Virulence Of Plant Pathogenic Fungi Research Articles

Regulatory insight for a Zn2Cys6 transcription factor controlling effector-mediated virulence in a fungal pathogen of wheat.

The regulation of virulence in plant-pathogenic fungi has emerged as a key area of importance underlying host infections. Recent work has highlighted individual transcription factors (TFs) that serve important roles. A prominent example is PnPf2, a member of the Zn2Cys6 family of fungal TFs, which controls the expression of effectors and other virulence-associated genes in Parastagonospora nodorum during infection of wheat. PnPf2 orthologues are similarly important for other major fungal pathogens during infection of their respective host plants, and have also been shown to control polysaccharide metabolism in model saprophytes. In each case, the direct genomic targets and associated regulatory mechanisms were unknown. Significant insight was made here by investigating PnPf2 through chromatin-immunoprecipitation (ChIP) and mutagenesis approaches in P. nodorum. Two distinct binding motifs were characterised as positive regulatory elements and direct PnPf2 targets identified. These encompass known effectors and other components associated with the P. nodorum pathogenic lifestyle, such as carbohydrate-active enzymes and nutrient assimilators. The results support a direct involvement of PnPf2 in coordinating virulence on wheat. Other prominent PnPf2 targets included TF-encoding genes. While novel functions were observed for the TFs PnPro1, PnAda1, PnEbr1 and the carbon-catabolite repressor PnCreA, our investigation upheld PnPf2 as the predominant transcriptional regulator characterised in terms of direct and specific coordination of virulence on wheat, and provides important mechanistic insights that may be conserved for homologous TFs in other fungi.

The cell cycle, autophagy, and cell wall integrity pathway jointly governed by MoSwe1 in Magnaporthe oryzae

The cell cycle is pivotal to cellular differentiation in plant pathogenic fungi. Cell wall integrity (CWI) signaling plays an essential role in coping with cell wall stress. Autophagy is a degradation process in which cells decompose their components to recover macromolecules and provide energy under stress conditions. However, the specific association between cell cycle, autophagy and CWI pathway remains unclear in model pathogenic fungi Magnaporthe oryzae. Here, we have identified MoSwe1 as the conserved component of the cell cycle in the rice blast fungus. We have found that MoSwe1 targets MoMps1, a conserved critical MAP kinase of the CWI pathway, through protein phosphorylation that positively regulates CWI signaling. The CWI pathway is abnormal in the ΔMoswe1 mutant with cell cycle arrest. In addition, we provided evidence that MoSwe1 positively regulates autophagy by interacting with MoAtg17 and MoAtg18, the core autophagy proteins. Moreover, the S phase initiation was earlier, the morphology of conidia and appressoria was abnormal, and septum formation and glycogen degradation were impaired in the ΔMoswe1 mutant. Our research defines that MoSWE1 regulation of G1/S transition, CWI pathway, and autophagy supports its specific requirement for appressorium development and virulence in plant pathogenic fungi.4YLJWdgoLcEF_sT4NWF7zpVideo

A key sphingolipid pathway gene, MoDES1, regulates conidiation, virulence and plasma membrane tension in Magnaporthe oryzae

Rice blast, caused by the plant pathogenic fungus Magnaporthe oryzae, is a destructive disaster all over the earth that causes enormous losses in crop production. Sphingolipid, an important biological cell membrane lipid, is an essential structural component in the plasma membrane (PM) and has several biological functions, including cell mitosis, apoptosis, stress resistance, and signal transduction. Previous studies have suggested that sphingolipid and its derivatives play essential roles in the virulence of plant pathogenic fungi. However, the functions of sphingolipid biosynthesis-related proteins are not fully understood. In this article, we identified a key sphingolipid synthesis enzyme, MoDes1, and found that it is engaged in cell development and pathogenicity in M. oryzae. Deletion of MoDES1 gave rise to pleiotropic defects in vegetative growth, conidiation, plant penetration, and pathogenicity. MoDes1 is also required for lipid homeostasis and participates in the cell wall integrity (CWI) and Osm1-MAPK pathways. Notably, our results showed that there is negative feedback in the TORC2 signaling pathway to compensate for the decreased sphingolipid level due to the knockout of MoDES1 by regulating the phosphorylated Ypk1 level and PM tension. Furthermore, protein structure building has shown that MoDes1 is a potential drug target. These findings further refine the function of Des1 and deepen our understanding of the sphingolipid biosynthesis pathway in M. oryzae, laying a foundation for developing novel and specific drugs for rice blast control.

