A GAL4-Like Protein is Involved in the Switch Between

8/4/2019 A GAL4-Like Protein is Involved in the Switch Between

http://slidepdf.com/reader/full/a-gal4-like-protein-is-involved-in-the-switch-between 1/12

The Plant Cell, Vol. 12, 1579–1589, September 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

A GAL4-like Protein Is Involved in the Switch betweenBiotrophic and Necrotrophic Phases of the Infection Processof Colletotrichum lindemuthianum on Common Bean

Marie Dufresne,

a,1 Sarah Perfect,

b

Anne-Laure Pellier,

a

John A. Bailey,

b

and Thierry Langin

a

a

Laboratoire de Phytopathologie Moléculaire, Centre de Recherche sur les Plantes, Université Paris-Sud, 91405 Orsay

Cedex, France

b

Department of Agricultural Sciences, Institute of Arable Crops Research, Long Ashton Research Station, University of

Bristol, Bristol BS18 9AF, United Kingdom

Random insertional mutagenesis was conducted with the hemibiotrophic fungus Colletotrichum lindemuthianum

,

causal agent of common bean anthracnose. Nine mutants that were altered in their infection process on the host plant

were generated. One of these, H433 is a nonpathogenic mutant able to induce necrotic spots on infected leaves rapidly.

These spots are similar to those observed during the hypersensitive reaction. Cytological observations showed that the

development of the mutant H433 is stopped at the switch between the biotrophic and the necrotrophic phases. This

mutant carries two independent insertions of the transforming plasmid pAN7-1. Complementation studies using the

wild-type genomic regions corresponding to the two insertions showed that one is responsible for the H433 phenotype.

Sequencing analysis identified a single open reading frame that encoded a putative transcriptional activator belonging

to the fungal zinc cluster (Zn[II]

2

Cys

6

) family. The corresponding gene was designated CLTA1

(for C. lindemuthianum

transcriptional activator 1). Expression studies showed that CLTA1

is expressed in low amounts during in vitro culture.

Targeted disrupted strains were generated, and they exhibited the same phenotype as the original mutant H433. Com-

plementation of these disrupted strains by the CLTA1

gene led to full restoration of pathogenicity. This study demon-

strates that CLTA1

is both a pathogenicity gene and a regulatory gene involved in the switch between biotrophy and

necrotrophy of the infection process of a hemibiotrophic fungus.

INTRODUCTION

Fungi, one of the most damaging groups of microorganisms

to plants, are characterized by a wide variety of infection

strategies, from strict biotrophy to necrotrophy. The ability

to utilize multiple modes of infection makes it difficult to pro-

vide a comprehensive explanation of the mechanisms in-

volved in successful infection of a host plant.

During the past decade, different strategies have been

developed to identify the fungal genes specifically involved

in infection of the host plant, the pathogenicity genes (Oliver

and Osbourn, 1995; Hensel and Holden, 1996; Hamer and

Holden, 1997). Among these strategies, random insertional

mutagenesis has been used to study several plant patho-

genic fungi (Bölker et al., 1995; Dufresne et al., 1998;Sweigard et al., 1998; Balhadère et al., 1999). A major ad-

vantage of such a strategy is that there is no a priori postu-

late about the function or expression of the tagged genes.

Moreover, functions have been identified more readily than

when a direct candidate approach is used. In Magnaporthe

grisea

, random insertional mutagenesis made it possible to

identify many pathogenicity genes that potentially encode

various enzymes acyltransferase, neutral trehalase, and the

catalytic subunit of a cAMP-dependent kinase (Sweigard et

al., 1998). Studies of pathogenicity genes generally yield a

large number of path

mutants, thereby confirming that

pathogenicity genes constitute a major part of the genome

of plant pathogenic fungi.

Colletotrichum lindemuthianum

is the causal agent of an-

thracnose on common bean. This interaction has been as-

sumed to fit the gene-for-gene hypothesis (Flor, 1971), butgenetic studies have been performed only for the plant part-

ner (Young and Kelly, 1996; Geffroy et al., 1998). Phenotypi-

cally, the infection of a resistant cultivar of common bean by

a corresponding avirulent strain of C. lindemuthianum (in-

compatible interaction)

leads to the

appearance of localized

necrotic spots typical of a classical hypersensitive response.

The infection process has been well documented in previ-

ous cytological studies (O’Connell et al., 1985; Bailey et al.,

1992). Basic compatibility, that is, interaction between a

1

To whom correspondence should be addressed. E-mail marie.

[email protected]; fax 44-1603-250024.



1580 The Plant Cell

susceptible cultivar of common bean and any virulent strain

of C. lindemuthianum

, begins with the adhesion of conidia

to aerial parts of the plant. Conidia then germinate and dif-

ferentiate to form an infection structure devoted to mechan-

ical penetration, the appressorium. After the penetration

step, the infection cycle is characterized by two successivephases. In the first phase, lasting 3 to 4 days, the fungus

grows biotrophically inside the infected epidermal cells. Dur-

ing this phase, referred to as the biotrophic phase, the fun-

gus differentiates infection vesicles and primary hyphae. The

second phase, which corresponds to the appearance of an-

thracnose symptoms, is completed 6 to 8 days after inocu-

lation. During this phase, the necrotrophic phase, the fungus

develops secondary hyphae that grow both intracellularly

and intercellularly and thus acts as a typical necrotrophic

pathogen. C. lindemuthianum

is classified as a hemi-

biotrophic fungus because of the succession of these two

phases during the infection cycle. Because the host plant is

not required for fungal growth, this pathogen can be manip-

ulated in vitro, in contrast to the case with strictly biotrophic(obligate) pathogens.

A random insertional mutagenesis technique was devel-

oped in our laboratory to identify essential virulence deter-

minants of the fungus (Dufresne et al., 1998). Nine less-

pathogenetic mutants were recovered from 1200 transfor-

mants tested. The molecular analysis of mutant strain H290

allowed us to identify the first C. lindemuthianum

pathoge-

nicity gene, CLK1

, which encodes a putative serine/threo-

nine kinase and is involved in the regulation of appressorium

function (Dufresne et al., 1998).

