein subunits SE2, S E 3 , STE4, GPA 1 and SE18, respectively, were
found not to be required for bath PHG in MAT& diploids and invasive growth in
haploids (Lui et al. 1 993; Roberts and Rnk, 1 994).
1- af PHG
PHG is induced by poor nitrogen sources - either prdine as the sole
nitmgen source or a low ammonia media. In S. cereuidae, the RAS (rat
sarcorna) pathway is thought to regulate various stress responses (Broach and
Deschennes, 1 990). RAS genes are part of the larger farnily of genes that
encode guanine nudeotide-binding proteins (Watson. 1992) and regulate
different signal transduction pathways in yeast. S. oereuisiae has two RAS
genes - RAS7 and RAS2 (see Uno, 1992) - which are activated with GTP by
the CDC25 protein. Ras proteins activate adenylate cydase which regulates the
intracellular secondary messenger CAMP, which in tum activates CAMP-
dependent protein kinases (see Uno, 1992; Marx, 1993).
To test the mode1 that nitrogen starvation might induce the RAS2 pathway
which le& to PHG, Gimeno et al. (1 992), looked at the effect of constitutively
activating the RAS2 signal transduction pathway on PHG, by transfoming
hapioid and diploid strains with a RAS2vd1g gene. This mutation destroys the O-
protein's GTP hydmlytic activity so that it remains active. iesulting in increased
intracellular CAMP levefs and incmacred sensitivity to nitmgen stantation. Gimeno
et al. (1 992), found that they could induce PHG (in diplob) on both nitmgen poor
and nitrogen rich media by expressing tMs adhiabcl RAS2 gene. consistent with
a mode1 for the involvement of the RAS signal tnnsdudion pathway in bringing
about a switch to PHG in response to nitiogen starvation. Anothei pogeibility
suggested was that Wvated RAS itsel may cause a change in the organismWs
ability to utilize or assimilate nîtrogen (Gimeno et al. 1992). It has recently b e n
suggested by Maikwardt et al. (1 995), t h a PHG is triggered by falli ng amino acid
pods within the cell, and that activateci RAS induces PHG by causing a decrease
in intracellular amino add pools.
Consistent with a nitrogen limiting switch for PHG, several mutations
which interfere with nitrogen utilization have been shown to stimulate
pseudohyphal development. The S M 3 gene (Gimeno et al. 1992) encodes a
component of the endoplasmic reticulum required for amino acid permeases,
including the proline permease. A shr3 mutant which fails to produce a functional
pennease and impairs amino acid uptake, was found to stimulate pseudohyphal
growth. The PPSl gene (Blacketer et al. 1994) encodes a
phosphoribosylpyrophosphate synthase, a key enzyme required for nitrogen
metabolism. This enzyme catalyzes the transfer of the terminal pyrophosphoryl
group of ATP to the C-1 carbon of ribose-5-phosphate. A mutation to this gene
also induced pseudohyphal growth (Blacketer et al. 1 994).
Several genes have been characterized which are invoived in cell
elongation, cell division and other motphological deteminants of PHG.
The PHDI gene (pseudohyphal deteminant), is believed to be a ceIl cycle
regulator contmlling cell cycle related processes such as cell wall biosynthesis
and cell separation. PHDl may have targets such se cydin and chitinase
encading genes (Gimeno and Fink, 1 994). When werexpressed, PHDl
intensifies and acceleiales the PHG response to nitmgen limitation in MATala
dipiob, and allows PHG to occur in nitrogen rich media and in liquid culture
(Gimeno and Fink, 1994). PHDl encodes for a putative transcription factor with
high sequeme similarity to the SM gene of As,ipergiIkrs n&&ns which 1s
20
invohmd in the regulation of conidiophon m~rphogenesis~ PHDl has a DNA-
binding motif related to those found in SW14 and MBP1 of S. cereWae. which
encode transcription factors regulating the cell cyde (Gimeno and Fink, 1994).
Genes implicated in the elongation of budding cells indude those involved
in the control of the cell division cycle. When mutated, cell division cyde mutants
CDC3, CDCIO, CDCI I and CDCIZ grow with an elongated cell phenotype. All
code for proteins that localire to the 10-nm filament iing, believed to be the bud
emergence site (see Blacketer et al. 1995). C. a/krns also has a structure
similar to the 10 nrn filament ring of S. oemvisiae. Antibodies raised against S.
cerevisiae CDC3, CDC10, CLXI 1 and CUCI2 gene products were found to
localize to the filament ring of C. albicans and to the mother bud junction
(Blacketer et el. 1995). Several other genes in S. mevisiae have b e n found to
produce elongated cell phenotypes when mutated. but for reasons unknown.
This would include those involved in cydin-dependent kinase aetivity,
ubiquitination-protein degradation, bud site seledion. the secretory pathway, the
signai transduction in response to high extemal osmolarity, protein kinases and
phosphatases. transcription factois SW14 and SWI 6, and the H+ ATPase (see
Blacketer et al. 1995).
Blacketer et al. (1 993, 1994, 1995) isolated 1 4 €LM (elongated
rnorphology) gene mutants identifid by a consüMhrely expmssed elongated cell
rnorphology on nitrogen rich media, which they pro- functioned to repress
PHG in inappropriate conditions. ELMI encoded a novel protein kinase
homolog, and two ELMgenes comsponded to previously charaderized genes
GRRI (encoding a muitifunctionaî protein, see Blacketer et a/. 1995) and CDCl2.
ELMI, €LW, €LM3 and COCI2 were found to constituitively express dl PHG
charsrcterMcs.
21
Humiôity was found to affect the extent of PHG response, with a humidity
of 55% resulting in the greatest PHG dltferentiation. Blaketer et al. (1 995),
suggested that the agar surface may be alteied in some way by the humkmy ot
the air, and that the inability of PHG forming strains to produce ?HG in liquid
media implicates the importance of surface contact.
PHG is induced by nitrogen staivation and is specific to MATa/MATa
diploids. PHG requires cell elongation, incornpîete cell sepration, invasive
growth and a unipolar W i n g pattern. All of these processes, except unipdar
budding. are induced by nitrogen staivation ( s e Gimeno and Fink, 1994).
Nitrogen starvation is the pfimary signal and rnay be transduced by a RAS2
adenylate cyclase/cAMP pathway (Gimeno et al. 1992). The protein kinase
cascade portion of the yeast phemrnone response pethway (MAP kinase
cascade) is also involved in the transduction of this signal (Liu et al. 1 993) and
the signal leading to the invasive growth of haploids (Roberts and Fink, 1994).
The protein kinase cascade is invohred in producing pseudohyphal cell shape,
but not in determining kidding pattern. A mode1 to explain the specificity of PHG
to MATa/a diploids, propoees that a matirtg type encoded represwr, a l a2,
represses the function of either or both Büû 3 and BUDl genes, allowing a
unipolar pattern of budding and thefefore PHG to occur under conditions of
nitrogen limitation (Chant and Hemkowitz, 1991). Pseudohyphel development
may function to put propagules into prwiously in80~~88/#e suôstrates (Blaaeter
et el. 1993). In addition to nitmgen starvation, humiaty aWor sutface contact
eppear to be impoclant in the expmssion of P M (Bladceter et al. 1995), and
aeiobic conditions are required for the PHG-lik gmwth tom seen in MATa and
MATa haploids (Wright et a/. 1993).
22
Control of dmorphism in Usfilago mayds
The genetic contrd of dimorphism in the coin smut U. rnaydis shows
remadable similarities to that of S. cemvisiae, despite the fact that U. maydis is a
Basidiornycete, and S. cereMsiae an Abcomycete. Ustihgo m a y a undergoes a
yeast to hyphal conversion when yeast-like haploids mate to fomi a hyphal and
pathogenic dikaryon, capable of infecting the host plant and indudng turnour
formation (see Banuett, 1995). This process is analogous to the S. cemwisiae
system where MATa and MATa haploiâs fom a diploid capable of PHG. While
with S. cemvisiee, nitmgen limitation is required for this conversion and is
considerd to be the primary signal in dimorphic switching, the situation is not as
dear with U. maydis ; unknown factors provided by the corn plant are required
for cantinued filamentous growth. A transient filamentous growth between
compatible haploids is possible without the unknown factors, and nitmgen
starvation may also stimulate the conversion in diploiâs heterozygous at the a
and b mating loci (Banuett and Herskowitz, 19943. Analogous to the invasive
growth phenotype of S. cerevisiae haploids, at low pH on synthetic medium
containing NH4+ ions, U. maydis haploids show fully filamentous growth identical
to that of diploids, and are therefoie capable of bypassing the mating requirement
for dimorphic switching under certain cultural conditions (Ruiz-Herrera et al.
1 995).
Ustirno mayas has two mating type loci a and b which are invdved in
dirorphic switching. The a locus governo cell fusion and filamentous gmwth,
and encodes components that are analogous to those found in the phemmone
response pathway of S. œrevisiee. The e locus has two alleles, a l and a2,
each ewoüng a pheromone preainroi (either Mal whidi is 40 amino adds long
and processeci to a 12 amino acid pheromone, or Ma2 which is 38 arnino aQds
long and is processed to 9 amino adds) and phemmone receptor (Mer Pral or
Pm), analogous to the S. cemvisiae a factor receptor encadecl by STE3 (-20%
identity with Step verws 24% identity between Pral and Pra2 themselves) (see
Banuett, 1 995). The existence of anU. mayas Ste* MAPKK homolog encoded
by W7 (Banuett and Herskowitz, 1 994a) and an Stem like effector homolog
(Banuett, 1995). suggests that the Pia receptors are likely to be linked to a G-
pmtein linkeâ MAP kinase cascade anaiogous to that tound in S. oerevisiae
(Banuett, 1995) (se6 Fig. 1). In S. œmvisiae, both pheromone and pheromone
receptor are requi red for mating, but do not function in pseudoh yphal formation.
However. in U. mayaYs, the continued functioning of these components is
required for the continued formation ot filamentous growth, suggesting that the a
locus has an autocrine or self-stimulatory mle in the formation of filaments
(Banuett , 1 995).
The activity of both a and b mating type loci are iequired for filamentous
gfowth (see Banuett, 1995). While the a locus fuMions extracellularly in the
mciling reaction and intracellulaily in an autocrine role, the b locus funclions
intracellulaily and is also required foi pathogenidty. The b locus is multiallelic
and encodes a pair of dissirnilar pioteins b€ and bW, which contain
homeodomain motifs (Banuett, 1995). Horneodomain proteins form a dess of
transcription factor. An active b cornpiex is fonned between M and bW proteins
of different allelic origin to fom a hetemdimer, while those pmsent in a haploid
cell am unable to dimerize (Kamper et ai. 1995). Thur, upon mating. a fundional
b mating type locus encoded, heteiodirneiic, puîativ8 transcription factor is
24
fomed, which functions to maintain the pathogenic filamentous fom. The
downstream targets of this putative transcription factor have not yet been
detemined.
Recent work by Ruiz-Herrera et al. (1 995). indicatm that filamentous
growth can be indu- in haplokb in both liquid and sdid media, and that the b
rnating type gene is not essential to this process under certain cultural conditions.
tt was found that an acid pH (optimally pH 3.0) induces filamentous growth
of haploids without the presence of a compatible haploid. Filament formation
under these conditions did not require activity of the b mating-type locus, as
determined by the induction of filamentous growth in nuIl mating-type b mutants.
Ruiz-Herrera el el. (1 995) founâ that yeast-like cells needed to be starved
and taken to the Go stage to be respnsive to pH. An acid pH in the media was
required both initially and continuously foi the transition; metabdic addification
of initially neutral meâia did not stimulate filamentous growth. The nitrogen
source was also found to be important in bringing about the transition.
Ammonium ions from NH4N& produceâ the best filamentous grawth, followed by
(NH4)2S04 and casamino ad&, while K N a did not stimulate hyphai gmwth.
Aeration of the media was aiso fwnd to stimulate hyphal development (Ruiz-
Henera et el. 1995).
The operation of the proton pump was suggested to be important in
causing the dimoiphic transition and not just intwnsic pH; both NH4+ containing
nitmgen sources, NH4Nq and (NH4)2w4, pmduced filamentous growth and
NH4+ ions are beliwed to require the adhrity of the paon antiport for theif
uptake which causes furthei addification of the media. the NO$ conteining
nitrogen source KNa, however, did not stimulate filamentous growth and does
25
not use the proton pump for uptake, but uses a proton sympart which reâuces
acid'i of the media. Fuithemore, acidifkation of the media was sensitive to
inhibitors of plasma membrane ATPase which is required for proton pump advity
(Ruiz-Herrera et al. 1 995).
Catabolite repression was also believed to be important in the formation of
mycelia. All caibon sources teaed (citrate, soibitol, xyîose, succinate, suciose
and glucose) stimulated hyphal growth when present at 1 % using (NH&S04 as
a nitrogen source. However, high concentrations of g lmse (5 and 10%)
reduced filamentous gmwth, while sudnate, which is poorîy utilized, generated
srnooth and regular hyphae (Ruiz-Herrera et al. 1995).
Cyclic AMP has also been shown to play a role in dimorphic switching in
U. maydis . The uecl gene of U. mayds was found to encode adenylyl cyclase,
the enzyme which pioduces the second messenger CAMP (Gold et al. 1994).
Cydic AMP binds to and inhibits the regulatoty subunit of cAMP-dependent
protein kinase (PKA), which is encoded for in U. m y d s by the ubcl gene (Gold
et al. 1994). This frees the catalytic subunit so that it may act to phosphorylate
various target proteins involved in dmoiphic switching. It is not currently known if
PKA ads to prornotes yeast gronRh or inhibit filamentous growth (Benu@tt, 1995).
Nitrogen levels and sources are known to be important in dimorphic
switching (ûanuett and Heikowitr, 1994; Ruiz-Herrera et el. 1995), and have
been suggested to directly affect CAMP levels (Banuett, 1995). Possible
invoîvernent of the proton pump or catabolite repmsbn in this CAMP pathway
have not been evaîuated.
A gene belonging to the hydrophobin family which was specifically
expnssed in the filamentous dikaryotic phase of U. maydls, was isolated using
differential display (Bolker et al. 1995). Hydmphobins are a secreted group of
proteins which f om a hydmphobic layer and in some cases are believed to play
a mle in the production of aerial hyphae.
Contml of dmrphism in CencAda albicans
Candd albitans demonstrates yeast and hyphal growth, but is also
capable of forming pseudohyphae (Shepherd, 1985). Unlike S. cerewisiae which
appears to require rnating for ?HG, C. albicans switches between yeast and
hyphal growth as a diploid. ln fact, a haploid sexual cyde is not known to exist
for this fungus (me Lui et al. 1994).
While the primary signal stimulating a yeast to PHG transition in S.
cemvisiae may be nitmgen stawation, the case is not as dear with C. albicans;
nitrogen starvation has reporteci to induce mainly yeast growih, and rich media to
stimulate hyphal growth (see Lui et al. 1994). However, as with S. cerevisiae, L-
proline. which is known to be a poor nitrogen source (Cooper, 1982), has been
shown to induce germ-tube formation (see Shepherd et al. 1985), and, in a
medium high in ammonium sulphate, to support yeest-like giowth in C. a1k.m~
(see Girneno et el. 1992). Proline induces the formation of a spedfc prdine
pertnease in C. albricans (see Shepherd et a/. 1985). C a m alMans also
responds to additional environmental aies; an add pH, or the addition of
glucose or cysteine to growth media stimulates a hyphaî to yaast conversion and
senrm stimulates a yeast to hyphal conversion (see Shepheid et al. 1985; Griffin,
1994; Lui et a' 1994 ). Changes in temperature may alao signai a dimorphic
switch. As in U. majds, plasma membrane H+ ATPase aelivity hm d m been
suggested to play a role in the dimorphism of C. al&kans (se0 Kaur and Mishra,
1994). Surface contact also plays a d e in the formation of hyphae in C.
albfcans, as it does with S. cerevisiae; an opaque colony growth f o n , can gmw
as a hypha in liquid culture, only when attached to the surface of a glass plate, or
layer of epithelial cells (Anderson et al. 1989).
Gemtic dissection of dimorphism is difficuit in C. eAbicans, since it has no
known sexual cycle and is diploid. However, transformation and
complementation of S. cerevisiae with C. albhrns genomic libraries prwides a
tractable method around this problem. Using this appmach. Lui et a/. ((1 994)
were able to identify several clones which enhanced PHG. They identifid the
CPH 1 gene (Candida pseudo h yp ha1 regulato r) , encoding a protei n homolog of
the S. wevisiae SE12 encoded transcription factor, which is required for PHG,
invasive gmwth, and mating in S. cereuisiae. When transformed into a S.
cemvisiae ste12 mutant, the C. alMcans CPHl protein was able to correct PHG
and mating defects. This suggested that the same MAP kinase casad8 may be
present in both organisms and that C. elbicens may have an undetected sexual
cyde (Lui et el. 1 994). In C. albicans CPHI double knockwt mutants, hyphal
growth was suppresed on solid media, but was still found in liquiâ media or
senim induced conditions, suggesting that C elMans migM have more than one
pathway to transmit the signal for yeast to hyphai conversion - fiiing with its
ability to respond to severai environmentai stimuli (Lui et al. 1 994).
A pH regulated gene PHRI (pH-responshre gene), which is required for
hyphaî growth, was oôtained from subtradive cDNA libmrim and found to
encode a ceIl surface pmtein with a glycos~phophatidyIimsitoI membrane
anchor (Fonzi et al. 1993). PHRI is expreaasrl at high pH where hyphal growlh
is stimulated, but is not expresseâ at acidic pH levek which favour the formation
of yeast-like growth. Disiuption of PHRl destmyed t k ability of the fungus to
28
gmw as a rnyceliurn. Pioteins with GPI anchois make up a diverse functional
gmup and no mechanism for its action in known, ôut mey play a structural role in
gerrn-tube formation or in the transduction of a signal fmm the environment
(Fonzi et a/. 1 993).