An auxiliary activity family 9 protein, VdAA91, is required for the penetration structure formation in Verticillium dahliae

Verticillium dahliae is a soil-borne fungal pathogen that can cause severe vascular wilt in many plant species; however, the role of some proteins secreted during infection needs to be explored. The fungal genome contains multiple auxiliary activity family 9 (AA9) genes that encode a conserved lytic polysaccharide monooxygenase domain, and some AA9 genes are required for the virulence of plant pathogenic fungi. To investigate the potential roles of AA9 genes, genome-wide identification and characterization were performed. A total of 20 AA9 genes were identified in the V. dahliae genome. According to a gene knockout experiment, VdAA91 is essential for the pathogenicity of V. dahliae. Furthermore, sequence analysis revealed that VdAA91 contains three conserved amino acid residues (His17, His114, and Tyr193) in the AA9 protein family. Subcellular localization and western blotting analyses revealed that VdAA91 is a secretory protein that is localized in the apoplast space of host cells. VdAA91 mutants showed defects in virulence and failures in root colonization based on observation of green fluorescent protein-labeled V. dahliae. We also observed penetration structure and its derived septin neck formation under microscopy. The VdAA91 mutants were incapable of producing penetration pegs and a VdSep5-GFP ring signal. Taken together, our results showed deletion of VdAA91 impairs the formation of the penetration structure and ultimately compromises V. dahliae colonization in the vascular bundles in the xylem of cotton.

Regulation of Autophagy Machinery in Magnaporthe oryzae.

Plant diseases cause substantial loss to crops all over the world, reducing the quality and quantity of agricultural goods significantly. One of the world’s most damaging plant diseases, rice blast poses a substantial threat to global food security. Magnaporthe oryzae causes rice blast disease, which challenges world food security by causing substantial damage in rice production annually. Autophagy is an evolutionarily conserved breakdown and recycling system in eukaryotes that regulate homeostasis, stress adaption, and programmed cell death. Recently, new studies found that the autophagy process plays a vital role in the pathogenicity of M. oryzae and the regulation mechanisms are gradually clarified. Here we present a brief summary of the recent advances, concentrating on the new findings of autophagy regulation mechanisms and summarize some autophagy-related techniques in rice blast fungus. This review will help readers to better understand the relationship between autophagy and the virulence of plant pathogenic fungi.

A Calcineurin Regulator MoRCN1 Is Important for Asexual Development, Stress Response, and Plant Infection of Magnaporthe oryzae.

The calcium/calcineurin signaling pathway plays a key role in the development and virulence of plant pathogenic fungi, but the regulation of this signaling pathway is still not clear. In this study, we identified a calcineurin regulator MoRCN1 in the plant pathogenic fungus Magnaporthe oryzae and found it is important for virulence by regulating the calcineurin pathway. MoRCN1 deletion mutants were severely decreased in colony growth and conidia formation. More importantly, the deletion of MoRCN1 led to a significant reduction in virulence due to defects in appressorium formation and invasive growth. The ΔMorcn1 mutants were more sensitive to different stresses and induced host ROS accumulation, suggesting a role of MoRCN1 in stress adaptation. We found that MoRCN1 directly interacted with the calcineurin catalytic subunit MoCNA and affected its protein stability, which was therefore important for regulating the calcineurin pathway. Transcriptome analysis showed that MoRCN1 significantly activated 491 genes and suppressed 337 genes in response to calcium ion, partially overlapped with the MoCRZ1-bound genes. Gene Ontology and KEGG pathway analyses indicated that MoRCN1-regulated genes were enriched in stress adaptation, lipid metabolism, and secondary metabolite biosynthesis, reflecting a function of MoRCN1 in host cell adaptation. Altogether, these results suggest MoRCN1 functions as a regulator of the calcium/calcineurin signaling pathway for fungal development and infection of host cells.