We report here the cytological and molecular character-

ization of one nonpathogenic mutant, designated H433. We

first show that this path

mutant is blocked during the infec-

tion process at the switch between biotrophy and necrotro-

phy. Then, we demonstrate that the mutant phenotype is

related to the interruption of the CLTA1

gene (for C. linde-

muthianum

transcriptional activator 1), which encodes a pu-

tative GAL4-like transcriptional activator. These studies

allowed us to identify and characterize a regulatory gene in-

volved at the transition between biotrophic and necrotrophic

phases, a key step in the infection process of the hemi-

biotrophic fungus C. lindemuthianum

.

RESULTS

Macroscopic Characterization of the H433

Mutant Phenotype

The mutant strain H433 was assessed in vitro for radial

growth, sporulation, and spore morphology. No significant

differences were observed when compared with wild-type

strain UPS9 (data not shown), demonstrating that the H433

strain is specifically altered for pathogenicity. When inocu-

lated on the susceptible cultivar of common bean La Victoire

(excised leaves or plantlets; see Methods), the mutant strain

was unable to produce anthracnose symptoms even after a

long incubation (Figure 1). Moreover, more detailed obser-

vations revealed that infection by mutant H433 reproducibly

led to the appearance of small necrotic spots similar to

those observed in a hypersensitive response

3 days afterinoculation. These necrotic spots were also observed after

inoculating another susceptible cultivar of common bean,

P12S, with strain H433 (see Methods). Figure 2 illustrates

the similarity of symptoms induced on two near-isogenic

lines of common bean (see Methods), the susceptible line

P12S (Figure 2A) and the resistant line P12R (Figure 2B) af-

ter inoculation with mutant strain H433.

These results show that mutant strain H433 displays a

double phenotype: it is a nonpathogenic strain, and it is able

to induce on the two susceptible cultivars tested local ne-

crotic reactions similar to those observed during an incom-

patible interaction.

Figure 1. Pathogenicity Tests of Mutant Strain H433 on the Sus-

ceptible Cultivar La Victoire of Common Bean.

(A) Excised cotyledonary leaves were inoculated with wild-type

strain UPS9 and mutant strain H433. Leaves were photographed 7

days after inoculation.

(B) Eight-day-old plantlets were spray-inoculated with either wild-

type strain UPS9 or mutant strain H433. Photographs were taken 7

days after inoculation.



Regulation of Biotrophy/Necrotrophy Transition 1581

H433 Is Blocked at the Switch between the Biotrophic

and Necrotrophic Phases

To determine the step altered in the mutant strain during the

infection process, we first assessed mutant strain H433 for

appressorium formation on the hypocotyl epidermis. For

wild-type strain UPS9 and mutant strain H433, the majority

of conidia germinated and produced appressoria (data not

shown), showing that H433 is not altered at this critical step

of the infection process.

To study the next steps of the infection cycle, we con-

ducted cytological observations with hypocotyl segments of

the cultivar La Victoire. Bright-field microscopic observa-

tions confirmed the induction of cell death, as visualized by

brown epidermal cells (Figure 3A). The brown color corre-

sponds to the accumulation of phenolic compounds after

cell death (O’Connell et al., 1985). These observations also

confirmed the formation of normal-looking appressoria (Fig-

ure 3A). Nomarski differential interference contrast observa-

tions revealed that appressorium function appears to be

normal because

infection vesicles and primary hyphae de-

velop and grow normally in infected epidermal cells beneath

appressoria (Figure 3B). However, even after a long incuba-

tion (14 days after inoculation; Figure 3B), no secondary hy-

phae were visible. After the same length of incubation, wild-

type strain UPS9 had invaded infected tissue with second-

ary hyphae (data not shown).

Such observations were confirmed by comparing the hy-

phal development of wild-type strain UPS9 and H433 ininfection time courses of hypocotyl segments of the suscep-

tible cultivar La Victoire. Mean values of hyphal length for

different points of the infection time course (Figure 3C)

clearly indicate that the hyphal growth of mutant strain H433

is inhibited at 3 to 4 days after inoculation, compared with

the extensive hyphal development of strain UPS9. This inhi-

bition kinetics is consistent with the previous observations

that only primary hyphae were differentiated by the mutant

strain H433.

We have thus demonstrated that mutant strain H433 is

blocked after primary hyphae differentiation, at the transition

between the biotrophic and necrotrophic phases, which cor-

relates with the nonpathogenic phenotype. Infection by this

mutant strain is further characterized by the appearance of

small necrotic spots, similar to hypersensitive lesions, on thetwo susceptible cultivars of common bean that were tested.

Molecular Analysis of the pAN7-1 Integration Pattern

and Recovery of Sequences Flanking the Insertion Loci

DNA gel blot experiments were conducted to determine the

integration pattern of the vector pAN7-1 (Punt et al., 1987),

Figure 2. Pathogenicity Tests of Mutant Strain H433 on Two Near-

Isogenic Cultivars of Common Bean.

For both cultivars, excised cotyledonary leaves were inoculated with

mutant strain H433. Photographs were taken 7 days after inoculation.

(A) Susceptible cultivar P12S.

(B) Resistant cultivar P12R.

Figure 3. Development of Fungal Infection Structures during Inter-

action of Susceptible Cultivar La Victoire with Mutant Strain H433.

(A) and (B) Leaves before and after, respectively, treatment with

chloral hydrate. AP, appressorium; HP, primary hyphae; VI, infection

vesicle. Bars in (A) and (B) 10 m.

(C) Time course of hyphal growth (see Methods). Square data points

are mutant strain H433; circles are wild-type strain UPS9.



1582 The Plant Cell

which was used in transformating wild-type strain UPS9 to

generate the mutants. Two hybridizing bands were detected

after KpnI restriction digestion (KpnI does not cut within the

transforming plasmid) of genomic DNA from the H433 mu-

tant strain, indicating that the vector had inserted in at least

two different loci in the genome (data not shown). Theshorter KpnI hybridizing band (

12 kb) is referred to here as

the first locus and is designated 433.1. The longer hybridiz-

ing band (

12 kb) is the second locus and is designated

433.2. The number and lengths of hybridizing bands ob-

served with HindIII and EcoRI restriction digests confirmed

this result and are consistent with the insertion of a single

copy at the 443.1 locus and with tandem integration of at

least two copies at the 433.2 locus (data not shown).