Contml of Omorphism in Mucor spp.
In Wmrspedes. hyphal growth ocairs in an aerobic environment and is
considered to be the normal form of growth . The yeast form occurs under
vadous types of stress, the most general of which is a microaerobic environment
in the presence of a hexose sugar (Ruiz-Herrera, 1993). Oxygen is the primary
moiphopoietic effector and acts independently of the respiratory chain (Borgia et
el. 1985). Dirnorphic switching is believed to be comrolled by an unknown môox
agent which senses oxygen levels (Salado-Hemandez and Ruiz-Herrera, 1 993).
Cydic AMP also plays a role in Mucorspecies ôimoiphism, with high levels
conelating with yeast-like growth (see Orkwski, 1995). A current mode1
suggests that the polyamine putresine, whose level increases during the yeast-
hyphal transition, acts antagonistically with CAMP to control dimorphism t hroug h
a DNA methylation transcriptional control (see Odowdri, 1 995); putresine, which
is formed from omithine by the enzyme omithine decarboxylase (ODC),
decmases CAMP levels by inhibiting adenylyl mase activity, while CAMP itself
represses the formation of ODC. During the yeast-hyphal transition, elevated
levels of putresine are proposed to activate genes controlling polar gIowlh by
effeding their demethylatkn. Reduction of putresine adhiity by
diaminobutanone, a cornpetitor of ODC, has been shown to Mock the yeast-
hyphal transition and the demethylation of nudear DNA that occun duiing the
devdopment of hyphae (see OikwsW, 1995). Ahcor l i ~ ~ ~ m o u s has three RAS
homologues, MRASI, MiUS2and AURAS3 MtUSl ib transcrlbed in gem
tubes and yeasts convetting to hyphae, while MRAS3 is tmsciibeâ in the yeast
form. MRAS2 appears not to be transciibed (see Oilowski, 1995). MRASl may
be a functional equivalent of the S. cemdsise WS2 gene and MRAS3 the
equhralent of S. mevlsae RASI. Oilowski (1 995), suggests that MMSl may
ôe repiessed under microaerobic conditions. and that a fermentable hexose
sugar derepresses MRAS3 to allow yeast-like growth to occur. It is not known if
either MRASl or MRAS3 activates adenylyl cydase activity (Oilowski, 1995).
ConttuI of dmrphism in Ophiostome ulmi
The cultural conditions required to stimulate dimorphic switching in
Ophiostoma ulmi have been determined. and several biochemical studies
suggest some of the genetic mechanisms that might be at work in this organism.
However, apaa frorn the meiotic analysis of a nonsponilating isolate by Richards
(1 994) and his finding that nonsponilation is likely under the control of a single
nuclear locus, there are no published studies on the genetics of dimorphism in O.
ulmi. Ophiostoma ulmi switches between yeast-like and hyphal forms as a
hapkid, while traditionally the other dimorphic fungi discussed have been
characterited as either switching from yeast-like to hyphal growth while
undergdng a haploiâ to diploidldikaryotic transition, or am dipîoid t hroug hout the
switch. Exceptions now indude the haploid switching of S. œrevisiae to the
invasive form (RobeRs and Fink, 1994) to PHG (Wright et al. 1993), and of U.
mawjs hapkids to a filamentous fonn at acidk pH (Ruiz-Herrera et a/. 1995).
The effect of arlarial andions on the dimorphic switching of O. u/mi wem
investigated by Kulkami and Nkkerson (1 981 ). The use of proline as the $018
nitrogen source in a defined liquid media wae found to sthlate yeast-like
grMh, as was the absence of a nitrogen source. ûther nitrogen sources tested,
(NH4)2S04, NHdCI, arginine and asparagine, al1 stimulated hyphal gmwth by
thernselves or in combination with proline. These results suggested that yeast-
like gmwth migM result from the absence of ammonium ions (Kulkarni and
Nickerson, 1989). The possible rdes played by pH and catabolite repression in
this process, as suggested by the woik of Ruiz-Herrera ef el. (1995) in U. maydis,
are difficutt to assess, since pH levels were artifiiaily maintained above pH 3.0
and glucose levels were invariably kept high at 20%. An affect of pH on cel
mrphology was noted, however, as hyphai fragmentation and production of
chlamydospores was reported when pH was allowed to dmp below 3 with media
containing either (NH4)2S04 or NH4CI. The effect of proline in bringing about
yeast-like gmwth may be important to the pathogenidty of 0. ulmi; resistant and
immune dm trees have been shown to have high levels of proline in their nylem
sap (up to 23% of total nitrogen) while susceptible trees have only trace amounts
(Singh and SmaJley, 1969).
A f ~ l e for the second messenger CAMP, in the dimorphic suvitchhg of O.
ukni was suggesteâ by the work of Brunton and G a d (1 989). Intracellular levels
of CAMP were found to incmase during the switch from yeast-like to hyphal
gmwth, and exogenously added nudeosides and nudeotides (induding dibutryryl
CAMP, a lem polar derivative of cAMP which enters the cell more readily) or
yeast extract, were found to promote hyphal g M h . Inhibition of CAMP
phosphodioaterase (POE) br8akôown of CAMP to AMP by theophylline and
cafîeine, also promoted hyphal gmwth (Brunton and Gadd, 1989). Cydic AMP
albicans, BBlaomyoes dematitids and P m d d e s bbresiliensis (Odowski,
1995). Given the alrnost ubiquitous invohrement of CAMP in the dimoiphic switch
of other fungi, il rnust be considered probaMe that such a genetic mechanism
controlling dimorphism is also at woik in O. ulmi.
The role of another second rnessenger system in 0. uhidirnorphism, the
inositol lipid signal pathway, was suggested by the woik of Muthukumar and
Nickerson (1984). and 6~n t0n and Gadd (1991). In this second messenger
system, a cell receptor activates th8 enzyme phospholipase, which functions ta
hydrolyze inositd phospholipids in the plasma membrane, to form inositol
triphosphate and diacylglycerol (see Watson et al. 1 992). Both of these
molecules then act as or through secondeiy messmgers in the cell. The C l s t
rndecule, inositoi triphosphate, causes the release of C s + into the cytoplasm by
binding to Ca*+containing intracellular vesides. The released Ca2+ acting as a
second messenger, binds to calmodulin which then regulates the activity of
several enzymes, including a Caz+CaM-dependent protein kinase. The other
second mesenger molecule, diacylglycerol, remains in the membrane and
activates protein kinase C (PKC), which phasphoiylates various proteins (se0
Watson et el. 1992).
The possiMe rde of this systern in the dimocphic switching of O. ulmi was
imestigated by tieating cells with exogenody supplied Cs+, Ca*+ antagonists
( M l 3 ) and chelaton (EGTA), and six airnodulin inhibitois (see Muthukumar
and Nidcenon, 1 984). Exogenously supplied Ca2+ stimulated hyphal growth,
white the Ca2+ and calmodulin inhibitois all inhibited hyphal growlh, consistent
with the invohrement of a Ca*+-CaM interaction in the fonatkn of hyphal gmwth
(Muthukumar and NUcerson, 1 984). The dmoduîin inhibitor (R24571) wes also
32
founâ to inhibit hyphal growth by Brunton and Gadd (1 991). while the effect of the
PKA activatoi, phorbd myristate acetate (PMA), was to M o r e hyphal growth,
consistent with PKC imolvement and the BCfivity of the second messenger
diacylglycerol in the formation of hyphal growth. lntracellular Ca2+ uptake was
found to be highest in geminating cells, while the exogenous addition of the
other secondary messenger, inositol triphosphate, stimulated hyphal growth
(8runton and Gadd, 1991). These results were consistent with the invohrement
of a inositol lipid signal pathway; however, genetic determination of the existence
of this pathway and identification of both its signal and receptor remain to be
ascertained.
Cytosolic Ca2+ levels may also be modulated by the plasma membrane
H+ -ATPase; by studying the effect of ATPase inhibitors on the uptake of
exogenoudy addeâ Cs+, Gadd and Bninton (1 992) found the uptake of
exogenously addeâ Cazt to be dependent on the electrochemical pmton gradient,
which is generated acioss the plasma membrane by the H+ -ATPase. Ca*+
-calmodulin may also regulate CAMP levels thmugh Ca*+ dependent adenylate
cydase and CAMP phosphocliesterase, thereby linking these second messanger
pathways (see Gadd and Brunton, 1992).
Finally, it seem probable that 0. uld rnay utilire components
homdogous to those of the S. œreVr.Sim pheturnone mponse pathway for
dirnorphic switching. In each of S. csrevisiae, U. mayds and C. aMcans, som
cornponents of this pathway appear to play non-mating roles in dimorphic
switching. A mle for these components in 0. ulmi dimorphism seems likely,
given the existence of the CPHI gene in C. tiWuuw, wh&h encodes a homdog
to the S. œmMsiee SE12 encoded transdpion &toi; C. a M a n s hes no
33
known sexual stage and theoretically no use for this gene in a mating function.
The existence of homologous pheromone msponse pathway components in O.
uhnl must remain open to speculation.
Transformation may be defined as the genetic transfer of pure extractecl
DNA from one organism into the cellls of another, which brings about a
hereditary change in the second cell type. DNA-mediateci transformation of a
filamentous fungus was first reported for Neumspon, c m s a (Mishra and Tatum,
1973). Since then, transformation systems have b e n developed for numemus
filamentous fungi (Fincham, 1 989; Fi nkelstein, 1992).
Severai st rategies for t ransfoimation of fungi have k e n developed
induding the addition of DNA to protoplasts. electroporated cells, or to intact cells
made permeable by treatment with alkali cations such as Li+ (Finkelstein, 1992),
by ballistic delivery into cells using a gene gun, or by the vigorws agitation of
DNA and cells wit h glas beads (Costanzo and Fox, 1 988). Most transformations
of filamentous fungi utilize a strategy similar to that first used with S. mmvisiae
(Hinnen et al, 1 978), where pmtoplasts (cells devoid of cell walls) are
enzymatically pioduced and resuspnded in an osmticaily supporting media.
The transforming DNA is then added to the protoplasts along with Ca&, which
facilitates DNA uptake by neutralizing the negativeiy charged phosphates in the
DNA and protoplast membranes. DNA uptake is effected by the addition of the
fusogen pdyethylene glycol (PEG), which causes protoplmts to dump and may
fadlitate trapping ot DNA (Fincham, 1989). Regeneration of protoplast cell walls
ocarrrr on an osrnotically balanced m d u m and the rwîting œlls are screened
to identify transfomed cells. Typical strategies loi the seldon of transtonnants
are based on complementation of auxotrophic mutations to piototmphy, dnig
34
resistance conferred by the incorporation of a drug resistance gene into the
vector DNA. or visual seleclion of Bgalactosidase activity on X-gal containing
media by incorporation of the Iacïgene into the vector DNA.
Traditional methods of mutagenesis typically use either chernical
compounâs, such as ethyl methanesulfonate, nitrosoguanidine, or UV radiation to
alter the DNA in a living cell. The mutagenized cells are then screened to iôentify
mutants with altereâ characteristics, such as temperature sensitivity, auxotrophy,
morphological or developrnental changes, etc. With these rnethods, the location
of the mutation is unmarked.
However, using transfonational mutagenesis, a DNA vector is
transfomed into a cell, where it integrates into the chromosomes and serves as a
DNA tag. In targeted transformationai mutagenesis, the transforming vector
contains regions of known homology to the genornic DNA and will preferentially
integrate into those sites in the chromosome to which it shares homology. This
technique may be used to replace one ailele with another (gene replacement) or
c m be used to produce nuIl alleles (gene disruption), in cases where the
expression and function of a gene is b ing studied or moâified. With random
transformationai mutagenesis, however, the transfoming vector DNA contains no
known homology to the genomic DNA of the cell being transformed. Integration
of the vector into the chromosomes should occur randomly and interrupl, alter or
destioy, the activity of various genes or genetic elements (Royer et al. 1991).
Vectois used in random transformational mutagenesis typically contain an
antibiotic gene such as hygiomydn phos(rhotmn8feram (hph), to allow foi the
selection of transfomants They aîso contain a bacîetiai antibiotk *ne, wch as
chloramphenicol aoetyltransterase (CAT), and an Ml 3 odgin of replication to
35
facilitate their retrievaî by plasmid IBSCU~ (Orbach et al. 1988) in E. ooli. Using
plasmid rescue, genomic DNA from the transformant is digested with a restriction
enzyme which does not cut the vector, but arts into the flanking genomic DNA.
Ugadion of genomic DNA. followed by transformation into E. coi with antibiotic
seledon, allowç the drculafited recombinant plasmid to be retrieved. The
recombinant plasmid contains portions of the intemipted gendgenetic element,
which can then be used to screen a wild type library and retrieve clones
containing the conesponding uninterrupted genelgenetic element.
A high frequency transformation system was developed for 0. ulmi by
Royer el al. (1991) using the protoplast CACI2/PEG methd of DNA delivery.
Protoplasts were generated from the predominantîy uninudeate haploid yeast-
like celk of O. ulmi (Brasier, 1 988) using the Trichodema viw enzyme
preparetion Novozyme 234, which contains a cornplex mixture of hydrd ytic
enzymes, indudng 1 &glucanase and chitinase (Fincham, 1989). One of the
transformation vectors used in this study, pPS57, contained no known homology
to 0. ulmi genomic DNA and was detemined to integrate randomly, suggesting
its suitability for random transformation J mutagenesis (Royer et al. 1 991 ). The
vector contained the hygromycin phosphotransferase gene (hph) driven by the
isopenicillin N synthese promoter of PeniCiIbum chlysogemm for seledion of
transfonnants on hygmrnycin containing media. An Ml3 origin of replication and
chlommphenicol acetyltransferase (CAT) gene #rom E. CON allowed the
intempted gene/genetic sequences to ô8 retrieved in E. W B by plasmiâ rescue.
Udng pPS57, random transformationai mutagenesis was subseqwntly
perfonned on O. u/mi to generate the d l morphology mutant (Royer et al.
1991, 1997).
Objectives
The pilmary objective of this study was to futther investigate the use of
transfomational mutagenesis as a means of generating mutants with an aîtered
dimorphic distribution, using the aggressive VA30 strain of Ophiostom ulmi.
A concunent objective was to characterize any mutants rewvered with
respect to the following questions:
U Are the mutants tagged by the transforming DNA or are they
nontagged?
ii/ Are chmmosomal length polymorphisms or other chromosomal
alterations açsociated with any of the mutants studied?
iii/ Are changes in DNA methylation patterns involved in switching fmm
hyphal to budding cell morphologies?
iv/ What an, the other relevant ôiological characteristics of the mutants
(0.g.. colony and micmscopic morphology, radial growth rates, etc.)?
The technique was evaluated utilizing the nonhomologous randornly-
integrating vector, pPS57, with the aggressive VA30 strain of 0. ulmi. The
purpose of the transformational mutagenesis, was to generate mutants defective
in their ability to grow predominantly in either b-ng-yeast or hyphal forms.
ulmi
For this study, the aggmssive VA3û strain wae chosen to allow mutants
aff8C1ed in their dimrphic distribution to k evaluated for changes in vinilence
37
with host plants at a Iater date. The integrative plasrnid uæd in this study,
pPS57, had previously b e n used to effidently transfoim the MH75 strain to
hygromycin resistance with a reportedly random pattern of integration as
determined by CHEF and RFLP analysis (Royer et el. 1991). Part of the work
containeci within this thesis was an attempt to establish a screening strategy for
the recovery of VA30 dimorphic mutants.
Ophiostoma ulmi as a "model dïmorphc tbqp~s'
Their were several reasons for undertaking a genetic study of fungal
dimorphism in O. ulmi. Firstly , it provided a tractable systern : Ophiosforna ulmi
grows vegetatively as a haploid and can be readily mated to produce meiotic
progeny (Holmes 1977; Asina and Hubbes 1980; Brasier 1981 ; Marshal et al.
1982), it can be tiansformed efficiently (up to 4 x 103 transformantslpg DNA per
1 o7 protoplasts, Royer et al. 1 991 ), budâng-yeast or hyphal phases can be
induced in the hapldd state by altering cultural conditions (Kulkarni and
Nickerson, 1981), and it presents less of a safety concern than the human
dimorphic pathogens. Secondly, an understanding of the genetic bads of
dimorphic growth could help define the ide that dimorphism plays in determining
Oplriostoma uhnivirulence on the host plant. The relationship ôetween
dimorphic growth and pathogenicity has been motivation in the past for the study
of fungal dimorphism in both plant and human dimoiphic pathogens.
C&ss~*~t ion of mutants
In this study, a genetic anaîyds d âimorphic mutants pmducecl by
transformation ai mutagenesis experiments, was canied out in order to dassify
38
the mutants as having arisen either by the insertion of pPS57 vedor into a
gene/genetic element which causes the obseweâ mutant phenotype (a tagged
mutation), or having arisen by another rnechanism (a nontagged mutation).
A nurnber of dimorphic mutants were recovered and partiaîly analyzed.
Most of the genetic analysis undertaken, was directed towards a single O. ulmi
transformant, designated yml for "yeast mutant 1 ." The yml mutant had a stable
phenotype for six months, but m a n to produce fast growing sectom with a fluffy
morphology, unlike the phenotype anbicipated from a simpîe reversion mutation.
Another objecîive of this study, was to attempt to idenüfy the nature of the
mutationls causing the ymt and ymt sector phenotypes, both @or to, and
following the meiotic analysis results.
Anabsis for reamngements and chromosoma/ allerations
Sectors were examined for possible rearrangement of pPS57, using
Southern analysis of yml and several yml sector isolates, with a*P-labelled
pPS57 as a probe. Wild type VA30, yml mutant, and yml sectom were also
examined for chromosomal differences, using contour-clamped homogeneous
electric field (CHEF) gel analysis of ethidium bromide stained chromosomal DNA
(chDNA) patterns. and by Southem analysis of CHEF gels using pPS57 and the
cloned rDNA sequence of SchizophyIIum commune as 3Waûelled probes.
