Divergent CAMP Signaling Pathways Regulate Growth and Pathogenesis in Rice Blast Fungus

8/12/2019 Divergent CAMP Signaling Pathways Regulate Growth and Pathogenesis in Rice Blast Fungus

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The P lant Ce ll, Vol. 10, 13611373, Augus t 1998, ww w.p lantcell.org 1998 America n S oc iety of P lant P hysiologists

Divergent cAMP Signaling Pathways Regulate Growth andPathogenesis in the Rice Blast FungusMagnaporthe grisea

Kiichi Adachi and J ohn E. Hamer1

Depa rtment of B iological S ciences , P urdue University, Wes t Lafa yette, Indiana 47906-1392

cAMP is involved in signaling appressorium formation in the rice blast fungusMagnaporthe gr isea.

However, null muta-

tions in a protein kinase A (PKA) catalytic subunit gene, CPKA

, do not block appressorium formation, and mutations in

the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium for-

mation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To

clarify cAMP signaling in M. grisea

, we have identified strains in which a null mutation in the adenylate cyclase gene

(

MAC1

) has an unstable phenotype such that the bypass suppressors of the Mac1

phenotype (

sum

) could be identi-

fied. sum

mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form

appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppres-

sor mutation (

sum1-99

) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA

(

SUM1

), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the cat-alytic subunit gene, CPKA

, encodes the only detectable PKA activity in M. grisea.

Because CPKA

is dispensable for

growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these pro-

cesses. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea

is regulated by

adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis

or pathogenesis.

INTRODUCTION

cAMP is a ubiquitous secondary messenger in prokaryotic

and eukaryotic ce lls. A we ll-chara cte rized intrace llular ta rget

of c AMP in eukaryotic ce lls is the reg ulato ry subunit of pro-

tein kina se A (P KA; Ta ylor et a l., 1990). P KA is a tetra meric

holoenzyme that is composed of two regulatory subunits

and two ca talytic subunits. B inding o f cAMP to the regula-

tory subunits releases the ca talytic kinase subunits to phos-

phorylate target proteins involved in cAMP-regulated

processes. In fungi, cAMP regulates both metabolism and

morphogenesis (reviewed in Pall, 1981). For example, in

Saccharomyces cerevisiae

, cAMP regulates ca rbon metabo -

lism (reviewe d in Theve lein, 1994), c ell cy cle pro gres sion

(Matsumo to e t a l., 1982; Tokiwa et a l., 1994), a nd p se udo-

hyphal growth (Lorenz and Heitman, 1997). In filamentous

fungi, such as Neurospora c rassa

, c AMP s igna ling p lay s im-

portant roles in hyphal tip growth, conidiation, and carbonmet a bo lism (Terenz i et a l., 1974, 1979; Bruno e t a l., 1996).

More recently, cAMP signaling also has been found to be

importa nt in funga l pathog enes is (Kronsta d, 1997).

Chronic and widespread throughout most of the worlds

rice growing areas, rice blast disease, caused by Magna-

porthe grisea

, is a persistent threat to world rice production

(Ou, 1980). Asexua l spores (co nidia ) infec t plant ce lls b y g er-

minating a nd d ifferentiating a d ome-sha ped c ell ca lled a n ap -

pres so rium (reviewe d in Ta lbot, 1995), whic h allow s d irec t

penetration of the plant cell wall. Germinating conidia also

form appressoria efficiently on inert hydrophobic surfaces in

a droplet of water (Hamer et al., 1988). In many strains, ap-

pressoria are much less efficiently formed on hydrophilic sur-

fac es (G ilbe rt et al., 1996). The exa ct m ec hanism b y w hich

the germ tubes of M. g risea

sense surface hydrophobicity is

not known. However, the secretion and self-assembly of a

hydrophobin protein encod ed b y the MPG1

gene ma y provide

a s ensing mec hanism for surfac e hydrophob icity (Talbot e t

al., 1993, 1996; Beckerman and Ebbole, 1996). Appresso-

rium formation ca n be inhibited by yeas t extract a nd b y high

concentrations of the S. cerevisiae

factor pheromone(Beckerman et al., 1997). Although the biochemical effects

of these additions are not known, they may perturb signaling

mechanisms that trigger appressorial differentiation. When

germinated on a hydrophilic surface, appressorial differenti-

ation can be induced by the addition of cAMP, its soluble

analogs, or inhibitors of cAMPphosphodiesterase (Lee and

Dea n, 1993). These findings s ugg es t that increa se s in the in-

trac ellular co ncentration of c AMP ma y a ct a s pa rt of an ea rly

signa ling e vent in appress orium formation.

1

To w hom co rrespo ndence should be a ddress ed. E-mail [email protected]; fax 765-496-1496.


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1362 The P la nt C ell

The role of the c AMP s ignaling pathw ay in a ppress orium

formation in M. grisea

has been investigated by assessing

the phenotypes o f gene knockout mutants in CPKA

, a gene

encoding a putative catalytic subunit gene (Mitchell and

Dean, 1995; Xu et al., 1997), and MAC1

, a gene encoding

adenylate cyclase (Choi and Dean, 1997; this study). How-ever, mutations in these genes give dramatically different

phenotypes, neither of w hich spec ifica lly a ffects appresso -

rium differentiation. For example, cpkA

null mutants ca n s till

form a ppress oria but fa il to penetra te plant ce lls. Thus, it is

not clear whether cAMP mediates its e ffect on a ppress orium

formation directly through PKA regulation. In addition to af-

fecting appressorium formation, mac1

null mutants have

pleiotropic defects in growth, sporulation, and mating abil-

ity, whereas cpkA

mutants have no effect on these pro-

ces ses . The obse rvation that exoge nously add ed c AMP ca n

overcome Mac1

defects in appressorium formation contin-

ues to suggest a role for cAMP signaling in appressorium

formation.

To c larify further the relations hip of cAMP signa ling t ogrowth and pathogenesis, we performed additional studies

on MAC1

and PKA regulation in M. grisea.

We functiona lly

characterized MAC1

and identified strains in which the

Mac1

phenotype is unstable. Among the bypass suppres-

sors of the mac1

mutation, we identified a specific mutation

in the reg ula tory s ubunit of P KA. This muta tion co mplete ly

restores the g rowth and m orphology of mac1

muta nts . This

mutation also allows appressorium formation on nonhydro-

phobic surfac es and in the presence o f yeas t extrac t. These

findings, together with biochemical studies with cpkA

mu-

tants, a llow us to co nclude tha t cAMP mediates its effect on

app ress orium forma tion through P KA, mo st likely through a

ca ta lytic s ubunit hig hly diverge nt from CP KA. Thus, c AMP

signaling for growth a nd pa thogenesis in M. grisea

diverges

at the level of P KA regulation.