A marker rescue strategy was used to recover the se-

quences flanking each integration site. For 433.1, the re-

striction enzyme KpnI was used to subclone a 2.0-kb

BamHI-HindIII fragment, designated SF433.1 (for sequences

flanking locus 433.1); for 433.2, because of the integration

pattern in tandem, we used the restriction enzyme HindIIIand recovered only one side of the locus, a 1.1-kb HindIII-

PstI fragment, designated SF433.2. Both fragments were

used as probes for restriction digests of UPS9 genomic

DNA. The simple hybridizing patterns were consistent with

the presence of a unique copy of each of these sequences

in the genome of C. lindemuthianum

(data not shown). The

probes SF433.1 and SF433.2 were then used to screen a

UPS9 genomic library to recover complete regions corre-

sponding to those of the wild-type strain. Long EcoRI frag-

ments of 11.0 and 8.0 kb, for the loci 433.1 and 433.2,

respectively, were subcloned. Restriction maps and location

of pAN7-1 insertions are shown in Figures 4A and 4B.

Complementation of the Insertion at Locus 433.2

Partially Restores Pathogenicity

To determine which integration event of vector pAN7-1—at

locus 433.1, locus 433.2, or both loci—was responsible for

the H433 mutant phenotype, we set up functional comple-

mentation experiments. Two functional complementation

plasmids, designated p433.1Phleo and p433.2Phleo, were

constructed by introducing the corresponding 11.0- and

8.0-kb EcoRI fragments into the unique EcoRI site of vec-

tor pAN8-1, which confers phleomycin resistance (Mattern

et al., 1988). H433 protoplasts were independently trans-

formed with the constructed plasmids p433.1Phleo andp433.2Phleo. For each transformation experiment, we re-

covered transformants carrying both the mutant allele from

the recipient strain H433 and the complete wild-type allele

from the transforming plasmid.

To establish whether complementation by either of the

two genomic regions could restore pathogenicity, we inocu-

lated excised primary leaves of the susceptible cultivar La

Victoire with spore suspensions of each type of transfor-

mant (at least four strains of each type), wild-type strain

UPS9 (Figure 5A), and the original mutant strain H433

(Figure 5B). All of the strains transformed with plasmid

p433.1Phleo (e.g., T433.1.2; Figure 5C) had the same mutant

phenotype as that of recipient strain H433, demonstrating

that the integration event at locus 433.1 is not responsible

for the mutant phenotype. In contrast, the strains trans-formed with plasmid p433.2Phleo (e.g., T433.2.19; Figure

5D) partly recovered pathogenicity on excised primary

leaves: reduced anthracnose symptoms were evident on

leaves, with a slight delay of 24 hr for symptoms to develop,

as compared with symptom development on leaves inocu-

lated with UPS9. All of the strains transformed with plasmid

p433.2Phleo also retained the ability to induce the appear-

ance of small necrotic spots, as did the original mutant

H433 (data not shown). These results demonstrate that the

integration event at locus 433.2 is responsible for the mutant

phenotype. The same results were obtained by using a 4-kb

HindIII subclone (see Figure 4). This fragment was therefore

selected for further analysis.

Mutant H433 Is Affected in a Gene Encoding a Putative

Zn(II)

2

Cys

6

Transcriptional Activator

The 4130-bp HindIII fragment was sequenced on both

strands. The SF433.2 fragment was used as a probe to

screen a cDNA library generated from wild-type strain UPS9

grown on rich medium (see Methods). Two independent

cDNAs were also recovered and sequenced on both

strands. Nucleotide sequence analysis revealed the exist-

ence of an open reading frame of 2526 bp (including introns)

Figure 4. Location of pAN7-1 Insertions in the Wild-Type GenomicRegions Corresponding to Loci 433.1 and 433.2.

(A) Restriction map of the 433.1 locus.

(B) Restriction map of the 433.2 locus. The two boldface H’s indi-

cate the location of the 4-kb HindIII fragment used in the comple-

mentation experiments.

B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S, SacI; X, XhoI; Xb,

XbaI. For each locus, the location of the insertion event is indicated

by the arrowhead. The locations of the recovered flanking se-

quences SF433.1 and SF433.2 are indicated by bars.




that potentially could encode a 746–amino acid polypeptide

having substantial homology with fungal transcriptional acti-

vators of the GAL4 family. This gene was designated

CLTA1

. Its nucleotide and deduced amino acid sequences

are presented in Figure 6.

The gene contains five introns, all of which exhibit the ex-

pected splice sites (Ballance, 1986). The putative transcrip-

tion start point is located 722 bp upstream of the initiation

codon, as determined by identical 5

ends for the two

cDNAs. The insertion event of vector pAN7-1 in the mutant

strain H433 occurred 303 bp upstream of the initiation

codon of the CLTA1 gene. This site is between the putative

transcription start point and the initiation codon of the

CLTA1

gene.Within the putative 746–amino acid polypeptide, three

major domains that are relatively conserved among the

many fungal transcriptional activators belonging to the zinc

cluster family can be identified. As shown in Figure 7, the first

one is a typical zinc binuclear cluster domain (Zn[II]

2

Cys

6

do-

main) located at the N terminus of the protein between resi-

dues 21 and 50 and may be a DNA binding domain. The

second, named MHR (for middle homology region), is less

conserved but is mostly specific for proteins belonging to the

zinc cluster family (Schjerling and Holmberg, 1996). The MHR

is found between amino acids 279 and 318 of the CLTA1

protein. The third domain, located at the C terminus of the

protein, is a possible activation domain that covers at least

95 amino acids from residue positions 605 to 700. In most

proteins of the zinc cluster family, the activation domain ispredominantly acidic. In the CLTA1 protein, this region is

glutamine, histidine, and proline rich (accounting for 39, 23,

and 25% of the amino acid content, respectively). Further

sequence analysis indicated that a putative dimerization ele-

ment (Suarez et al., 1995) is present between amino acids

67 and 80. By considering the first two protein domains,

CLTA1 seems to be more closely related to the Aspergillus

nidulans transcriptional activator UaY (Suarez et al., 1995),

having 56.7% identical residues within the zinc cluster do-

main (Figure 7) and 39.5% identity within the MHR. These

sequence features indicated that the CLTA1 protein is a po-

tential transcriptional activator belonging to the fungal zinc

cluster family.

