The key gluconeogenic gene PCK1 is crucial for virulence of Botrytis cinerea via initiating its conidial germination and host penetration

The process of initiation of host invasion and survival of some foliar phytopathogenic fungi in the absence of external nutrients on host leaf surfaces remains obscure. Here, we demonstrate that gluconeogenesis plays an important role in the process and nutrient‐starvation adaptation before the pathogen host invasion. Deletion of phosphoenolpyruvate c arboxyk inase gene BcPCK1 in gluconeogenesis in Botrytis cinerea, the causative agent of grey mould, resulted in the failure of the ΔBcpck1 mutant conidia to germinate on hard and hydrophobic surface and penetrate host cells in the absence of glucose, reduction in conidiation and slow conidium germination in a nutrient‐rich medium. The wild‐type and ΔBcpck1 conidia germinate similarly in the presence of glucose (higher concentration) as the sole carbon source. Conidial glucose‐content should reach a threshold level to initiate germination and host penetration. Infection structure formation by the mutants displayed a glucose‐dependent fashion, which corresponded to the mutant virulence reduction. Exogenous glucose or complementation of BcPCK1 completely rescued all the developmental and virulence defects of the mutants. Our findings demonstrate that BcPCK1 plays a crucial role in B. cinerea pathogenic growth and virulence, and provide new insights into gluconeogenesis mediating pathogenesis of plant fungal pathogens via initiation of conidial germination and host penetration.     

Peroxisomal fission is induced during appressorium formation and is required for full virulence of the rice blast fungus

Peroxisomes are involved in various metabolic processes and are important for virulence in different pathogenic fungi. How peroxisomes rapidly emerge in the appressorium during fungal infection is poorly understood. Here, we describe a gene, PEF1, which can regulate peroxisome formation in the appressorium by controlling peroxisomal fission, and is required for plant infection in the rice blast fungus Magnaporthe oryzae. Targeted deletion of PEF1 resulted in a reduction in virulence and a delay in penetration and invasive growth in host cells. PEF1 was particularly expressed during appressorial development, and its encoding protein was co‐localized with peroxisomes during appressorial development. Compared with the massive vesicle‐shaped peroxisomes formed in the wild‐type appressorium, the Δpef1 mutant could only form stringy linked immature peroxisomes, suggesting that PEF1 was involved in peroxisomal fission during appressorium formation. We also found that the Δpef1 mutant could not utilize fatty acids efficiently, which can improve significantly the expression level of PEF1 and induce peroxisomal fission. As expected, the Δpef1 mutant showed reduced intracellular production of reactive oxygen species (ROS) during appressorium formation and induced ROS accumulation in host cells during infection. Taken together, PEF1‐mediated peroxisomal fission is important for fungal infection by controlling the number of peroxisomes in the appressorium.     

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (h yphal b ranching-related f actor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

The response regulator BcSkn7 is required for vegetative differentiation and adaptation to oxidative and osmotic stresses in Botrytis cinerea

The high‐osmolarity glycerol pathway plays an important role in the responses of fungi to various environmental stresses. Saccharomyces cerevisiae Skn7 is a response regulator in the high‐osmolarity glycerol pathway, which regulates the oxidative stress response, cell cycle and cell wall biosynthesis. In this study, we characterized an Skn7 orthologue BcSkn7 in Botrytis cinerea. BcSKN7 can partly restore the growth defects of S. cerevisiae SKN7 mutant and vice versa. The BcSKN7 mutant (ΔBcSkn7‐1) revealed increased sensitivity to ionic osmotic and oxidative stresses and to ergosterol biosynthesis inhibitors. In addition, ΔBcSkn7‐1 was also impaired dramatically in conidiation and sclerotial formation. Western blot analysis showed that BcSkn7 positively regulated the phosphorylation of BcSak1 (the orthologue of S. cerevisiae Hog1) under osmotic stress, indicating that BcSkn7 is associated with the high‐osmolarity glycerol pathway in B. cinerea. In contrast with BcSak1, BcSkn7 is not involved in the regulation of B. cinerea virulence. All of the phenotypic defects of ΔBcSkn7‐1 are restored by genetic complementation of the mutant with the wild‐type BcSKN7. The results of this study indicate that BcSkn7 plays an important role in the regulation of vegetative differentiation and in the response to various stresses in B. cinerea.     