RESULTS

An Unstable Allele ofMAC1

in Strain Guy11

A candidate gene for the M. grisea

adenylate cyclase was

identified from a genomic cosmid library by using a probe

generated by polymerase chain reaction (PCR) amplification

with primers de signed to amplify the c onserved c ata lytic do-

main (see Methods). A 13.4-kb EcoRI fragment was sub-cloned, and sequencing of 8.7 kb of DNA around the

putative catalytic site identified an open reading frame of

7151 nucleotides encoding a 2160amino acid protein with

significa nt similarity to funga l ade nylate cyc las es . The DNA

sequence has GenBank accession number AF006827. A

second MAC1

seq uence from strain 70-15 (G enBa nk acc es-

sion number AF012921) and characteristic features of this

ge ne ha ve be en published (Choi and Dea n, 1997). This s e-

quence differs from AF006827 at 13 nucleotide positions,

resulting in three amino acid changes. All nucleotide poly-

morphisms in AF006827 we re reconfirmed by se q uencing.

To te st whe ther MAC1

could function in a well-character-

ized cAMP s igna ling pa thwa y, w e introduced MAC1

into the

N. crassa crisp-1

(

cr-1

) mutant. cAMP signaling is essential

for growth polarity (hyphal extension) in N. crassa

(Bruno et

al., 1996). cr-1

mutants have a drama tica lly reduced c olonial

growth rate (Figure 1), which can be rescued by the applica-

tion of exogenous cAMP. Introduction of the MAC1

gene to-

gether with a plasmid conferring hygromycin B resistance

efficiently rescued the cr-1

phenotype, w hereas disruption

of MAC1

in the cons erved c ata lytic d omain prevented c om-

plementation (Figure 1). DNA gel blot analysis was used to

confirm integration events (data not shown). We conclude

that MAC1

encodes a typical membrane-associated fungal

adenylate cyclase and can function in the polarity signalingpathway of N. crassa.

A mac1

null mutant was created by one-step gene re-

placement (Figure 2A). Among 160 initial hygromycin-resis-

tant transformants, spore PCR (Xu and Hamer, 1995)

identified 26 putative null mutants.

mac1

::

Hph

strains w ere

confirmed by the absence of MAC1

cod ing se q uences (Fig-

ure 2B, probe 1), the alteration of flanking DNA sequences

(Figure 2B, probe 2), a nd the introduction of the hygromyc in

B phosphotransferase gene (

HPH

) (Fig ure 2B , pro be 3). One

Figure 1. The MAC1Gene Complements Colonial Growth of the N.

crassa cr-1Mutant.

The cultures we re mad e on Vogel minima l aga r medium (Davis a nd

de Serres, 1970) and incubated at 25C for 3 days. Compared with

the wild type (top left), the cr-1mutant e xhibits drama tica lly reduced

grow th with tight clusters o f con idia (top middle). This ab norma l

morphology was restored by the addition of 5 mM cAMP in the me-

dium (top right) and also by introduction of the MAC1gene (bottom

left). The MAC1ge ne disrupted in the ca talytic doma in w ith Tn1000

loses the ability to rescue the growth defect of the cr-1mutant (bot-

tom right).


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Divergent cAMP S ignaling in Fungal Pa thogenesis 1363

Figure 2. Disruption of the M. grisea MAC1Gene.

(A)The MAC1locus a nd ta rgeting knockout vecto r. The blac k and w hite a rrows indicate the pos itions and transcriptional directions o f the MAC1

and HPHgene s, respe ctively. Restriction enzyme cleava ge sites a re abb reviated as follows : E, EcoRI; K, KpnI; N, NcoI; and X, Xba I. Integration

by homologous recombination produces a mutated locus in which the 3 tw o-thirds po rtion o f the MAC1 gene containing the leucine-rich re-

peats (amino ac ids 700 to 1374; hatc hed b ox) and ca talytic d oma in (amino ac ids 1751 to 1994; shad ed b ox) are deleted. The pos itions of threeprobes for DNA gel blot a nalysis are d epicted w ith stippled lines.

(B)DNA gel blot analysis of MAC1knockout trans formant s. The ge nomic DNAs extra cte d from wild-type stra in G uy11 (lane 8), MAC1knockout

trans formant s DA127, DA128, DA133, DA138, DA154, and DA156 (lanes 1 to 6), and ec topic inse rtiona l mutant DA50 (lane 7) were dige ste d w ith

KpnI and sepa rated in a 0.7% aga rose g el. The blot wa s probed with a 1.8-kb XbaI frag ment of the MAC1ge ne (Probe 1), stripped and reprobed

with 600-bp NcoI-KpnI fragment in the right flanking region (Probe 2), and then stripped and reprobed with the 600-bp PCR product from the

HPHg ene (P robe 3). The HPHprimers were described in Xu and Hamer (1996).

(C) Colony morphology of the MAC1knockout transformant and a suppressor mutant. Wild-type strain Guy11 (top left) and MAC1knockout

transformant DA128 (top right) were c ultured on o atmea l aga r medium for 10 days . A sporulating s ecto r arose after DA128 (botto m left) wa s cul-

tured for 15 days . The se ctor wa s s ingle-spore isolated on oa tmeal ag ar medium and c ultured for 10 days (botto m right).


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MAC1

Requirements for Appressorium Formation Are

Suppressed by sum

Mutations

Infection structure (appressorium) formation was assayed

on hydrophobic plastic coverslips or the hydrophilic side of

G elBond film (Tab le 1) in the a bs enc e o r pres ence of d iffer-ent co nce ntrations o f exog enous ly ad de d c AMP (Tab le 2).

Appressorium formation was scored after 24 hr of incuba-

tion (see Method s). The w ild-type strain G uy11 forms a p-

pressoria efficiently on hydrophobic surfaces but does not

form app resso ria efficiently on hydrophilic s urfac es . As p re-

viously repo rted, mac1

mutants are deficient for appresso-

rium formation (Tab le 1) and prod uced ge rm tubes that

failed to undergo appressorium formation and remained

somewhat undifferentiated (Figure 3). However, the appres-

sorium-deficient phenotype is reminiscent of mutations in

the Mpg 1 hydropho bin (Talbot et a l., 1993). For b oth pheno-

types, germ tubes curl and hook repeatedly and appear to

grow wider but do not form appressoria. In contrast, when

mac1

mutants w ere incubated on a hydrophilic surfac e, thegerm tubes were very similar to germ tubes of the wild-type

strain Guy11. They w ere thin, grew straight, a nd rema ined

undifferentiated.