Insertion of pAN7-1 at the 433.2 Locus Is Responsible

for the H433 Mutant Phenotype

Functional complementation experiments demonstrated

that the integration event at locus 433.2 is responsible for

blocking the H433 mutant at the switch between biotrophic

and necrotrophic phases. Final disease symptoms induced

by complemented strains on the susceptible cultivar La Vic-

toire were not as severe as those that occurred with the

wild-type strain (Figure 5). Therefore, we cannot exclude

that another mutation exists. If so, it would occur indepen-

dently of the insertion of vector pAN7-1 and would lead to

this additional mutant phenotype, which is masked by the

blockage between biotrophy and necrotrophy in the H433

mutant. To demonstrate that the insertion event in the

CLTA1

gene alone is responsible for the mutant phenotype,

we conducted gene disruption experiments. Two gene dis-

ruption vectors, p433.2A:hygro and p433.2B:hygro, contain-

ing the hygromycin resistance gene cassette flanked on

both sides by homologous sequences, were constructed as

shown in Figures 8A and 8C, respectively. The location of

the hygromycin resistance cassette in p433.2A:hygro (Fig-

ure 8A) was chosen to reproduce the insertion event at lo-

cus 433.2 in mutant strain H433. In p433.2B:hygro, an

internal fragment of the CLTA1

coding sequence was de-

leted and replaced by the hygromycin resistance cassette togenerate a null mutant allele (Figure 8C). These disruption

vectors were used independently to transform protoplasts of

wild-type strain UPS9. For each transformation experiment,

60 hygromycin-resistant transformants were arbitrarily se-

lected for DNA gel blot analysis. After transformation with

p433.2A:hygro, three of the 60 hygromycin-resistant trans-

formants analyzed were found to carry the mutant allele

introduced by transformation, as determined by the switch

of the 4.0-kb fragment present in the genomic DNA of

Figure 5. Pathogenicity of Complemented Transformants on the

Susceptible Cultivar La Victoire of Common Bean.

Excised cotyledonary leaves were inoculated using the following

strains. Photographs were taken 7 days after inoculation.

(A) Wild-type strain UPS9.

(B) Mutant strain H433.

(C) Complemented strain T433.1.2.

(D) Complemented strain T433.2.19.



1584 The Plant Cell

wild-type strain UPS9 to a 6.5-kb fragment in the disrupted

strains (Figure 8B). For the second disruption plasmid,

p433.2B:hygro, two of the 60 hygromycin-resistant transfor-

mants analyzed were disrupted (Figure 8D).

Excised primary leaves of the susceptible cultivar La

Victoire were inoculated with spore suspensions of the dis-rupted strains (e.g., R433.2.16, a p433.2A:hygro trans-

formant; Figure 9C), corresponding ectopic transformants,

wild-type strain UPS9 (Figure 9A), and mutant strain H433

(Figure 9B). The three disrupted strains obtained with the

p433.2A:hygro plasmid had exactly the same phenotype as

mutant strain H433 (R433.2.16; Figure 9C), whereas the ec-

topic transformants retained their ability to induce anthrac-

nose symptoms (data not shown). Identical results were

observed with disruptants resulting from transformation with

the p433.2B:hygro plasmid (data not shown). Therefore, dis-

ruption of the CLTA1

with either of the two mutant alleles is

sufficient to reproduce the H433 mutant phenotype.

Complementation of CLTA1

-Disrupted Strains Leads to

Complete Restoration of Pathogenicity

Complementation of H433 did not fully restore pathogenic-

ity, suggesting that the mutant possessed a virulence defect

independent of the phenotype of the clta1

mutant. Conse-

quently, similar functional complementation experiments us-

ing the 4-kb HindIII fragment (see Figure 4) were performed

with a disrupted strain (R433.2.41, a p433.2A:hygro trans-

formant) as a recipient strain. Complemented transformants

were recovered and assessed for pathogenicity on excised

cotyledonary leaves of the susceptible cultivar La Victoire.

Complemented transformants were able to induce theappearance of anthracnose symptoms as rapidly and exten-

sively as did the wild-type strain (data not shown). The dif-

ference between complementation of H433 and R433.2.41

is discussed later.

Figure 6. Nucleotide and Predicted Amino Acid Sequences of the

C. lindemuthianum CLTA1 Gene.

Nucleotides are numbered with reference to the putative transcrip-

tion start point (indicated by the asterisk). The location of the pAN7-1

insertion is shown by the arrow. The locations of the forward primer(1821 to 1847) and the reverse primer (2267 to 2244) are underlined.

Introns are presented in lowercase letters. The putative Zn(II)2Cys6

DNA binding domain is indicated in boldface. The nucleotide se-

quence of the C. lindemuthianum CLTA1 gene has GenBank acces-

sion number AF190427.

Figure 7. Sequence Alignment of Zn(II)2Cys6 Domains from CLTA1

and from Other Fungal Zinc Cluster Transcriptional Activators.

Identical residues are indicated by asterisks and similar residues by

dots. The UaY and FACB genes were isolated from A. nidulans

(Suarez et al., 1995; Todd et al., 1997). The AMDR gene was isolated

from A. oryzae (Wang et al., 1992). The GAL4 gene was isolated

from Saccharomyces cerevisiae (Laughon and Gesteland, 1984). Se-

quence alignment was determined by using the CLUSTALV algo-

rithm (Higgins et al., 1992).