Anawsis of DAU methyletion
Wild type VA30, yml and yml secîors wem examined for evidence of
DNA methylation. Regulation of fungal dimorphism by a DNA methylation
mechanism has been suggested for M w r m. (Cano et al. 1 988; Oilowski.
1995), and in C. alWins, levels of S-rnethykyt6sine (5mC) have b e n found to
be greater in the yeast phase than in the hyphaî phase (Russell et al. 1 987).
39
Transforming vector DNA has also been shown to becorne subject to methylation
in the Ascomycete Neumslipora cmssa (Bull and W~ott@n, 1984; Pandit and
Russo, 1992).
Materials and Methods
Materials
Unless specified, all chernicals were obtained from either Sigma Chernical Co.,
Fisher Çcientific, or BDH (Toronto, Ont.) and were of reagent grade. Restriction
enzymes were purchased from eithei BiofCan (Mississauga, Ont.) or Pharmacia.
Strains and cuituml cionditions
Ophiostoma ulmi strain VA30, originally isolated in Viiginia, was oMained fmm
S. Takai (Sauit Ste. Marie, Ont.). Strain MH75, isolated In Toronto in 1975, was
obtained from M. Hubbes (Toronto, Ont.). Strains 173, 175, M3096 and PPi used
for mating crosses, were also obtained from M. Hubbes. Strains CESS1 GK, W2
and W2-TOL alço used for mating crosses, were oôtained from L. Bernier (Univ.
Laval, Quebec City). CESS16K was originally obtained from S. Takai, W2 fiom
C. Brasier (UK) and W2-TOL from J. Webber (UK). Stock cultures of VA30,
MH75,173,175, M3096, PPi, CESS1 GK, W2, WPTOL, and VA30 derived
muîants yml, yml sectoi, 26,158,94-7-27-1,94-7-27-2,94-7-27-3,94-8-4-3,
94-8-4-5,94-8-4-6,99-8-8-20 and 94-8-8-30 are meintained in 10%
dirnethylsulforide (DMSO) at =7O0C. For -70 aC preseivation of yml , sarnples
were taken from the thid seilal transfer after purification from original
transtorrnetion plates (ym l C, J, X, Y, 2). For sectored isolates of ym l (ym l Jb,
M, O, 0, hyphaî tips wen transferred h m the maipins of the tastest giowing
patt of the sectored colony and serially transfened twice. Material fmrn this last
transfer was preserved at -70 OC.
Working cultures were streaked out from -70°C stocks ont0 complete
media (CM) agar plates (see below) and allowed to grow for 5 to 7 days at 25OC.
then resîmaked onîo fresh CM agar and stored et 25% in the dark. Complete
media contained per Mer of double didilled water: 1g KH2P04; 0.59 MgSO,
*7H20; O. 1 g CaCI, 2Hz0; 500 pg H3BO3 ; 400 pg ZnSO, -7H20; 400 pg
MnSO. *7H20; 200 pg Na2Mo04 .2H,O; 200 pg FeCIS -6H20; 40 pg CuS04
5H20; 100 pg pyridoxine-HCI; 20g sucrose; 1 . l g proline as a nitrogen soum.
For complete media (CM), (NH4),S04 at 1.32gA was used as a nitrogen source in
place of proline, and 5 g/L of malt extract (Difco , Detrol, MI) and 5 g/L of yeast
extract (Difco) were added. For CM plates, Bacto-agar (Difco) was added at
20g/L. For minimal media plates, agarose (Sigma) was adâed at 20glL.
For protoplasüng experiments, where budding-yeast cell cultures of
O. ulmi were required, 100 ml cultures of liquid minimal medium in 250 ml
Eilenmeyer tasks were inoculated with 5 mm diameter hyphal plugs taken from
the margin of an adively growing CM culture of VA30 or MH75. Cultures were
typically set up in tripliate or in batches of up to 9 flesks when larger amounts of
material were required, and grown at 25OC in the da& on a shaker at 150 rpm for
7 to 9 days.
Phsrnid pPS57
Plasmid pPS57 was obtained from P. Skatrud (Eli Lilly Corp.). Plasmid
pPS57 consists of the hygromycin phoephotransfeiase (hm) gene of Eschetjchia
coi fused to the isopenicillin N synthetaee promoter of Penia'lum chrysoq8nurnI
the chlorarnphenicd acetyltransfera$a gene ot E. COI, and the Ml 3 ongin of
replication. Phsrnid vector DNA was used in both circulai and linear forms in
transformation expeiiments. Vector DNA was isolatecf using an alkaline lysis
pmtocol (see section on DNA isolation below). Unearized vedor was prepared
by Hind III (Sigma) digestion. foltowed by phenoVchlorofom and chkrofom
extractions, ethanol precipitation, and resuspension in ddH8.
Pmtq&sting of Ophiostome ulmi mIIs
Piatoplasts were generated from O. u/mi budding-yeast cells using the
procedure developed by Royer et al. (1 991 ). Prior to protoplasting, cell densities
of VA30 or MH75 100ml budding-yeast cell liquid media cultures, were rneasured
with a haemocytometer. Cells were collectd ont0 steiile 1.2pm membranes
(Gelman) using vacuum filtration with a Nalgene filter apparatus. Solutions were
sterilized either by autodaving or by filtration through 0.2prn disposable syringe
filters (Uniflo). Filter apparatuses were sterilized by autodari ng . Fitters were
removed with forceps and cells releaseâ into 50 ml polypropylene centrifuge
tubes (Falcon) containing 50ml of 25mM 2-mercaptoethanol. 5mM NeEDTA.
Cells were pretreated in this solution by gentle agitation for 20 minutes on an
Mams Nutator (Becton, Dickinson. Parsippany, N.J.) and centrituged in an IEC
centrifuge for 10 minutes on setting 7. The pelleted cells were washed with 50 ml
of filtei sterilized 1 M h@S04, then pelleted again at setting 7 for 10 min. Cells
were digested with 10 ml of filter sterilized Novozyme 234 (1 0 rng/ml) in 1 M
MgS04 for 2 hrs with agitation on the Nutator et m m temp. Centrifugation for 5
minutes at setting 2 separateci reddual unprotoplasted cells from the protoplasts
which remained in the supernatant. Supematants were transferreâ to new tubes
and six volumes of 0.6 M KCI edded to m v e i pmtopiasts by centrifugation foi
20 minutes at setting 1 on an IEC centrifuge. Pellets were washed with STC
buffer (1 M sorbitol, 25 mM f RIS-HCl pH 7.5,50 mM CaCld, opun for 15 min. on
setting 1 and resuspended in STC buffer. Memurement of protopiast dendly
42
was made using a haemocytometer, and cell density aâjusted to give a final
concentration of approximately 1 x 108 protoplest per 1 ml of a solution
containing 800 pl STC ,200 pl PEG-TC (PEG 4000(66%) in 25 mM TRIS-HCI,
pH 7.5,50 mM CaCIJ and 10 pl of dimethylsulfoxide (DMSO). Aliquots of 200 FI
(2 x 1 O7 protopiasts) were placed into Eppendorl tubes and fiozen at -70°C.
Tmsîbmation
Transformation reactions and no-DNA control reactions were cartied out in
50 ml polypropylene centrifuge tubes following the prooedure developed by
Royer et al. (1 991). Aliquots of protoplagts (2 x 107 protopiastS/20Opl) were
removeâ from -70 O C storage and thawed on ice. Circular or Hind III linearized
pPS57 vector DNA (1 pg) was added to the protoplasts, followed by 1 pl 2-
mercaptoethanol and 50pI of PEG-TC solution. No-DNA transformation cuntrols
contained ail of the above components, except vector DNA, for which sterile
ddHIO was substituted. Controls were incubated at 22OC for 20 minutes followed
by the addition of 2.5ml of PEG-TC solution in aliquots of one drop. 0.5ml and
2ml. Tubes were incubated for 20 minutes at m m temperature followed by the
sequential addition of 1 , 5 and 30ml of STC. Protoplasts were pelleteci by
centrifugation at setting 3 in an IEC centrifuge for 20 minutes and the pellet
resuspenôed in 1 ml of CM containing 0.6M sucrose. Sampîes were incubated for
3-5 hm at room temperature. Aliquots of I O @ or 1 OOCJ of the transfomation
reaaion were pîated out onto CM agar mntaining 0.6M suciose and hygmmycin
at 200Cie/ml, and spiead over the surlace diredly, or more oommonly, an overlay
technique was utilized to allow for a more even wparation of the colonies;
aliquots of the transformation mix were added to Sml of CM containing 0.6M
suciose and low rnelting point agame (7.5gA) at 45%, mixed, then ovedaid
onto CM plates containing 0.6M sucrose and hyglomydn at 200pglml. The
following controls were performed:
V To determine the total number of viable protoplasts and unprotoplasted cells
in transformations and no-DNA transformation contrds, 1 O@ of a 100% dilution
of the reactions were plated ont0 CM plates containing 0.6 M suciwe, without
hygromycin, using a CM overlay which contained 0.6M sucrose.
ii/ To determine the number of unprotopiasted budding-yeast cells remaining
(control for protoplasting reaction), 10pl of a 1 OOx dilution of the
transformation reaction and no-DNA contiols were piated ont0 CM plates
which did not contain 0.6M sucrose as an osrnoticum, using a CM overlay
also without 0.6M sucrose.
iiü To detemine the etfectiveness of hygromycin for the selection for
transformants, 10pl or 100@ of the no-DNA control reactions were plated ont0
CM plates containing both 0.6M sucrose and hygromycin at 200pgiml, using a
CM overlay which contained 0.6M sucrose.
Screening strategy lor reoovery of dmorphic mutants on solH media
To select for 0. ulmi mutants with a budding-yeast cell mutant phenotype,
transformation plates were initially exarnined for the presence of budding-yeast
cell colonies. However, with the detemination that selection on CM plates
containing hygromyan (2Wpg/ml) and 0.6M sucrose induced budding-yeast
gmwîh in VA30 and VA30 protoplast regenerates, the screening strategy for
selection of mutants haâ to be altered. Subsequently, a screening strategy was
adopteci whereby transformation plates were obsenred for the presence of
coknies consisting only of krdding-yea8t cells which were growing fagtei than
those found on the no-DNA control plates. These cdonies weie transfened to
numbered grided CM plates withwt hygmrnydn seldon using an inoculation
loop, and examined for continued krdding-yeast cell growth for 7-10 days.
Colonies which reveited back to a wild phenotype were discaideô. while those
which continued to display a predominantly budding-yeast cell phenotype on CM
plates were selected and puilfied (see below).
A second strategy for the recovery of yeast-like mutants was also
empîoyed. Over a peiiod of approximately 3 weeks, most colonies wem
observeci to accumulate pigment with aga Yeast-iike colonies which meintained
an unpigrnented appearance were transfened to CM plates and obsented for
continuing budding-yeast cell growt h.
To select for hyphal mutants with a reduced budding-yeast cell gmwth.
hyphal colonies were transfened from transformation plates and grown on CM
plates. Colonies were examined under a dissecting microscope (Zeiss) and
regular light microscopes (Zeiss) for reduced buMing-yeast cell gmwth. After
purification (see below), selected colonies were grown up in minimal media (MM)
liquiâ shake culture and examined for the relative amounts of yeast to hyphal
gmwth using VA30 as a control.
DNA isolations
For the routine production of O. ulmi material used in DNA isolations.
1 Wrnl CM liquid aiitures were collecteci in 50ml (Falcon) polypropylene tubes by
centrifugation in an IEC centrifuge on the high setting foi 10 minutes. Matefial
was collected and placed ont0 miradoth, wrapped in a layer of cheeædoth and
pîaced into liquid nitrogen. Samples were lyophilized foi 3 to 4 days and stored
with silico gel desiccant at -20°C. DNA was iwiated using a miniprep piocedure
(Zolan and Pukkila, 1986).
Plasmid pPS57 was transformeci into DH5a competent cells (Gibco BRL)
using a bacterial transformation procedure (Sambmk et al. 1989) and isolated
using an alkaline lysis protocol (Maniatis et al. 1982).
Restfiction digesiions, gel electrophoresis and Southem anaîysis
Restriction digestions were carried out using restriction endonudeases
(BioiCan or Pharmada) following the manufecturers instructions with suppîied
buffeis. DNA electrophoresis was performed using 0.8% agarose gels with fBE
buffer and gels stained with ethidium bromide for viewing and photography.
Transfer of DNAs was petformeci with GeneScreenPlus (Dupont) m r d i n g to
the manufadureh instructions. Hybridizations were peiformed overnight at 6S°C
(Maniatis et al, 1982). Radioadive DNA probes were prepared using a ni&-
translation kit (Bet hesda Research Labo ratones, Gai the rsôurg , MD).
Meiotic anaîysis
Matings were petformed as outlined by Bernier (1 988). E h Sapwood
Agar (ESA) phtes weie used for rnating reactions. Elm wood was obtained from
M. Hubôes (Toronto, Ont.) and milled through a mesh screen. The milled wood
was pmautoclaved for 20 minutes and inôividual plates prepared by mixing 3g of
milled elm wood with 5ûml ddH,O and 1g of agar. This was autodaved for 20
minutes, allowed to cool and paured into single Petri dishes. Plates were aîkwed
to sit ovemight in a transferhood with laminar flow to reduce surface moisaire.
W n g s were initially perfomied by pledng 5 mm plugs (rom both parent cultures
on the plate surlace. Later matings wem perforrned by allowing a 5 mm plug
from the fernale parent strein to colonize the center of the plate for 10 days.
Budding-yeast ce8 suspensions of the mutants being analyzed were prepared in
46
ddH20 and then spread over the surface of the plate containing the female
parent. The following controls were performed:
V Mutants were meied to wild type VA30 as a negatieve contmls.
iV The female parent strain was mated with itself as a negative contml.
iiü VA30 was mated with the female parent strain as a positive control.
Plates were exarnined for the presence of peritheda on a weeWy basis
using a dissecting scope. Perithecia usually appeared after 4-6 weeks for
succassful matings and ascospores were collected as follows; using a dissecting
scope in a transfer hood, sterilized pins were touched to the exudeâ spores at the
tops of individual perithecia and the spores from 5 perithecia cornbined into 2ml
sterile MH20 or 1 perithecium/200pI where fewer peritheda were available.
Dilutions were made (10X) and 200@ aliquots plated onto CM and CM containing
hygromycin at 400 vg/ml. Plates were examined for wiid type and mutant
colonies and observations recorded induding the number of each colony type.
Between 3 and 7 days, individual colonies frorn CM plates Wthout hygromycin
were transferred to CM plates diviâed by a grid pattern, and allowed to grow for 3
days. Transfers from colony margins were made and replated ont0 individual CM
plates. Cultures were grown for 10 days at 2S°C in the daik befoie being used
for DNA isolations. Stocks (-70°C) were prepared fo i 50 ymlXW2-TOL progeny.
CHEF electmphoresis and Southem awsis
Protoplasts were prepared as describeci above, and embedded in low
melting preparative grade agarose plugs (BioRad) at a concentration of 4x1@
protoplasts/ml, then treated and stoied in 50mM EDTA at 4% (Emmpelli.
personal communication). CHEF gels were run using a CHEF-OR II system
(BioRad, Mississauga, Ont.) in 0.5% T6E butfer at 14%. Gels were macle with
0.6% chiornosornai grade agamm (BioRad) and eîectrophoresed at 40V with a
47
swi?ch interval mmp of 50 to 110 minutes over 250 hours. A chmmosomal DNA
pnparation of Schizosacchammyœs pombe strain 972 (BioRed) was wed as a
mdeailar sire standard. Following dectrophoresis, geb were stained with
etMdium bmmide and photographed using a gel digitizer (BioRad). Transfer to
Genescreenflus (New England Nudear) in 0.4N NaOH, was pertormed as
indicateâ in the GenescieenPlus manuai (Red and Mann, 1985). Hybridlzatlons
weie performed ovemight at 65OC (Maniatis et al. 1982). Radioadive DNA
probes wem prepaied using a ni&-translation kit (Bathesda Reseaich
Laboratories, Gaitherskirg. MD).
DNA methylaton measurements
For Msp VHpa II isoschizorner analyses, DNAs (1 5pg) weie digested
ovemight at 37OC, and electmphoresed on 0.7% T E agarose gels. For 5.
azacytidne experiments, CM plates containing Bazacytidine at Opglml, 1 Opg/ml,
50Cig/ml, 100pghnl and SOOpgiml, in triplicate, were innoculated with 5 mm
diameter hyphal piugs taken frorn the margin of actively growing cultures of
VA30, yml and yml sectoi. Cultures were grown f o i 9 days ai 25OC in the da&,
and observed for phenotypic differences. Three measurements of cdony
diameter were made for each plate.
Hygmmycin oontaining sel8CIion media aitets VA30 phenutype
Initial transformation experiments were perforrned with strain VA30 using
drcularized pPS57 vector. Transformed VA30 protoplasts were plated onto CM
solid media containing 0.6M sucrose and hygrornycin at 200pg/ml (CM+O.GM
sucrase+hyg.2ûô~g/mI), wit h a CM+O.GM sucrose agame overlay . Unexpectedly, numemus yeast-like colonies composed entirely or sometimes
parily of budding-yeast cells, were observed on transfomation plates. These
colonies had a shiny smooth and convex shape, similar in appearance to certain
yeast or bacterial colonies. Transformation plates also containecl colonies with a
hyphal phenotype more typical of VA30, and colonies of intermediate
morphologies containing some hyphal and budding-yeast forms of growth.
