MAP Kinase and cAMP Signaling Pathways Modulate the pH-Induced ...

CURRENT MICROBIOLOGY Vol. 49 (2004), pp. 274 –281 DOI: 10.1007/s00284-004-4315-6

Current Microbiology An International Journal © Springer Science⫹Business Media, Inc. 2004

MAP Kinase and cAMP Signaling Pathways Modulate the pH-Induced Yeast-to-Mycelium Dimorphic Transition in the Corn Smut Fungus Ustilago maydis Alfredo D. Martıńez-Espinoza,1 Jose´ Ruiz-Herrera,2 Claudia G. Leoń-Ramı´rez,2 Scott E. Gold1 1

Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274, USA Departamento de Ingenierıá Gene´tica, Unidad Irapuato, Centro de Investigacioń y de Estudios Avanzados del Instituto Politećnico Nacional, Apartado Postal 629, 36500 Irapuato, Gto., Mexico 2

Received: 2 February 2004 / Accepted: 15 April 2004

Abstract. Acid pH induces the yeast-to-mycelium transition in haploid cells of Ustilago maydis. We tested two signal transduction pathways known to be involved in dimorphism for roles in acid-induced filamentation. In wild-type cells intracellular cAMP levels were reduced under acid growth. A mutant defective in the regulatory subunit of PKA, ubc1, failed to respond to acid induction on solid medium, but in liquid medium showed a mycelial phenotype at acid pH. Mutants in the pheromone-responsive MAP kinase pathway lost the capacity to grow as mycelium at acid pH, while a mutant in the pheromone response-transcriptional regulator, prf1, behaved as wild-type. Filamentation by both ubc1 and prf1 mutants was inhibited by addition of cAMP. A putative MAP kinase cascade adaptor protein gene, ubc2, complemented a previously identified myc mutant strain defective in pH-induced myceliation. These results indicate that pH-dependent dimorphism is regulated by two known signaling pathways but that an effector for cAMP signaling alternative to Ubc1 is present in U. maydis and that Prf1 is not the sole downstream target of MAP kinase signaling.

Ustilago maydis (DC.) Corda is a worldwide smut pathogen of maize (Zea mays L.). The haploid fungus grows in the form of a saprophytic budding yeast. An obligate pathogenic dikaryotic mycelium is produced after mating of compatible haploid cells. The mycelium invades the plant and eventually forms diploid teliospores that fill the tumors induced in the infected plant [3, 7, 25]. Mating, yeast-to-mycelium transition, and pathogenicity are regulated by the a and b mating-type loci [19, 20, 30, 53]. Cyclic AMP plays an important role in morphogenesis and virulence in many fungi, including Ophiostoma ulmi [8], Ustilago hordei [35], Candida albicans [9], Histoplasma capsulatum and Blastomyces dermatitidis [42], and Mucor spp. [46]. Additionally, cAMP levels regulate appressorium formation in Magnaporthe grisea [33] and pseudohyphal growth of Saccharomyces cerevisiae [21, 36]. For a review see Lengeler et al. [34]. Cyclic AMP also plays a significant role in the dimorphism of U. maydis. Several Correspondence to: S.E. Gold; email: [email protected]

genes, including those encoding adenylate cyclase (uac1) [22], the regulatory subunit of the cAMP-dependent protein kinase (ubc1) [22], and adr1, the major cAMP-dependent protein kinase (PKA) encoding gene [16], have been cloned and their participation in morphogenesis and virulence demonstrated. Signaling through the mitogen activated protein (MAP) kinase cascade is also important for pathogenicity and dimorphism in a variety of fungi [2, 12, 14, 34, 57]. The MAP kinase cascade is important in the filamentous growth and virulence of U. maydis [1, 4, 39, 40]. Several genes, such as those encoding the MAP kinase ubc3/kpp2 [40, 45], the MAPKK kinase ubc4 [1, 5], and the MAPK kinase fuz7/ubc5 [1, 5], were identified as genes whose mutation suppresses the filamentation associated with uac1 mutants unable to produce cAMP. These ubc genes were also required for full virulence. These data suggest interplay between the cAMP pathway and the MAP kinase cascade in filamentous growth and virulence in U. maydis [1, 6, 23, 29, 31, 39, 41, 45].