Expression Analysis of the CLTA1 Gene in Fungal

Cultures from the Wild-Type and Mutant Strains

The expression of the CLTA1 gene was first examined in

pure cultures of wild-type strain UPS9 grown in either com-

plete or nitrogen-depleted medium. The latter condition was

tested because it reflects leaf conditions at the beginning of

a pathogen’s infection cycle. Expression has been demon-

strated in several cases in which pathogenicity or avirulence

genes were shown to be induced under nitrogen or car-

bon starvation conditions (Talbot et al., 1993; Van den

Ackerveken et al., 1994). RNA gel blots prepared with either

Figure 8. Gene Disruptions of the 433.2 Locus.

(A) p433.2A:hygro contains the hygromycin resistance gene cassette (a 3638-bp XhoI-SpeI fragment from the pAN7-1 plasmid demonstrated byPunt et al. [1990] to be sufficient to confer hygromycin resistance) flanked with border sequences of the wild-type genomic locus. The left and

right flanking regions are 260 bp (HindIII-SnaBI fragment) and 3.4 kb (XhoI-HindIII fragment), respectively.

(B) DNA gel blot analysis of the disrupted transformants recovered after transformation with p433.2A:hygro. Total genomic DNAs of the wild-

type strain (lane 1), one ectopic transformant (lane 2), and the two disrupted transformants obtained (lanes 3 and 4) were digested with HindII

and probed with a 363-bp KpnI-SacI fragment, indicated as probe in (A).

(C) p433.2B:hygro was constructed by replacing a 759-bp SacI-HindIII fragment located at the 3 end of the CLTA1 open reading frame by the

hygromycin resistance gene cassette (a 2704-bp SacI-HindIII fragment from the pAN7-1 vector).

(D) DNA gel blot analysis of the disrupted transformants recovered after transformation with p433.2B:hygro. Total genomic DNAs of the wild-

type strain (lane 1), two ectopic transformants (lanes 2 and 3), and the two disrupted transformants obtained (lanes 4 and 5) were digested with

HindIII and probed with a 363-bp KpnI-SacI fragment, indicated as the probe in (C).

As expected in both disruption experiments, the native 4.0-kb band of strain UPS9 remained in the ectopic transformants but was no longer

present in the disrupted strains. B, BamHI; H, HindIII; K, KpnI; P, PstI; S, SacI; Sn, SnaBI; X, XhoI. Homologous recombination through a double

crossover event (denoted by crossed lines) results in the replacement of a part of the wild-type region by the hygromycin resistance gene ( hph ).

The curved lines represent the genome. The small arrowheads indicate the location of the pAN7-1 insertion sites. Asterisks in (A) and (C) denote

borders of the restriction fragments deleted in the disruption vectors. Numbers at left in (B) and (D) indicate lengths in kilobases.



1586 The Plant Cell

20

g of total RNA or 2 g of mRNA were probed with the

363-bp KpnI-SacI internal fragment. No hybridization signal

was observed in total RNA, even after long exposure, in any

sample (data not shown). Thus, we concluded that expres-

sion of the CLTA1 gene was too weak to allow detection with

total RNA. A weak hybridization signal corresponding to the

expected size, 3.0 kb, was observed in mRNA (Figure 10A).

Signal intensity was not markedly different under the two

growth conditions previously described (Figure 10A, lanes 1

and 2), indicating that CLTA1 is expressed constitutively at a

very low level in pure cultures of wild-type strain UPS9 and is

probably not induced under nitrogen starvation.

Because of its location within the promoter region, the in-

sertion event within the CLTA1 gene (Figure 6) could have

led either to a null mutation or to a deregulation of expres-

sion. We thus investigated the consequences of such an

event on the expression of the CLTA1 gene in mutant strain

H433. RNA gel blot analysis was conducted with poly(A)

RNA of mutant strain H433 grown on complete medium. No

hybridization signal was detected, indicating that the gene

does not appear to be expressed in the mutant strain (Figure

10A). Using the more sensitive method of reverse transcrip-

tion–polymerase chain reaction (RT-PCR), no amplification

product specific for the CLTA1 gene was obtained from total

RNA of the mutant strain, whereas product was clearly ob-

served when total RNA of wild-type strain UPS9 (Figure 10B)

was amplified. These results confirm that the insertion eventof vector pAN7-1 in H433 resulted in nonexpression of the

CLTA1 gene in pure fungal cultures.

DISCUSSION

In this study, we have characterized a nonpathogenic mu-

tant derived from C. lindemuthianum, designated H433. This

nonpathogenic strain is able to induce local necrotic reac-

tions in susceptible cultivars of common bean. These reac-

tions are similar to those resulting from an incompatible

interaction between an avirulent strain of C. lindemuthianum

and a corresponding resistant genotype of common bean.

Cytological studies have shown that this mutant strain is

unable to differentiate secondary hyphae, which allow

necrotrophic growth in a basic compatible interaction

(O’Connell et al., 1985; Bailey et al., 1992). H433 is therefore

a mutant blocked at the transition between biotrophy and

necrotrophy.

An interesting aspect to the phenotype is the ability

of mutant strain H433 to induce on susceptible cultivars lo-

cal necrotic spots very similar to hypersensitive lesions

(O’Connell et al., 1985). The ability of biotrophic pathogens

not to induce host cell death in a compatible interaction has

been discussed by Morel and Dangl (1997). Three hypothe-

ses can be used to explain the mutant phenotype of strain

H433. The ability to induce local necrotic spots could result

from (1) the deregulated production of an elicitor molecule

(which is nonspecific because the same reaction was ob-

served with different susceptible genotypes of common

bean); (2) a defect in the suppression of defense responses

leading to cell death; or (3) the inability of the fungus to

achieve its developmental program within the plant, allowing

the completion of nonspecific host defense responses.

These hypotheses will be tested in future work.Molecular analyses allowed us to identify the CLTA1

gene, which encodes a potential 746-residue polypeptide

and is responsible for the phenotype observed in H433.

Gene disruption experiments demonstrated that inactivation

of CLTA1 results in the exact same phenotype as that of

mutant strain H433, proving that CLTA1 is a pathogenicity

gene. Expression of CLTA1 in pure cultures of the fungus is

very low. Preliminary experiments did not detect any induc-

tion during infection. In mutant strain H433, the insertion

Figure 9. Pathogenicity of the CLTA1 Disruptants on the Susceptible Cultivar La Victoire of Common Bean.

