Shijie Zhang

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.

High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

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.

Effectors and Effector Delivery in Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae is one of the most destructive fungal diseases of rice and a model for studying fungal-plant interactions. The fungus penetrates plant cells with appressoria and develops the narrow primary invasive hyphae (IH) and, subsequently, the bulbous secondary IH. As a hemibiotrophic pathogen, biotrophic IH are enclosed in the extra-invasive-hyphal membrane (EIHM) produced by the plant cells [1]. Like many other fungal pathogens [2], M. oryzae secretes effector proteins to manipulate plant immunity and physiology to promote infection.

Differences between appressoria formed by germ tubes and appressorium-like structures developed by hyphal tips in Magnaporthe oryzae.

Melanized appressoria are highly specialized infection structures formed by germ tubes of the rice blast fungus Magnaporthe oryzae for plant infection. M. oryzae also forms appressorium-like structures on hyphal tips. Whereas appressorium formation by conidial germ tubes has been well characterized, formation of appressorium-like structures by hyphal tips is under-investigated. In a previous study, we found that the chs7 deletion mutant failed to form appressoria on germ tubes but were normal in the development of appressorium-like structures on artificial hydrophobic surfaces. In this study, we compared the differences between the formation of appressoria by germ tubes and appressorium-like structures by hyphal tips in M. oryzae. Structurally, both appressoria and appressorium-like structures had a melanin layer that was absent in the pore region. In general, the latters were 1.4-fold larger in size but had lower turgor pressure than appressoria, which is consistent with its lower efficiency in plant penetration. Treatments with cAMP, IBMX, or a cutin monomer efficiently induced appressorium formation but not the development of appressorium-like structures. In contrast, coating surfaces with waxes stimulated the formation of both infection structures. Studies with various signaling mutants indicate that Osm1 and Mps1 are dispensable but Pmk1 is essential for both appressorium formation and development of appressorium-like structures on hyphal tips. Interestingly, the cpkA mutant was reduced in the differentiation of appressorium-like structures but not appressorium formation. We also observed that the con7 mutant generated in our lab failed to form appressorium-like structures on hyphal tips but still produced appressoria by germ tubes on hydrophobic surfaces. Con7 is a transcription factor regulating the expression of CHS7. Overall, these results indicate that the development of appressorium-like structures by hyphal tips and formation of appressoria by germ tubes are not identical differentiation processes in M. oryzae and may involve different molecular mechanisms.

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.

The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum.

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.

FgKin1 kinase localizes to the septal pore and plays a role in hyphal growth, ascospore germination, pathogenesis, and localization of Tub1 beta‐tubulins in Fusarium graminearum

The Kin1/Par-1/MARK kinases regulate various cellular processes in eukaryotic organisms. Kin1 orthologs are well conserved in fungal pathogens but none of them have been functionally characterized. Here, we show that KIN1 is important for pathogenesis and growth in two phytopathogenic fungi and that FgKin1 regulates ascospore germination and the localization of Tub1 β-tubulins in Fusarium graminearum. The Fgkin1 mutant and putative FgKIN1(S172A) kinase dead (nonactivatable) transformants were characterized for defects in plant infection, sexual and asexual reproduction, and stress responses. The localization of FgKin1 and two β-tubulins were examined in the wild-type and mutant backgrounds. Deletion of FgKIN1 resulted in reduced virulence and defects in ascospore germination and release. FgKin1 localized to the center of septal pores. FgKIN1 deletion had no effect on Tub2 microtubules but disrupted Tub1 localization. In the mutant, Tub1 appeared to be enriched in the nucleolus. In Magnaporthe oryzae, MoKin1 has similar functions in growth and infection and it also localizes to septal pores. The S172A mutation had no effect on the localization and function of FgKIN1 during sexual reproduction. These results indicate that FgKIN1 has kinase-dependent and independent functions and it specifically regulates Tub1 β-tubulins. FgKin1 plays a critical role in ascospore discharge, germination, and plant infection.

Thioredoxins are involved in the activation of the PMK1 MAP kinase pathway during appressorium penetration and invasive growth in Magnaporthe oryzae

In Magnaporthe oryzae, the Mst11-Mst7-Pmk1 MAP kinase pathway is essential for appressorium formation and invasive growth. To determine their roles in Pmk1 activation and plant infection, we characterized the two thioredoxin genes, TRX1 and TRX2, in M. oryzae. Whereas the Δtrx1 mutants had no detectable phenotypes, deletion of TRX2 caused pleiotropic defects in growth, conidiation, light sensing, responses to stresses and plant infection progresses. The Δtrx1 Δtrx2 double mutant had more severe defects than the Δtrx2 mutant and was non-pathogenic in infection assays. The Δtrx2 and Δtrx1 Δtrx2 mutant rarely formed appressoria on hyphal tips and were defective in invasive growth after penetration. Pmk1 phosphorylation was barely detectable in the Δtrx2 and Δtrx1 Δtrx2 mutants. Deletion of TRX2 affected proper folding or intra-/inter-molecular interaction of Mst7 and expression of the dominant active MST7 allele partially rescued the defects of the Δtrx1 Δtrx2 mutant. Furthermore, Cys305 is important for Mst7 function and Trx2 directly interacts with Mst7 in co-IP assays. Our data indicated that thioredoxins play important roles in intra-cellular ROS signalling and pathogenesis in M. oryzae. As the predominant thioredoxin gene, TRX2 may regulate the activation of Pmk1 MAPK via its effects on Mst7.