The mac1

mutants co uld b e induced to form appressoria

in the prese nce of increas ing c onc entrations o f cAMP (Tab le

2 and Figure 3). These co ncentrations o f cAMP also re-

stored appressorium formation to the wild-type strain when

it w a s g erminate d o n a hy drop hilic surfa ce (Ta ble 2). Al-

though appresso rial as sa ys w ere done w ith conidia from 10-

day-old cultures, the small fraction of appressoria produced

from the mac1

mutants is likely due to the presence of

spores conta ining suppressor mutations.

sum

mutations dramatically restored the ability of mac1

strains to form appres so ria o n hydrophob ic s urfac es (Tab le

1 and Figure 3). In addition, unlike wild-type strains, mac1

strains carrying sum

mutations also were able to form ap-

transformant (DA50; Figure 2B, lane 7) contained an intact

MAC1

gene, a nd the targeting vector presumab ly inserted at

an ectopic chromosomal site. As expected, loss of adeny-

late cyclase had a dramatic effect on growth, asexual mor-

phogenesis, and mating ability.

mac1

::

Hph

strains had

reduced colonial growth rates, reduced conidiation, and a

highly mosaic colony appearance on oatmeal agar medium

(Figure 2C ). Trans forma nts w ith simila r phenoty pes w ere re-

covered when cAMP was included in the protoplast regen-

eration med ium.

After 10 to 15 days of growth, the rapidly growing and

evenly sporulating sectors from

mac1

::

Hph

stra ins (Fig ure

2C) were recovered. Single-spore isolations from these sec-tors resulted in the growth of strains with wild-type patterns

of sporulation, growth rate, and colony appearance. An inde-

pendently generated collection of

mac1

::

Hph

strains cre-

ated with a different gene disruption vector produced

identical results, that is,

mac1

::

Hph

strains with slow-

growing co lony phenotypes that eventually produced more

rapidly growing sectors (data not shown). Sectors were ob-

served on a variety of culture media (oatmeal agar, V8 juice

agar, and minimal and complete agar media). DNA gel blot

analysis of several wild-type-like colonies derived from

mac1

::

Hph

strains confirmed the presence of the gene

disruption event (data not shown). Genetic crosses of one

wild-type-like c olony d erived from a

mac1

::

Hph

strain to a

wild-type tester showed that the original

mac1::Hphcolony

phenotype could be recovered from segregating progeny.

We c onclude that these mac1::Hphstrains of strain Guy11

ca n give rise to s pontaneous b ypas s suppressing mutations

that restore wild-type-like growth. We designated these

strains mac1::Hph sum(for suppressors of Mac1). We se-

lected three independent suppressor mutants designated

DA61, DA67, and DA99 for further analysis. Each was from

an independent mac1::Hphtransformant.

Table 1. Appress orium Formation in mac1a nd S uppressor Mutants

of M. grisea

Surface Property

S tra in G enotype Hydrophobic Hydrophilic

G uy11 Wild type 90.6 4. 0 a 0.9 1. 3

DA128 mac1::Hph 1.2 1.8 0.4 0. 8

DA154 mac1::Hph 1.7 2.1 0.0 0. 0

DA156 mac1::Hph 0.0 0.0 0.3 0. 8

DA61 mac1::Hph sum-61 98.3 1.2 97.3 0. 6

DA67 mac1::Hph sum-67 95.3 0.6 95.0 1. 0

DA99 mac1::Hph sum1-99 97.0 1.0 95.7 1. 5

a The percentag e of germinated conidia that formed appress oria w as

qua ntitated after 24 hr. The mea n and s tanda rd deviation we re

calculated from three independent experiments. Each experiment

consisted of three replicates, and 100 germinated conidia were ex-

amined p er replica te.

Table 2. cAMP Resc ues the Appress orium Formation Defect in

mac1 Mutants of M. griseaa

S tra in No cAMP 10 mM c AMP 50 mM cAMP

G uy11 0.7 1. 2b 36.3 8.0 90.7 2. 5

DA127 4.0 1.0 61.3 9.2 96.7 2. 5

DA128 0.7 0.6 57.3 4.0 95.7 0. 6

DA129 1.0 1.0 69.3 3.1 91.0 3. 6DA154 0.3 0.6 81.7 6.8 89.0 3. 0

DA156 0.7 0.6 76.7 4.2 92.0 2. 6

a Hydrophobic p las tic cove rslips (Fisher) were used for the mac1mu-

tants; pieces of the hydrophilic side of GelBond were used for wild-

type s train G uy11.b The percentag e of germinated conidia that formed appress oria w as

qua ntitated after 24 hr. The mea n and s tanda rd deviation we re

calculated using combined data of at least three replicates. One

hundred g erminated conidia we re examined per replicate.


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pres so ria e fficiently on hyd rophilic surfac es (Ta ble 1 and

Figure 3). Thus , sum mutations not only restore wild-type

growth characteristics to mac1strains but also restore and

enhanc e appres so rium formation. These findings s ugg es t

that the suppression of the requirement for adenylate cy-

clase during appressorium formation leads to an inability to

discriminate hydrophobic and hydrophilic surfaces. Adeny-

late cyclasedeficient strains containing sum mutations did

not form appressoria when germinated on agar-solidified

media and did not differentiate appressoria in liquid culture(da ta not show n).

Appressorium formation in M. griseaca n be repressed by

the addition of 2% yeast extract, and this suppression has

been show n to b e ma ting-type depend ent (Tab le 3 a nd Fig-

ure 3). The suppres sion phenome non is propose d to b e me-

diated by small pheromone-like peptides present in yeast

extract beca use the S. cerevisiae factor also suppresses

appresso rium formation in a mating-type d ependent manner

(Be ckerma n et al., 1997). Tw o perce nt yea st e xtract e ffectively

suppressed appressorium formation in strain Guy11 (MAT1-2)

but did not suppress appressorium formation in strain

AFTH3 (MAT1-1) (Ta ble 3). Ade nylate cy cla se deficient m u-

tants conta ining sum-99, sum-67, and sum-61muta tions (all

strains are isogenic with Guy11 and therefore with MAT1-2)

were no longer sensitive to mating-type-dependent inhibi-

tion of a ppress orium formation by ye as t extrac t (Tab le 3 and

Figure 3). In yeast extract, appressoria differentiated from

the tips of highly bra nched ge rm tubes (Figure 3).