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A.D. Martıńez-Espinoza et al.: Signal Transduction Pathways in Ustilago maydis Dimorphism Table 1. U. maydis strains used Strain name

Relevant genotype

2/9 1/2 1/9 1/68 1/53 2/40 1/52 2/58; FB1⌬kpp2 3/25 3/6 ⌬fuz7; 2/38 CLB1 CL233 CL211 HA99 HA87 ER22 gpa3Q206L 001-12 (Phlr)

a2b2 a1b1 (521) uac1⫺::ble ubc1⫺ uac1⫺, ubc1⫺ ⌬ubc2::hygB; oliR uac1⫺ubc2⫺ (ts) ⌬ubc3::nat uac1⫺, ubc3⫺ uac1⫺, ubc4⫺ ⌬fuz7/ubc5⫺ myc⫺ myc⫺ myc⫺ ⌬prf1, a1b1 ⌬prf1, a2b2 gpa3Q206L adr1-1 cpka

Proposed function affected

Reference

Adenylate cyclase rPKA rPKA Putative adaptor protein Putative adaptor protein MAPK MAPK MAPKKK MAPKK Unknown Unknown Putative adaptor protein Transcript. Regulator Transcript. Regulator G protein constitutive cPKA

Gold et al. [23] Kronstad and Leong, 1989 Gold et al. [22] Gold et al. [22] Gold et al. [22] Mayorga and Gold [41] Mayorga and Gold [39] Mayorga and Gold [40], Muller et al. [45] Mayorga and Gold [39] Mayorga and Gold [39] Banuett and Herskowitz [4], Andrews et al. [1] Martinez-Espinoza et al. [38] Martinez-Espinoza et al. [38] Martinez-Espinoza et al. [38], this work Hartmann et al., 1996 Hartmann et al., 1996 Regenfelder et al. [49] Durrenberger et al. [16]

In addition to genetic determinants, there are several environmental conditions that promote the yeast-to-hypha transition in U. maydis, including nutrient and nitrogen deprivation [4, 28], exposure to air [22], and acid pH [50]. In this report we focus on the phenomenon of pH-induced fungal morphogenesis, an area most well studied in the saprophyte Aspergillus nidulans [13]. Haploid U. maydis cells grown on neutral medium (pH 7) display yeast growth, whereas on acid media (pH 3) they grow as filaments [50]. The relationship between pH and dimorphism in U. maydis is comparable to that described for C. albicans although the outcome is reversed: acid pH inhibits mycelial growth in C. albicans [54]. In C. albicans several genes have been shown to play roles in pH-induced dimorphism [17, 18, 44, 48, 51, 52]. In U. maydis initial steps toward understanding the pH-induced dimorphism have been initiated. Martinez-Espinoza et al. [38] isolated mutants unable to carry out the dimorphic transition from yeast to mycelium normally induced at acid pH. To further elucidate the mechanisms controlling pH-regulated dimorphic transitions in U. maydis, we present morphological, physiological, and genetic analyses to determine the roles of the cAMP and pheromone-responsive MAP kinase signaling cascades in production of acid-induced filamentation. Materials and Methods Strains, media, and culture conditions. The strains used in this work are described in Table 1. Strains were grown either in liquid or on solid potato-dextrose-broth/potato-dextrose-agar (Difco), YEPS [39], or synthetic minimal medium (MM) or complete medium (CM) [26]. To perform experiments on solid media at different pH (pH 3 or