Colonies were also observed on "no-DNA" transformation control plates (also
CM4.6M sucmse+hyg.200pg/mI with an overlay), however, theæ were
extremely slow growing, yeast-like, of a uniform sin, and nevei fast growing or
partly hyphal, li ke the colonies found for the actual transfomation readions. On
control plates not containing hygromyan (CM+O.BM sucrose = total viable
piotoplasb and unprotoplasted celb), al1 colonies, either from the no-DNA control
reaction or the transformation reaction, were of a phenotype msemMing that of
wild type strain VA30, but with a greater proportion of budding-yeast ceils than
what would typically be found for VA30 on CM plates not wntaining 0.6M
sucrose. Several of the slow gmwing yeast-like colonies were transfened ftom
transformation plates (CM4.6M sucr~se+hyg.200~ml) to CM plates. All
reveited to the fast growing, mainly-hyphel phenotype of wild type strain VA30,
suggesting that these colonies were not Wng-yeast mutants, and that the
CM4.6M sucrose+hyg.2ûûpg/ml seledive media had induced budding-yeast
growth. When cell suspensions of the budding-yeast cells were diluted and
plated onto CM plates. only colonies with a normal VA30 phenotype wem
obsewed, demonstrating that no contaminant was present.
By plating VA30 and three of the hygromycin resistant budding-yeast cell
VA30 protoplast regenerates onto CM, CM4.6M sucicwe, and
CM+hyg.200pg/ml plates, it was found thai both high levels of sucrose (O.BM),
and to a much greater extent, hygmmycin at 200 pgirnl, caused kidding-yeast
cell gmwlh to occur. This result was surprising given that the identical
transformation protocol and selection conditions had been used with drain
MH75, and that no phenotypic changes or induction of yeast-like growth of
protoplast regenerates on transformation plates had been reported (Royer et al.
1991 ).
The growth of wiîd type strains VA30 and MH75 was examined on CM
plates, and on CM plates containing hygromycin at 2Wpg/ml- the concentration
of hygmnydn used by Royer et al. (1 991) for the seleclion of MH75
transformants. Hygromydn at 2ûûpglml induced slow budding-yeast growth in
MH75 as well as VA30 (Fig .2A).
The slow rate of growth of many of the colonies on transformation plates
sug-sted that a large number may have been unstaôîe transfomants or
transfomants with poor hph expression. To determine if some of the cdonies
wen unstable transformants, the growth of 104 randomly selecled colonies with
varying growth rates and morphologies was evaîuated under continued
hygmmyan seledon for a 30 day peiiod. Cdonies were selected from
Fig. 2. Cornpison of mutant and wild type colony growth. Colony morphologies and radial growth rates wen examined on complete medium (CM), CM containing hygromycin at 2 0 I(
ml, and minimal medium (MM) plates after 15 days of growth at 2S°C. A; wild type strains VA30 and MH75. B; mutants col1 and yml.
transformation plates where the cirwlar plasrnid, pPS57, had been uHlized in the
transfomation reactions. Colonies from transformation plates and
nontransformed VA30 (in tripkate), were initidly grown on CM plates containing
hygromycin at 200pg/ml, and 0.5cm plugs of 7 day old cdonies transfened to CM
plates and CM plates containing hygromycin at 200pglrnl. Radial gmwîh was
assessed after 3,9 and 30 days, and compareci to nontransformed strain VA30.
On CM plates, el1 104 cdonies and the VA30 nontransformed control reverted to
a wiîd type fast and predominantly hyphal fonn of growth (Table l), confirming
that the hygrornycin-containing selection media had induced the mutant
phenotypes, and that none were caused by actual mutations. Under cantinued
hygromycin seledion, a polarization of growth and cdony morphologies
occuned; by day 30, 84 of the 104 cdonies displayed a very slow rate of yeast-
like growth similar to that of the rontransfoimed VA30 control (Table l), while
only 20 cdonies developed a fast growing hyphai rnorphology. The numerical
results of Table 1 and the obsenred progression of growth foi individual colonies
(data not shown) suggested that the majority of slow and fast growing yeast-like
colonies were likely to be unstable transformants which originally contained
vector DNA (presumabîy unintegrated), but then loat the vector with the hph gene
over the 30 dey period, while those identified on transformation plates as being a
mixture of yeast and hyphal growth or of a hyphal mophology (1 9 of the 104
cdonies) wen originally stable transfomants or had staüely integrateâ pPS57
durlng the 30 day pend of growth; the later is suggested to have ocaiired in one
isolate, since 19 colonies were hyphal on original transformation plates, and this
increased to 20 by day 9 (Taôîe 1 ). When colonies were grown on CM plates
without hygromydn selective preswre for six deys prkr to transfer to
CM+hyg.200pg/ml piatm, 21 fast giowing hyphai cdonies were oôtained (Table
1. far right column), suggesting that selecîive pmmm by hygmmycin toi the
Table 1. Phenotypes of colonies on transformation plates. and upon transfer to CM plates with and without hygromycin, observed at various time periods.
Number and phenotype of donies on:
Original CM CMthyg. CMthyg. CM+hyg. W h y g . der Tnnsforrnation Plate 3 daya 3 dam 9 days 30 deys 6 dayi growth
(pbmtYP@) on CM. O days
integlation of plasmid was negligible this late after the transformation and
Southem analysis was performed on 12 of 104 cdonies to definitively
identify which colony types wem transformants and which were not, and
detemine if any of the slower growing colonies may have been transfonants
which poorly expressed the hph gene. Four slow giowing, yeast-like colonies,
two fast growing, yeast-like colonies, one slow growing, partly hyphal cdony, and
five fast growing, mainly hyphal colonies were selected. Colonies were single
spore purified and material for DNA extraction was gmwn in CM liquid media, as
described in the Materials and Methods. Undigesteâ DNAs from the 12 colonies
and VA30 negative control were probed with 3*P-laôelled pPS57 (Fig. 3). None
of the slow or fast growing yeast-like colonies contained inserts (lanes 4 through
9 and 22 through 27 respectively). Only the slow growing, partly hyphal colony
(colony 1 54, lanes 1 2 and 1 3) and, surpiisingl y, only 2 of the 5 fast growing
hyphal colonies (colony 128, lanes 10 and 1 1, and colony 157, lanes 14 and 15)
contained an inser?.
VA30 versus MH75 translbrmaîion; tmnslbmtion with cimlar and linear vector,
and ~~ Iec t ion with varying concentratons of hygmmyun
A transformation expriment cornparhg strains MH75 and VA30 under
different transformation and selection conditions was performed dong with no-
DNA contrd reactions. There were several masons for pedoming this
experiment: Fimtly, to detemine why VA30 protoplast regenerates were affeded
by hygromycin, while Royer et al. (1991) reported no effects with protoplast
regeneiates of MH75. Secondly, the optimal levd of hygmmyun for selection of
VA30 transfomnts might be different than what had been detennined for MH75.
Signilicantly dffeient levels of hygromycin wnsitivity have, foi instance, b n
2324 a26 2728 . " .
. . U.lkb- ' "." . ', o. .
Lt B= œ- . - 4.4 kb-
. . . . ' - .
Fig. 3. Southem hybridization of undigested DNAs from colonies isolated from transformation plates, probeâ with 3*P-labelled pPS57. Uncestricted DNAs (5 pgllane and 30pg/lane), were run out on a 0.7% agarose TBE gel at 45V. DNA was transfemd to a nylon membrane and hybridized with pPS57. The membrane was given a 2 d2y exposure. Top panel; ethidium bromide stahed gel. Bottom panel; gel probed with '*P-labelled pPS57. Lane 1. Hind III digested lambda marker; lane 2, Spg VA30 nontransfonned negative contml; lane 3.30pg VA30; lane 4.5pg colony 100; lane 5. 30pg colony 100; lane 6.5pg colony 101; lane 7.30pg colony 101; lane 8.5pg colony 103: lane 9,30/cg colony 103; lane 10.5pg colony 128; lane I l , 30pg colony 128; lane 12, Syg colony 154; lane 13,30pg colony 154; lane 14.5pg colony 157; lane 15.30 pg colony 157; lane 16,5yg colony 158; lane 17.30pg colony 158; lm 18.5 yg colony 169; lane 19,30pg colony 169; lane 20.30pg colony 170; lane 21,51(g colony 170; lane 22.30pg colony 189; lane 23,5yg colony 189; lane 24,30pg colony 190; lane 25,5pg colony 190; lane 26,30/cg colony 191 ; Lane 27, Spg colony 191 ; lane 28, Hind III digested lambda market.
55
repotîed to occur amongst strains of Aspergi//us nidvlens (Leong et al, 1987).
Thirdly, the effects of transfonning with a drailar versus a linear vector, and of
using an agarose oveilay dunng selection Ath strain VA30 wuld also need to be
evaluat ed.
Strains MH75 and VA30 were transforrned with circular and Hind III
linearized pPS57. Selection was performed at four concentrations of
hygromycin: CM plates containing O.6M sucrose Ath 5ôpg/ml, 100pg/mI,
200pg/ml, and 500 Wrnl of hygromycin. each with and without CM+O.GM
sucrose agarose ovedays. Transformation plates were initially examined after 7
days. Most of the colonies on VA30 transformation plates and no-DNA contml
plates (CM+O.GM sucmse+hyg.200pg/mI) wem small in size, and consisted of
budding-yeast cells, while colonies on MH75 transformation plates and no-DNA
controls were more hyphal. However, when MH75 plates were reexarnined on
day 9, colonies were obseived to have becorne largely composed of budôing-
yeast cells also.
The results of this experiment. scored after 1 O days of growth, are given in
Table 2. The total number of pmtoplast regenerates (and nonprotoplasteci cells)
per plate, was similar for each transformation reaction, as determined by colony
counts on CM plates containing O.6M sucrose (hygmycin Opg/ml). Slow
growing budding-yeast colonies were numerowr or confluent for both strains at
50pg/ml and 100pghnl hygrornydn setection (fable 2). Hygromydn at a
concentration of 200pg/ml prevented the growth of most nontransfomeci
pmtoplast regenerates on MH75 plates (31 slow growing yeast colonies with an
oveiley and 53 without, on MH75 no-DNA control plates). By cornparison,
nontransformed VA30 protoplast regenerates wem found to be less sensitive to
hygromycin and wem still able to giow at 2ûôpgiml hygmmycin, howwer, their
rates of growth were edremdy slow (confluent with an oveilay, and 61 2 without
Tabk 2. Number and morphology of cdonies growing up on MH75 and VA30 transformation plates. utilizing boUl drcular and linearized pPS57 vector, and with varying levels of hygromydn seledion.
Transformation Select ion Conditions Conditions
hygro mydn hygm y d n hygroydn hygrornydn hygromydn W m ? SOpg/mI 1 owfnl 2dOpgîml s w d m l
57
on VA30 no-DNA control plates). Hygrornycin et a concentration of 5ôôpgiml
was required to completely prevent the growth of al1 nontransformed protoplast
regmerates on no-DNA control plates for both strains. This level of selection
also swerely reduced the number of putative transfonnants growing on
transformation plates (Table 2).
A greater number of colonies with a hyphaî moiphology and iewer
colonies with a slow growing yeast colony morphology were obtained when
transformations were performed with linearized pPS57 vector instead of drarlar
pPS57 vector for both VA30 and MH75 strains. When linearized pPS57 vector
was used, 177 hyphal and inestimable (but not confluent) slow growing yeast-like
colonies were obseived for VA30 using an oveilay and selecting with 200pgIml
hygrornycin, while 15 hyphal and confluent slow g W n g yeast colonies were
observed with circular pPS57 (Table 2). Similariy for strain MH75, utilizing
linearized plasmid, 154 hyphal and 162 slow gmuing yeast colonies were
obtained using an overlay and 200pg/ml hygromydn seleclion, while only 18
hyphal but 1273 fast growing yeast colonies were oôtained when cirailar pPS57
was used (Table 2). The reduction in the number of slow or fast growing yeast
colonies, and the concurrent increase in hyphal colonies which was oôtained with
linearized vector, potentially reflects a greater efficiency of stable transformation
with linearized plasmid.
Plating of protoplasts without agarose overlays $ave less deairable results.
Colonies were pooily separated making the seledion of individual colonies
diffgilt. Scoring was irnpeded and offen msulted in inestimable colony cwnts
(TaMe 2).
58
Screeniing sttategy lor m v e r y of dimorphc mutants
To select O. ulmi mutants with e predorninantly budding-yeast cell mutant
phenotype, transformation plates were examined for colonies which consisted
almost entirely of budding-yeest cells, and which grew faster than coknies found
on no-DNA contrd plates. These were transfened to numbereô, CM plates
containing grids. and examined for continued budding-yeast cell growth ove? the
course of 7-1 0 days. Colonies which reverted to a wild type hyphallbudang-
yeast growth were discarded, while those which continued to display a
piedominantly budding-yeast growth were selected and single spore purified
(Materials and Meth&).
A second strategy for the recovery of mutants with a predorninantly yeast-
like gmwth was also empîoyed. Over a period of approximately 3 weeks, most
colonies accumulated pigment with age. Yeast-like colonies which maintained a
healthy non-pigmented apparence were transfened to CM plates and obsewed
for continued budding-yeast cell growth.
To select hyphal mutants with a reduced budding-yeast cell phase. hyphal
colonies wsre transferreâ from transformation plates to CM plates. Colonies
were examined for reduced budding-yeast cell gmwth with compound and
dismcting light microscopes.
Number of translbmtion expenhmts petibmied end puretive tmsîbmants
scmned
Twelve transformations (one transformation reaction utilires 2x1 07
protoplasts) dong with control readions, wem peitomied with stiain VA3û on CM
plates. Seven expen'rnents wen perfomed with circular pPS57 vwor and,
following the resuits of the previously describecl expriment, five ewperiments
utilized a Hind III linearized vectoi with 2CQ@rnl hygromycin aelecüon and an
59
oveilay. If only fast growing. hyphal cdonies an, considered, the total number of
putative transformants scieened on solid media plates was approximated to be
8655. This estimation L based on the number of hyphal colonies counted on 3
plates (one tenth of a transformation reaction/plate) fmm 3 separate experiments,
using li nearized vedor (mean î standard deviation of 1 51 *35 colonies) , and 2
plates from 2 experiments using circulai vectoi (1 7f2 cdonies). However, if dl
colonies larger than those seen on no-DNA transformation controls are
consiâered, the number of putative transformants screened on solid media plates
would be greatet than 64,000. The Southem analysis of putative transformants
described previously (see Fig. 3) suggested that only a portion of the hyphal
colonies (3 of 6) and none of the slow or faster gmwing, yeast-like colonies (4
slow, 2 fast) were actually transformants; these rearlts may apply where a
circular vector is utilized in transformation mactions, but it is not known if a similar
situation exists for colonies derived h m transformation experiments where a
linearized vector is utilized. However, Southem analyds of fast growing, yeast-
like mutants generated fmrn transformation experiments, using the linearized
plasmid pPS57, does suggest that a greater proportion of colonies (of any
morphology) may contain stable integiations of pPS57 (see below).
Four mutants which displayad a predominantly budding-yeast cell
phenotype were generateâ eaily on in a series of transformation expeiiments
which utilized circulai pP S57 vector. These mutants wem selected on
transformation plates as fast gmwing yeM-like colonies which had remeined
heaithy in appearance and diâ not accumulate pigment ovei an extended period
of tirne, and were designateci yml, y&, ym3 and yml (yeast mutanis 1 thmugh
4. see Table 3). Mutant yml fortned upraised Mged masses composed almost
entirely of kidding-yeast cells on the plate's surface, and grew invasively into the
agar plate with hyphae (see Figs. 4 and 5). The mutants ym2, ym3 and ym4 had
smooth colony morphologies and aîso grew invasively into the agar plate surface.
On CM plates, al1 four mutants had a reâuced rate of raâiaî growth: 2.3f0.1 cm,
2 .M. 1 cm. MM. 1 cm and 2.2iû.0 cm for ym 1, ym2, ym3, and yrn4
respectively, compared to VA30 at 7.6f0.l cm over a 9 day period (see Table 4.
top). All four mutants grew slightly faster than VA30 on CM+hyg.POO pg/ml
plates: O.8f0.l cm for VA30, versus 1 . W . 0 cm, 1 .M. 1 cm, 1 .W. 1 cm , and
1 .W. 1 cm for ym 1, ym2, ym3 and ym4, respectively. When plated ont0
agarose minimal media, VA30, ym2, ym3 and ym4 grew quickly over a 9 day
period (3.6#). 1 cm, 2 . M . 1 cm, and 1 .W. 1 cm, respectively), while ym l
showed minimal growlh (O.W.3 cm) (Table 4, top). This initially suggested that
yml might be an auxotmph, and proviâed evidence that the yml mutation was
likely different from the col1 mutation of strain MH75, which grew well on minimal
medium (Fig. 28).
Growth characteristics in IiquM culture were examined. In CM liquid non-
shaking culture (conditions which favour hyphal growth), VA30 grew as a mixture
of budding-yeast and hyphal forms (Fig. 6). An extembve mat of submerged
m ycelium trappeâ and held budding-yeest cells, so that little kidding-yeast
growth was obsû~ûd, unless the cuîture was sheken vigorously. Hyphal growth
was paiticulady reduced with yml(Z), which formed a looculant budding-yeast
cell precipitate, while ym2, ym3 and ym4 consisted of a mixture of budang-yeast
and hyphal gmwths (Fig. 6).
Micmscopic analysis was peilormeâ on 9 day dd colonies of VA30 and
yml growing on CM plates. Ail surface growth contained within a 5mmxSrnm
agar Modc taken from the periphery of the donies, was removed by scraping
Figo 40 G corn piete plates. l!
rowth of wiM type strain VA30 and budding-yeast mutants yrnl, ym2, y n J and medium (CM), CM containing hypmycin at 200pg/ml, and minimal medium 5 days of growth at 25.C.
Fig. 5. Comparison of Ophiostomu ulmi wild type strain VA30 and yml budding-yeast mutant colony morphologies on complete med- ium (CM) plates, after 15 àays of growth at 25OC.
Tabbk 4. Radial gmwth extension of strain VA30 and generated mutants after 9 days of growth on complete medium (CM), complete medium containing hygmmycin at ZOOpg/ml, and minimal medium (MM) plates.