BcHnm1, a predicted choline transporter, modulates conidial germination and virulence in Botrytis cinerea

Previous network-based comparative genomic analysis between major lifestyles of fungal plant pathogens highlighted that HNM1, a predicted choline transporter, is part of the necrotroph core-genome's functions. In this work we have generated and characterized deletion mutants and developed complemented strains for the HNM1 homolog (Bchnm1) in the necrotrophic model fungal plant pathogen Botrytis cinerea. The Bchnm1 deletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of the Δbchnm1 deletion mutants was illustrated by reduced necrotic colonization of B. cinerea on tomato and French bean leaves. The role of BcHnm1 in germination was also supported by qRT-PCR results that illustrated increased Bchnm1 transcript levels during the early infection stages (at 16 h post inoculation) of the WT strain on tomato plant leaves, and during conidia germination (in-vitro). In line with the predicted function of BcHnm1 in choline transport, Δbchnm1 deletion mutant showed an attenuated choline import capacity. The potential role of choline in the WT B. cinerea was further demonstrated by an increase in conidia germination (by 100%) in the presence of 1 mM exogenous choline while growth in the presence of hemicholinium-3, an inhibitor of choline transporter, showed 40% inhibition in germination. In contrast to the WT, exogenous choline and the inhibitor did not affect conidia germination in the Δbchnm1 deletion mutants. Collectively, this study shows for the first time that BcHnm1, a predicted choline transporter, is important for conidial germination, germ tube elongation, response to exogenous choline, and virulence in plant pathogenic fungi.

The key iron assimilation genes ClFTR1, ClNPS6 were crucial for virulence of Curvularia lunata via initiating its appressorium formation and virulence factors.

Iron is virtually an essential nutrient for all organisms, to understand how iron contributes to virulence of plant pathogenic fungi, we identified ClFTR1 and ClNPS6 in maize pathogen Curvularia lunata (Cochliobolus lunatus) in this study. Disruption of ClNPS6 significantly impaired siderophore biosynthesis. ClFTR1 and ClNPS6 did mediate oxidative stress but had no significant impact on vegetative growth, conidiation, cell wall integrity and sexual reproduction. Conidial germination delayed and appressoria formation reduced in ΔClftr1 comparing with wild type (WT) CX-3. Genes responsible for conidial germination, appressoria formation, non-host selective toxin biosynthesis and cell wall degrading enzymes were also downregulated in the transcriptome of ΔClftr1 and ΔClnps6 compared with WT. The conidial development, toxin biosynthesis and polygalacturonase activity were impaired in the mutant strains with ClFTR1 and ClNPS6 deletion during their infection to maize. ClFTR1 and ClNPS6 were upregulated expression at 12-24 and 48-120 hpi in WT respectively. ClFTR1 positively regulated conidial germination, appressoria formation in the biotrophy-specific phase. ClNPS6 positively regulates non-host selective toxin biosynthesis and cell wall degrading enzyme activity in the necrotrophy-specific phase. Our results indicated that ClFTR1 and ClNPS6 were key genes of pathogen known to conidia development and virulence factors.

Effects of iron on the asexual reproduction and major virulence factors of Curvularia lunata

Curvularia lunata (Wakker) Boed, the causal agent of Curvularia leaf spot, is an important fungal pathogen in maize. Iron deficiency or excess plays a crucial role in mycelial growth and virulence of plant pathogenic fungi. In this study, the following pathogenic characteristics of C. lunata under different Fe3+ concentrations were investigated: mycelial growth, conidiation, conidium germination, virulence in maize, toxin and cell wall degrading enzymes (CWDEs) activity. The results showed that iron had a limited impact on the mycelial growth, while it had an obvious impact on mycelial biomass, conidiation, and the conidial germination at a concentration of 40–50 μM Fe3+. Exogenous supplementary of Fe3+ also significantly enhanced the pathogenicity of C. lunata CX-3 strain on maize inbred line Huangzaosi. However, toxin activity was independent of Fe3+ concentration. Exogenous iron application improved polygalacturonase (PG) activity in the early stages of infection, and the activity of cellulase (Cx), pectin polymethyl galacturonase (PMG), polygalacturonic acid transeliminase (PGTE) and pectin methyl transeliminase (PMTE) after 72 h of infection in maize.