pH 7), media were prepared as described in detail in Ruiz-Herrera et al. [50]. For pH 3 minimal medium, NH4NO3 and other salts were dissolved in water at a 2⫻ concentration, adjusted to pH 2.75 using HCl and autoclaved. Separately, a mixture of 2% glucose and 4% agar was autoclaved. When cooled, equal volumes of the two solutions were mixed. For pH 7 minimal medium preparation was similar to that for pH 3 medium except that KNO3 was substituted for NH4NO3 as the nitrogen source [50] and pH was adjusted to 7 using NaOH. The effect of different nitrogen sources on the media has been thoroughly reviewed [50]. For charcoal-containing media, charcoal (Sigma, St. Louis, MO) was included in the glucose-agar solution. For minimal medium pH 3 or pH 7 supplemented with cAMP, appropriate amounts of filter-sterilized 1 M cAMP were added to a final concentration of 25 mM. For liquid media the preparation was the same but agar was excluded. Induction of mycelial growth. Mycelial growth was induced as previously described [50]. Briefly, cells were grown for 48 h in 15 mL PDB at 30°C. One-tenth volume was then inoculated into 15 mL of fresh medium and grown with shaking on a rotary shaker (250 rpm) for an additional 18 –20 h at 30°C. Cells were centrifuged and washed twice with sterile distilled water (SDW). Cells were resuspended in 15 mL SDW and incubated for 2–3 h at 30°C. Cells were then washed twice and the pellet resuspended in 1.5 mL of SDW, and incubated on ice for 15 min. Cells were counted on a hemacytometer and 1 ⫻ 105 cells were immediately placed in 5 mL of the corresponding medium (MM pH 3, MM pH 7, MM-cAMP pH 3, or MM-cAMP pH 7) that had been maintained at 37°C. The transfer of cells from ice to 37°C was previously shown to be important for filament induction [50]. Cells were then incubated at 28°C with continuous shaking and cell morphology was recorded at specific intervals microscopically (see below). The mycelial index was calculated as described in Martinez-Espinoza et al. [38]. Briefly, for these calculations the ratios between cell length, width, the number of pseudohyphal-like cells in each hypha, and the number of branches were recorded relative to the wild-type cells. Mycelial cells were given a value of 1.0 and yeast cells a value of 0. One hundred cells were observed for each treatment.

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CURRENT MICROBIOLOGY Vol. 49 (2004)

Fig. 1. Colony morphology of several U. maydis signaling mutants grown on acid or neutral pH solid media. Strains used are listed in Table 1 and were: WT, (1/2); gpa3Q206L, (ER22 gpa3Q206L); uac1⫺, (1/ 9); ubc1⫺, (1/68); adr1⫺, (001-12 (Phlr)); ubc2⫺, (2/40); ubc4⫺, (3/6 - uac1⫺, ubc4⫺); ubc5⫺, (⌬fuz7; 2/38); prf1⫺, (HA87). Microscopy and photography. Colony preparations were visualized with a Zeiss Axioplan Universal microscope (Carl Zeiss, Microscope Division, Oberkochen, Germany) and photographed with Kodak TMAX 100 film (camera set at ASA 200). Slides were scanned with a Nikon Coolscan (Nikon, Melville, NY). Images were compiled with the software application Photoshop 4.0 (Adobe Systems, Mountain View, CA). cAMP quantification. To quantify cellular cAMP levels, an immunoassay kit was used (Biomol, Plymouth Meeting, PA). Cells were grown in 5 mL PDB medium for 20 h at 30°C. A 1 mL sample was centrifuged and 300 mg of glass beads (465– 600 ␮m in diameter, Sigma) were added to the cell pellet. The pellet-containing tubes were quickly immersed in liquid nitrogen and stored at ⫺70°C. These samples were labeled as “harvest time”. Immediately after filament induction, a second sample was withdrawn, labeled “Time 0”, and frozen as described above. Adjusted amounts of cells were then separated into the different pH media, and incubated accordingly. Periodically, samples were withdrawn and frozen as above. To obtain extracts, cells were thawed on ice, vortexed at 4°C for 2 min and placed on ice for 1 min. This procedure was repeated 6 times to ensure complete cell breakage. Samples were centrifuged at 4°C, the supernatants were collected separately and kept on ice. Protein concentration was determined using Bradford reagent (BioRad, Hercules CA). Supernatant volumes containing 50 –100 ng of protein were used for immunoassay according to the manufacturer’s instructions and cAMP content recorded per milligram protein. DNA manipulations. U. maydis myc mutants belonging to different complementation groups [38] were transformed independently with cloned ubc1, ubc2, ubc3, ubc4, or ubc5 genes [1, 22, 40, 41]. Transformations were performed using protoplasts and ARS-containing selfreplicating plasmids as described in Tsukuda et al. [56]. Transformants that showed a mycelial phenotype on acid media were recorded.