Colony diameter (cm). on: Culture
VA30
yml ym2 ym3 vm4
VA30
yml yml sector 26 158 94-7-27- 1 94-7-27-2 94-7-2793 94-8-4-3 94-8-4-5 94-8-46 94-8-8-30
Fig. L Liquid c u l ~ r e growth of Ophiostom ulmi strain VA30 and mutants yml(Z), yml(0) sector, ym2, y d and ym4. Complete medium (CM) liquid cultures (lOml), w e n grown for 9 days without agitation at 25 O C .
66
and viewed under a ligM micmscope with 10X and 4OX objectives.
Miciophotographs of fields of view, which pmviâed a representative view of the
relative amounts of budding-yeast and hyphal growth forms, are shown in Fig. 7.
VA30 consisted of an extensive rneshwoik of hyphae and budding-yeast cells.
Mutant yml consisted of rnostly budding-yeast cells. The few hyphae presem
were shorter and much less developed than those of VA30.
Southern analysis was performed on yml, ym2, ym3 and ym4 to
detemine if they were transformants. Undigested and Bgl II digestecl DNAs were
transfened to a nylon membrane and probed with 32P-labelled pPS57. VA30
DNA was used as a negative control, and DNA from the ml1 transformant (Royer
et al. 1991) was used as a positive control and for size comparison. *P-Labelled
plasmid pPS57 hybridized to a single 15.7 kb Bgl II fragment of BQ/ II digested
ym 1 DNA (see Fig. a), indicating the presence of no more than 3 copies of the
vector at one insertion site. Plasmid pPS57 hybridized to high molecular weight
DNA (>23kb) in the undigesteci yml DNA lane, consistent with a chromosumai
integration. The mutants ym2, ym3 and ym4. while similar in appearance and
growth characteristics to yml, were detemined not to be transformants by
Sout h m analysis (Fig. 8).
8y transferring yeast-like colony mutants from transformation plates onto
CM plates, and selecting for donies which maintained their mutant phenotypes
without hygromycin selection, 8 budding-yeast mutants and 1 hyphaî mutant
were recovered and purified for further examinatlon (see Table 3 for summary of
mutants, Figs. 9 and 10 for colony morphologies, and Table 4 for linear gmwth
rates of colonies). Budding-yeast mutants wen designateci 91-7-21- 1,
21-2, 94-7-27-3, 94-û-4-3, 944-4-5, 94-84-20, and 94-8-8-30. W~th the
exception of 26, al1 were generated in transfomation experiments w k r e Hind III
Iinearized pPS57 had been utilized. All of these mutants, as originally
Fie. 8. Southem hybridization of undigested and Bgl II digested DNA of Ophiostoma ulmi budding-yeast mutants yml, ym2, y& and ym4. prokd with 32P-laôelled pPS57. DNAs ( 15 pg/ lane) were run out on a 0.7% agarose TBE gel at 45V. Top panel; ethidium bromide stained gel. Bottom panel; gel probed with 32P-Iaklled pPS57. Lane 1, Hind II1 digested lambda marker; lane 2, undigested VA30 nontransfonned negative control; lane 3, VA30 Bgl II digest; lane 4, undigested coll positive control; lane 5, coll Bgl II digest; lane 6. ym I undigested; lane 7, yrnl Bgl Il digest; lane 8, ym2 undigested; lane 9, ym2 Bgl II digest; lane 10, ym.3 undigested; lane 1 1, y n J Dg111 digest; lane 12, ym4 undigested; lane 13, ym4 Bgl II digest.
Fig. 9. Colony morphologies and radial growth rates of O. ulmi nontransfomeci mutants. Colonies wen grown on complete medium (CM), CM containing hygmmycin at 200pglrni and minimal medium (MM) plates for 9 &ys ai 2S°C.
10. Colony morphologies and radial gmwth rates of 0. ulmi tnuisfomed mutants mies were gmwn on complete medium (CM), CM containing hygromycin at 200p minimal medium (MM) plates for 9 days at 2S°C.
71
characterlzed, exhibited reduced hyphal growth on CM plates, and consisteâ of a
greater proportion of budding-yeast cells than wild type VA30, as detennined by
microscopie analysis. Mutant 26 proâuced only budang-yeast cells when grown
on CM plates containing hygmrnycin at 200pg/ml, while al1 other mutants
displayed some degree of hyphal growth, however limited, on hygromycin
containing media. Mutant 26, which had been maintaineâ on CM+hyg.2Wpg/ml
plates continuously for over 3 months (26 hyg.), displayed a greater amount of
budding-yeast growth than 26 maintained without hygromycin selection, when
transfened to CM plates. The only hyphal mutant recovered, 94-8-4-6, formed
small, tight colonies of highly branched hyphae on CM plates (Fig. 10). Initially,
budding-yeast growlh was extremely reâuced and almost completely absent foi
94-8-4-6, however, it was occasionally observed to mvert to a more wild type
mixture of yeast and hyphal forms of gmwth and lose its frequent branching
phenotype (see section on instability below).
In CM liquid non-shaking culture (Fig. 1 1 ), mutants 26, 94-7-27-1, 94-70
27-2, and 94-7-27-3 contained some hyphal growth and a flocculant yeasty
predpitate. Mutants 94-8-4-3 and 94-8-4-5 contained signifcantly more hyphal
growth, and hyphai mutant 94-8-4-6 had no detectabîe budding-yeast growth.
Mutants 94-84-30 and 94-8-8-30 were sirnilar to VA30. Mutant 26 maintained on
CM+hyg.200bg/ml plates (26 hyg.) contained significantly less hyphal growth and
more budding-yeast gmwth than 36 maintained without hygromydn.
Southern analysis was perforrneà on Bgl II digested and undigestecl DNAs
probed with UP-labellecl pPS57 (Figs. 12a and 12b). Plasmid pPS57 hybrldited
to a 19.1 kb Bgl II fragment of Bgl II dgested 94-7-27-3 DNA, a 22 kb 89111 fragment of 94-8-49 DNA, a 20 kb Bgl II fragment of 94-84-5 DNA, and a 19.1
kb @Ill fragment of 91-8-44 DNA. Mutants 26, 91-7-27-1, 94-7-27-2 and96
84-30 were detemined not to be transformants (Taüe 3). Plasmid pPS57
Fig. II. Liquid culture growth of Ophiostuma ulmi strain VA30 and mutants 26,26 maintained on hygmmycin. 94- 7-2 7- 1.94- 7-2 7-2.94- 7-2 7-3,94-8-4-3.94-8-4-5.94-84-6.94-8-8-20, and 94-8-8-30. Complete medium (CM) liquid cultures (100ml). were grown for 9 days without agitation at 25OC.
"Y!. ' ' I ., k W / &-uu, L **. #
0 !
I
23.1 kb- / i 9 ,
9.4 kb- !
6.6 kb - 4.4 kb -
2.3 kb- 2.0 kb -
Fig. 12a. Southem hybndization of Bgl II and H j d III digested DNA of Ophiustom ulmi mutants pmbed withuP-laklled pPS57. DNA (15 &lane) was mn out on a 0.7% agame TBE p l at 45V. Lane 1. Hind III digested lambda marker; lane 2, ynr l Bg1 II digest positive control ; lane 3. Bgl II di gested VA30 nontransformed negative contrd; lane 4, VA30 Hind III digest; lane 5, 26 Bgl Il digest; lane 4 26 Hind I l i digest; lane 7.94-7-21-1 Bgl II digest; lane 8.94-7-27-1 Hind III digest; lane 9.94-7-27-2 Bgl II digest; lane 10,947-27-2 Hind III digest; lane 11.94-7-27-3 Bgl II digest; lane 12.94-7-27-3 Hind III digest; lane 13.94-8-4-3 Bgl II digest; lane 14.94-8-4-3 Hind III digest; lane 15.91-84-5 Bgl II digest; lane 16.94-8-45 Hind III digest; lane 17. W-8-46 Bgl I l digest; lane 1 8.94-846 Hind III digest; lane 19,94-88-191 II digest; lane 20,948-8-1 Hid nI digest; lane 21. Hind III digested lambda marker; lane 22, pPSS7 linearized with Hind III p s i tivc contd and marlcr.
Fig. 12b. Southem hybridization of Bgl II and Hind III digested DNA of Ophiostm ulmi mu- tants probed with "P-labelled pPS57. DNA (ISpg/iane) was run out on a 0.7% ûgarose TBE gel at 45V. Lane 1, Hind III digested lambda market; lane 2, yml Bgl II digest positive control; lane 3,948-8-20 Bgl II digest; lane 4, P4-8-8-20 Himi III digest; lane 5.94-8-8-39 Bgl II digest; lane 6,948-8-30 Hind III digest; lane 7, ynul Bgl II digest; lane 8, ym3 Hind III digest; lane 9, ym4 Bgl II digest; lane 10, ym4 Hind III digest; lane 11, Hind III digested lambda marker; lane 12. pPS57 linearized with Hind III positive control and marker.
75
hybfidized to high molecular weight (~23kb) unrestricted DNA in transfomants
(Figs. 1 2a and 1 2b), indicating that transfomants likely contained chromosomal
integrations and not an autonomously replicating plasmid.
Severai changes in phenotype were noted when cuitures were set up for
microscopie analysis (see section on instability). Mutants 94-7-2793 and 94-8-8-
30 looked similar to wild type VA30, consisting of a mixture of budding-yeast and
hyphal growth. Mutant 94-8-4-3 consisted mainly of budding-yeast cells, mutant
94-8-45 unexpectedly consisted almost entirely of hyphae, and 94-8-4-6
consisted of a mixture of hyphae and budding-yeast cells.
Mutant 158 was one of 3 colonies that displayed a fast growing hyphal
growth on hygromycin containhg CM plates (Table 4). kR did not contain a
pPSS7 insert as determined by Southern analysis (Fig. 3). On solid media
plates, 1% displayed a near wild type phenotype, consisting of a mixture of
budding-yeast and hyphai cells (Fig. 9). After approximately 3 weeks on a CM
plate. 158 was observed to form dramatic upraised, convoluted masses of
budding-yeast cells (Fig. 13). These masses were not t o r d evenly throughout
the entire culture, kit at distinct points, and continued to increase in number and
sire (up to 4mm above the surface of the plate) as the colony aged. 158 was
puiifieâ twice, by plating out suspensions of budding-yeast cells from upraised
masses ont0 CM plates, and ælecting single colonies. When yeast celis derived
from the purified culture were streaked ont0 CM plates, the resulting growth was
at first yeast-like and resulted in the production of small convoluted ridges of
budding-yeast cells. These cells soon aftennaids produceû a normal looking,
fast growing mycelia. which grew out to cover the plate and l&ed much the
sarne as it had on the original tmnsfonnation plate. After approximately thme
weeks, masses of amvoluted budding-yeast cells wen again obeenred to fom et
distinct points on the plate even though the culture had been puiified by
Fige 13. Convoluted growth of mutant 158. Mutant 158 was grown on a complete medium (CM) plate for 7 weeks at 2S°C.
77
selecting single colonies derived from single yeast cells frorn the convoluted
mass of cells.
Mutant 158 grew well on CM+hyg.200pg/ml plates, yet was found not to
contain a pPS57 insert. To determine il 158 migM have b e n an u n M e
transformant that had lost pPS57 when it was gmwn up foi DNA isolation in CM
liquid culture, the following expertment was performed; 158, VA30 (negative
control) and ym 1 (positive control) were gmnrn in CM liquid media (1 00ml in
tripikate) for DNA isdations wit h varying levels of hygromycin selection: Opgf ml,
1 Opg/ml, 5ûpg/ml and 100pg/ml. VA30 grew poorly at 1 OCig/rnl. while yml and
158 grew well at 100jqfml hygromycin. DNA samples from the cultures were
içolated, digested with Bgl II. transferred to a nylon membrane, and Southern
analysis was pedormed using 3*P-labelled pPS57 (Fig. 14). Plasmid pPSS7
hybridized to a 15.7kb Bgl II fragment of Bgl II cut yml DNA (positive control) as
expected. and with uniform intensity for al1 levels of hygromycin selection. 158
did not hybridize to pPS57 at any of the levels of hygrornycin selection (Fig. 14
midae panel), suggesting that 158 was not transformed, and that 158 did not
loæ an autonomously repîicating pPS57 plasrnid during the growlh of material in
CM liquid culture for DNA isolation. Restriction digests of 158 DNA with 801 II producd an ethidium bromide (EthBr) stained banding pattern of repetitive DNAs
that was distindy âifferent than that of VA30 (Fig. 1 4 top panel). To detemine if
158 was a derivative of VA30, the membrane îmm the above expriment was
stripped and probed with the *P-labelled iDNA iepeat of ScnitophyIkrm
aommune. A different banding pattern was obsenred for 158 (mg. 1 4 bottom
panel), suggesting that 158 was most certainly a contaminent.
StabiYty of mutants
Ol the many cdonies scrwned which ap~eared to have Feduced hyphal
itmt 158. Muiant 158 almg with VA30 (negative control) and ym l (positive grown at a range of hygromycin conceniratioris. DNAs wctc run out on a 0.7% TBE gci at 4SV. Top pnnei; cthidium bromide stnincd pl . Mlddk pud; gel pmkd with "P-laklîcd pPS57. truK 1, Hind Ill digeotcd lambda marker, lane 2 and 3, Bgl II d i ~ t c d VA30 grown at Opglml d 10pghnl hygromycin mpaaively; Iancs 4 through 7, Bgl II digestcd yml gmwn at Op@ml, 1 Opgid. 5ûpg/mi and 1 Oûpglrnl bygromycin rcspciively ; lanet 8 h g h 1 1. Bgl II digestal 158 grown at Olglml, IOpghi, Sûpglrnl and 10ûpgIrnl hy gromycia mpectively ; lane 12, Bgl II digcslcd pPS7 c0llt.d; lane 13. Hind Ili digestcd lambda marlrer. httm plW(; gel probai with sP-lakllcd pR1 rDNA prok of SchirophyIIwn corrrmy~.
79
growth, only the 14 mutants listed in Table 3 were found to be mitotically stable
between the time they were recovered from transfomation plates, and the time
they were g m up for DNA isolations. Of these 14 mutants, both transformants
and nontransfomants. many eventually showed some degree of reversion to a
more wild type phenotype, either between the time they had been identifid as
mutants and transferred to -70°C, or after having been removed from -70% for
preparation of material used in various manipulations. This variability was also
obwtved repeatedly with the wll mutant of MH75. Revetsion to a near wild type
VA30 motphology was observed for 94-7-27- 1,9407-27-2 and 94-8-4-3 (see Figs.
9 and IO), and a reversal of the alteration of dimorphic distribution occurred for
budding-yeast mutant 94-8-4-5, which began to grow almost constituitively as a
rn ycelium on CM plates (see Fig . 1 0) . Mutant 94-84-6 was also observed to
occasionally ieveit to a more wild type phenotype, and IOS~ its distinctive
compact colony and extensive hyphal branching phenotype.
Sectodng of yml mutant
Over a period of 6 months (four serial transfeis from the original
transformation plate), ym l remained unchanged when grown either with or
without hygrornycin selection, continuing to grow mainly as a budding-yeast. The
yml plate cultures from the fouith transfei grew nomaIIy at Irst, but then some
plates developed distincü y diff erent "sectored" growth rno~hologies. In al1
instances, sectors consisteci of upiaiseâ areas of fast growing fluffy giowth, with
increesed derelopment of aerial hyphae. The morphology of some of these
sectors is illustrateci in mg. 15. Non sectord yml is shown in the top mw, lefi.
The 4 colonies in the second and third rows were dl derkred from the same plate
of non sectoied ym l and are of the same age. In =me instances, the entire
colony grew normaîly at fia, then became fluffy (Fig. 15 top row, iigM and
Fig. 15. Sectored growth of budding-yeast mutant yml on complete medium (CM) plates. A; yml. B through H; sectors ofyml.
bottom mw fight). Some sectors were dramatically pie shaped (Fig. 15 second
row Mt), while othem occurred later in the gmwth of the colony and spread at the
margin (Fig. 15 second row right, third mw left. and bottom row Mt). Sectord
growth was never observed to revert back to the slow growing budding-yeast
phenotype of ym 1.
The linear growth rates of yml, yml(0) sector. and VA30 were compred
on CM, CM+hyg.ZOOCie/rnl and MM plates (Fig. 16). On CM plates, the radial
growth of yml sector was equal to or typicelly greater than that of VA30 (6.3I0.2
cm versus 6.4I0.2 cm, respectively, in Fig. 16, and 8.2130.2 cm versus 7.3îû.1
cm, respectively for an eailier erperiment) versus 2.3I0.3 cm for yml. On
minimal media. yml sector grew well (5.4I0.3 cm), while yml grew very pooiiy
(1.2M.2 cm). The yml sectoi grew very well on CM+hyg.200pg/rnl plates
(6.9I0.2 cm) compared to yml (1.3M.3 cm) (Fig. 1 6 and Table 4).
Microphotographs were taken of ym 1 (O) sector (Fig. 17). Compared to VA30,
ym 1 sector had a reduced budding-yeast phase and consisteci of a greater
proportion of hyphae which were long and robust.
Sectois were examined for alterations in the site of pPS57 insertion, or
the number of insertions. Fig. 1 8 illustrates the hybridization of Bgl II and Hind III
digested ym 1 mutant and yml sector DNA with 32P-labelled pPS57. Bgl Il does
not cut within pPS57, while Hind Ill cuts pPS57 once. NO differences were seen
between yml and ymt sector. In each instance, a dngle Bgl II fragment of
approximately 15kb hybridized to pPS57, indicethg neither an increase noi a
decrease in the number of veclor insertions et this one site. A change in the
insertion site or number of insertion sites was also not indicated. However,
rearrangement and reinsertion at another location of a laiger fragment containing
the entire 15 kb Bgl II fragment could have ocaimd. This possibility w88 rot
investigated given the iesults of the rneiotic anaîysib of yml(0) -or, which
Fig. 16. C( gmwn on ( (rn agm
~lony morphologies and radial growth rates of VA30, yml and yml sector. Colonies were :ornplete medium (CM), CM containing hygromycin at 200pgfml and minimal medium plates for 9 âays at 2S°C.