MoWhi2 regulates appressorium formation and pathogenicity via the MoTor signalling pathway in Magnaporthe oryzae

Magnaporthe oryzae causes rice blast disease, which seriously threatens the safety of food production. Understanding the mechanism of appressorium formation, which is one of the key steps for successful infection by M. oryzae, is helpful to formulate effective control strategies of rice blast. In this study, we identified MoWhi2, the homolog of Saccharomyces cerevisiae Whi2 (Whisky2), as an important regulator that controls appressorium formation in M. oryzae. When MoWHI2 was disrupted, multiple appressoria were formed by one conidium and pathogenicity was significantly reduced. A putative phosphatase, MoPsr1, was identified to interact with MoWhi2 using a yeast two-hybridization screening assay. The knockout mutant ΔMopsr1 displayed similar phenotypes to the ΔMowhi2 strain. Both the ΔMowhi2 and ΔMopsr1 mutants could form appressoria on a hydrophilic surface with cAMP levels increasing in comparison with the wild type (WT). The conidia of ΔMowhi2 and ΔMopsr1 formed a single appressorium per conidium, similar to WT, when the target of rapamycin (TOR) inhibitor rapamycin was present. In addition, compared with WT, the expression levels of MoTOR and the MoTor signalling activation marker gene MoRS3 were increased, suggesting that inappropriate activation of the MoTor signalling pathway is one of the important reasons for the defects in appressorium formation in the ΔMowhi2 and ΔMopsr1 strains. Our results provide insights into MoWhi2 and MoPsr1-mediated appressorium development and pathogenicity by regulating cAMP levels and the activation of MoTor signalling in M. oryzae.     

The actin‐regulating kinase homologue MoArk1 plays a pleiotropic function in Magnaporthe oryzae

Endocytosis is an essential cellular process in eukaryotic cells that involves concordant functions of clathrin and adaptor proteins, various protein and lipid kinases, phosphatases and the actin cytoskeleton. In Saccharomyces cerevisiae, Ark1p is a member of the serine/threonine protein kinase (SPK) family that affects profoundly the organization of the cortical actin cytoskeleton. To study the function of MoArk1, an Ark1p homologue identified in Magnaporthe oryzae, we disrupted the MoARK1 gene and characterized the ΔMoark1 mutant strain. The ΔMoark1 mutant exhibited various defects ranging from mycelial growth and conidial formation to appressorium‐mediated host infection. The ΔMoark1 mutant also exhibited decreased appressorium turgor pressure and attenuated virulence on rice and barley. In addition, the ΔMoark1 mutant displayed defects in endocytosis and formation of the Spitzenkörper, and was hyposensitive to exogenous oxidative stress. Moreover, a MoArk1‐green fluorescent protein (MoArk1‐GFP) fusion protein showed an actin‐like localization pattern by localizing to the apical regions of hyphae. This pattern of localization appeared to be regulated by the N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins MoSec22 and MoVam7. Finally, detailed analysis revealed that the proline‐rich region within the MoArk1 serine/threonine kinase (S_TKc) domain was critical for endocytosis, subcellular localization and pathogenicity. These results collectively suggest that MoArk1 exhibits conserved functions in endocytosis and actin cytoskeleton organization, which may underlie growth, cell wall integrity and virulence of the fungus.     

Perilipin LDP1 coordinates lipid droplets formation and utilization for appressorium-mediated infection in Magnaporthe oryzae

Lipid droplets (LDs) serve as one of the major reservoirs in conidia of Magnaporthe oryzae and are quickly utilized during appressorium formation. Here, we identified a gene, LDP1, encoding a perilipin that is important for LD formation and utilization during appressorium maturation. LDP1 is highly expressed in conidium and immature appressorium. Disruption mutants of LDP1 were significantly reduced in virulence, due to appressorial turgor reduction and difficulty in penetration. LDs were significantly reduced in the Δldp1 mutant, indicating LDP1 was required for LDs formation. LDP1 was colocalized with the LDs in conidium and immature appressorium but was gradually separated during appressorium maturation. A typical intracellular triacylglycerol lipase, TGL1-2, was clearly separated with LDs in conidium and immature appressorium but was well colocalized with LDs during appressorium maturation. The subcellular localization of TGL1-2 was affected by LDP1. These data suggested that LDP1 was bound to LDs for protecting from utilization in conidia and at the early appressorium stage but was separated from LDs for lipase entering and degradation. LDP1 was phosphorylated by CPKA at Thr96, which was essential for its localization and functions. These data indicate perilipin LDP1 can coordinate LD formation and utilization for appressorium-mediated infection of M. oryzae.     