Results Colony morphology of mutants affected in their cAMP and MAP kinase signaling pathways. Colony

morphology of mutants belonging to the different signaling pathways was evaluated on solid media adjusted to neutral and acidic pH (Fig. 1). On neutral medium (pH 7), all strains used formed yeast colonies except for strains 1/9 (uac1 mutant) and adr1, which were mycelial. On acid medium (pH 3), the wild-type strains 1/2 and 2/9 had a strong mycelial phenotype. Single MAP kinase cascade mutant strains such as 2/40 (⌬ubc2; deleted for a putative adaptor protein of the MAP kinase module) formed yeast colonies (Fig. 1), as did 2/38 (⌬fuz7/ubc5, deleted for MAPKK), and 2/58 (⌬ubc3/kpp2; deleted for MAPK). We tested prf1 (a downstream transcriptional regulator of the pheromone response pathway) mutants to evaluate the role of this transcription factor in pH signaling. HA87 (prf1, a1b1) exhibited a mycelial colony phenotype at pH 3. Mutants affected in the cAMP signaling pathway were also evaluated. As noted above, the adenylate cyclase mutant 1/9 (uac1⫺), as well as the adr1⫺ strain, had a strong mycelial phenotype as expected, independent of the pH of the growth medium. Strain 1/68 (ubc1⫺; mutant for the PKA regulatory subunit) produced smooth and shiny yeast colonies with no visible filaments at either pH 7 or pH 3 (Fig. 1). Double mutants affected in both a MAP kinase pathway component gene and in uac1 [39] were tested for their response to acid pH media. Strains 1/52 (uac1⫺, ubc2⫺), 3/25 (uac1⫺, ubc3⫺) and 3/6 (uac1⫺, ubc4⫺) (affected in the adaptor, MAPK, or MAPKKK coding genes, respectively) formed yeast colonies with a “frosty” phenotype [39], characterized by very short filaments on the surface of the colonies (Fig. 1) Strain

A.D. Martıńez-Espinoza et al.: Signal Transduction Pathways in Ustilago maydis Dimorphism

