Chenfang Wang

Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum.

As in other eukaryotes, protein kinases play major regulatory roles in filamentous fungi. Although the genomes of many plant pathogenic fungi have been sequenced, systematic characterization of their kinomes has not been reported. The wheat scab fungus Fusarium graminearum has 116 protein kinases (PK) genes. Although twenty of them appeared to be essential, we generated deletion mutants for the other 96 PK genes, including 12 orthologs of essential genes in yeast. All of the PK mutants were assayed for changes in 17 phenotypes, including growth, conidiation, pathogenesis, stress responses, and sexual reproduction. Overall, deletion of 64 PK genes resulted in at least one of the phenotypes examined, including three mutants blocked in conidiation and five mutants with increased tolerance to hyperosmotic stress. In total, 42 PK mutants were significantly reduced in virulence or non-pathogenic, including mutants deleted of key components of the cAMP signaling and three MAPK pathways. A number of these PK genes, including Fg03146 and Fg04770 that are unique to filamentous fungi, are dispensable for hyphal growth and likely encode novel fungal virulence factors. Ascospores play a critical role in the initiation of wheat scab. Twenty-six PK mutants were blocked in perithecia formation or aborted in ascosporogenesis. Additional 19 mutants were defective in ascospore release or morphology. Interestingly, F. graminearum contains two aurora kinase genes with distinct functions, which has not been reported in fungi. In addition, we used the interlog approach to predict the PK-PK and PK-protein interaction networks of F. graminearum. Several predicted interactions were verified with yeast two-hybrid or co-immunoprecipitation assays. To our knowledge, this is the first functional characterization of the kinome in plant pathogenic fungi. Protein kinase genes important for various aspects of growth, developmental, and infection processes in F. graminearum were identified in this study.

Multiple Plant Surface Signals are Sensed by Different Mechanisms in the Rice Blast Fungus for Appressorium Formation

Surface recognition and penetration are among the most critical plant infection processes in foliar pathogens. In Magnaporthe oryzae, the Pmk1 MAP kinase regulates appressorium formation and penetration. Its orthologs also are known to be required for various plant infection processes in other phytopathogenic fungi. Although a number of upstream components of this important pathway have been characterized, the upstream sensors for surface signals have not been well characterized. Pmk1 is orthologous to Kss1 in yeast that functions downstream from Msb2 and Sho1 for filamentous growth. Because of the conserved nature of the Pmk1 and Kss1 pathways and reduced expression of MoMSB2 in the pmk1 mutant, in this study we functionally characterized the MoMSB2 and MoSHO1 genes. Whereas the Momsb2 mutant was significantly reduced in appressorium formation and virulence, the Mosho1 mutant was only slightly reduced. The Mosho1 Momsb2 double mutant rarely formed appressoria on artificial hydrophobic surfaces, had a reduced Pmk1 phosphorylation level, and was nonresponsive to cutin monomers. However, it still formed appressoria and caused rare, restricted lesions on rice leaves. On artificial hydrophilic surfaces, leaf surface waxes and primary alcohols-but not paraffin waxes and alkanes- stimulated appressorium formation in the Mosho1 Momsb2 mutant, but more efficiently in the Momsb2 mutant. Furthermore, expression of a dominant active MST7 allele partially suppressed the defects of the Momsb2 mutant. These results indicate that, besides surface hydrophobicity and cutin monomers, primary alcohols, a major component of epicuticular leaf waxes in grasses, are recognized by M. oryzae as signals for appressorium formation. Our data also suggest that MoMsb2 and MoSho1 may have overlapping functions in recognizing various surface signals for Pmk1 activation and appressorium formation. While MoMsb2 is critical for sensing surface hydrophobicity and cutin monomers, MoSho1 may play a more important role in recognizing rice leaf waxes.

Different Chitin Synthase Genes Are Required for Various Developmental and Plant Infection Processes in the Rice Blast Fungus Magnaporthe oryzae

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.

A MADS-box transcription factor MoMcm1 is required for male fertility, microconidium production and virulence in Magnaporthe oryzae

Appressorium formation is a key step in the infection cycle of Magnaporthe oryzae. Mst12 is a transcription factor essential for appressorium penetration and invasive growth. In this study we used the affinity purification approach to identify proteins that physically associate with Mst12. One of the Mst12‐interacting genes identified was MoMCM1, which encodes a MADS‐box protein orthologous to yeast Mcm1. MoMcm1 interacted with both Mst12 and Mata‐1 in yeast two‐hybrid assays. Deletion of MoMCM1 resulted in the loss of male fertility and microconidium production. The Momcm1 mutant was defective in appressorium penetration and formed narrower invasive hyphae, which may be responsible for its reduced virulence. In transformants expressing MoMCM1–eGFP fusion, GFP signals were observed in the nucleus. We also generated the Momcm1 mst12 double mutant, which was defective in penetration and non‐pathogenic. On hydrophilic surfaces, germ tubes produced by the double mutant were severely curved, and 20% of them formed appressoria. In contrast, the Momcm1 or mst12 mutant did not form appressoria on hydrophilic surfaces. These results suggest that MoMCM1 and MST12 have overlapping functions to suppress appressorium formation under non‐conducive conditions. MoMcm1 may interact with Mst12 and MatA‐1 to regulate germ tube identity and male fertility respectively.