Fig. 17. Microphotogniphs of wild type strain VA30 and mutant y m l ( 0 ) sector. Pîate A; VA30. 10X objective. Plate B; yml (O) sector, 1OX objective. Colonies were grown on complete media (CM) plates for 9 days and examined by taking surface scrapings from 5mm X 5mm agar blocks taken from colony rnargins.
Fig. 18. Sourhem analysis of pPSS7 insertion in yml and yml secton. Bgl II and Hinû III digested yml and yml scctor DNAs (15 pghne) werc run out on a 0.7% agarose TEE gel at 45V, and pmbcd ~ith~~P-labt l led pPSS7. Top ml; ethidium bromide stained gel. Bottom -1; gel probcd with nP-labclled pPSS7, Lane 1, Hind III digestcd lambda market; lant 2, Bgl II digested VA30 negative control; lane 3, Hind III digested VA30 negative control; lanes 4 and 5, yml(C) Bgl II and Hind II digests ttspectively (panially degradcd); lanes 6 and 7, yml(K) Bgl II and Hinû III digests; lanes 8 and 9, yml (M) scctor Bgl II and Hind 111 digests; lanes 10 and 1 1, yml (O) scctor Bgl II and Hind Il1 digests; lanes 12 and 13, yml (P) sector Bgl II and Hind III digests; lanes 14 and 15, yml( W) Bgl II and Hind II1 digests; lanes 16 and 17, yml(X) Bgl II and Hinâ II1 digests; lanes 18 and 19, yml(Z) Bgl II and Hind Il1 digests; lane 20, Hind II1 digcsted lambda mariter; Iant 2 1, empty ; lme 22, pfS57 linearizcd w itb Hind II1 positive control and rnarker.
indicateâ that the sector phenotype did not cosegmgate with pPS57 (see section
on meiotic analysis below). Restriction endonudease analysis of yml and ym1
sector DNA with Hind III resolved a single band of 5.38 kb, equivaîent to the size
of the linearlzed vector pPS57 itself. The intensity of this band remained the
same for yml and yml sector supporting the Bgl II digest data, which indicated
that there was neither an increase nor a decrease in the number of pPS57
insertions.
Melotlc Anaîysis of Mutants
Mating reactions
VAN, ym 1 0 , ym1 (O) sectoi, 94-7-27-3, 94-8-4-3, 94-8-4-6 and 158
were rnated with three O. ulmistrains which are North American race of the
aggressive biotype and 6 mating type: CESS 16 K, W2 and W2-TOL (obtained
from L. Bernier, Quebec City). B mating type strains were used as fernale
cultures in crosses on Elm Sapwood Agar (ESA) plates, with the appropriate
negative and positive cont rols (Materials and Methods) . Perit hecia were
obtained for the VA30 positive conttol and each of the mutants crossed with W2-
T a , but with none of the negative contd crosses. Perithecia were obtained
after 4 to 6 weeks for all mutants.
MeioHc anahsis of yml
A successful mating of ymlX W2-TOL was performed îwice. Progeny
were either distindly like the m'Id type parents, or veiy dow growing and yeast-
like, es per the yml parental strain on CM plates. In fact. yml-like progeny were
geneially slower gIomng and more yeast-like than the yml parant itself. No
colonies of intemiediate size or morphological characteristics were ever
86
observed. From the first mating, a total of 120 single spon pmgeny wen
collected from CM plates; 67 had a nomal wild type phenutype while 53 had a
distinct yml mutant phenotype. From the second mating, a total of 118 pmgeny
were collected; 72 were normal and 46 wen of e yml mutant phenotype. The
yml mutant phenotype was inherited in an approximately 1 :1 ratio in the yml X
W2-TOL progeny (0.005~p<0.01).
The ratio of normal to mutant phenotype progeny on CM plates, with and
without hygrornycin selection, was insufticient to deariy demonstate
cosegregation of the vector with the mutant phenotype. ît was necessary to
isolate DNA from single spore progeny and determine the presence of pPS57 by
Southern analysis, and not rnerely rneasure hygmmycin resistance, as had been
done with the col1 mutant (Royer et al. 1997). This was necessary because of
three factors: Firstly, there was the possibility that some of the yml-like pmgeny
would sector, making it difficult to distinguish mutant from wild type phenotypes.
Secondly, transiorming DNA could be subjed to DNA modification - either repeat
induced point mutation ("RIPn; Selker et al. 1987; Selker, 1990) or methylation
induced prerneiotically ("MIP"; Rossignol and Picard, 1991) - and lead to a failure
to transmit hygromycin resistance through to the progeny. RIP has been
suggested to account for the low levels of transmission of hygmmycin iesistance
to the progeny of some of the MH75 transformants analyzed by Royer et al.
(1 991 ) in instances where more than one copy of the vector DNA had been
integrated. RIP or MI? of pPS57 (possiôly present in more than one copy) in the
yml X W2-TOL progeny was a concem. Thirdly, when analyzing progeny Ath a
ym 1 mutant phenotype, it was difficult to distinguish hygmmydn sensitivity from
hygmmydn resistance, because of the very siow rate of gmwth of many of the
progeny on plates with or without hygmmydn.
87
Fi@ ot the 120 single spore prageny from the first yml X W2-TOL mating
were purified (see Materials and Methods) and DNAs exarnined for the presence
of pPS57 by Southem hybridization (Figs. 19a and 19b). Greater than half of the
pmgeny with a yml mutant phenotype âid not contain a pPS57 insert (1 2 of 20,
see Table 5). indicating that the ym 1 mutant phenotype dM not cosegregate with
the vector, and therefore that yml was likely not a tagged mutant.
Linear gmwth rates for progeny with a yml mutant phenotype were
distinctly âifferent from those with a wild type phenotype on CM plates; overall
average colony diameter was 1 . M . 9 cm for yml-like progeny, veisus 5 . M .4
cm overall cdony diameter for those displaying a wild type phenotype (TaMe 5).
Radial growth rates on CM+hyg.400pg/ml plates was a reasonaûie indicator of
the presence of pPS57 in the progeny; progeny with a yml mutant phenotype
which contained pPS57 grew only slightly faster glowth on hygrornycin containing
plates than those not containing pPS57 (0.9M.3 cm containing pPS57, versus
0.6H.2 cm without pPS57) (Table 5). However, for those colonies displaying a
wild type phenotype on CM plates, the presence of pPS57 conferrd an ability to
gmw significantly faster on CM+hyg.400Ciglml plates (5.0f0.8 cm with pPS57,
versus 0.7f0.2 cm without pPS57; see Table 5).
Prior to the detemination that yml was unlikely to be a tagged mutant, a
considerable effort was put forth to analyze yml and retrieve the gene/genetic
element interrupted by the pPS57 insetlion. This seemd a masonable appmach
at the time given the great length of time which lapsed between the determination
t hat ym l was a transformed mutant, a d analysis of meiotic progeny (because of
an inaôility to successfully mate yml with any of the strains initially obtained for
this purpose). Secondly, given that Royer el a/. (1997) hed determinecl their
single a / l buôôing-yeast mutant to have ben tagged, it seemeâ piobable that
yml might be a tagged mutation as well. Thersfoie, the following woik woe
Fig. 1%. Southem anal ysis of Bgl II digested yrnl X W2-TOL progeny probed with xP-labelled pPS57. DNAs (15pgIlane) were run out on a 0.7% TBE gel at 4SV. Top panel; ethidium bromide stained gel. Bottom panel; gel pmbed w ith 32P-labelled pPS57. Lane 1, Hind III digested lambda marker; lane 2, Bgl II digested pPS57 (22ng) positive control; lane 3, Bgl II digested VA30 nega- tive control; lane 4, Bgl II digested W2-TOL negative contml; lane 5, Bgl II dipsted yml positive control; lanes 6 through 34, Bgl II digcsted yml X W2-TOL progcny # 1 through 29.
Fig. 1%. Southem analysis of Bgl II digested yml X W2-TOL progeny with 32P- labelled pPS57. DNA ( 1 Spgllane) was nin out on a 0.7% TBE gel at 45V. Top pne l ; ethidium bromide stained gel. M o m panel; gel probed with 32P-labelled pPS57. Lane 1. Hind III digested lambda marker; lane 2. Bgl II digested pPS57 (22 ng) positive control; lane 3, Bgl II digested VA30 negative contml; lane 4, Bgl II digested W2-TOL negative contml; lane 5. Bgl Il digested yrnl positive control; lanes 6 through 26, Bgl II digested ym I X W2-TOL progeny 1 30 through 50.
Tabh 5. Summary of meiotic analysis for yml X W2-TOC progeny.
Number and pPS57 Colony diameter (cm) on: phenotype of present ProgenY
CM CM+ hyg.400wg/ml
20 yml mutant 12 no 1.6f0.4 ~ Y W 2.Wû.8
30 Wild type 13 no 5.W.9 17 yes 5.Sf1.2
91
perlonned in an attempt to retrieve the gendgenetic element intempted by
pPS57:
i/ Mutant yml was examined for the piesence of the chloramphenicol
acetyltransferase (CAT) gene and Ml 3 origin of replication by Southem
analysis, both of which are required for successful retrieval of the vector in
E. coli by plasmid rescue.
iif Attempts at pîasmid rescue with yml were made, along with wlt as a
positive control.
iiil A partial yml genomic library was constructed in Lambda FIX-II, and
screened with pPS57.
Meiotic malyss of ym 1 sector
ym l(0) sector was mated with W2-TOL Nice. On CM plates, progeny
were either similar to those obtained in the VA30 X W2-TOL positive control
cross. or had a fluffy and fast growing phenotype typical of yml(0) sector. (The
VA30 X W2-TOL positive control cmssing produced colonies of a unifom
phenotype that did not display ftuffy growth to the extent seen in the yml sector
rnating). Progeny with a slow growing,yml budding-yeast cell moiphology were
never obærved. suggesting that the mutation whidi causes the yml phenotype
occuned at the same locus or was dosely linked to the locus which caused the
ym l(0) secior phenotype.
To detemine if yml sector was a tagged mutation, a total of 39 randomly
seleded single spore isolates were examined. A wilâ type phenotype was
obsewed for 23 isolates. white 16 had a fluffy yml sector phenotype on CM
plates. Measurements of colony diameters were made a m i 7 days of gmwth on
CM plates both with and without hygromydn et 4ûOpg/ml (Table 6). Single spore
isolates with a fluffy yml sector phenotype grew quicûly on CM plates with a
Table 6. Summary of meiotic analysis for yml sector X W2-TOL progeny.
Phenotype pPS57 Colony diameter (cm) on: and number presenta of progeny
CM CM+ hyg.400pg/ml
23 Wild type 19 no 5.6îû.8 0.6M. 1 4 yes 5.6f6.4 4.6k1.8
a The presence ol p f S57 is Mened h m greater Ynear W h rates on CM+hyg.4ôûp@ml pîates.
93
mean colony diameter of 7.0f1.3 cm, versus 5.6I0.8 cm for those with a wild
type phenotype. Based on the results of the ymlXW2TOL progeny, a fast rate of
growth on CM+hyg.4Wpg/ml plates was lnfened to indicate the presence of
pPS57. Approrirnately haH of the progeny with a sectoi phenotype (7 of 16, see
Table 6), grew pooily on CM+hygAOOpg/ml plates (0.W.1 cm) indicating that
the yml sector phenotype did not cosegregate with the vector, and therefoie that
yml sector was also not a tagged mutation.
Meiotic anaîysis of other mutants
Three of the five transformed dimorphic mutants (94-7-27-3, 04-8-4-3 and
94-8-4-6) remained mitotically stable and wen mated with W2-TOL. All of the
progeny from the 94-7-27-3 X W2-TOL mating had a normal appearance when
grown up on CM plates both with and without hyg.40Opg/rnl. No colonies with a
94-7-27-3 parental budding-yeast mutant phenotype were obsetved. However,
when growing 94-7-27-3 from -70°C storage prior to the mating reaction, the 94-
7-27-3 phenotype had becorne increasingly like the VA30 wild type . Progeny from the 94-8-4-3 X W2-TOL mating grown on CM plates had a
normal colony appearance. Only slow growing budding-yeast colonies were
observed on CM+hyg.400~g/ml plates, but when transferred to CM plates, al1
grew with a normal VA30 phenotype.
Progeny from the 94-8-46 X W2-TOL mating grorni up on CM plates
were almost al1 nomial. Only a few colonies with a 84-84-6 mutant phenotype
were obsenred amongst hundreds of nonnal lod<ing colonies. No colonies grew
up on CM+hyg.400pg/ml plates.
94
CHEF &ct~~phomsls and Southem ~ ~ l y s i s
Having established by meiotic analysis that the mutation/s resp~nsible for
causing the yml and ym1 sector phenotypes were not âirectly tagged by the
insertion of vector pPS57, yml and yml sector were exarnined for chromosomal
aiterations. CHEF (aontourclamped homogeneous electiic field) gel anelysis
was perfonned on yml and yml sector to:
il determine if chromosomal poiymrphismr were 8ssociated with the
formation of the yml mutant or sectoring in the yml mutant.
iü confirrn that pPS57 had integrateâ into a chromosome and was not
replicating autonomously as a high moleculai weight digomer, and
determine into which chromosome integration had occurred.
iiil determine if changes in the rDNA tract were associated with the
formation of the ym 1 mutant or sedoring in the yml mutant.
Separation of chromosomal DNAs (chDNAs) by CHEF gel electrophoresis
was performed on wild type VA30, three separate cultures of yml, which had
been maintained on CM plates for several months (ymlC, ym1 J and yml Y), and
three yml sector isolates (yml Jb, ym lM and ymlû). lsolate WPTOL and six
pmgeny from the yml X WBTOL rnating with either normal or yml mutant
phenotypes (with and without pPS57 insertions), were also exarnined to
determine if a partiailar chromosomal polymorphisrn segregated with the yml
mutant phenotype. A karyotypic pattern consisting of at least six chromosomal
bands, ranging from greater than 5.7 Mb to less than 3.5 Mb, was msohred for all
isolates except sector ymlJb (Fig. 20, top panel). The pattern oûtained for the
VA strain was similar to that observed by others (Dewar and Bernier, 1993;
Bowden et al. 1994). For the yml Jb sector isdate, chromosome II appean, to
have been lost and a unique chONA between chDNA II1 and IV gained (Fig. 20,
top panel, lane 7 position of the a m ) , perhape through brmbge and b s of
CHEF ge
III
hybridized with3p-lakikd pff57. L.m 1; Schi;osa~~horr,myccs pornbr &cr; Iw 2 VA30; lue 3. W2-TOL; lane 4. yml(C): lanc 5. yrnl(J); lane 6, yml(Y); lane 7, yml(Jb) scctor; lonc 8, yml(M) scctor, lnnc 9, yml (0 ) sector. lancs 10 through 1 S. yrnl X W2-TOL progay. Bottom pud; gel hy bridizcd wiih 32P-labtlkd pR 1 rDNA probe oï SchizophyIInm commune.
96
part of chromosome II. It is difficult to identify this chromosome as being uniquely
present in ymf Jb from the ethidium bmmide stained gel alone, due to the poor
separation of chromosomes in this region. When the chDNAs were blotted ont0
Genescreen Plus and hybridized with SP-la$elled pPS57 (mg. 20. rniddle panel),
pPS57 appeared to have integrated into chDNA IV (the fourth largest
chiornosorne) in each of the yml and yml sector isolates. The position of the
band which hybridized to 32P-labelled pPS57 (chONA IV) was lowest in the
ymiJb lane (lane 7), while the ethidium bromide stained gel dearly shows the
putatively unique chONA of yml Jb to ocaipy a higher position than chDNA V of
yml Y and ymlM in adjacent lanes (see Fig. 20, middle panel position of anow).
This strongly suggests that the chDNA between chDNA III and chDNA N is
unique to sector ym 1 Jb. Altematively, this band may be present in the other
lanes, but not well resolveû, and the rONA tract may have transposed fmm this
chromosome to chDNA IV in dl isolates except ymlJb. Given the relatively weak
staining of some of the chromosomal bands, additional CHEF separations would
be required to confirm th8 loss of chromosome II, and Southern analyses with
probes residing on chromosome II would be required to determine if the unique
chromosome between chDNA III and chDNA IV in isolate ym1Jb was derived
ffm chromosome II. Polymorphisms were not deteded in any of the other
sectoreâ isolates. However, chromosornal separation in the e3.5 Mb range was
insutricent to definitively rule out the existence of additional polymorphisms. A
thorough investigation would require severcil CHEF gels to be cun undei âifferent
conditions, or different separation techniques to be utilized, such as PACE
(programmable autonomously controlled electrodes) PFAGE, with which supefior
separation of the O. u/mi ùaryoîype has been achieved (Dewar and Bernier.
1 995).
To detenine if changes in the rûNA chiornosorne, such as translocation
of the rDNA tract to other chromosomes, or large expansions or contractions of
the rûNA tract were assodatecl with yml or ym1 sector phenotypes, the same
CHEF gel was stripped and hybridized with the 32P-labelled iDNA probe which
mitains the entire cassette for the ribosomal genes of SchizophyI/um commune
(Bucùner et al, 1988); variation in the rûNA chromosome has. for example,
been observeci after transformation, and changes in rDNA tract length have been
conelated with alteration of growth rates in K/uyveromyc8s W i s ; C. a-ns and
S. cerevisiae (see Zolan, 1995). A single band of approxirnately the same sire
in each lane corresponding to chDNA IV, hybridized to the rûNA probe (Fig. 20,
bottorn panel). Therefore, transposition of rONA to a different chromosome or
large changes in rONA tract length did not appear to be associated with yml
sector formation, or with the formation of the original yml mutation.