PKA Activity inM. grisea

PKA has been implicated in the regulation of appressorium

formation in M. g risea(Lee a nd De an, 1993; Mitchell and Dea n,

1995); however, there have been no direct measurements of

PKA activity. Null mutations in CPKA do not block appres-

sorium formation and do not affect growth and sexual or

as exual morphoge nesis. Thus, the grow th and a ppressorium

Figure 3. Appress orium Forma tion of Wild-Type St rain Guy 11, mac1::Hph Strain DA128, and mac1::Hph sum1-99 Strain DA99 on Hydro-

phobic a nd Hydrophilic Surfaces .

The effects of 2% yeast e xtrac t (YE; for Guy11 and DA99) and 50 mM cAMP (cAMP; for DA128) on appressorium formation on hydrophobic

surfaces are indica ted. The loss of a ppressorium formation in DA128 was restored by the sum1-99mutation in DA99. Appressorium formation

assays performed with mac1::Hph sum strains DA61 and DA67 gave identical results. Germinating conidia and appressoria were photo-

graphed using differential interference co ntras t microsco py. Arrow head s indicate appress orium formation.


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formation defects of mac1 mutants may be mediated by

ca talytic subunit ge nes o ther than CPKA.Although multiple

PKA catalytic subunit genes exist in fungi, only a single reg-ulatory subunit occurs in S. cerevisiae (Tod a e t a l., 1987a ,

1987b). Thus, summutations may alleviate the effects of the

loss of adenylate cyclase by altering the single regulatory

subunit of P KA and a ffecting the a ctivity of multiple ca talytic

subunits.

To tes t this hypo thesis, w e a ss ay ed P KA ac tivity in protein

extracts made from wild-type and mutant strains of M.

grisea (see Methods). Figure 4A shows that exponentially

growing hyphae from M. g risea contain a cAMP-stimulated

protein kinase activity that phosphorylates a model PKA

substrate (Kemptide). Assays with strains DA61 and DA67

showed that neither strain contained significant levels of

cAMP-independent PKA activity, as might be expected from

mutants in the regulatory subunit of PKA. In addition, bothstrains contained a cAMP-stimulated activity similar to that

of the w ild typ e. In contra st, s train DA99 conta ining the sum-

99 mutation showed elevated levels of cAMP-independent

PKA activity. However, PKA activity in this strain still could

be e nhanced by the a dd ition of cAMP. sum-99mutants con-

sistently had lower levels of cAMP-stimulated PKA activity

than d id G uy11. Strikingly different results we re ob tained from

extrac ts p repared from 8-hr germinated co nidia (Figure 4B).

PKA levels were undetectable for strain DA99, regardless of

the presenc e or a bse nce o f cAMP. This s ugges ts that the

sum-99muta tion inhibits P KA ac tivity in ge rminating spo res.

To c onfirm that CPKA encodes a biochemically active

catalytic subunit and to search for additional PKA activities,

we mea sured P KA levels in strains co ntaining va rious a lleles

of cpkA in both g ermlings a nd a ctively grow ing my ce lia. Two

mutant alleles in strains I27 and DF51 were created by gene

replacements and are presumed to be null (Mitchell and

Dea n, 1995; Xu et a l., 1997). A third a llele in st rain pTH4 is

an insertional mutation at the 3 end of the gene that has

phenotype s s imilar to the g ene replac ement alleles (Xu et a l.,

1997). All three cpkA mutants were dramatically deficient in

PKA activity, and residual PKA activity could not be de-

tected reliably in mycelia (Figure 4C). Similar results were

ob tained w ith ge rmling extrac ts (da ta no t show n). These re-

sults suggest that CPKA encodes the major PKA in M.

griseathat could be a ssa yed.

sum-99Identifies a Mutation in the Regulatory Subunit

of PKA

To de termine whe ther sum-99 contained a mutation in the

regulatory subunit of PKA, we cloned the regulatory subunit

gene by using PCR primers designed around conserved

amino acid motifs in fungal regulatory subunit genes (see

Methods). An amplified DNA fragment with significant ho-

mology to fungal PKA regulatory subunits was used to identify

clone s from c DNA a nd g eno mic libra ries . Three full-leng th

cDNAs a nd a genomic clone were se quence d. The gene

(designated SUM1) structure is very similar to that of the N.

crassa regulatory subunit gene mcb (Bruno et al., 1996).

Tha t is, one intron is in the c od ing reg ion (the po sition ide nti-cal to the intron of mcb), w hereas two introns are in the 5un-

translated region (Figure 5A). DNA gel blot a nalysis s how ed

that SUM1is a single-co py ge ne (da ta no t show n). The ope n

reading frame encodes 390 amino acids and shows high

similarity with regulatory subunits from other fungi (Figure

5B). Pairs of oligonucleotide primers were used to amplify

the SUM1 coding region from DA61, DA67, and DA99 (see

Methods). As expected, DA61 and DA67 carry a wild-type

SUM1g ene. Howe ver, DA99 co ntains a single (T-to-G) mu-

ta tion in the first c AMP b inding d oma in (Figure 5C ). The m uta-

tion c hang es an invariant leucine at pos ition 211 to a rginine.

Because a mutation in the regulatory subunit of PKA re-

verts the growth a nd a ppress orium-defec tive phenotypes in

mac1mutants, a n ad ditional ca talytic s ubunit gene must ex-ist that cannot be detected in our PKA assays. Additional

catalytic subunit genes could not be identified by DNA gel

blot analysis (Mitchell and Dean, 1995). We performed an

extensive P CR-bas ed sc reen for additional ca talytic subunit

genes. Although many different protein kinaserelated se-

quences were detected, we failed to detect additional PKA-

related sequences in CPKA-deleted strain (data not shown).