Involvement of FgERG4 in ergosterol biosynthesis,vegetative differentiation and virulence in Fusarium graminearum

The ergosterol biosynthesis pathway is well understood in Saccharomyces cerevisiae, but currently little is known about the pathway in plant‐pathogenic fungi. In this study, we characterized the Fusarium graminearum FgERG4 gene encoding sterol C‐24 reductase, which catalyses the conversion of ergosta‐5,7,22,24‐tetraenol to ergosterol in the final step of ergosterol biosynthesis. The FgERG4 deletion mutant ΔFgErg4‐2 failed to synthesize ergosterol. The mutant exhibited a significant decrease in mycelial growth and conidiation, and produced abnormal conidia. In addition, the mutant showed increased sensitivity to metal cations and to various cell stresses. Surprisingly, mycelia of ΔFgErg4‐2 revealed increased resistance to cell wall‐degrading enzymes. Fungicide sensitivity tests revealed that ΔFgErg4‐2 showed increased resistance to various sterol biosynthesis inhibitors (SBIs), which is consistent with the over‐expression of SBI target genes in the mutant. ΔFgErg4‐2 was impaired dramatically in virulence, although it was able to successfully colonize flowering wheat head and tomato, which is in agreement with the observation that the mutant produces a significantly lower level of trichothecene mycotoxins than does the wild‐type progenitor. All of these phenotypic defects of ΔFgErg4‐2 were complemented by the reintroduction of a full‐length FgERG4 gene. In addition, FgERG4 partially rescued the defect of ergosterol biosynthesis in the Saccharomyces cerevisiae ERG4 deletion mutant. Taken together, the results of this study indicate that FgERG4 plays a crucial role in ergosterol biosynthesis, vegetative differentiation and virulence in the filamentous fungus F. graminearum.     

Endocytic protein Pal1 regulates appressorium formation and is required for full virulence of Magnaporthe oryzae

Endocytosis plays key roles during infection of plant-pathogenic fungi, but its regulatory mechanisms are still largely unknown. Here, we identified a putative endocytosis-related gene, PAL1, which was highly expressed in appressorium of Magnaporthe oryzae, and was found to be important for appressorium formation and maturation. Deletion of PAL1 significantly reduced the virulence of M. oryzae due to defects in appressorial penetration and invasive growth in host cells. The Pal1 protein interacted and colocalized with the endocytosis protein Sla1, suggesting it is involved in endocytosis. The Δpal1 mutant was significantly reduced in appressorium formation, which was recovered by adding exogenous cAMP and 3-isobutyl-1-methylxanthine (IBMX). Moreover, the phosphorylation level of Pmk1 in Δpal1 was also reduced, suggesting Pal1 functions upstream of both the cAMP and Pmk1 signalling pathways. As a consequence, the utilization of glycogen and lipid, appressorial autophagy, actin ring formation, localization of septin proteins, as well as turgor accumulation were all affected in the Δpal1 mutant. Taken together, Pal1 regulates cAMP and the Pmk1 signalling pathway for appressorium formation and maturation to facilitate infection of M. oryzae.     

The farnesyltransferase β-subunit RAM1 regulates localization of RAS proteins and appressorium-mediated infection in Magnaporthe oryzae

Post-translational farnesylation can regulate subcellular localization and protein–protein interaction in eukaryotes. The function of farnesylation is not well identified in plant pathogenic fungi, particularly during the process of fungal infection. Here, through functional analyses of the farnesyltransferase β-subunit gene, RAM1, we examine the importance of protein farnesylation in the rice blast fungus Magnaporthe oryzae. Targeted disruption of RAM1 resulted in the reduction of hyphal growth and sporulation, and an increase in the sensitivity to various stresses. Importantly, loss of RAM1 also led to the attenuation of virulence on the plant host, characterized by decreased appressorium formation and invasive growth. Interestingly, the defect in appressoria formation of the Δram1 mutant can be recovered by adding exogenous cAMP and IBMX, suggesting that RAM1 functions upstream of the cAMP signalling pathway. We found that two Ras GTPases, RAS1 and RAS2, can interact with Ram1, and their plasma membrane localization was regulated by Ram1 through their C-terminal farnesylation sites. Adding a farnesyltransferase inhibitor Tipifarnib can result in similar defects as in Δram1 mutant, including decreased appressorium formation and invasive growth, as well as mislocalized RAS proteins. Our findings indicate that protein farnesylation regulates the RAS protein-mediated signaling pathways required for appressorium formation and host infection, and suggest that abolishing farnesyltransferase could be an effective strategy for disease control.     