Excised cotyledonary leaves were inoculated with the following strains. Photographs were taken 7 days after inoculation.

(A) Wild-type strain UPS9.

(B) Mutant strain H433.

(C) Disrupted strain R433.2.16.




event of vector pAN7-1 led to nonexpression of CLTA1 in

pure cultures. Complementation of the mutant strain by the

corresponding region led to weak anthracnose symptoms,

whereas full restoration of pathogenicity was observed

when a CLTA1-disrupted strain was the recipient. These re-

sults show that most likely another mutation in mutant strainH433 (possibly the second pAN7-1 insertion), which does

not itself affect the infection process, does not allow the full

recovery of wild-type pathogenicity.

These results indicate that the CLTA1 gene encodes a pu-

tative GAL4-like transcriptional activator. The deduced

amino acid sequence of the CLTA1 protein suggests that it

contains the three functional domains characteristic of the

GAL4-like family of transcriptional activators (Schjerling and

Holmberg, 1996): the C6 zinc cluster involved in DNA bind-

ing, an MHR, and a possible transcriptional activation do-

main. In Fusarium solani f sp pisi, another Zn(II)2Cys6protein, CTF1, involved in transcriptional activation of a cuti-

nase-encoding gene, was identified (Li and Kolattukudy,

1997). However, gene replacement experiments still need to

be performed to demonstrate its role in fungal pathogenicity.

Identifying the CLTA1 gene will lead to identification of the

regulatory pathway and of the genes controlling the transi-

tion between biotrophy and necrotrophy. Most of the fungal

transcriptional activators belonging to the zinc cluster family

control catabolic pathways: FACB in A. nidulans controls

the expression of acetate utilization genes (Todd et al.,

1997), NIT4 in Neurospora crassa mediates nitrate induction

(Yuan et al., 1991), and UaY in A. nidulans regulates purine

utilization (Suarez et al., 1995). During the infection process

of hemibiotrophic fungi, entry into the necrotrophic phaseinvolves major nutritional changes, in particular the ability of

the pathogen to utilize plant constituents by activating cata-

bolic pathways. One hypothesis is that the putative CLTA1

transcriptional regulator controls genes that allow necro-

trophic growth on infected plant tissues.

In C. lindemuthianum, two mutants obtained by random

insertional mutagenesis have been characterized. Their

genes encode a serine/threonine protein kinase and a puta-

tive GAL4-like transcriptional activator (Dufresne et al., 1998;

this study). Each mutant was demonstrated to be affected at

a key step of the infection process: the first, H290, is affected

at the penetration step, whereas H433 is blocked at the

switch between biotrophy and necrotrophy. Both studies

demonstrate that random insertional mutagenesis is a pow-

erful tool to isolate pathogenicity determinants of plant

pathogenic fungi. A comparative analysis with results from

the other pathosystems should lead to a better knowledge of

the infection processes of aerial fungal pathogens.

METHODS

Strains, Media, and Growth of Colletotrichum lindemuthianum

Colletotrichum lindemuthianum UPS9 (Fabre et al., 1995) was used

as the wild-type recipient strain in this study. Growth conditions andmedia are as described in Dufresne et al. (1998).

Bean Cultivars and Near-Isogenic Lines

The common bean ( Phaseolus vulgaris ) cultivar La Victoire (Tezier,

Valence-sur-Rhone, France) was used as the susceptible cultivar in

all pathogenicity assays. A pair of near-isogenic common bean lines,

P12S and P12R, differing only by the resistant gene present in P12R,

Figure 10. CLTA1 Expression in the Wild Type, Mutant Strain H433,

and Corresponding Complemented or Replaced Strains.

(A) RNA gel blot analysis. Samples (2 g per lane) were fractionatedon a formaldehyde–agarose gel and transferred to a nylon mem-

brane (see Methods). The blot was hybridized with the 363-bp KpnI-

SacI fragment. Lanes 1 and 2, wild-type strain UPS9 grown in com-

plete or minimal medium, respectively. Lanes 3 to 6, samples ex-

tracted from cultures grown in complete medium: lane 3, mutant

strain H433; lane 4, complemented strain T433.2.19; lane 5, another

H433-complemented strain; and lane 6, replaced strain R433.2.16.

The 3.3 at left indicates the length of the hybridizing band in kilo-

bases.

(B) Ethidium bromide–stained RT-PCR products corresponding to

CLTA1-specific transcripts. These experiments were conducted

with 4 g of total cellular RNA. For fungal samples, total cellular

RNA was extracted from culture grown in complete medium, except

for those in lanes 3 and 5, which correspond to the wild-type strain

UPS9 and the mutant strain H433, respectively, grown in nitrogen-

starved medium. Two amplification products were observed, one

corresponding to the genomic DNA amplification product (447 bp)

and the other corresponding to the mRNA amplification product

(398 bp). Lane 1, susceptible cultivar La Victoire (plant negative con-

trol); lanes 2 and 3, wild-type strain UPS9 grown in complete or min-

imal medium, respectively; lanes 4 and 5, mutant strain H433 grown

in complete or minimal medium, respectively; lane 6, replaced strain

R433.2.16; and lane 7, genomic DNA of the wild-type strain UPS9

(DNA control). Numbers at left denote the lengths of the different

amplification products in base pairs.



1588 The Plant Cell

were used to demonstrate the similarity of the symptoms induced by

mutant strain H433 on susceptible and resistant cultivars (Geffroy et

al., 1998).

Infection Assays, Light Microscopy, and Induction of

Appressorium Formation

The screening procedure for identification of the altered pathogenic-

ity mutants, the pathogenicity tests, and the assays for induction of

appressorium formation were performed as previously described

(Dufresne et al., 1998). For cytological observations, infection as-

says with excised hypocotyl segments were used as described in

O’Connell et al. (1985). Hypocotyl segments of the susceptible culti-

var La Victoire were inoculated with a spore suspension of the mu-

tant strain H433 at a concentration of 5 103 spores mL1. Strips of

epidermal tissue were sampled from beneath the site at which inoc-

ulation droplets were applied and were directly mounted in distilled

water for bright-field microscopy.