We conclude that a highly divergent and sum1-99affected

activity plays a role in cAMP-stimulated appressorium for-

mation and g rowth morphogenesis in M. grisea.

sumMutations Only Partially Suppress the

Pathogenicity Defect in the Adenylate Cyclase Mutant

The sum1-99mutation and the undefined sum-61and sum-

67 mutations restore normal growth morphology and ap-

pressorium formation to adenylate cyclasedeficient mu-

tants of M. grisea.To d etermine w hether these s uppresso rs

fully restored pathogenicity, we conducted conidial spray in-

oculations and leaf injections of rice cultivars CO39 (Figure

6) and Sariceltik (data not shown) with wild-type strain

Table 3. Effect of Yea st Extract on Appress orium Formation o f

mac1Suppressor Mutants of M. griseaa

S tra in Ma ting Type No Additive Yea s t Extra c t

Guy11 MAT1-2 92.3 2. 9b 0.0 0. 0

AFTH3 MAT1-1 96.3 2.3 62.0 4. 5DA61 MAT1-2 92.7 3.1 90.0 3. 6

DA67 MAT1-2 97.0 1.7 90.0 1. 0

DA99 MAT1-2 97.3 1.2 92.7 3. 1

a Hydrophobic plastic c overslips (Fisher) were use d for the a ss ays .b The percentag e of germinated conidia that formed appress oria w as

qua ntitated after 24 hr. The mean a nd sta ndard de viation were ca l-

culated using combined data of at least three replicates. One hun-

dred ge rminated co nidia we re examined per replica te.


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Guy11 and isogenic derivatives DA50 (ectopic integration

event), DA154 (mac1::Hph), DA99 (mac1::Hph sum1-99),

DA61 (mac1::Hph sum-61), and DA67 (mac1::Hph sum-

67). As expected, adenylate cyclasedeficient strain DA154

was nonpathogenic when compared with control inocula-

tions, and only rare pinpoint lesions were observed occa-sionally. Surprisingly, strain DA99 only showed a modest

increase in pathogenicity (Figure 6). Fewer and smaller le-

sions in DA61, DA67, and DA99 we re co nsistently obs erved

compa red with Guy11 and DA50. Lesion co unts o n sec ond

leaves of rice cultivar Sariceltik showed that Guy11 pro-

duced 10.1 lesions (S D9.8; n 58) and DA50 produced

12.1 lesions (S D13.2; n53). In co ntrast, DA61 produce d

2.4 lesions (S D3.3; n 59), DA67 produced 0.8 lesions

(S D 1.2; n 57), and DA99 produced 4.4 lesions (S D

6.3; n52). Leaf sheath injection assays also showed that

strains carrying sum mutations were still reduced in patho-

genicity (data not shown).

DISCUSSION

Recent studies have demonstrated that fungal pathogens of

plants and animals require genes encoding components of

highly conserved signal transduction pathways for patho-

ge nesis. These include co mponents of heterotrimeric G pro-

teins (Gao and Nuss, 1996; Alspaugh et al., 1997; Kasahara

and Nuss, 1997; Liu and Dean, 1997; Regenfelder et al.,

1997), MAP kinas es (Ba nuett a nd Herskowitz, 1994; Xu a nd

Hamer, 1996), and components of cAMP signal transduction

pathways (Gold et al., 1994; Mitchell and Dean, 1995; Xu et

al., 1997). Although these pathway components have been

well studied in model genetic systems, such as S. cerevi-siae, their role in fungal pathogenesis is novel and some-

what unexpected. Components of these pathways with

specific effects on pathogenesis provide opportunities for

the development of novel antifungal drugs and crop-pro-

tecting chemicals.

cAMP Signaling Pathways for Fungal Growth

and Pathogenesis

A role for c AMP s igna ling d uring patho ge nesis o f the rice blast

fungus has been confirmed by an analysis of phenotypes

Figure 4. P KA Activity in Wild-Type and Mutant S trains of M. grisea.

PKA activity was monitored in mac1::Hph sum mutants ([A] a nd

[B]) and cpkA mutants (C). Extrac ts w ere prepa red from myc elia ([A]

and [C]) and germlings (B), and PKA activity was determined with

() or without () the addition of 1 M cAMP. Enzyme activity was

monitored b y ge l electrophoresis (phospho rylated subs trate migrat-

ing toward the anode) and quantitated using spectrofluorometry

(see Methods). The g el wa s photog raphed on a UV trans illuminator.

PKA activity is expressed in nanomoles of phosphate transferred to

a subs trate per minute per milligram o f protein. Da ta are representa-

tive o f two independ ent experiments with nea rly identical results.

(A) and (B) G uy11 (w ild ty pe), DA61 (mac1::Hph sum-61), DA67

(mac1::Hph sum-67), a nd DA99 (mac1::Hph sum1-99) were tested .

(C) I27, pTH4, a nd DF51 a re various cpkAmutants (see text for de-

tails).


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Figure 5. Molecular Cloning a nd Analysis of the P KA Regulatory Subunit Gene (SUM1) from M. grisea.

(A)Schematic diagram of the SUM1 gene. The boxes and b oldface lines indica te exons a nd introns, respec tively. Nucleotide s eq uencing of

three cDNA clones identified two introns within the 5 untranslated region a nd o ne intron w ithin the c oding region. The sta rt positions of the

cDNA clones a re marked by a rrow s. The tw o c AMP b inding doma ins a nd the interaction site with the P KA ca talytic s ubunit a re represented by

boxes with widely spa ced and closely spa ced diago nal lines, respectively (Taylor et al., 1990). The nucleotide seq uence from S ac I to Xba I has

GenB ank ac ces sion number AF024633.

(B) Amino a cid se quenc e a lignment o f the P KA regulato ry subunit g enes from fungi. Se q uences are a ligned using the C LUSTAL W program(Thomps on et a l., 1994): Mg , M. grisea; Nc, N. crassa (Bruno et al., 1996); Be, Blastocladiella emersonii (Marques and Gomes, 1992); Um,

Ustilago maydis (Gold et al., 1994); Sp, Schizosaccharomyces pomb e(DeVoti et al., 1991); and Sc, S. cerevisiae(Tod a et a l., 1987a). Identica l

amino ac ids are highlighted o n a blac k background; similar amino ac ids are show n on a g ray bac kground. Ga ps introduce d for the alignment a re

indicated by d ots. The a mino a cid pos itions are indica ted a t left. The tw o c AMP b inding do mains a re underlined. The interac tion s ite w ith the

PKA catalytic subunit is double underlined.

(C)Point mutation found in the PKA regulatory subunit gene of strain DA99 (mac1::Hph sum1-99). The co don a nd a mino a cid subs titutions are

underlined . The pos ition of the p oint mutation is indica ted b y an a ste risk in (A).


