Cdc42 is required for proper growth and development in the fungal pathogen Colletotrichum trifolii

Cdc42 is a highly conserved small GTP-binding protein that is involved in regulating morphogenesis in eukaryotes. In this study, we isolated and characterized a highly conserved Cdc42 gene from Colletotrichum trifolii (CtCdc42), a fungal pathogen of alfalfa. CtCdc42 is, at least in part, functionally equivalent to Saccharomyces cerevisiae Cdc42p, since it restores the temperature-sensitive phenotype of a yeast Cdc42p mutant. Inhibition of CtCdc42 by expression of an antisense CtCdc42 or a dominant negative form of CtCdc42 (DN Cdc42) resulted in appressorium differentiation under noninductive conditions, suggesting that CtCdc42 negatively regulates pathogenic development in this fungus. We also examined the possible linkage between CtCdc42 and Ras signaling. Expression of a dominant active Cdc42 (DA Cdc42) in C. trifolii leads to aberrant hyphal growth under nutrient-limiting conditions. This phenotype was similar to that of our previously reported dominant active Ras (DA Ras) mutant. Also consistent with our observations of the DA Ras mutant, high levels of reactive oxygen species (ROS) were observed in the DA Cdc42 mutant, and proline restored the wild-type phenotype. Moreover, overexpression of DN Cdc42 resulted in a significant decrease in spore germination, virtually no hyphal branching, and earlier sporulation, again similar to what we observed in a dominant negative Ras (DN Ras) mutant strain. Interestingly, coexpression of DA Cdc42 with DN Ras resulted in germination rates close to wild-type levels, while coexpression of DN Cdc42 with the DA Ras mutant restored the wild-type phenotype. These data suggest that CtCdc42 is positioned as a downstream effector of CtRas to regulate spore germination and pathogenic development.     

The COT1 homologue CPCOT1 regulates polar growth and branching and is essential for pathogenicity in Claviceps purpurea

Claviceps purpurea, the ergot fungus, is a common grass pathogen attacking exclusively young ovaries. Its pathogenic development involves an early phase of directed growth (with strictly suppressed branching) towards the floral vascular tissue. Since Ser/Thr protein kinases of the NDR family have been shown to be involved in polar growth and branching in fungi, we have analyzed a C. purpurea homologue of the Neurospora crassa cot-1 gene, cpcot1. It encodes a functional homologue of COT1 since it can fully complement the N. crassa cot-1 mutant phenotype. Delta cpcot1 mutants are significantly impaired in vegetative growth properties: they are characterized by hyperbranching, reduced growth rate, and decreased conidiation. Infection studies on rye plants and isolated ovaries show that the delta cpcot1 mutants are apathogenic; microscopical analyses indicate a very early block, probably in penetration. Thus CPCOT1 is not only involved in polarity and branching and hence oriented growth in the host tissue as expected, but it is essential for the initiation of infection.     

The small GTPase Rac and the p21-activated kinase Cla4 in Claviceps purpurea: interaction and impact on polarity, development and pathogenicity

Claviceps purpurea , the ergot fungus, is a highly specialized pathogen of grasses; its colonization of host ovarian tissue requires an extended period of strictly polarized, oriented growth towards the vascular tissue. To understand this process, we study the role of signalling factors affecting polarity and differentiation. We showed that the small GTPase Cdc42 is involved in polarity, sporulation and in planta growth in C. purpurea . Here we present evidence that the GTPase Rac has an even stronger and, in some aspects, inverse impact on growth and development: Δ rac mutants form coralline-like colonies, show hyper-branching, loss of polarity, sporulation and ability to penetrate. Functional analyses and yeast two-hybrid studies prove that the p21-activated kinase Cla4 is a major downstream partner of Rac. Phosphorylation assays of MAP kinases and expression studies of genes encoding reactive oxygen species (ROS)-scavenging and -generating enzymes indicate a function of Rac and Cla4 in fungal ROS homoeostasis which could contribute to their drastic impact on differentiation.     

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase,is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.     

Polygalacturonase is a pathogenicity factor in the Claviceps purpurea/rye interaction

Claviceps purpurea is a biotrophic, organ-specific pathogen of grasses and cereals, attacking exclusively young ovaries. We have previously shown that its mainly intercellular growth is accompanied by degradation of pectin, and that two endopolygalacturonase genes (cppg1/cppg2) are expressed throughout all stages of infection. We report here on a functional analysis of these genes using a gene-replacement approach. Mutants lacking both polygalacturonase genes are not affected in their vegetative properties, but they are nearly nonpathogenic on rye. Complementation of the mutants with wild-type copies of cppg1 and cppg2 fully restored pathogenicity, proving that the endopolygalacturonases encoded by cppg1 and cppg2 represent pathogenicity factors in the interaction system C. purpurea/Secale cereale, the first unequivocally identified so far in this system.     

Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthracnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, including germination and subsequent differentiation, resulting in appressorium formation. Our previous studies with pharmacological effectors of signaling pathways have suggested the involvement of cyclic AMP (cAMP)-dependent protein kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) subunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene replacement. Southern blot analysis with C. trifolii genomic DNA suggested that Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RNA from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactivated by gene replacement, the transformants showed a small reduction in growth relative to the wild type and conidiation patterns were altered. Importantly, PKA-deficient strains were unable to infect intact alfalfa (host) plants, though only a slight delay was observed in the timing for conidial germination and appressorial formation in the Ct-PKAC disruption mutants. Moreover, these mutants were able to colonize host tissues following artificial wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely due to a failure in penetration. Our results demonstrate that PKA has an important role in regulating the transition between vegetative growth and conidiation, and is essential for pathogenic development in C. trifolii.     

Proper regulation of cyclic AMP-dependent protein kinase is required for growth, conidiation, and appressorium function in the anthracnose fungus Colletotrichum lagenarium

Colletotrichum lagenarium, the casual agent of anthracnose of cucumber, forms specialized infection structures, called appressoria, during infection. To evaluate the role of cAMP signaling in C. lagenarium, we isolated and functionally characterized the regulatory subunit gene of the cAMP-dependent protein kinase (PKA). The RPK1 gene encoding the PKA regulatory subunit was isolated from C. lagenarium by polymerase chain reaction-based screening. rpk1 mutants, generated by gene replacement, exhibited high PKA activity during vegetative growth, whereas the wild-type strain had basal level activity. The rpk1 mutants showed significant reduction in vegetative growth and conidiation. Furthermore, the rpk1 mutants were nonpathogenic on cucumber plants, whereas they formed lesions when inoculated through wounds. A suppressor mutant showing restored growth and conidiation was isolated from a rpk1 mutant culture. The rpkl-suppressor mutant did not show high PKA activity, unlike the parental rpk1 mutant, suggesting that high PKA activity inhibits normal growth and conidiation. The suppressor mutant, however, was nonpathogenic on cucumber and failed to form lesions, even when inoculated through wounds. The rpk1 and suppressor mutants formed melanized appressoria on the host leaf surface but were unable to generate penetration hyphae. These results suggest that proper regulation of the PKA activity by the RPK1-encoded regulatory subunit is required for growth, conidiation, and appressorium function in C. lagenarium.     

The NADPH oxidase Cpnox1 is required for full pathogenicity of the ergot fungus Claviceps purpurea

The role of reactive oxygen species (ROS) in interactions between phytopathogenic fungi and their hosts is well established. An oxidative burst mainly caused by superoxide formation by membrane-associated NADPH oxidases is an essential element of plant defence reactions. Apart from primary effects, ROS play a major role as a second messenger in host response. Recently, NADPH oxidase (nox)-encoding genes have been identified in filamentous fungi. Functional analyses have shown that these fungal enzymes are involved in sexual differentiation, and there is growing evidence that they also affect developmental programmes involved in fungus-plant interactions. Here we show that in the biotrophic plant pathogen Claviceps purpurea deletion of the cpnox1 gene, probably encoding an NADPH oxidase, has impact on germination of conidia and pathogenicity: Deltacpnox1 mutants can penetrate the host epidermis, but they are impaired in colonization of the plant ovarian tissue. In the few cases where macroscopic signs of infection (honeydew) appear, they are extremely delayed and fully developed sclerotia have never been observed. C. purpurea Nox1 is important for the interaction with its host, probably by directly affecting pathogenic differentiation of the fungus.     

Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans

Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.     

The small GTPase BcCdc42 affects nuclear division, germination and virulence of the gray mold fungus Botrytis cinerea

The small GTPase Cdc42 plays a central role in various processes in eukaryotic cells including growth, differentiation and cytoskeleton organization. Whereas it is essential in the yeast Saccharomyces cerevisiae, its role in filamentous fungi differs, due to the complementing, partly overlapping function of Rac. We analyzed the role of the Cdc42 homologue in the necrotrophic, broad host range pathogen Botrytis cinerea. Deletion mutants of bccdc42 showed various growth abnormalities; the mutants had reduced growth rate and hyphal branching, they produced fewer conidia, which were enlarged and misshapen and had germination defects. Additionally, the mutants were impaired in sclerotia development. Cytological studies indicate that at least part of this phenotype could be attributed to disturbed control of nuclear division: conidia and hyphae of the mutant showed twofold higher nucleus/cytoplasm ratio compared to wild type cells. Apart from these effects on vegetative growth and differentiation, Δbccdc42 strains were attenuated in penetration and colonization of host tissue, confirming that BcCdc42 – though being not essential like in yeast – is involved in important developmental processes in B. cinerea.     

