Wenhui Zheng

A Homeobox Gene Is Essential for Conidiogenesis of the Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae starts its infection by the attachment of pyriform conidia on rice tissues, and severity of the disease epidemic is proportional to the quantity of conidia produced in the rice blast lesions. However, the mechanism of conidial production is not well understood. Homeodomain proteins play critical roles in regulating various growth and developmental processes in fungi and other eukaryotes. Through targeted gene replacement, we find that deletion of HTF1, one of seven homeobox genes in the fungal genome, does not affect mycelial growth but causes total defect of conidial production. Further observation revealed that the Deltahtf1 mutant produces significantly more conidiophores, which curve slightly near the tip but could not develop sterigmata-like structures. Although the Deltahtf1 mutant fails to form conidia, it could still develop melanized appressoria from hyphal tips and infect plants. The expression level of HTF1 is significantly reduced in the Deltamgb1 G-beta and DeltacpkA deletion mutant, and the ACR1 but not CON7 gene that encodes transcription factor required for normal conidiogenesis is significantly downregulated in the Deltahtf1 mutant. These data suggest that the HTF1 gene is essential for conidiogenesis, and may be functionally related to the trimeric G-protein signaling and other transcriptional regulators that are known to be important for conidiation in M. oryzae.

A Conserved Homeobox Transcription Factor Htf1 Is Required for Phialide Development and Conidiogenesis in Fusarium Species

Conidia are primary means of asexual reproduction and dispersal in a variety of pathogenic fungi, and it is widely recognized that they play a critical role in animal and plant disease epidemics. However, genetic mechanisms associated with conidiogenesis are complex and remain largely undefined in numerous pathogenic fungi. We previously showed that Htf1, a homeobox transcription factor, is required for conidiogenesis in the rice pathogen Magnaporthe oryzae. In this study, our aim was to characterize how Htf1 homolog regulates common and also distinctive conidiogenesis in three key Fusarium pathogens: F. graminearm, F. verticillioides, and F. oxysporum. When compared to wild-type progenitors, the gene-deletion mutants in Fusarium species failed to form conventional phialides. Rather, they formed clusters of aberrant phialides that resembled elongated hyphae segments, and it is conceivable that this led to the obstruction of conidiation in phialides. We also observed that mutants, as well as wild-type Fusaria, can initiate alternative macroconidia production directly from hyphae through budding-like mechanism albeit at low frequencies. Microscopic observations led us to conclude that proper basal cell division and subsequent foot cell development of macroconidia were negatively impacted in the mutants. In F. verticillioides and F. oxysporum, mutants exhibited a 2- to 5- microconidia complex at the apex of monophialides resulting in a floral petal-like shape. Also, prototypical microconidia chains were absent in F. verticillioides mutants. F. graminearum and F. verticillioides mutants were complemented by introducing its native HTF1 gene or homologs from other Fusarium species. These results suggest that Fusarium Htf1 is functionally conserved homeobox transcription factor that regulates phialide development and conidiogenesis via distinct signaling pathways yet to be characterized in fungi.

Retromer Is Essential for Autophagy-Dependent Plant Infection by the Rice Blast Fungus

The retromer mediates protein trafficking through recycling cargo from endosomes to the trans-Golgi network in eukaryotes. However, the role of such trafficking events during pathogen-host interaction remains unclear. Here, we report that the cargo-recognition complex (MoVps35, MoVps26 and MoVps29) of the retromer is essential for appressorium-mediated host penetration by Magnaporthe oryzae, the causal pathogen of the blast disease in rice. Loss of retromer function blocked glycogen distribution and turnover of lipid bodies, delayed nuclear degeneration and reduced turgor during appressorial development. Cytological observation revealed dynamic MoVps35-GFP foci co-localized with autophagy-related protein RFP-MoAtg8 at the periphery of autolysosomes. Furthermore, RFP-MoAtg8 interacted with MoVps35-GFP in vivo, RFP-MoAtg8 was mislocalized to the vacuole and failed to recycle from the autolysosome in the absence of the retromer function, leading to impaired biogenesis of autophagosomes. We therefore conclude that retromer is essential for autophagy-dependent plant infection by the rice blast fungus.

Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in Fusarium graminearum

Rab GTPases represent the largest subfamily of Ras-related small GTPases and regulate membrane trafficking. Vesicular transport is a general mechanism that governs intracellular membrane trafficking along the endocytic and exocytic pathways in all eukaryotic cells. Fusarium graminearum is a filamentous fungus and causes the devastating and economically important head blight of wheat and related species. The mechanism of vesicular transport is not well understood, and little is known about Rab GTPases in F. graminearum. In this study, we systematically characterized all eleven FgRabs by live cell imaging and genetic analysis. We find that FgRab51 and FgRab52 are important for the endocytosis, FgRab7 localizes to the vacuolar membrane and regulates the fusion of vacuoles and autophagosomes, and FgRab8 and FgRab11 are important for polarized growth and/or exocytosis. Furthermore, both endocytic and exocytic FgRabs are required for vegetative growth, conidiogenesis, sexual reproduction, as well as pathogenesis and deoxynivalenol metabolism in F. graminearum. Thus, we conclude that Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in F. graminearum.

AVR1-CO39 Is a Predominant Locus Governing the Broad Avirulence of Magnaporthe oryzae 2539 on Cultivated Rice (Oryza sativa L.)

Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.