New Insight Into Pathogenicity and Secondary Metabolism of the Plant Pathogen Penicillium expansum Through Deletion of the Epigenetic Reader SntB.

Penicillium expansum is one of the most harmful post-harvest pathogens of pomaceous fruits and the causal agent of blue rot disease. During infection, P. expansum produces the toxic secondary metabolites patulin and citrinin that can impact virulence and, further, render the fruit inedible. Several studies have shown that epigenetic machinery controls synthesis of secondary metabolites in fungi. In this regard, the epigenetic reader, SntB, has been reported to govern the production of multiple toxins in Aspergillus species, and impact virulence of plant pathogenic fungi. Here we show that deletion of sntB in P. expansum results in several phenotypic changes in the fungus including stunted vegetative growth, reduced conidiation, but enhanced germination rates as well as decreased virulence on Golden Delicious apples. In addition, a decrease in both patulin and citrinin biosynthesis in vitro and patulin in apples, was observed. SntB positively regulates expression of three global regulators of virulence and secondary metabolism (LaeA, CreA, and PacC) which may explain in part some of the phenotypic and virulence defects of the PeΔsntB strain. Lastly, results from this study revealed that the controlled environmental factors (low temperatures and high CO2 levels) to which P. expansum is commonly exposed during fruit storage, resulted in a significant reduction of sntB expression and consequent patulin and citrinin reduction. These data identify the epigenetic reader SntB as critical factor regulated in post-harvest pathogens under storage conditions and a potential target to control fungal colonization and decaying of stored fruit.

Phospholipid homeostasis plays an important role in fungal development, fungicide resistance and virulence in Fusarium graminearum

Phospholipids are major structural components of all cell membranes and participate in energy storage, signal transduction and environmental adaptability in eukaryotes. To date, the enzymes involved in phospholipid biosynthesis have been well characterized in budding yeast. However, their functions in filamentous fungi are largely unclear, especially their contribution to the interaction between phytopathogenic filamentous fungi and plants. In this study, we identified 10 phospholipid biosynthesis-related genes and genetically analyzed their functions in the Fusarium head blight pathogen Fusarium graminearum. The results of this study indicate that phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are critical for fungal vegetative growth. The biosynthesis of PE and PC is largely dependent on FgPsd2, FgCho2 and FgOpi3 in the de novo pathway of phospholipid biosynthesis in F. graminearum. Phospholipid biosynthetic gene mutants showed abnormal conidiation, increased sensitivity to fungicides and the oxidative stress agent H2O2, and defective endocytosis, especially the ΔFgpsd2, ΔFgcho2 and ΔFgopi3 mutants. Importantly, this study shows for the first time that the de novo pathway of phospholipid biosynthesis is required for mycotoxin production and full virulence in plant pathogenic fungi.

Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat.