The FgHOG1 Pathway Regulates Hyphal Growth, Stress Responses, and Plant Infection in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a destructive disease of wheat and barley worldwide. In a previous study of systematic characterization of protein kinase genes in F. graminearum, mutants of three putative components of the osmoregulation MAP kinase pathway were found to have distinct colony morphology and hyphal growth defects on PDA plates. Because the osmoregulation pathway is not known to regulate aerial hyphal growth and branching, in this study we further characterized the functions of the FgHog1 pathway in growth, pathogenesis, and development. The Fghog1, Fgpbs2, and Fgssk2 mutants were all reduced in growth rate, aerial hyphal growth, and hyphal branching angle. These mutants were not only hypersensitive to osmotic stress but also had increased sensitivity to oxidative, cytoplasm membrane, and cell wall stresses. The activation of FgHog1 was blocked in the Fgpbs2 and Fgssk2 mutants, indicating the sequential activation of FgSsk2-FgPbs2-FgHog1 cascade. Interestingly, the FgHog1 MAPK pathway mutants appeared to be sensitive to certain compounds present in PDA. They were female sterile but retained male fertility. We also used the metabolomics profiling approach to identify compatible solutes that were accumulated in the wild type but not in the Fghog1 deletion mutant. Overall, our results indicate that the FgSsk2-FgPbs2-FgHog1 MAPK cascade is important for regulating hyphal growth, branching, plant infection, and hyperosmotic and general stress responses in F. graminearum.

A Novel Transcriptional Factor Important for Pathogenesis and Ascosporogenesis in Fusarium graminearum

Fusarium head blight or scab caused by Fusarium graminearum is an important disease of wheat and barley. The pathogen not only causes severe yield losses but also contaminates infested grains with mycotoxins. In a previous study, we identified several pathogenicity mutants by random insertional mutagenesis. One of these mutants was disrupted in the ZIF1 gene, which encodes a b-ZIP transcription factor unique to filamentous ascomycetes. The Δzif1 mutant generated by gene replacement was significantly reduced in deoxynivalenol (DON) production and virulence on flowering wheat heads. It was defective in spreading from inoculated florets to the rachis and other spikelets. Deletion of the ZIF1 ortholog MoZIF1 in the rice blast fungus also caused reductions in virulence and in invasive growth. In addition, the Δzif1 mutant is defective in sexual reproduction. Although it had normal male fertility, when selfed or mated as the female in outcrosess, the Δzif1 mutant produced small, pigmented perithecia that were sterile (lack of asci and ascospores), suggesting a female-specific role for ZIF1 during fertilization or ascus development. Similar female-specific defects in sexual reproduction were observed in the ΔMozif1 mutant. When mated as the female, the ΔMozif1 perithecia failed to develop long necks and asci or ascospores. The ZIF1 gene is well conserved in filamentous ascomycetes, particularly in the b-ZIP domain, which is essential for its function. Expression of ZIF1 in Magnaporthe oryzae complemented the defects of the ΔMozif1 mutant. These results indicate that this b-ZIP transcription factor is functionally conserved in these two fungal pathogens for plant infection and sexual reproduction.

The MAT Locus Genes Play Different Roles in Sexual Reproduction and Pathogenesis in Fusarium graminearum

Sexual reproduction plays a critical role in the infection cycle of Fusarium graminearum because ascospores are the primary inoculum. As a homothallic ascomycete, F. graminearum contains both the MAT1-1 and MAT1-2-1 loci in the genome. To better understand their functions and regulations in sexual reproduction and pathogenesis, in this study we assayed the expression, interactions, and mutant phenotypes of individual MAT locus genes. Whereas the expression of MAT1-1-1 and MAT12-1 rapidly increased after perithecial induction and began to decline after 1 day post-perithecial induction (dpi), the expression of MAT1-1-2 and MAT1-1-3 peaked at 4 dpi. MAT1-1-2 and MAT1-1-3 had a similar expression profile and likely are controlled by a bidirectional promoter. Although none of the MAT locus genes were essential for perithecium formation, all of them were required for ascosporogenesis in self-crosses. In outcrosses, the mat11-1-2 and mat11-1-3 mutants were fertile but the mat1-1-1 and mat1-2-1 mutants displayed male- and female-specific defects, respectively. The mat1-2-1 mutant was reduced in FgSO expression and hyphal fusion. Mat1-1-2 interacted with all other MAT locus transcription factors, suggesting that they may form a protein complex during sexual reproduction. Mat1-1-1 also interacted with FgMcm1, which may play a role in controlling cell identity and sexual development. Interestingly, the mat1-1-1 and mat1-2-1 mutants were reduced in virulence in corn stalk rot assays although none of the MAT locus genes was important for wheat infection. The MAT1-1-1 and MAT1-2-1 genes may play a host-specific role in colonization of corn stalks.