A prelirninary analysis was perfoned to determine if DNA methylation
was açsociated Wh the formation of the yml mutant, yml sectors, or involved in
the control of dimorphic growth in general:
i/ Isdates ym 1 , ym 1 sector, and several other transfomants were
examineci b r methylation of the pPS57 vector and flanking genomic DNA,
using the restridion enzyme isoschizomer pair Hps II end 1.
ii/ Isolates yml , yml sector, and VA30 were examined for changes in
methylation of their rDNA repeats, using the Hpa II end Msp I
isoschizomer pair.
iiil lsdates yml, yml sedor and VA30 wen tmated with the methylation
inhibitor 5-azacytidine, and examined foi phenotypic changes.
98
Anabsis ofpPSS7 methyleîon w'th Hps 11 and Msp 1 isoschizomers
Prior to the results of the yml and ym l s a o r meiotic analyses, a DNA
methylation mode1 was proposed which might account for the occurrence of the
yml and yml sector. Methylation of vector DNA has been reported to oocur
during the asexuaî cyde after transformation in the Ascomycete Neumspom
cmsa (Bull and Wootten. 1984; Pandit and Russo, 1992) when more than one
copy of the vector has been integrated. It was hypothesized that pPS57 repeats
and 0. ulmi DNA, which flanks the site of insertion. may have become
methylated in yml when it was transfomied. If this genomic DNA was involved in
dimorphic control, then its methylation (the promoter region in particulai), could
have resulted in the yml phenotype, and demethylation in the yml sWor
phenotype. Methylation at the promoter region of a gene, has been shown to be
suffident to repress gene activity; for instance, methylation of specific DNA sites
have been found to inhibit the binding of "CREW, the CAMP-responsive element
binding factor (see Levine et al. 1992), a transcription factor which may itseH be
expected to play a role in 0. ulmi dimorphism. (Note that for this methylaüon
mode1 to be valid, pPS57 would have had to been either tagged or be8n very
closely linked to the mutation responsible for causing the yml phenotype, and
therefore this mode1 was considered invalid after the resuL of the yml meiotic
anaîysis were obtained).
To test for the methylation of pPS57 and flanking O. ulmi genomic DNA,
ONAS from VA30, yml and ym l sectors wwe digested with the isoschizomer pair
Hpb, M and Msp I, electrophoresed and probed with labelled pPS57.
Restiidon of DNAs with this pair of enzymes distingukhee the internai
unmethylated C from a 5-methyîcytdne (5-mC) in the sequence S CSniCGG 3';
Hpa 11 will not deave at this site when the internai C is rnethyîated, while Msp I
deaves this site mgardless of its methylation status. A number of fragments
hybridized to pPS57. However, no ôifferences in hybddization banding patterns
wen obsenred between the isoschizomer pair for any of the yml or ymI sector
isolates (Fig. 21). This suggested that the pPS57 inseWs and the Hpa IUMsp I
fqments spenning the insert(s) and flanking O. ulml DNA were not differentially
methylated at the intemal C position at any of the âigested CCGG sequences for
the isolates examined. Several other transformed mutants were also examined
(mg. 22). No differences in Hpa II and Msp I digestion patterns in any of the
isolates were observed, suggesting that pPS57 inserts and flanking genomic
DNA were not differentially methylateâ at these CpG ôinudeotide sites.
Anal)sis of rDhlA methyktion with Hpa II and M s p I isoschizomets
In other fungi which have been studed, the rONA repeat has usually been
shown to be methylated, and may be dfferentially methylated in various
developrnental stages or morphological foms (see Magill and Magill, 1 989). The
possiôility of modulation of rDNA gene expression by DNA methylation was
examined in ymf, yml sector and severel other dimorphic mutants. Hpa II end
Msp 1 restricted DNAs from VA30, yml and yml secton were electrophomsed
and probed with the 32P labelled heterologous rDNA probe pR1 of Schizophy//um
commune. A number of fragments hybiidized to pR1, however, no differences in
hybriâization ôanding patterns or €thBr stained gel digest patterns wem
observed (Fig. 23). These results suggested that differential methylation of rûNA
migM not be important in sedored gmwth or dlmoiphic switching. (Note; no
differences in hybndzation banding patterns were obsetved for yml end yml
sector DNA isdates; however, the signais wen weak and requim further
and ysis).
werc nui out on a 0.7% TBE gel aî 4SV. ?iDp p W ; eihidium bnnnidc sîaincd gel. Batîan pucl; ficl pobcd wiihJ2P-labcllcd pfSS7. Lam 1, HM III digcsiui lamùda d e r ; lane 2, Hpp II digestcd VAW. lsac 3, Msp 1 digestcd VA30; lanc 4, H' II digestcd pi557 (M ng); lane 5, Msp 1 digcsicd pPSS7 (50 ng): lane 6, Hpcl II digcstcd yml(C): lanc 7, Msp 1 digwtcd yml(C); lanc 8, Hsp 11 digestcd yml(K); laoe 9, Msp 1 digestcd yml(K); loac 10, H p II digestcd yml(M) sector; lanc II, Msp I digestcd yml(M) sector, lane 12, H p II digcsted ynil(0) sector; lane 13, Msp 1 digestcd yml(O) sector, lane 14, Hpo II digcsted yml(P) secior, lane 15, Msp 1 d i g d yml(P) scctor; lanc 16, H p II digcmd yml(W); lant 17, Msp [ digesicd yml(W); tanc 18, Hpa II digestal yml(X); lanc 19, Msp II digcstaî yml(X); lanc 20, H p II digestcd yml(Z); l ~ a c 2 1, Msp II digcsted yml(Z); lane 22, Hind III dipicd lambâa mariter.
Fis. 22. Analysis of pPS57 rnethylation in Ophiostom ulmi strain VA30 transfumants. H' II and Msp 1 isoschizomer digested DNAs (15pgAane) wece nin out on a 0.7% TBE gel at 45V. Top panel; ethidiurn bromide stained gel. Botton panel; gel pcobed with nP-labelled pPS57. Lane 1, Hind III digested lambda marker; lane 2, Hpu II digcsted VA30; lane 3, Msp 1 digested VA30; lane 4, Hpa II digested pPSS7 (Song); lane 5, Msp 1 digested pPS57 (50ng); lane 6, Hpa 11 digested 94-7-27-3; lane 7, Msp 1 digested 94-7-27-3; lane 8, H p II digested 94-8-43; lane 9, Msp 1 digested 94-8-43; lane 10, Hpo II digested 94-8-45; lane 1 1, Msp 1 digested 94-8-45; lane 12, H p II digested 94-8- 4-6, lane 13, Msp 1 digested 94-8-4-6; lane 14, H p II digested 94-8-8-30; lane 1 5, Msp I digested 94-8-8-30.
Fig. 23. Analysis of rDNA methyiation in Ophiostornu ulmi svain VA30 and VA30 transfomana. Hpa II and Msp 1 isoschizomer digestcd DNAs (15&lane) w e n run out on a 0.7% TBE p l at 4SV. Top panel; ethidium bromide stained gel. Bottom panel; gel probcd with XP-labelled pR 1 rDNA probe of Schizophyllum commune. Lane 1, Hid III digested lambda marker; lane 2, Hpu II digest4 VA30: lane 3, Msp 1 digestcd VA30; iane 4, Hpo 11 digested pPSS7 (50 ng); lane 5. Msp 1 dipsted pPS57 (50 ng); lane 6, Hpo II digested 94-7-27-3; lam 7, Msp 1 digested 94-7-27-3; lane 8, H p II digested 94-8-4-3; lane 9. Msp I digested 9-48-43; lane 10, Hpo II digested 94-8-45; lane 1 I , Msp 1 digestcd 94-84-5; lane 12. H p 11 digested 94-84-6; lane 13, Msp 1 digestcd 94-8-44; lane 14, H p II digestcd W-8-&JO; lane 15, Msp 1 digestcd 94-8-8-30.
1 O3
Analysis of DNA mefhyhtion with 5-az-ine
The in hibitor of DNA methylation, 5=azacytidine, was used to investigate
the d e of DNA methylation in the formation of yml, yml sector and regulation of
dimorphic switching. Wild type VA30, yml (2) and yml(0) sector were gmwn (in
tripkate) on CM plates containing 5-azacytidine at concentrations of OpM 1 OpM,
WpM, 100pM and 500pM. Plates were examined both macroscopically and
microscopically for alterations in colony morphoîogy over a three-week petîod.
No changes in colony morphology or altered dimorphic distribution were
observed for any of the cultures, at any concentrations of 5-azacytidine. Radial
gmwîh rates were recorded after 8 days of growth, by taking 3 measuremenls of
colony diameter for each of the triplicate plates. The radial growth of VA30
declined steadily with increasing 5-azacytidine concentration; 7.3M.1 cm on CM
plates without Sazacytidine, to 5.5I0.6 cm on CM plates containing 5ûOpM 5-
azacytidine (Fig. 24). Mutant yml(Z) remained unchanged a 2.W.0 cm. while
the colony diameter of yml(0) sector decreased only marginally; 8.Zîû.2 cm
wit hout 5-azacytidine, to 8.M0.4 cm wit h 500pM Sazacytidine.
Discurslon
In the present study, the technique of insedional mutageneds, as developed
by Royer et al. (1 991), was used to generate and begin a gnetic analysis ot
dimorphic mutants in O. ulmiarain VA30.
Flg. 24. Effect of the methylation inhibitor 5-azacytidine, on radial growth rates of VA30, ym l and ym l sector. Colonies were grown on complet0 medium (CM) plates containing 5-azacytidine at Opg/ml, 1 Opglml, SOpg/ml, 100pglml and 500pg/ml in triplicate for 9 days at 2S°C.
Fig. 25. Effect of agar concentration on the growth of VA30 on various solid media. VA30 was grown on complete medium (CM), complete medium which did not contain yeast extract and minimal medium (MM), at three concentrations of agar: IO@. 20gk and Mg&. Plates were photographed after 9 days of growth at 25'C.
106
Induction of buMing-yeast gmwh by hygmmycin
Hygromycin was found to stmngly induce yeast-like gmwh in O. ulmi
strain VA30. High levels of sucrose (0.6M). piesent as an osmotic stabilizer in
the transformation plates and the oveilay, was dso found to induce budding-
yeast growth in VA30. but to a much lesser extent than what had b e n obsened
wit h hygromycin. H ygrom ycin is a broad spectwm arninoglycoside antibiotic
which has been shown to inhibit pmtein synthesis by interfering with translocation
in eukaryotic translation (Cabanes et al. 1978; Gonzalez et al. 1978) and causes
translational misreading (Singh et al. 1 979). Hygromycin may have induced
yeest-like gmwt h in O. ulmi by activating a cellular stress response, (possibly
through a heat shock mechanism), or by reducing the level/activity of a protein
required for hyphal growth.
The gmwth of cultures on CM plates containing hygrornycin was also
found to induce a lasting alteration to the phenotypes of cultures. The
nontransformed mutant 26, was sirnilady affected by continueci culturing on
hygromycin containing media Mutations may have ocairred to affect the cell
wall to allow the antibiotic to be excluded or to be more effiiently transpoRed
from the cell.
Transformation status of colonies appem'ng on transîbmtion pletes
Yeast-like colonies originally identifieci on transformation plaies as being
either slow or fast growing, were not erpected to contain pPS57 inserts, since
they usually reverted to a slow nontransfomecl rate of growlh by day 30 on CM
plates containing hygromycin. Howevei, only 3 of the 6 hyphal colonies
containeci pPS57 (Fig. 3). ît is unlikely that the vedoi was beiow levels of
detection by Southem analysis; when a blot containing equaîly loaded col1 DNA
(believeâ to amtain 1 or 2 copies of pPS57), and 158 &/ II digested DNAs wen
1 O7
piobed with 32P-labelled pPS57, the cd1 mutant gave a dearîy visible signal,
while no signals wem detected for 158. While 158 was likely a contaminent,
some of the other colonies might have amtained an autonomously replidng
vector, which may have been lost duiing g m h in CM liquid media. Some
nontransformed colonies may have becorne mistant to hygromycin by mutations
induced by transcient or unstable interactions with the transfoning DNA.
Additionally, it has been suggested that transformation conditions may stimulate
the induction of repair processes, and lead to a hypermutabie state where the
stnngent controls over the karyotype are relared and lead to mutations (Zolan,
1995); however, no specific mechanism has been identified h m .
Transformation parameters for strain VA30
Induction of budding-yeast growth by hygmrnycin and 0.6M suciose
required an investigation of the screening strategy used to identify yeast-like
mutants using VA30. Considerable variability in hygromydn sensitivity amongst
strains, has for instance, been found to ocçwr in AspetpiIlus nidulans (Cullen et
al. 1987) - VA30 and MH75 strains may also have b e n variable in their
sensitivity. The resuHs in table 2 indicate that hygromycin strongly induces
budding-yeast growth in MH75, but only atter a 9 dey period subsequent to
plating of the protopiasts. Strain VA30 dearly showed yeasl-like gmwth as won
as colonies were visible on transformation plates.
Transformation experiments Wh strain VA30 should be perlomied with
linearized pPS57 vector in order to maximize the number of stable integrants.
Most yeast-like colonies are likely to be untransfonwed or unstabie transformants
which contain autonomously replicating vector, while hyphal colonies am more
likely to be stable transfomants which have integrated the plmrnid into one or
more chromosomes, and am expressing I@J Bdivhy wfkiontly to wercome the
1 O8
budding-yeast indudng effects of hygromydn. An overlay of agarose is required
for better separation of colonies. Selection of transformants with 200pg/ml
hygromydn gave optimal results by allowing reasonabk seledion of
transfomiants, while maintaining the sunrivel of transformants expressing low
levels of hph activity. ûuerall, the transformation parameters optimized by Royer
et ai. (1991) for MH75 were found to be suitaMe for VA30, and were adopted for
this study.
SeIeciion of dimorphc mutants
Selection of transformed colonies with a predominantl y yeast-like growt h,
was accomplished by transfernng budding-yeast colonies, which were larger than
those found on the no-DNA control pla!es, to CM plates without hygromyun.
Only a small proportion of these transfened colonies had a piedominantly
budding-yeast phenotype on CM plates. These were subsequently purifieci, and
examined for mitotic stability. This screening stmtegy was not optimal. since it
would not identify transformed yeast-like mutants with pooi hph expression, or
transformed yeast-like mutants with an intrinsically very slow rate of growth.
The second strategy which was concunently employed to identify colonies
with a predominently yeast-like growth, was based on the obsenration that neaily
al1 of the slow gmwing, yeast-like caionies becme dafkiy pigmented with tirne.
Pigmented colonies were likely to be either non-transformants, unstable
transfonnants, or transformants with poor hph expression. Fast growing hyphal
colonies overgrew the plates in a few weeks, obaiarring some of these potential
slow gmwing mutants. However, this additional strategy led to the identification
of 4 mutants; yml, ym2, ym3 and ym4.
The straiegy for the selection of mutants gmwing predominantly as a
myoelium may not have ben as serioudy aflecîed by the selednre media
1 O9
Colonies on transformation plates with reduced budding yeast growth were
transfened to CM plates and examineci under a dssecling andlor compound light
microscope. Only one predominantly hyphd colony was iecovered: 94-84-6.
Selection by this method required that colonies have the ability to grow
predominantly in the hyphal phase on CM plates containing both hygromydn and
0.6M sucrose. A more effective screen for hyphal mutants might utilire CM
minus yeast extract, with a lower concentration of agar, such as 1.5% (1% plates
would likel y be too watery); these conditions strongly favour yeast-like growth in
the wild type (Fig. 25). Yeast-like mutants would likewise be more effectively
selected, by pîating protoplasts onto CM with 4% agar. For efficient scieening of
budang-yeast mutants, the use of a vector other than pPS57 is required. An
altemate osmotic stabilizer should be used, since 0.6M sucrose altered the
dimorphic distribution. The effect of sucrose on the dimorphic distribution may
have been caused by osmotic stress, an increase in caibon source
concentration, perhaps thiough cetabolite repression, or alteration of the
caibonhitrogen ratio.
The overall nurnber of stable transformants screened was ditficuit to
estimate accurately. Southem analysis indicated that when transfomations were
performed with cimlar vector. that few slow or fast growing yeast-like colonies
and only some hyphal colonies were likely to be stable transformants (Fig. 3).
However, transformation with linear vector produced more staôîe transformants
than circularized vector; this wes reflected in the greater proportion of hyphal
coknies and lower proportion of yeast-like cdonies oblained with lineaiited
vector (TaMe 2). Royer et al. (1991 ), ieported that Hneerlration ot pPS57
increased the frequency of stable transformation (number of transformant$ per
microgram of pPS57 DJA per IO7 protoplmts) from 3W15.3 to W11.7 in an
eailiei stuây with MH75, and that linearization al= notinnahly reduced unstable
110
transfonants. With optimized conditions, Royer et al. (1 991) reported
transformation frequendes as high as 4000. Estimated transformation
frequendes obtained in this study using drwlar and linearized vector (1 7-
and 151 CH35 respedively). were comparable to those obtalned by Royer et al.
(1 991 ) and those of Bowden et crl. (personal communication). using MH75.
Based on the number of hyphal colonies growing on transfomation plates in
several experiments, an estirnated 8,655 colonies were screened.
Mutants isolated
The ym l budding-yeast mutant
A total of fourteen colonies with aitered dirnorphic distribution were
isdated using the selection rnethods of this study. Fow colonies with a
predomi nantl y budding-yeast cell phenotype, designated ym 1, ym2, ym3 and
ym4, were nonpigmented and grew faster than the wikl type on hygromycin
containing media. Mutant yml may have a nutritional or metabdic defect which
severely limits gmwth on a nutrient poor medium. Altematively, the added level
of metabolic stress experienced by yml on minimal media may induce a stronger
metabolic stress signal, which transduces into a decreased rate of growth, and a
stronger induction of the budding-yeast phase. The yml mutation was different
than the MH75 defived col1 mutation isolateci by Royer et el. (1 991 ); cdl had a
smooth colony phenotype and grew well on minimal media plates (Fig. 1).