Carbon Catabolite Repression in Filamentous Fungi

Carbon Catabolite Repression (CCR) has fascinated scientists and researchers around the globe for the past few decades. This important mechanism allows preferential utilization of an energy-efficient and readily available carbon source over relatively less easily accessible carbon sources. This mechanism helps microorganisms to obtain maximum amount of glucose in order to keep pace with their metabolism. Microorganisms assimilate glucose and highly favorable sugars before switching to less-favored sources of carbon such as organic acids and alcohols. In CCR of filamentous fungi, CreA acts as a transcription factor, which is regulated to some extent by ubiquitination. CreD-HulA ubiquitination ligase complex helps in CreA ubiquitination, while CreB-CreC deubiquitination (DUB) complex removes ubiquitin from CreA, which causes its activation. CCR of fungi also involves some very crucial elements such as Hexokinases, cAMP, Protein Kinase (PKA), Ras proteins, G protein-coupled receptor (GPCR), Adenylate cyclase, RcoA and SnfA. Thorough study of molecular mechanism of CCR is important for understanding growth, conidiation, virulence and survival of filamentous fungi. This review is a comprehensive revision of the regulation of CCR in filamentous fungi as well as an updated summary of key regulators, regulation of different CCR-dependent mechanisms and its impact on various physical characteristics of filamentous fungi.

Septins are involved in nuclear division, morphogenesis and pathogenicity in Fusarium graminearum

Septins are GTP-binding proteins that regulate cell polarity, cytokinesis and cell morphogenesis. Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating diseases worldwide. In this study, we have functionally characterized the core septins, Cdc3, Cdc10, Cdc11 and Cdc12 in F. graminearum. The loss of FgCdc3, FgCdc11, FgCdc12, but not FgCdc10, mutants showed significant reduction in growth, conidiation and virulence. Microscopic analyses revealed that all of them were involved in septum formation and nuclear division. Moreover, disruption of septin genes resulted in morphological defects in ascospores and conidia. Interestingly, conidia produced by ΔFgcdc3, ΔFgcdc11 and ΔFgcdc12 mutants exhibited deformation with interconnecting conidia in contrast to their parent wild-type strain PH-1 and the ΔFgcdc10 mutant that produced normal conidia. Using yeast two-hybrid assays, we determined the interactions among FgCdc3, FgCdc10, FgCdc11 and FgCdc12. Taken together, our results indicate that septins play important roles in the nuclear division, morphogenesis and pathogenicity in F. graminearum.

Updated Insight into the Physiological and Pathological Roles of the Retromer Complex

Retromer complexes mediate protein trafficking from the endosomes to the trans-Golgi network (TGN) or through direct recycling to the plasma membrane. In yeast, they consist of a conserved trimer of the cargo selective complex (CSC), Vps26–Vps35–Vps29 and a dimer of sorting nexins (SNXs), Vps5–Vps17. In mammals, the CSC interacts with different kinds of SNX proteins in addition to the mammalian homologues of Vps5 and Vps17, which further diversifies retromer functions. The retromer complex plays important roles in many cellular processes including restriction of invading pathogens. In this review, we summarize some recent developments in our understanding of the physiological and pathological functions of the retromer complex.

Endosomal sorting complexes required for transport-0 is essential for fungal development and pathogenicity in Fusarium graminearum

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The vacuolar protein sorting (Vps) protein Vps27 is a component of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway during endocytosis. In this study, we investigated the function of FgVps27 using a gene replacement strategy. The FgVPS27 deletion mutant (ΔFgvps27) exhibited a reduction in growth rate, aerial hyphae formation and hydrophobicity. It also showed increased sensitivity to cell wall-damaging agents and to osmotic stresses. In addition, FgHog1, the critical component of high osmolarity glycerol response pathway, was mis-localized in the ΔFgvps27 mutant upon NaCl treatment. Furthermore, the ΔFgvps27 mutant was defective in conidial production and was unable to generate perithecium in sexual reproduction. The depletion of FgVPS27 also caused a significant reduction in virulence. Further analysis by domain-specific deletion revealed that the FYVE domain was essential for the FgVps27 function and was necessary for the proper localization of FgVps27-GFP and endocytosis. Another component of ESCRT-0, the FgVps27-interacting partner FgHse1, also played an important role in F. graminearum development and pathogenesis. Overall, our results indicate that ESCRT-0 components play critical roles in a variety of cellular and biological processes.

ESCRT‐0 complex is required for fungal development and pathogenicity in Fusarium graminearum

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The vacuolar protein sorting (Vps) protein Vps27 is a component of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway during endocytosis. In this study, we investigated the function of FgVps27 using a gene replacement strategy. The FgVPS27 deletion mutant (ΔFgvps27) exhibited a reduction in growth rate, aerial hyphae formation and hydrophobicity. It also showed increased sensitivity to cell wall-damaging agents and to osmotic stresses. In addition, FgHog1, the critical component of high osmolarity glycerol response pathway, was mis-localized in the ΔFgvps27 mutant upon NaCl treatment. Furthermore, the ΔFgvps27 mutant was defective in conidial production and was unable to generate perithecium in sexual reproduction. The depletion of FgVPS27 also caused a significant reduction in virulence. Further analysis by domain-specific deletion revealed that the FYVE domain was essential for the FgVps27 function and was necessary for the proper localization of FgVps27-GFP and endocytosis. Another component of ESCRT-0, the FgVps27-interacting partner FgHse1, also played an important role in F. graminearum development and pathogenesis. Overall, our results indicate that ESCRT-0 components play critical roles in a variety of cellular and biological processes.