1/53 (uac1⫺, ubc1⫺), a mutant in the PKA regulatory subunit in a uac1⫺ background, formed smooth and shiny yeast colonies with no visible filaments. Upon addition of cAMP to solid MM medium at pH 3 or pH 7 (with or without charcoal), colony morphologies of all strains tested with the exception of adr1 were yeasts. Additionally, as reported previously, the monomorphic mutants CL1B and CL233 [38] produced yeast colonies on all solid media tested. Cell morphology of cAMP and MAP kinase signaling pathway mutants grown in liquid media. Cell morphology of the different signaling pathway mutants was evaluated by growing strains in liquid medium adjusted to pH 3 or pH 7. Mycelial growth was induced as described [49; see Materials and Methods]. At pH 7, the wild-type strains 1/2 and 2/9 grew with the typical polar budding pattern of U. maydis. Single mutants affected in the MAP kinase cascade, including ⌬fuz7/ubc5, ⌬ubc3/ kpp2, and ⌬ubc2, displayed a wild-type budding phenotype. Strains with mutations in the cAMP pathway showed cell morphologies similar to those observed on solid media. The uac1 and adr1 strains defective in cAMP-dependent protein kinase activity formed cells with a filamentous morphology. Strain 1/68 (ubc1⫺) grew in the form of multiple budding cells, as observed previously at neutral pH [39]. The prf1 deletion strain HA99 grew as budding yeasts at this pH. The double mutant strains affected in both uac1 and members of the MAP kinase pathway—1/52 (uac1⫺, ubc2⫺), 3/25 (uac1⫺, ubc3⫺) and 3/6 (uac1⫺, ubc4⫺)— grew as a mixture of normal budding and branching pseudohyphallike joined budding cell clusters as reported earlier [39]. At this pH, strain 1/53 (uac1⫺, ubc1⫺) showed a multiple budding form similar to that described by Gold et al. [22]. The monomorphic strains CL1B and CL233 [37] grew by typical sporidial budding. Wild-type strains became filamentous when grown at pH 3. The long, slender, branched mycelium had a central thicker cell (mycelial index of 1.0 according to Martinez-Espinoza et al. [38]. Mutants affected in members of the MAP kinase pathway presented diverse morphological phenotypes when grown in liquid media. The ⌬ubc2 strain grew in the form of bud-like cells similar to wild-type sporidia (mycelial index of 0.1). Most of the ⌬ubc3/kpp2 cells appeared budding, but approximately 20% of the cells had an incipient elongated phenotype (mycelial index of 0.2) perhaps due to partial redundancy of function as postulated for the relatively weak effects on pathogenicity in this mutant [40]. The cell phenotype of strain ⌬fuz7 was more elongated than other MAP kinase pathway mutant strains tested (mycelial index of 0.4). Strains with mutations affecting the cAMP pathway

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showed the following phenotypes at pH 3: as expected, strains 1/9 (uac1⫺) and adr1⫺ grew elongated and branched filaments (mycelial index of 1.0). Surprisingly, strain 1/68 (ubc1⫺) formed long and distorted mycelial cells (mycelial index of 0.8), resembling those of the wild-type on acidic medium. The ⌬prf1 mutant HA87 grew as filaments with short branches (mycelial index of 0.7). Double uac1 and MAP kinase pathway mutants [39] exhibited differing phenotypes. Strain 1/52 (uac1⫺, ubc2⫺) formed budding cells joined in chains (mycelial index 0.2) that were generally more elongated than wildtype cells grown at neutral pH. Strain 3/25 (uac1⫺, ubc3⫺) grew in the form of budding cells with a slightly elongated shape (mycelial index of 0.2). Strain 1/53 (uac1⫺, ubc1⫺) displayed an elongated and distorted mycelial phenotype (mycelial index of 0.6), although cells were shorter and less branched than wild-type strains. As reported previously [38], myc mutants CLB1 and CL233 grew strictly in the budding form. Effect of cAMP on U. maydis colony and cell morphologies. To evaluate the effect of exogenous cAMP on colony and cell morphologies, 25 mM cAMP was added to solid or liquid media. All strains with the exception of adr1⫺ displayed a yeast phenotype when cAMP was added to solid media at neutral or acid pH. In liquid pH 7 medium amended with cAMP, all strains tested including wild-type, with the exception of adr1⫺, appeared as short chains of budding cells (4 – 6 per cluster) reminiscent of the ubc1 mutant. A notable difference was ⌬ubc2, which exhibited less conspicuous multiple budding groups (2 or 3 cells per cluster) and accentuated lateral budding. As reported earlier, the adr1 mutant remained filamentous at all times. Upon addition of cAMP to the pH 3 medium, all strains except adr1⫺ grew as laterally budding cells. There was some variation among the strains regarding multiple bud cluster size, but most had 5–7 joined cells per cluster. cAMP quantification during morphogenesis. Since exogenous addition of cAMP has a profound effect on the morphology of U. maydis [22], we investigated whether endogenous levels of cAMP were correlated with cell morphology. It is known that adenylate cyclase and phosphodiesterase tightly regulate cellular cAMP levels in various organisms [15] by its synthesis and/or degradation, respectively. High levels of cAMP were detected when wild-type cells were grown for 20 h in neutral medium (Fig. 2). When cells were prepared for mycelial growth induction cAMP levels decreased sharply. When cells were transferred to acid medium, cAMP levels continued to drop to undetectable levels. Cell morphology remained yeast-like early after placement in acid mycelial induction medium. Between 3 and 6 h post-