9/13


ated, similar to N. crassa adenylate cyclase (Flawia et al.,

1977). Similar to mutations in MAC1, a null mutation in the

M. griseaG protein s ubunit ge ne MAGBhas pleiotropic ef-

fects on growth, sporulation, appressorium formation, and

pathog enicity, and a ppressorium formation ca n be restored

in magB null mutants by the addition of cAMP (Liu andDea n, 1997). Thes e findings are c ons iste nt with the po ss ibil-

ity that a denylate cyc las e in M. griseamay be a ctivated by a

membrane-as soc iated heterotrimeric G protein.

Appress orium forma tion is inhibited in a ma ting-type-spe -

cific ma nner by yea st extract a nd factor (Beckerman et al.,

1997). This inhibition c a n be o verco me b y the sum1-99m u-

tation, suggesting that the inhibition acts at the level of

cAMP generation. Interestingly, the addition of the factor

also is known to repress adenylate cyclase in yeast (Liao

a nd Thorner, 1980). The req uireme nt for a hyd ropho bic s ur-

face for appressorium formation is also overcome by the

sum1-99 mutation. We propose that cAMP activates multi-

ple PKAs through an interaction with the regulatory subunit

of P KA. S pec ificity of c AMP s igna ling fo r either grow th orpathog enesis is imparted by the ac tion of ac tivated ca talytic

sub units. The finding tha t the sum1-99 mutation inhibits

CPKA-encoded PKA activity in germlings but allows appres-

sorium formation is consistent with our previous findings

that CPKAis largely dispens ab le for app ress orium formation

but ne ce ss ary fo r plant penetration (Xu et a l., 1997). The o b-

servation that the sum1-99mutation restores normal growth

imparted by null mutations in the adenylate cyclase gene

(MAC1) (Choi and Dean, 1997; this study). However, mac1

mutants a lso d isplay a wide variety of de fects in growth and

morphoge nesis, demo nstrating tha t cAMP has wider roles in

grow th regulation. The finding of m utations tha t suppres s

the growth defects of mac1 mutants but do not restore fullpathogenicity demonstrates that there are divergent cAMP

signaling pa thways for growth a nd pa thogenes is. Two lines

of evidence suggest that divergence occurs at the level of

PKA regulation. First, mutations in a PKA catalytic subunit

gene (CPKA) have spe cific e ffects on pa thogenes is a nd, un-

like mutations in MAC1, do not affect axenic growth and

sexual or asexual morphogenesis (Mitchell and Dean, 1995;

Xu et al., 1997). Second, a suppressor of the pleiotropic

Mac1 phenotype, sum1-99, identifies the regulatory sub-

unit of PKA. Although this mutation restores growth and

conidiation, it alters appressorium formation and does not

restore full pa thog enicity.

Ba sed on the phenotypes of cpkA null mutants, we sug-

gested that an additional PKA(s) may exist in M. grisea(Xu andHamer, 1996; Xu et al., 1997). For example, when assayed

over 24 hr, cpkAmutants retained an ability to be stimulated

to form appressoria by cAMP. However, we could find no

other PKA activity in cpkA null mutants, and DNA gel blot

ana lysis (Mitchell and Dea n, 1995) failed to d etec t a dd itiona l

catalytic subunit genes. Studies with yeast suggest that

these findings do not rule out the existence of additional

PKA catalytic subunit genes. S. cerevisiae contains three

ca talytic subunit ge nes: TPK1, TPK2, and TPK3(Tod a et a l.,

1987b). Tpk2 a ctivity co uld not b e de tec ted in ass ay s, eve n

when overexpressed in a tpk1tpk3strain. Detecting low

levels of Tpk3 ac tivity req uires o verexpres sion in a tpk1

tpk2ba ckground (Mazon et a l., 1993). TPKgenes are par-

tially redundant for growth, and a triple tp knull mutant of S.

cerevisiae is very slow growing. Overexpression of a PKA-

related kinase gene, SCH9, can functionally rescue a triple

TPKde letion mutant (Tod a et a l., 1988) and suppres s ma ny

of the defects imparted by mutations in the Rasadenylate

cyclase signaling pathway. A related gene, sck1, exists in

Schizosaccharomyces pombe. This g ene c an a lso res cue

defects associated with the PKA signaling pathway and ap-

pears functionally related to the pka1-encoded catalytic

sub unit g ene (J in et al., 1995). Although other c AMP targe ts

apart from PKA have been identified in yeast (e.g., Muller

and Bandlow, 1994), the growth defects of the mac1 mu-

tants were suppressed by a mutation in the regulatory sub-

unit of P KA. Thus, it rema ins p os sible tha t a divergent c AMP-dependent catalytic subunit gene(s) or PKA-related genes

exist in M. g risea that are not functionally redundant with

CPKA.A sea rch for these g enes is in progress .

Our findings are compatible with a model in which a sur-

face-activated signal, such as assembly of the Mpg1 hydro-

phob in (Talbo t et a l., 1993, 1996; Bec kerma n a nd Ebb ole,

1996), results in the activation of adenylate cyclase (Figure

7). The find ing tha t MAC1 can complement the cr-1 muta-

tion of N. crassa suggests that MAC1 is membrane associ-

Figure 6. Rice Infection Ass ays .

Rice seedlings of cultivar CO39 were spray inoculated as indicated

below. Typica l leaves were co llected 7 da ys a fter inoculation a nd

photographed.

(A)Gelatin solution.

(B)Co nidial suspens ion of w ild-type s train G uy11.

(C)Co nidial suspens ion of ec topic integration trans formant DA50.

(D)Co nidial suspension of mac1::Hphmuta nt DA156.

(E)Conidial suspension of mac1::Hph sum 1-99muta nt DA99.


10/13


and morphogenesis but not pathogenicity leads us to pro-

pose the existence of additional cAMPSum1regulated

activities required for growth, conidiation, and appressorium

morphogenesis.

Suppressors of themac1Mutation

Suppressors of adenylate cyclase mutations have also been

identified in S. cerevisiae and Ustilago maydis (Matsumoto

et al., 1982; Gold et al., 1994). In these studies, suppressor

mutations frequently define the gene for the regulatory sub-

unit of PKA. Surprisingly, suppressor mutations were not

identified in mac1 mutants generated by Choi and Dean

(1997). One pos sibility is tha t the different g enetic b ac kground

employed in their studies may have masked the effects ofsuppressor mutations. We note that the mac1g ene-replac e-

ment mutant created by Choi and Dean retained the highly

conserved catalytic domain. Interestingly, studies with the

homologous CYR1gene in S. cerevisiaeshowed that inser-

tion/deletion mutations tha t did not inac tivate the c ata lytic

do ma in did no t crea te null muta nts (Suz uki et a l., 1990). The

deletion of the catalytic domain in our gene-replacement

constructs may have provided a stronger growth selection

for suppressor mutations.