For microscopic observations using Nomarski differential interfer-

ence contrast, strips were cleared in a solution of chloral hydrate

(2.5 g mL1 ) before being mounted in distilled water. Hyphal devel-opment was measured as follows. For each strain, wild-type strain

UPS9 and mutant strain H433, nine hypocotyl segments of La Vic-

toire plantlets were inoculated with a spore suspension at a concen-

tration of 5 105 spores mL1. Subsequently, for each hypocotyl,

one infection site was arbitrarily selected for microscopic examina-

tion. At each infection site, the length of hyphae produced from 270

appressoria was measured 2, 3, 4, 6, and 8 days after inoculation.

Nucleic Acid Manipulation and Fungal Transformation

Genomic DNA extractions were performed according to Ross (1995).

DNA gel blot analysis was conducted as described in Dufresne et al.

(1998). Total cellular RNA was extracted according to the procedure

described in Vallélian-Bindschedler et al. (1998). RNA samples wereseparated by formaldehyde–agarose gel electrophoresis and trans-

ferred to nylon membranes N (Amersham France SA, Les Ulis,

France) by capillary elution (Sambrook et al., 1989). Hybridizations

were conducted as previously described (Dufresne et al., 1998).

mRNA was isolated from total RNA by using magnetic Dynabeads

oligo(dT)25 (Dynal, Compiègne, France) according to the manufac-

turer’s instructions.

Reverse transcription–polymerase chain reaction (RT-PCR) exper-

iments were conducted with the Ready-To-Go RT-PCR beads kit

(Pharmacia Biotech S.A., Saint-Quentin en Yvelines, France). Each

reaction used 4 g of total RNA and was conducted according to the

manufacturer’s instructions with 2 mM MgCl2 (final concentration).

The two primers—forward, 5-cgcagtacgccatgttggaccccgtcgc-3;

and reverse, 5-GCCCAATAAAGGACGCTGCGTCAGG-3 —were de-

signed for specific amplification of CLTA1 transcripts and were usedat an annealing temperature of 60C.

Construction and Screening of the cDNA Library

A C. lindemuthianum cDNA library was constructed in vector ZAP

Express by using the ZAP-cDNA Synthesis Kit (Stratagene, La Jolla,

CA) and mRNA of wild-type strain UPS9 grown in liquid potato dex-

trose medium. cDNA synthesis was conducted according to the

manufacturer’s instructions. After in vitro packaging with the Giga-

packII Gold Packaging Extract Kit (Stratagene), recombinant phages

were infected in Escherichia coli XL1Blue-MRF (Sambrook et al.,

1989). The complexity of the library before amplification was 1.2

106 recombinants. For amplification steps and screening, E. coli

Q358 (Sambrook et al., 1989) was used. The number of plaque-form-

ing units plated for the screening was approximately three to four

times the initial number of recombinants. Screening was performedaccording to standard procedures (Sambrook et al., 1989).

DNA Sequencing and Sequence Analysis

Nucleotide sequences of the genomic region and cDNA of the

CLTA1 gene were determined by using ABI PRISM Big Dye Termina-

tor cycle sequencing (Perkin-Elmer Corp., Norwalk, CT). Sequencing

reactions were separated on an Applied Biosystems (Foster City, CA)

AB377 autosequencer. To search for homologies with known se-

quences in the data banks, we used the BLAST program (Altschul et

al., 1990). Multiple sequence alignments were determined using the

CLUSTAL V software (Higgins et al., 1992).

ACKNOWLEDGMENTS

We thank John P. Morrissey and Richard Laugé for critical reading of

the manuscript and the anonymous reviewers for their helpful advice.

This work was supported by the Centre National pour la Recherche

Scientifique and the Université Paris-Sud.

Received January 10, 2000; accepted June 14, 2000.

REFERENCES

Altschul, S.F., Gish, G., Miller, W., Myers, E.W., and Lipman, D.J.

(1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410.

Bailey, J.A., O’Connell, R.J., Pring, R.J., and Nash, C. (1992).

Infection strategies of Colletotrichum species. In Colletotrichum

Biology, Pathology and Control, J.A. Bailey and M.J. Jeger, eds

(Wallingford, UK: CAB International), pp. 88–120.

Balhadère, P.V., Foster, A.J., and Talbot, N.J. (1999). Identification

of pathogenicity mutants of the rice blast fungus Magnaporthe

grisea by insertional mutagenesis. Mol. Plant-Microbe Interact.

12, 129–142.

Ballance, D.J. (1986). Sequences important for gene expression in

filamentous fungi. Yeast 2, 222–236.

Bölker, M., Böhnert, H.U., Braun, K.H., Görl, J., and Kahmann, R.

(1995). Tagging pathogenicity genes in Ustilago maydis by restric-tion enzyme–mediated integration (REMI). Mol. Gen. Genet. 248,

547–552.

Dufresne, M., Bailey, J.A., Dron, M., and Langin, T. (1998). CLK1,

a serine/threonine protein kinase–encoding gene, is involved in

pathogenicity of Colletotrichum lindemuthianum on common

bean. Mol. Plant-Microbe Interact. 11, 99–108.

Fabre, J.V., Julien, J., Parisot, D., and Dron, M. (1995). Analysis of

diverse isolates Colletotrichum lindemuthianum infecting common

bean using molecular markers. Mycol. Res. 99, 429–435.




Flor, H.H. (1971). Current status of the gene-for-gene concept.

Annu. Rev. Phytopathol. 9, 275–296.

Geffroy, V., Creusot, F., Falquet, J., Sévignac, M., Adam-

Blondon, A.-F., Bannerot, H., Gepts, P., and Dron, M. (1998). A

family of LRR sequences in the vicinity of the Co-2 locus for

anthracnose resistance in Phaseolus vulgaris and its potential use

in marker-assisted selection. Theor. Appl. Genet. 96, 494–502.

Hamer, J.E., and Holden, D.W. (1997). Linking approaches in the

study of fungal pathogenesis: A commentary. Fungal Genet. Biol.