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Fig. 2. cAMP levels of a U. maydis wild-type strain 1/2 grown at pH 3 or pH 7. Bud, budding; Fil, filamentous; H, harvest; T0, time 0. Data are averages of two independent experiments. Table 2. cAMP levels in the gpa3Q206L and uac1 mutants

inoculation, progressive elongation and branching of cells occurred. During this period cAMP levels remained low. At 12 h post-inoculation in acid medium a wellformed mycelium was observed that correlated with low levels of cAMP. In neutral media, cAMP levels were similar to those detected in acid media at early time periods. During the first 30 min, cAMP was at the limits of detectability. However, after 1 h, cAMP levels progressively increased. After 24 h, cAMP levels were similar to those found at the beginning of the experiment. In this neutral medium only budding cells were observed. For comparison we tested strain 1/9 (uac1) and constitutive gpa3Q206L strains [49] which were expected to have reduced and elevated cAMP levels, respectively. In all media tested the adenylate cyclase mutant (uac1) had cAMP levels at the margin of detectability while the constitutive gpa3Q206L strain had cAMP levels comparable to those found in the wild-type strains (Table 2). Complementation of myc mutants. Several myc mutants belonging to the previously described four complementation groups [38] were transformed using the five cloned ubc genes (ubc1⫺, ubc2⫺, ubc3⫺, ubc4⫺, and ubc5⫺) to determine whether any of these mutants were defective in previously isolated genes influencing dimorphism. When ubc2 was introduced into the strain CL211,

cAMP levelsa gpa3Q206L Time Harvest T0 1 min 10 min 60 min 6h 8h 12 h 24 h

uac1

pH 7

pH 3

pH 7

pH3

80 10 7 5 20 35 50 60 90

80 10 5 5 7 10 14 20 25

6.5 10 5 0 5 5 12 10 18

6.5 10 7 0 0 5 9 10 10

pmol/mg protein. Averages of four independent experiments. SE ⫾ 3.0.

a

hundreds of transformants were recovered on hygromycin plates. All transformants recovered were restored to the wild-type phenotype and thus displayed a filamentous phenotype when grown on acid media (data not shown). None of the additional myc⫺ strains corresponding to the complementation groups discussed in Martinez-Espinoza et al. [38] were complemented by any of the ubc genes.


Discussion Our results indicate that acid pH induction of filamentous growth in U. maydis is influenced by well-characterized signal transduction pathways: the pheromone-responsive MAP kinase cascade, and the cAMP-dependent protein kinase pathway. These results show that mycelium formation on solid acidic medium (pH 3) requires all the known components of the MAP kinase module. In contrast to wild-type strains that form mycelial colonies at pH 3, yeast colonies were produced by strains bearing mutations in genes encoding a MAPKKK, MAPKK, or MAPK. A mutant affected in a putative MAP kinase cascade adaptor protein encoded by the ubc2 gene [41] also failed to grow as mycelium. A more complex set of phenotypes was observed for mutants of the cAMP pathway on solid media. The gpa3Q206L mutant (constitutively active Gpa3) behaved similarly to wild-type strains. A uac1 mutant was filamentous at pH 7 and at pH 3, whereas the ubc1 mutant produced yeast colonies at both pH values. The adr1 mutant was filamentous at both neutral and acid pH and in all media tested. When evaluated on acidic media, double mutants (uac1 ubc3; uac1 ubc4; uac1 ubc5) produced a “frosty” morphology, which is a yeast colony with short sparse filamentation. This phenotype is similar to that previously described for these strains at neutral pH [39]. In liquid media, wild-type strains formed filaments upon induction by acid pH, but the effect was overridden by the exogenous addition of 25 mM cAMP, indicating that at this concentration cAMP represses the mycelial induction initiated by acid pH. The addition of cAMP to wild-type strains resulted in a multiple budding phenotype at both neutral and acid pH. In acid liquid media the adenylate cyclase mutant formed mycelium constitutively. This effect was repressed by the addition of cAMP as previously reported in neutral media [22]. The adr1 mutant grew myceliallike at all times, including when cAMP was added to the media, consistent with the epistatic effect of the mutation. Interesting results were obtained with the ubc1 mutant (rPKA) grown in liquid media. This strain produced multiple budding cell clusters at pH 7 but grew as mycelium under acid conditions. However, when exogenous cAMP was added to the acid media the ubc1 mutant was returned to the multiple budding phenotype. These results suggest that a second rPKA may be present in U. maydis. Additionally, since the ubc1 mutants grew as yeast on solid media at pH 3, different signaling systems appear to operate in solid or liquid media. Another important observation was that prf1 mutants produced mycelium in solid and liquid media upon