In addition to mutations in the PKA regulatory subunit

gene in yeast, s uppress ors of the Ras-depe ndent cAMP sig-

naling pathway defects in S. cerevisiaeinclude mutations in a

cAMP phosphodiesterase gene SRA5 (Wilson and Tatchell,

1988) and the overexpression of a protein kinase gene

SCK1/SCH9 (Tod a et a l., 1988) and a transc ription fac torgene SOK2(Ward et al., 1995). In N. crassa, muta tions in the

hah gene elevate the levels of intracellular cAMP and sup-

press the colonial growth of cr-1mutants (Murayama et al.,

1995). Other s uppresso r mutations identified in our stud y re-

main undefined. Neither sum-61 nor sum-67 elevates the

levels of PKA activity, and neither mutation occurs in the

SUM1gene . These findings sugg est tha t sum-61and sum-67

act independently of PKA. Like sum1-99, both suppressors

restore growth and morphogenesis but not pathog enicity to

mac1muta nts. Although summutations allow abundant ap-

pressorium formation on hydrophilic surfaces, none of the

sum mutations permitted appressorium formation on agar

surfaces or in liquid media. Studies have suggested that

surface hardness may also be a signal for appressorial dif-ferentiation (Xiao et a l., 1994). This hypothes is ca n b e te ste d

using the strains containing summutations.

The sum1-99mutation alters an invariant leucine to argi-

nine in the first c AMP b inding do ma in (site A) of the reg ula -

tory s ubunit of P KA. This mutation a llow s an increa se in PKA

ac tivity in the a bs enc e of c AMP, b ut PKA a ctivity still can b e

pa rtially s timulate d b y the ad dition of c AMP. These bio-

chemical findings sugg est tha t the sum1-99encoding regu-

latory subunit may be inefficient at inhibiting the catalytic

subunit. P revious mutagenes is studies w ith other orga nisms

did not identify this leucine residue (Su et al., 1995). Analysis

of the crystal structure of the bovine regulatory subunit

shows that the homologous leucine residue (L-203) is con-

served in cAMP binding domain A but does not contact the

cAMP molecule (Su et al., 1995). Rather, nearest-neighbor

ana lysis show s that it aids in forming a network of contacts

with conserved residues that make up the cAMP binding

poc ket in do ma in A.

The finding tha t the sum1-99 mutation restores growth

morphology and appressorium differentiation but not full patho-

ge nicity sug ge sts a hierarchica l req uirement for cAMP /P KA

signaling in M. grisea.Low P KA ac tivity ma y be sufficient for

growth and morphogenesis but insufficient for pathogenic-

ity. However, an extensive mutational analysis of the PKA

regulatory subunit gene, bcy1, in S. cerevisiae shows that

the se verity of the physiologica l defect imparted by a regula-

tory subunit mutation does not correlate with the level ofPKA activity (Cannon et al., 1990; Zaremberg and Moreno,

1996). These stud ies s ugg es t that regulatory sub unit muta -

tions c an impart a variety of phenotypes ba sed on their bind-

ing affinity to catalytic subunits. For example, the sum1-99

enco ding regulatory subunit ma y not s epa rate efficiently from

the catalytic subunit in the presence of cAMP, thus reducing

PKA activity even in the presence of cAMP. In addition, the

sum1-99mutation causes a drama tic loss of P KA ac tivity in

germinating spores. Although the basis for this inhibition is

Figure 7. A Model for Divergent cAMP Signaling during Early

Stages of Infection-Related Morphogenesis in M. grisea.

X and Y represent PKA catalytic subunits other than CPKA that are

involved in growth and morphogenesis of M. grisea.Yeast extract

and the factor presumably interact with the membrane-associated

MAT1-2 recepto r and inhibit c AMP signa ling upstrea m o f c AMP

generation.


11/13


not known, it highlights the diverse ways in which regulatory

subunit mutations can affect PKA activity.

Finally, the sum1-99 mutation may allow PKA activity in

the holoenzyme complex and thereby alter PKA affinities for

spe cific sub strate s. Thus, PKA sub strate s phos phoryla ted in

the mac1::Hph sum1-99 background restore growth andapp ress orium forma tion b ut not full pathog enicity. The un-

fulfilled requirement for cAMP signaling in pathogenicity

may reflect the need for increased carbohydrate mobiliza-

tion fo r efficient pe netration a nd/or a lterations in funga l me-

tabolism to accommodate growth in plant cells. In U. maydis,

accurate PKA regulation imparted by the regulatory subunit

of P KA is ne ce ss ary for pa thog enicity (G old et a l., 1997).

METHODS

Fungal Strains and Methods

Magnaporthe g risea wild-type strains Guy11 and its descendant

4375-R-26 were used for MAC1and SUM1cloning. Guy11 also was

used for disruption of the MAC1ge ne. AFTH3, c arrying the MAT1-1

mating-type allele, was used for sexual crosses. Fungal mainte-

nance , sexua l cross es, trans formation, and DNA iso lations w ere per-

formed as previously described (Crawford et al., 1986; Sweigard et

al., 1992; Talbot e t a l., 1993). Rice infection as sa ys were pe rformed

using cultivar CO39 and Sariceltik, as previously described (Xu and

Hamer, 1996; Xu et a l., 1997). For the q uantitative a nalysis of pa tho-

genicity, 2-week-old seedlings of cultivar Sariceltik were sprayed

with 12 mL of conidial suspension (2 104conidia per mL in 0.25%

gelatin solution), and lesions on second leaves were counted 7 days

after inoculation. Neurosp ora crassas trains w ere kindly provided b y

M. Pla ma nn (University of Miss ouri, Ka nsa s C ity). The c ulture meth-

ods for Neurospora strains are described in Davis and de Serres(1970), a nd trans formations w ere performed as des cribed by Vollmer

and Yano fsky (1986).