21, 11–16.

Hensel, M., and Holden, D.W. (1996). Molecular genetic approaches

for the study of virulence in both pathogenic bacteria and fungi.

Microbiology 142, 1049–1058.

Higgins, D.G., Bleasby, A.J., and Fuchs, R. (1992). CLUSTAL V:

Improved software for multiple sequence alignment. Comput.

Appl. Biosci. 8, 189–191.

Laughon, A., and Gesteland, R.F. (1984). Primary structure of the Sac-

charomyces cerevisiae GAL4 gene. Mol. Cell. Biol. 4, 260–267.

Li, D., and Kolattukudy, P.E. (1997). Cloning of cutinase transcription

factor 1, a transactivating protein containing Cys6Zn2 binuclear clus-ter DNA-binding motif. J. Biol. Chem. 9, 12462–12467.

Mattern, I.E., Punt, P.J., and van den Hondel, C.A.M.J.J. (1988). A

vector of Aspergillus transformation conferring phleomycin resis-

tance. Fungal. Genet. Newsl. 35, 25.

Morel, J.B., and Dangl, J.L. (1997). The hypersensitive response and

the induction of cell death in plants. Cell Death Differ. 4, 671–683.

O’Connell, R.J., Bailey, J.A., and Richmond, D.V. (1985). Cytology

and physiology of infection of Phaseolus vulgaris by Colletotri-

chum lindemuthianum. Physiol. Plant Pathol. 27, 75–98.

Oliver, R.P., and Osbourn, A.E. (1995). Molecular dissection of fun-

gal pathogenicity. Microbiology 141, 1–9.

Punt, P.J., Oliver, R.P., Dingemanse, M.A., Pouwels, P.H., and

Van den Hondel, C.A.M.J.J. (1987). Transformation of Aspergil-

lus nidulans based on the hygromycin B marker from Escherichia

coli. Gene 56, 117–124.

Punt, P.J., Dingemanse, M.A., Kuyvenhoven, A., Soede, R.D.M.,

Pouwels, P.H., and Van den Hondel, C.A.M.J.J. (1990). Func-

tional elements in the promoter region of the Aspergillus nidulans

gpdA gene encoding glyceraldehyde-3-phosphate dehydroge-

nase. Gene 93, 101–109.

Ross, I.K. (1995). Non-grinding method of DNA isolation from

human pathogenic filamentous fungi using xanthogenates. Bio-

techniques 18, 828–830.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular

Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY:

Cold Spring Harbor Laboratory Press).

Schjerling, P., and Holmberg, S. (1996). Comparative amino acid

sequence analysis of the C6 zinc cluster family of transcriptional

regulators. Nucleic Acids Res. 24, 4599–4607.Suarez, T., Vieira de Quieroz, M., Oestreicher, N., and Scazzocchio,

C. (1995). The sequence and binding specificity of UaY, the spe-

cific regulator of the purine utilization pathway in Aspergillus nidu-

lans, suggest an evolutionary relationship with the PPR1 protein

of Saccharomyces cerevisiae. EMBO J. 14, 1453–1467.

Sweigard, J.A., Carroll, A.M., Farrall, L., Chumley, F.G., and

Valent, B. (1998). Magnaporthe grisea pathogenicity genes

obtained through insertional mutagenesis. Mol. Plant-Microbe

Interact. 11, 404–412.

Talbot, N.J., Ebbole, D.J., and Hamer, J.E. (1993). Identification

and characterization of MPG1, a gene involved in pathogenicity

from the rice blast fungus Magnaporthe grisea. Plant Cell 5, 1575–

1590.

Todd, R.B., Murphy, R.L., Martin, H.M., Sharp, J.A., Davis, M.A.,

Katz, M.E., and Hynes, M.J. (1997). The acetate regulatory gene

facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional

activator. Mol. Gen. Genet. 254, 495–504.

Vallélian-Bindschedler, L., Mosinger, E., Métraux, J.P., and

Schweizer, P. (1998). Structure, expression and localization of a

germin-like protein in barley ( Hordeum vulgare L.) that is insolubi-

lized in stressed leaves. Plant Mol. Biol. 37, 297–308.

Van den Ackerveken, G.F.J.M., Dunn, R.M., Cozijnsen, A.J.,

Voosen, J.P.M.J., Van den Broeck, H.W.J., and De Wit,

P.J.G.M. (1994). Nitrogen limitation induces expression of the

avirulence gene avr9 in the tomato pathogen Cladosporium ful-

vum. Mol. Gen. Genet. 243, 277–285.

Wang, X.W., Hynes, M.J., and Davis, M.A. (1992). Structural andfunctional analysis of the amdR regulatory gene of Aspergillus

oryzae. Gene 122, 147–154.

Young, R.A., and Kelly, J.D. (1996). Characterization of the genetic

resistance to Colletotrichum lindemuthianum in common bean dif-

ferential cultivars. Plant Dis. 80, 650–654.

Yuan, G.F., Fu, Y.H., and Marzluf, G.A. (1991). nit-4, a pathway

specific regulatory gene of Neurospora crassa, encodes a protein

with a putative binuclear zinc DNA-binding domain. Mol. Cell.

Biol. 11, 5735–5745.



DOI 10.1105/tpc.12.9.15792000;12;1579-1590Plant Cell

Marie Dufresne, Sarah Perfect, Anne-Laure Pellier, John A. Bailey and Thierry Langinon Common BeanColletotrichum lindemuthianumInfection Process of

A GAL4-like Protein Is Involved in the Switch between Biotrophic and Necrotrophic Phases of the

This information is current as of August 2, 2011

References http://www.plantcell.org/content/12/9/1579.full.html#ref-list-1

This article cites 26 articles, 7 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant PhysiologyandThe Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY

© American Society of Plant Biologists

http://www.plantcell.org/content/12/9/1579.full.html#ref-list-1



https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X


http://www.plantcell.org/cgi/alerts/ctmain




http://www.aspb.org/publications/subscriptions.cfm











Documents

A GAL4-Like Protein is Involved in the Switch Between