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induction by acid pH. To date, the only reported likely substrate of the MAP kinase Ubc3/Kpp2 is Prf1 [45]. Because ubc3 mutants grow as budding cells at pH 3, a substrate of the Ubc3 MAPK kinase alternative to Prf1 is likely involved in triggering filamentation. Furthermore, as prf1 mutants responded to the addition of cAMP, alternative transcription factors are also likely involved in downstream signaling of the PKA pathways. These results are consistent with those noted by Lee and Kronstad [32] in which epistasis experiments indicated that Ras2 may regulate filamentation via the pheromoneresponsive MAP kinase cascade including Ubc3, but not through the activation of Prf1. The results presented here indicate that cAMP levels, and therefore adenylate cyclase and/or phosphodiesterase activities, correlate with pH-induced morphogenesis in U. maydis. Low levels of cAMP were found in cells grown at acid pH in which filamentation occurred, whereas high levels of cAMP were observed in budding yeast cells grown at neutral pH. This observation was congruent with the induction of budding growth when exogenous cAMP was added to acid media. This may imply that under acidic pH conditions, adenylate cyclase is inhibited and de novo cAMP synthesis is curtailed. Alternatively, phosphodiesterase activity is increased and cAMP is degraded rapidly. The ability of CL211 to produce filaments at acid pH conferred by the cloned ubc2 gene shows that this particular complementation group described in MartinezEspinoza et al. [38] is related to the MAP kinase pathway. There are, however, other complementation groups (genes) described in that work that remained uncomplemented by any of the ubc genes, indicating that novel genetic mechanisms involved in pH-regulated morphogenesis in U. maydis remain to be identified. The maize leaf environment may provide acidic conditions contributing to the induction of filamentation in U. maydis. Plant signals have long been speculated to be involved in regulating morphological differences witnessed in fungus grown in vitro as opposed to in planta [2–5, 27]. Interestingly, the plant apoplastic pH decreases in response to elevated auxin levels to 4.5 to 5.0 and this pH can induce plant cell elongation [11, 47]. There are reports that U. maydis produces auxin [10, 24, 38, 56], suggesting that the fungus may indirectly precipitate a localized reduction in the pH of the plant tissue in which it grows; feeding back to the induction of filamentous growth. ACKNOWLEDGMENTS We express our thanks to Drs. Steve Denison and Steve Klosterman for critical review of the manuscript. We thank Drs. Flora Banuett, Regina Kahmann, and Jim Kronstad for generously providing U. maydis

280 strains used in this work. We also acknowledge support from the U.S. Department of Agriculture National Research Initiation Competitive Grants Nos. 99-35303-8635 and 01-35319-10139 and from the U.S. National Science Foundation Division of International Programs Grant Nos. INT-9802638 and INT-0203661 to S.E.G A.D.M.E. and J.R.H. acknowledge CONACYT for its financial support. A.D.M.E. thanks IFS for its financial support.


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