Meiotic analysis of yml established that pPS57 DNA did not cosegregate
with the mutant phenotype, and that the insertion of pFS57 into chromowmal
DNA likely did not directly cause the yml phenotype. It is possible that the vector
pPS57 may sUll Yag" the site of mutation. If, for instance, the yml phenotype
was caused by a chromosomrd break, and the vector joined to the bioken
111
fragment then rejoined to another chromosome, or to the bmken chiornosorne
greater than 50 map units away from the initial breakage site, then the vector
could Mill tag the mutated genelgenetic elernent. If tMs occured, then the yml
mutation could still be m v e r e d by pîasmid rescue. The ratio of mutant to wild
type progeny approximated 1 :1 (0.W5q~û.01) and may be consistent with the
inheritance of a single nuclear mutation. There were slightly more progeny with a
wild type phenotype than with a mutant phenotype (139 wiM type and 99 mutant).
Non-meiotic budding-yeast cells or mycelia from the W2-TOC fernale culture may
have contaminated the meiotic progeny to increase the apparent number of wild
type progeny. Secondly, fast growing wild type colonies rnay have obscured the
yrnl colonies which were extremely slow gnnnring.
Othe? mutants
Several ot her colonies wit h altered dimorphic distribution were isolated
but only partially characterized; eight budding-yeast cdonies and one hyphal
colony were obtained by transferfing yeast-like colonies from transformation
plates onto CM plates, and selecting colonies which maintained an altered
phenotype. Only five of the colonies were transformants as detennined by
Southern analysis. Linear gmwth on CM+hygm2ûûpg/ml plates was, as eitpected.
a good indication of transformation; only those colonies with a linear growth rate
significarttly greater than VA30 were tramforW. The size and intensity of the
Bgl II and M d III bands was greeler than what had been obseiued for yml
(which contained 3 copies of pPS57 at mm), indicating that pPS57 likely
integrata as tandem repeats in at least one site in each tmnsfomied cdony.
One intense trailing &Ill and H M III ôand was found for each of the colonies
with the exception of the Hind III digest of $4-8-4-5, for which two bands were
observed (Fig. 12a, lane 16). This result is amsistent with a tandem integrdon
112
of pPS57, where one Hind III restriction site had been left intact in the vector, or
mth a second site of chromosomal integration by a single copy of pPS57. ît
appears as if the Hind III restriction sites were lost upbn integration ot the Himl III
linearizeû vector in each case. since transformant DNAs digested with Hind III
and pmbed with pPS57 never resolved a band at 5.38kb. the expected sire of
pPS57. Loss of restriction sites in the linearized vector may indicate degradation
of vector ends by exonuclease activity or some aspect of the mmbination and
repair proces.
Meiotic analysis of the three stable transfomed cdonies 94- 7-27-3. 94-8-
4-3 and 94-84-6, suggested that none were tagged by pPS57; 947-2793
reverted to a wild type phenotype when cultural material was grown from -70°C
storage. Despite this phenotypic change, 94-7-27-3 was crossed with W2-TOL to
examine the nature of the progeny. However, no mutant progeny were obseived.
Similady, no mutant progeny were oôserved with the 91-8-4-3 X WBTOL rnating
on CM plates, and only a few colonies with a 94-8-4-6 mutant phenotype were
observed amongst hundreds of typical wild type colonies on CM plates. ît is
possiMe that the 94-8-4-6 phenotype may not have ben an inheritable genomic
mutation, and the few mutant cdonies observeci resuîted from mitotically derived
cells contaminating the sticky mass of meioticelly derivecl ascospores at the top
of the peritheda. Abmatively, the mutation causing the 94.8-4-6 phenotype
and those of 94-8-49 may have been poorîy tmnsmitted to their piogeny. Low
transmissibility of the phenotype to progeny may have ocarned. for example, if
the mutant phenotype had been caused by a c h r o m m a i aiteration such as a
brd<en chromosome, and loss of a c h r o m m a l fragment, the latter of which
could have been selected against. Mutations migM elso have been
mitochondriaî, in which case none of t k progeny would display the mutant
phenotype if the mitochondria were matemaîly inherited. Foi 94-843, the
I l 3
colony phenotype may have reverted to the wiM type h m the tirne the elmwood
agar plates were inoculated with spore suspensions of 94-8-4-3, until the time
meiosis occuned. Adâtionally, none of the progeny fmm the 94-86-3 cross
were obseived on h ygromycin containing plates (versus several hundred
colonies observeâ on CM plates with the same size of spom inoculum). Pwr
transmissibility of hph gene expression may have been caused by DNA
modification; judging frorn the intensity and size of the band obtained by probing
94-8-4-3 DNA with 32P-labelled pPS57 (Fig. 12a, lanes 14 and 15). the 91-84-3
mutant appeared to contain multiple copies of pPS57, and therefore may have
been subjed to DNA modification.
FuRh.r a ~ l y s l s of yml and yml secfor
Mutant yml was mitotically stable for appmximately six rnonths but then
began to fom sectors which had a flufiy appearance with a much reduced
budding-yeast phase, and were typically faster growing than the VA30 wild type,
(se8 Figs. 15 and 16 for colony morphologies on plates, Fig. 6 for liquM media
gmwth, and Fig. 17 for microphotographs). Sectors were never oôsenred to
revert back to a yml phenotype. The fornation of these sectors was of pafticular
interest, dnce ym 1 and ym 7 sector exhiôited the two extremes of dmorphic
distdôutions, were derived lineaily fmm VA30, and theiefore might underscore a
cornmon genetic mechanism invdved in morphk switching.
An alteration in the number of pP S57 inselts, or additionai mutation
through transposition of pPS57 to another chromosomal location, were
considered as possiôie rnodds to explain cudorlng, Moi to the r e w b of the
meiotic anelysis. The ability of yml -01 to grow wdl on CMthyg.2ûOpgIrnl
plates (mg. 16) initially suggested that yml sectom dd not lose the pPS7 insert
114
and Southem analysis of yml and yml sectors probed with 32Plabelled pPS57
confirmed that neither an inctease nor a decrease in the number of pPS57
insertions had ocaineci (Fig. 1 8).
Analysis of yml sector X W2-TOL piogeny also indicated that the pPS57
inseition did not tag the mutation responsiMe for the sector phenotype (Table 6).
MigM the presence of pPS57 in the yml mutant have predisposed il to sector
formation through an increased tendency to cause chromosornal reanangements
or karyotypic instability by homologous recombination? Recombination between
ectopic copies of pPS57 in yml would be extremely unlikely, since pPS57
integrated et a single chromosomal location with a single or at most two copies
(32P-labelled pPS57 hybridizing ta Bgl II digested yml DNA gave a weaker signal
than that of the ooll positive control, which may contain two copies of pPS57 at
m0St; S8û f ig. 8).
The meiotic progeny of yml sector X W2-TOL possessed phenotypes
which were either like the VA30 X W2-TOL control progeny, or the yml sector.
Slow growing yml-like progeny were not found in the ,200 progeny examined,
indicating that ym l and ym l sector were most likely caused either by two very
closely linked mutations, or by two mutations at the same locus. The yml sector
phenotype could have resulted from a chromosomal break, and the yml mutant
allele may have been lost in a chromosornai fragment.
Both wild type VA30 and yml sector grew predominantly in the hyphaî
fom on MM plates. The original yml mutant grew dowly, and predominanty in
the budding-yeast tom, on MM media plates (Ag. 16), yet did not have a
significantly reduced rate of groWh in CM liquid media (Fig. 6). Mnimal media
poorly supported growth with this metabolic mutetion - the gene/genetic element
mutated in yml may have had a rnetabdic fundion. Altematively, an added
metabolic stress irnposed on the yml mutant by minimal media may have
115
resulted in the transduction of a stronger signai to aller the contmls goveming
gmwth, resulting in both a reduction of growth rate, and an induction of the yeast
phase.
CHEF anaîysis
Pulsed field gel electrophoresis (CHEF) chromosomal separations of ymt
and yml sectors probed with 32P-labelled pPS57 esteMished that pPS57
integrated into chromosome IV, and that pPS57 diâ not exist as an autonomowrly
replicating , high moleculai weight oligomer (Fig. 20). Et hidium bromide stained
chromosomal separations of yml and yml -or wem similar to that obtained
for VA30, and did not indicate that chromosomal polymorphisms were associatecl
with the formation of the yml mutant or yml secîored gmwth. One of the three
sector isolates, ymlJb, exhibited a putative chromosomal pdymorphism (Fig. 20,
lane 7). Sectors of ym1 or yml itself may have had a genetic alteration which
predisposed the mutant to chromosomal breakage or othei chromosomal
instabilities. Examples of these instabilities would indude: Alteration in the
distribution or numbei of repetitive DNA spedes such as the iDNA repeats,
dispersed repetitive elements, or the ectivity of transposons or retrovinises.
Alternatively, the yml mutant may be in a metabolicaîly stmS88d hypermutaMe
state which promotes karyotypic instability. ln response to starvation or certain
other physidogical states, Zdan (1 995) has suggested that either the controls
goveming ùaryotypic stability becorne relexed, or DNA iepair hrnctions becorne
induced, leadng to phenotypic variability and chiorno8omal variation. Cdd
conditions for example, were found to induoe vadous types of cdony mutants in
C. abkms, some of which showed speciîk karyotypk alteraîions (Rustchenko-
Bulgac et al. 1990). Several of these C. dbicans cobnies are very sirnilai in
appearance to the O. ulm yml mutant whîch m y or may n a be signifiant.
I l 6
Aiterations to the rûNA chromosome were not observed in yml or yml
sector isolates at the level of resolution obtained hem. it was considered
possible that aiterations to the rDNA tract, induding translocation, expansion or
contraction, might have been assodated with either the slow growing yml mutant
or fast growing yml sector formation. Variation in the iDNA chromosome has,
for example, been obsenred after transformation, and changes in rûNA tract
length have been correlated with alteration of growth rates (see Zolan, 1995).
Growth-related amplification of rDNA sequences has been found to occur in
Kluyveromyces lactis; slow growing strains that experienced an increase in
growth rate, ais0 underwent an increase in the size of the rûNA duster. Fast
growing cuHures of C. ai'bicens and S. cemw'siae have dso been found to contain
more copies of iDNA than slow growing cultures (see Zdan, 1995). Changes in
chromosome size often involve the iDNA chromosome and variability of the
rDNA chromosome has been obseiveâ amongst several fungi examinai,
induding 0. ulmi (Dewar, personal communication, in; Zolan. 1995).
CHEF gel separations of ym1 and ym1 probed with the 32P-labelled pR1
rDNA probe of Schizophy/lum commune did not, however, indicate that
alterations to the iDNA tract haâ ocarrred (Fig. 20, bottom panel). Finer
detection methods would be required to iâentify srnall rûNA expansions or
contractions, or rearrangements within the same chromosome not detectabb by
the methods used hem.
Transformation conditions have also been suggested to cause the
induction of repair processes which resuît in karyotypic changes (Zolan, 1995),
and may suggest a mechanism for the generation of the nontagw mutations
obtained in the cunent study. Extensive karyotvpic aiteration duiing
transformation has, for instance, b e n reportecl to ocav in several fungi: A
nidulens (Xuei and Sketrud, 19944 C. alMans (6arton et al, 1994), Cqprin~S sp.
117
(Casselton, 1995), Coprinus dnereus (PuMla and Sknynia, 1993). and N.
cessa (PeiWns et al. 1 993). Peikins et al. (1 993) have ieported chromosomal
reanangements to occur in 10% or more of dl mitotically stable transfonnants,
but aîmost no rearrangements in unt ransformed control reactions, indicating that
the vector DNA itself must somehow be involved in the induction of
reanangements. The lad< of mutant colony phenotypes observeci on O. ulmi no-
DNA transformation control plates and t heir presence on t ransformatiocr plates
where pPS57 was included, is consistent with this hypothesis.
DNA methylation
A preliminary analysis of DNA methylation found no evidence for its
involvement in the formation of the yml mutant, yml secton, or in the control of
dimorphic growth in 0. ulmi. DNA methylation has generally been found to
conelate with gene inactivity and hypomethylation Wth gene activation, end is
believed to play a role in the regulation of gene expression and cellular
differentiation in eukaryotes (Magill and Magill. 1989). Regulation of fungel
dirnorphisrn by a DNA methylation mechanism has been sugg8!3Wd for Wear
spp. (Cano et al. 1988; Orlowski, 1995; see introduction and discussion below).
A correlation between levels of methylation and dimorphic switching has also
b e n found to occur in C. albicans, where 5-methylcytosine (5-mC) levels have
been reported to be about twofold lower in the hyphel form than in the yeast-like
fom (Russell et al. 1987). SmC is the only base found to be methyîatated in
eukaryotic DNA (see Selker, 1 993).
No evidence was bund for WgO methylation of vector DNA in O. uhni
transformanls. Methylation of transforming DNA ha$, for example, been found to
ocair in N. massa in instances wem more then one copy of the vectoi ha8
integrated (Bull and Wootten, 1984; Pandit and Russo, 1992). No S n i C p O
118
methylation of insert or flanking genomic DNA was detecteâ using Msp I and Hpa
II isoschizomer anaîysis (Fig. 21). More irnportantly, meiotic analysis of yml and
yml sector established that the site of vector insedon dkl not cosegregate with
these mutant phenotypes. Therefore, nelhei mutation nor epigenetic alleiation
of vector or flanking genomic DNA by a DNA methylation mechanism wuid have
been responsible for causing the obseived mutations.
Ym 1 and ym 1 sectors were examined for the diff erenti al met hylation of
their iDNA repeats. using H ' II and Msp I isoschizomer analysis. In a number
of fungi which have been studied, the rDNA repeat has wually been shown to be
highly methylated, but hypomethylation of rDNA has also ben observed (see
Magill and Magill, 1989; see Li and Horgen, 1993). A greater level of SmC
residues in rONA regions has also b e n repoRed to occur in ungeminated
versus germinating conidia of N. cmsa (see Magill and Magill, 1989). No
CsWGG methylation of iDNA repeats was detecîeâ in yml and yrnl sedois
(Fig. 23), nor was any methylation detected in the rDNA repeats of wild type
VA30 or in any other of the transformants analyzed (Fig. 24). It therefore
appean as if generation of the ym l and ym 1 sector phenotypes could not be
accounted for by modulation of iRNA gene expression through methylation. Nor
did a correlation exist between colony phenotype ( W i n g yeast cell growth in
yml versus hyphal growth in yml sector) and the degme of rûNA methyletion, as
measured using isoschizomer analysis.
The role played by DNA methylation in 0. ulmi dimorphism and the nature
of the yml and yml seclor mutationls, wes investigated using the DNA
methylation inhibitor 5-azacytidine. No alterations to VA30, yml or ym 1 sedor
phenotypes were obsenred on CM plates cmtaining 5-ataeytkline concentrations
of up to 50pM (Fig. 25), suggesting that DNA methylation might not be imoîved
in dimorphic switching or yrnl -or formation in 0. ukni. However, there was
119
no simple way to demonstrate that 5-azacytidine was entering the cells and was
able to effect DNA demethylation (part of the rationale for examining rûNA
methylation was to identify a rnethylated DNA sequence which could be used to
verify cytosine residue dernethylation in studies with 5azacytiâine). Since a
genelgenetic element of O. ulmi has not been demonstratd to be methylated
using isoschizomer analysis, detection of O. ulmi DNA demethylation by 5-
azacytidine activity would require measumment of 5mC levels in cultures before
and after 5-azacytidine treatment using a more sensitive technique such as
HPLC. Preliminaty HPLC measurement of methylation in O. ulmi (Horgen et al.
unpublished data) does indicate that 5-mC is present in low quantities, and that
greater levels of 5-mC may be present in the yeast phase than in the hyphai
phase (1.1 mole % and 0.6 mole % respectively). The reduction in growth rate
by VA30 with increasing concentrations of 5azacytiâine (Fig. 24), suggests that
Bazacytidine was likely entering the cell. In N. crassa, a final concentration of
30ûpM 5-azacytidine in plates easily induces the demethylation of cytosine
residues, yet does not result in any major inhibition of growth (Pandit and Russo,
1 992) and 25pM Bazacytidine is known to result in the kss of approrimately
90% of cytosine methylation (Cambareri and Kinsey. 1993). The same may not
be true of the 0. ulmi system - verifmtion of cytosine resMue demet hylation is
required. It may be signifîînt thad the linear growth rates of yml and yml sector
were not affecteâ by high levels of 5azecytidine. In Mucor spp. the polyamine
putresine has been proposad to regulate DNA methylation by indudng the
demethylation of genes during the yeast to hyphal transition (Cano et al. 1988).
The enzyme omlhine decarboxylase, which catelyzes the formation of putresine
from ornithine, was shown to be MOdceâ by daminohtanone (OA8). DA6 also
inhibits the yeast to hyphal tiansilkn, and, consistent with a DNA methyûation
mechanism for the contml of gene expression, the methylation inhibitor 5-
120
azacytiane was shown to counteract the effeds of DA0 and restore the yeast to
hyphal transition. supposedly by indudng DNA demethylation (Cano et al. 1988).
Surprisingly, in the Cano et al. stuây, the overail level of ONA methylation in
spores was so great. that Msp IIHpa II isoschizomer digestion of total DNA
proâuced extremely different distributions of DNA fragments, which was
consistent with a high degree of 5-mC DNA methylation. Ukewise, a treatment
with 2mM 5-azacytiâine resulted in identical distributions of lower molearlar
weight fragments, consistent with complete dernethylation of total DNA. Baseâ
on what is cunently known, it remains unclear whether DNA methylation plays a
prlmary role in controlling fungai dimoiphism. Methyîation and demethylation of a
single key gene's promoter region could facilitate a dimogW switch. The
number of DNA probes that could be screened for differentiaî methylation would
be unlimited, but few DNA probes are actually available. Therefore. the fiist step
in analyzing the role played by DNA methylation in dimorphic control, should be
to determine whether the methylation inhibitor 5-azacytidinr can induce a
dirnorphic switch. Higher concentrations of bazacytidine should be utilized, and
demethylation of cytosine residues verifid using HPLC.
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