MAC1andSUM1Cloning

A nested polymerase chain reaction (PCR) strategy was used to

clone the MAC1 and SUM1 genes. For MAC1, two degenerate

primer sets were synthesized to match the conserved catalytic do-

main of funga l ad enylate c yclases . The ca talytic d oma in of MAC1

was initially amplified with primers FP1 (5 -TNG TNTTYACNG -

AYATHAA-3) and RP4 (5-DATYTG NCC NCC RTC NGC -3) by using

Guy11 genomic DNA as a template. Nested P CR wa s performed with

primers FP 2 (5-ACNG ARG G NGAYG C NTTYATG -3) and RP3 (5-

AYNGG NCC RWARTARTC CAT-3). The P CR mixture w as heated to

94C for 3 min and amplified for 30 cycles under the following condi-

tions: 94C for 1 min, 50C for 2 min, and 72C for 3 min, with a final

extention cycle at 72C for 10 min. A 250-bp nested PCR product

wa s cloned into pGEM-T (P romega) and used to identify cosmid

clone 1F8 from a genomic cosmid library. A 13.4-kb EcoRI fragment

from 1F8 wa s s ubcloned into pUC18 a nd d esignated pKA1. The

8.7-kb DNA region containing the full-length MAC1 gene was se-

quenced. For SUM1 cloning, three degenerate primers were de-

signed based on the conserved domain of fungal protein kinase A

(P KA) regula tory s ubunits. The p rimers RF1 (5-G AYTAYTTYTAYG TN-

G TNGA-3) and RR4 (5-G C NARYTCNCC RAARWA-3) were used

with Guy11 geno mic DNA as a template. Nested P CR wa s performed

with primers RF2 (5-G ARYTNG C NYTNATG TA-3) and RR4. Thermal

cyc ling c onditions w ere identical to those used fo r MAC1except that

45C was used as an annealing temperature. SUM1cDNA and ge-

nomic clones were isolated from cDNA and genomic libraries (Xu and

Hamer, 1996) by using a 370-bp nested PCR product as a probe.Three cDNA clones w ere seq uenced. A 3.5-kb Sa cI frag ment from a

genomic clone was subcloned into pBluescript KS(Stratagene, La

J olla, CA), a nd the DNA sequence of the 2.9-kb Sa cI-Xba I region

containing the full-length SUM1gene w as determined.

All DNA seq uencing w as do ne using Tn1000mutagenesis to create

nested primer sites (Strathma nn et al., 1991), a nd dideoxy s eq uenc-

ing w as performed on a n ALF-Express DNA seq uencer (Pha rmacia)

by using a Thermo Seq uenase fluorescent-lab eled primer cycle se -

q uencing kit (Amersha m). To s ca n for mutat ions in the SUM1gene,

two PCR primers, RPKA1 (5-AC CG AG TG AAC CC G TTTATC-3) and

RP KA4 (5-C AG CATTCTTC AG CC TCTG -3), were designed to am-

plify the 1.3-kb co ding region. Thermoc ycling reac tions w ere per-

formed with Pfu polymerase (Stratagene) for high fidelity of DNA

synthesis. The 1.3-kb products were cloned into the S maI site of

pBluescript KS, and the DNA sequences were determined from atleast two independently amplified PCR clones.

Disruption of theMAC1Gene

The ge ne disruption vec tor pKA2 wa s c reated as follow s. The plas-

mid pKA1 was digested with NcoI to release 2.9- and 2.7-kb NcoI

fragments containing the 3 two-thirds of the MAC1coding region.

The HPH gene cassette was prepared from plasmid pCB1003

(Ca rroll et a l., 1994) by d iges tion with Hpa I. After blunt-end mod ifica -

tion of the NcoI sites of pKA1, the HPH cassette was inserted and

designated as pKA2. pKA2 was linearized with EcoRI and trans-

formed into Guy11. By using this construct, we expected complete

disruption of both leucine-rich repeats and the predicted catalyticdomain of the MAC1gene.

Appressorium Formation Assays

Conidia were collected from 10-day-old cultures on oatmeal agar

medium, filtered o nce through Miracloth (Calbioc hem-Nova biochem,

La J olla, C A), and ad justed to 2 104conidia per mL in water or 2%

yeast extract. Ninety microliters of conidial suspension was placed

on a hydrophobic plas tic co verslip (Fisher, P ittsburgh, P A), w hereas

50 L was used on a 1 1-cm piece of the hydrophilic side of G el-

Bond (FMC, Rockland, ME). Counts were performed after a 24-hr

incuba tion in a mo ist c hambe r at room tempe rature. For cAMP a ddition,

we prepared 10 co ncentrated stoc k solutions (100 and 500 mM) inwa ter and a dded them to co nidial suspension a t appropriate d ilutions.

PKA Assays

PKA assays were performed using a nonradioactive PKA assay kit

(Promega ) acco rding to the ma nufac turers sugg estions. P KA ac tivity

was visualized by agarose gel electrophoresis of a fluorescent PKA

mod el subs trate (Kemptide). To qua ntify P KA ac tivity, fluoresc ent


12/13


Kemptide w as releas ed from the aga rose gel acc ording to the ma n-

ufacturers suggestions, and the amount of fluorescent Kemptide

was determined in a spectrofluorometer. Concentrations of the

Kemptide were calculated from a standard curve generated with a

Kemptide phosphorylated with purified PKA catalytic subunit from

bovine heart. Hyphal extracts were prepared from mycelia grown in

co mplete liq uid me dium (Talbo t et a l., 1993) for 2 da ys w ith vigo rousshaking. Germling extracts were prepared from conidia germinated

for 8 hr in ste rile distilled wa ter. The extrac tion proce dure wa s d e-

scribed previously (Yang and Dickman, 1997). Protein concen-

trations were a djusted to 1.5 mg /mL by using the protein ass ay kit

(Bio-Rad), with BSA as a standard.

ACKNOWLEDGMENTS

We thank members of the Hamer laboratory, especially Drs. Martin

Urban a nd J in-Rong Xu (Novartis C rop Protec tion, Inc., Res earch

Triang le Pa rk, NC) for insightful disc uss ions. We grat efully a cknow l-

edg e the as sistanc e of J ane Einstein in collecting DNA seq uence in-

formation, Dr. Ralph Dean (Clemson University, Clemson, SC) for

providing information before publication, and Dr. Martin Dickman

(University of Nebras ka, Lincoln) for a dvice o n P KA as sa ys. K.A. w as

supported by a J apa n Soc iety for Promotion of Sc ience P ostdoc toral

Fellowship for Research Abroa d. This resea rch wa s s upported by a

National Sc ience Foundation grant a nd a P residential Fac ulty Fellow

Awa rd to J.E.H.

Received March 27, 1998; ac cepted J une 12, 1998.

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Divergent CAMP Signaling Pathways Regulate Growth and Pathogenesis in Rice Blast Fungus