Cross-kingdom RNAi is a well-documented phenomenon where sRNAs generated by host and pathogens may govern resistance or susceptible phenotypes during host-pathogen interaction. With the first example of the direct involvement of fungal generated sRNAs in virulence of plant pathogenic fungi Botrytis cinerea and recently from Puccinia striiformis f. sp. tritici, we attempted to identify sRNAs in Puccinia triticina (P. triticina). Four sRNA libraries were prepared and sequenced using Illumina sequencing technology and a total of ~ 1-1.28 million potential sRNAs and two microRNA-like small RNA (mil-RNAs) candidates were identified. Computational prediction of targets using a common set of sRNAs and P. triticina mil-RNAs (pt-mil-RNAs) within P. triticina and wheat revealed the majority of the targets as repetitive elements in P. triticina whereas in wheat, the target genes were identified to be involved in many biological processes including defense-related pathways. We found 9 receptor-like kinases (RLKs) and 14 target genes of each related to reactive oxygen species (ROS) pathway and transcription factors respectively, including significant numbers of target genes from various other categories. Expression analysis of twenty selected sRNAs, targeting host genes pertaining to ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors along with two pt-mil-RNAs by qRT-PCR showed distinct patterns of expression of the sRNAs in urediniospore-specific libraries. In this study, for the first time, we report identification of novel sRNAs identified in P. triticina including two pt-mil-RNAs that may play an important role in biotrophic growth and pathogenicity.

Hormetic Effects of Mixtures of Carbendazim and Iprodione on the Virulence of Botrytis cinerea.

Hormetic effects of fungicides on mycelial growth and virulence of plant pathogenic fungi have been reported, but the effects of fungicide mixtures on virulence hormesis of plant pathogens remain to be investigated. In this study, hormetic effects of mixtures of carbendazim and iprodione on the virulence of two carbendazim-resistant isolates of Botrytis cinerea were determined. Spraying carbendazim alone at 3 to 800 μg/ml exhibited hormetic effects on virulence to cucumber leaves, and carbendazim at 10 μg/ml had the maximum stimulation of 16.7% for isolate HBtom451. Spraying iprodione alone at 0.0001 to 0.0625 μg/ml exhibited hormetic effects on virulence, and iprodione at 0.025 μg/ml had the maximum stimulation of 18.7% for isolate HBtom451. However, spraying simultaneously carbendazim at 800 μg/ml and iprodione at 0.0625 μg/ml showed inhibitory effects on virulence to cucumber leaves. The mixture of carbendazim at 3 μg/ml and iprodione at 0.0001 μg/ml had much higher virulence stimulations than either fungicide at the same concentration alone. The maximum stimulation for the mixtures occurred at 10 and 0.0005 μg/ml for carbendazim and iprodione, respectively, and these concentrations were much lower than the concentration of their respective fungicide alone eliciting the maximum stimulations. The maximum stimulation amplitude for the mixture was slightly higher than that of each fungicide alone. These results demonstrated that carbendazim and iprodione mainly had dose-additive rather than amplitude-additive interactions when sprayed simultaneously with regard to virulence stimulations. Studies on virulence stimulations for mycelia treated with fungicide in potato dextrose agar showed that the maximum stimulation for the mixtures occurred at concentrations much lower than the concentration of carbendazim alone, indicating a dose-additive interaction when compared with carbendazim hormesis. Studies on potential physiological mechanisms of hormesis showed that increased tolerance to H2O2 may be one of the mechanisms for virulence hormesis for the mixtures of iprodione and carbendazim. These studies will advance our understanding of hormesis of fungicide mixtures.

The TOR signaling pathway regulates vegetative development and virulence in Fusarium graminearum

The target of rapamycin (TOR) signaling pathway plays critical roles in controlling cell growth in a variety of eukaryotes. However, the contribution of this pathway in regulating virulence of plant pathogenic fungi is unknown. We identified and characterized nine genes encoding components of the TOR pathway in Fusarium graminearum. Biological, genetic and biochemical functions of each component were investigated. The FgFkbp12-rapamycin complex binds to the FgTor kinase. The type 2A phosphatases FgPp2A, FgSit4 and FgPpg1 were found to interact with FgTap42, a downstream component of FgTor. Among these, we determined that FgPp2A is likely to be essential for F.graminearum survival, and FgSit4 and FgPpg1 play important roles in cell wall integrity by positively regulating the phosphorylation of FgMgv1, a key MAP kinase in the cell wall integrity pathway. In addition, the FgPpg1 interacting protein, FgTip41, is involved in regulating mycelial growth and virulence. Notably, FgTip41 does not interact with FgTap42 but with FgPpg1, suggesting the existence of FgTap42:FgPpg1:FgTip41 heterotrimer in F.graminearum, a complex not observed in the yeast model. Collectively, we defined a genetic regulatory framework that elucidates how the TOR pathway regulates virulence and vegetative development in F.graminearum.