The sorting nexin FgAtg20 is involved in the Cvt pathway,non‐selective macroautophagy,pexophagy and pathogenesis in Fusarium graminearum

The sorting nexin Atg20/Snx42 plays an important role in autophagy. The wheat head blight pathogen Fusarium graminearum contains an FgAtg20 protein orthologous to Saccharomyces cerevisiae Atg20/Snx42, but its function remains largely unknown. Here, we report a role for FgAtg20 in regulating morphogenesis and fungal pathogenicity. Cytological observation and Western blot analysis revealed that ΔFgAtg20 mutants are defective in vacuolar transport and proteolysis of GFP‐FgAtg8, indicating that FgAtg20 is required for non‐selective macroautophagy. Furthermore, we found that FgATG20 is necessary for the maturation of FgApe1, an indicator of the cytoplasm‐to‐vacuole targeting (Cvt) pathway. Immunoblot analysis displayed lower level of FgPex14, a peroxisomal integral membrane protein in ΔFgAtg20 mutants, suggesting that pexophagy is impaired. Furthermore, we demonstrate that FgAtg20 forms a complex with FgAtg1, FgAtg11, FgAtg17 and FgAtg24. When considered together, we conclude that FgAtg20 plays a critical role in vegetative growth, conidiation and pathogenicity of the head blight pathogen, and is involved in the Cvt pathway, non‐selective macroautophagy and pexophagy.     

PTPμ‐dependent growth cone rearrangement is regulated by Cdc42

PTPμ is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G‐proteins were involved in PTPμ‐dependent signaling. The stimulation of retinal cultures with purified PTPμ resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTPμ stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTPμ‐induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTPμ signaling, we utilized dominant‐negative mutants of Cdc42 and Rac. Dominant‐negative Cdc42 blocked PTPμ‐induced rearrangement, while wild‐type Cdc42 and dominant‐negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTPμ homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone. © 2003 Wiley Periodicals, Inc. J Neurobiol 56:199–208, 2003     

A Pmk1-interacting gene is involved in appressorium differentiation and plant infection in Magnaporthe oryzae

In the rice blast fungus Magnaporthe oryzae, the PMK1 mitogen-activated protein (MAP) kinase gene regulates appressorium formation and infectious growth. Its homologs in many other fungi also play critical roles in fungal development and pathogenicity. However, the targets of this important MAP kinase and its interacting genes are not well characterized. In this study, we constructed two yeast two-hybrid libraries of M. oryzae and screened for Pmk1-interacting proteins. Among the nine Pmk1-interacting clones (PICs) identified, two of them, PIC1 and PIC5, were selected for further characterization. Pic1 has one putative nuclear localization signal and one putative MAP kinase phosphorylation site. Pic5 contains one transmembrane domain and two functionally unknown CTNS (cystinosin/ERS1p repeat) motifs. The interaction of Pmk1 with Pic1 or Pic5 was confirmed by coimmunoprecipitation assays. Targeted gene deletion of PIC1 had no apparent effects on vegetative growth and pathogenicity but resulted in a significant reduction in conidiation and abnormal germ tube differentiation on onion epidermal cells. Deletion of PIC5 led to a reduction in conidiation and hyphal growth. Autolysis of aerial hyphae became visible in cultures older than 4 days. The pic5 mutant was defective in germ tube growth and appressorium differentiation. It was reduced in appressorial penetration and virulence on the plant. Both PIC1 and PIC5 are conserved in filamentous ascomycetes, but none of their orthologs have been functionally characterized. Our data indicate that PIC5 is a novel virulence factor involved in appressorium differentiation and pathogenesis in M. oryzae.     

The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development

The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.     

Identification of vib-1, a locus involved in vegetative incompatibility mediated by het-c in Neurospora crassa

A non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.     

Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell–cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell–cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and β-catenin, adherens junctional proteins, at the cell–cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell–cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell– cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell–cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell–cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell– cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell– cell adhesion.     

The effect of gene tubC on the vegetative growth of benomyl-resistant strains of Aspergillus nidulans

Abstract Two benomyl-resistant mutants, benD3 tubC41 and benD4 tubC42 , of Aspergillus nidulans were isolated after UV treatment. The tubC mutations permitted good conidiation of these strains in culture media containing benomyl and were responsible for increasing their benomyl resistance levels. This implies that β3-tubulin, a product of the tubC gene, in addition to being involved in fungal conidiation, participates in the vegetative growth of the fungus. The tubC gene was located in linkage group I.