The HDF1 Histone Deacetylase Gene Is Important for Conidiation, Sexual Reproduction, and Pathogenesis in Fusarium graminearum

Head blight caused by Fusarium graminearum is an important disease of wheat and barley. Its genome contains chromosomal regions with higher genetic variation and enriched for genes expressed in planta, suggesting a role of chromatin modification in the regulation of infection-related genes. In a previous study, the FTL1 gene was characterized as a novel virulence factor in the head blight fungus. FTL1 is homologous to yeast SIF2, which is a component of the Set3 complex. Many members of the yeast Set3 complex, including Hos2 histone deacetylase (HDAC), are conserved in F. graminearum. In this study, we characterized the HDF1 gene that is orthologous to HOS2. HDF1 physically interacted with FTL1 in yeast two-hybrid assays. Deletion of HDF1 resulted in a significant reduction in virulence and deoxynivalenol (DON) production. The Δhdf1 mutant failed to spread from the inoculation site to other parts of wheat heads or corn stalks. It was defective in sexual reproduction and significantly reduced in conidiation. Expression of HDF1 was highest in conidia in comparison with germlings and hyphae. Deletion of HDF1 also resulted in a 60% reduction in HDAC activity. Microarray analysis revealed that 149 and 253 genes were down- and upregulated, respectively, over fivefold in the Δhdf1 mutant. Consistent with upregulation of putative catalase and peroxidase genes, the Δhdf1 mutant was more tolerant to H(2)O(2) than the wild type. Deletion of the other two class II HDAC genes had no obvious effect on vegetative growth and resulted in only a minor reduction in conidiation and virulence in the Δhdf2 mutant. Overall, our results indicate that HDF1 is the major class II HDAC gene in F. graminearum. It may interact with FTL1 and function as a component in a well-conserved HDAC complex in the regulation of conidiation, DON production, and pathogenesis.

FgSKN7 and FgATF1 have overlapping functions in ascosporogenesis, pathogenesis and stress responses in Fusarium graminearum

Fusarium head blight caused by Fusarium graminearum is one of the most destructive diseases of wheat and barley. Deoxynivalenol (DON) produced by the pathogen is an important mycotoxins and virulence factor. Because oxidative burst is a common defense response and reactive oxygen species (ROS) induces DON production, in this study, we characterized functional relationships of three stress-related transcription factor genes FgAP1, FgATF1 and FgSKN7. Although all of them played a role in tolerance to oxidative stress, deletion of FgAP1 or FgATF1 had no significant effect on DON production. In contrast, Fgskn7 mutants were reduced in DON production and defective in H2 O2 -induced TRI gene expression. The Fgap1 mutant had no detectable phenotype other than increased sensitivity to H2 O2 and Fgap1 Fgatf1 and Fgap1 Fgskn7 mutants lacked additional or more severe phenotypes than the single mutants. The Fgatf1, but not Fgskn7, mutant was significantly reduced in virulence and delayed in ascospore release. The Fgskn7 Fgatf1 double mutant had more severe defects in growth, conidiation and virulence than the Fgatf1 or Fgskn7 mutant. Instead of producing four-celled ascospores, it formed eight small, single-celled ascospores in each ascus. Therefore, FgSKN7 and FgATF1 must have overlapping functions in intracellular ROS signalling for growth, development and pathogenesis in F. graminearum.

Histological and cytological characterization of adult plant resistance to wheat stripe rust

AbstractWheat cultivar Xingzi 9104 (XZ) possesses adult plant resistance (APR) to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). In this study, histological and cytological experiments were conducted to elucidate the mechanisms of APR in XZ. The results of leaf inoculation experiments indicated that APR was initiated at the tillering stage, gradually increased as the plant aged and highly expressed after boot stage. The histology and oxidative burst in infected leaves of plants at seedling, tillering and boot stages were examined using light microscopic and histochemical methods. Subcellular changes in the host–pathogen interactions during the seedling and boot stages were analyzed by transmission electron microscopy. The results showed that haustorium formation was retarded in the adult plants and that the differentiation of secondary intercellular hyphae was significantly inhibited, which decreased the development of microcolonies in the adult plants, especially in plants of boot stage. The expression of APR to stipe rust during wheat development was clearly associated with extensive hypersensitive cell death of host cells and localized production of reactive oxygen species, which coincided with the restriction of fungal growth in infection sites in adult plants. At the same time, cell wall-related resistance in adult plants prevented ingression of haustorial mother cells into plant cells. Haustorium encasement was coincident with malformation or death of haustoria. The results provide useful information for further determination of mechanisms of wheat APR to stripe rust. Key message The expression of APR to stipe rust in wheat cultivar Xingzi 9104 (XZ) was clearly associated with extensive hypersensitive cell death of host cells and the localized production of reactive oxygen species.