Control of glucosylceramide production and morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum.

The contribution of plasma membrane proteins to the virulence of plant pathogenic fungi is poorly understood. Accordingly, the objective of this study was to characterize the acyl-CoA dependent ceramide synthase Bar1 (previously implicated in plasma membrane organization) in the wheat pathogen Fusarium graminearum. The role of Bar1 in mediating cell membrane organization was confirmed as ΔBAR1 mutants failed to display a distinct sterol-rich domain at the hyphal tip. The ΔBAR1 mutants were non-pathogenic when inoculated onto wheat heads, and their in vitro growth also was severely perturbed. ΔBAR1 mutants were incapable of producing perithecia (sexual fruiting structures) and only produced macroconidia (asexual spores) in the presence of NaCl. Sphingolipid analyses indicated that Bar1 is specifically necessary for the production of glucosylceramides in both F. graminearum and Aspergillus nidulans. Interestingly, glucosylceramides appear to mediate sensitivity to heat stable antifungal factor (HSAF), as, in addition to ΔBAR1 mutants, a glucosylceramide synthase deficient mutant of Yarrowia lipolytica is also resistant to HSAF.

A nitrogen response pathway regulates virulence in plant pathogenic fungi

Virulence in plant pathogenic fungi is controlled through a variety of cellular pathways in response to the host environment. Nitrogen limitation has been proposed to act as a key signal to trigger the in planta expression of virulence genes. Moreover, a conserved Pathogenicity mitogen activated protein kinase (MAPK) cascade is strictly required for plant infection in a wide range of pathogens. We investigated the relationship between nitrogen signaling and the Pathogenicity MAPK cascade in controlling infectious growth of the vascular wilt fungus Fusarium oxysporum. Several MAPK-activated virulence functions such as invasive growth, vegetative hyphal fusion and host adhesion were strongly repressed in the presence of the preferred nitrogen source ammonium. Repression of these functions by ammonium was abolished by L-Methionine sulfoximine (MSX) or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR (Target Of Rapamycin), respectively, and was dependent on the bZIP protein MeaB. Supplying tomato plants with ammonium rather than nitrate resulted in a significant delay of vascular wilt symptoms caused by the F. oxysporum wild type strain, but not by the ΔmeaB mutant. Ammonium also repressed invasive growth in two other pathogens, the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. Our results suggest the presence of a conserved nitrogen-responsive pathway that operates via TOR and MeaB to control infectious growth in plant pathogenic fungi.

A nitrogen response pathway regulates virulence functions in Fusarium oxysporum via the protein kinase TOR and the bZIP protein MeaB.

During infection, fungal pathogens activate virulence mechanisms, such as host adhesion, penetration and invasive growth. In the vascular wilt fungus Fusarium oxysporum, the mitogen-activated protein kinase Fmk1 is required for plant infection and controls processes such as cellophane penetration, vegetative hyphal fusion, or root adhesion. Here, we show that these virulence-related functions are repressed by the preferred nitrogen source ammonium and restored by treatment with l-methionine sulfoximine or rapamycin, two specific inhibitors of Gln synthetase and the protein kinase TOR, respectively. Deletion of the bZIP protein MeaB also resulted in nitrogen source-independent activation of virulence mechanisms. Activation of these functions did not require the global nitrogen regulator AreA, suggesting that MeaB-mediated repression of virulence functions does not act through inhibition of AreA. Tomato plants (Solanum lycopersicum) supplied with ammonium rather than nitrate showed a significant reduction in vascular wilt symptoms when infected with the wild type but not with the DeltameaB strain. Nitrogen source also affected invasive growth in the rice blast fungus Magnaporthe oryzae and the wheat head blight pathogen Fusarium graminearum. We propose that a conserved nitrogen-responsive pathway might operate via TOR and MeaB to control virulence in plant pathogenic fungi.