Deubiquitinase Ubp3 regulates ribophagy and deubiquitinates Smo1 for appressorium-mediated infection by Magnaporthe oryzae

The Ubp family of deubiquitinating enzymes has been found to play important roles in plant-pathogenic fungi, but their regulatory mechanisms are still largely unknown. In this study, we revealed the regulatory mechanism of the deubiquitinating enzyme Ubp3 during the infection process of Magnaporthe oryzae. AUBP3 deletion mutant was severely defective in appressorium turgor accumulation, leading to the impairment of appressorial penetration. During appressorium formation, the mutant was also defective in glycogen and lipid metabolism. Interestingly, we found that nitrogen starvation and rapamycin treatment induced the ribophagy process in M. oryzae, which is closely dependent on Ubp3. In the ∆ubp3 mutant, the ribosome proteins and rRNAs were not well degraded on nitrogen starvation and rapamycin treatment. We also found that Ubp3 interacted with the GTPase-activating protein Smo1 and regulated its de-ubiquitination. Ubp3-dependent de-ubiquitination of Smo1 may be required for Smo1 to coordinate Ras signalling. Taken together, our results showed at least two roles of Ubp3 in M. oryzae: it regulates the ribophagy process and it regulates de-ubiquitination of GTPase-activating protein Smo1 for appressorium-mediated infection.     

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.     

MoOpy2 is essential for fungal development,pathogenicity, and autophagy in Magnaporthe oryzae

The development and pathogenicity of the fungus Magnaporthe oryzae, the causal agent of destructive rice blast disease, require it to perceive external environmental signals. Opy2, an overproduction-induced pheromone-resistant protein 2, is a crucial protein for sensing external signals in Saccharomyces cerevisiae. However, the biological functions of the homologue of Opy2 in M. oryzae are unclear. In this study, we identified that MoOPY2 is involved in fungal development, pathogenicity, and autophagy in M. oryzae. Deletion of MoOPY2 resulted in pleiotropic defects in hyphal growth, conidiation, germ tube extension, appressorium formation, appressorium turgor generation, and invasive growth, therefore leading to attenuated pathogenicity. Furthermore, MoOpy2 participates in the Osm1 MAPK pathway and the Mps1 MAPK pathway by interacting with the adaptor protein Mst50. The interaction sites of Mst50 and MoOpy2 colocalized with the autophagic marker protein MoAtg8 in the preautophagosomal structure sites (PAS). Notably, the ΔMoopy2 mutant caused cumulative MoAtg8 lipidation and rapid GFP-MoAtg8 degradation in response to nitrogen starvation, showing that MoOpy2 is involved in the negative regulation of autophagy activity. Taken together, our study revealed that MoOpy2 of M. oryzae plays an essential role in the orchestration of fungal development, appressorium penetration, autophagy and pathogenesis.     

Infection Process of Botrytis cinerea on Eucalypt Leaves

This study aimed to elucidate the infection process of Botrytis cinerea on eucalypt leaves. Tests were conducted to evaluate the influence of leaf side (adaxial or abaxial), leaf age and luminosity on conidial germination, appressorium formation and grey mould (GM) severity. The adaxial and abaxial surfaces of detached eucalypt leaves were inoculated with a conidial suspension of B. cinerea and kept under constant light or dark. Subsequently, the adaxial surface of young and old leaves was inoculated and kept in the dark. To evaluate the percentage of conidia germination and appressorium formation, leaf samples were collected 6 hours after inoculation (hai), clarified (alcohol and chloral hydrate) and evaluated under a light microscope. The severity of GM was assessed 10 days after inoculation. For scanning electron microscopy analysis, samples were collected from 2 to 168 hai. A higher percentage of conidia germination (92%) and GM severity (21%) occurred on the adaxial surfaces of leaves kept in the dark. There was no statistical difference between the surfaces of young and old leaves for conidia germination. No appressorium was formed by B. cinerea. The GM severity on young leaves (17.3%) was 34 times higher than on old leaves (0.5%). The micrographs showed germinating conidia emitting 1–4 germ tubes in samples at 4 hai. The fungus penetration occurred through intact leaf surfaces, and both extra‐ and intracellular colonization of the mesophyll cells by the hyphae of the pathogen were observed at 120 hai. Sporulation occurred on the adaxial and abaxial surfaces (macronematous conidiophores) and below the epidermis (micronematous conidiophores).     

Involvement of a putative response regulator Brrg-1 in the regulation of sporulation,sensitivity to fungicides,and osmotic stress in <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

The response regulator protein is a core element of two-component signaling pathway. In this study, we investigated functions of BRRG-1 of Botrytis cinerea, a gene that encodes a putative response regulator protein, which is homologous to Rrg-1 in Neurospora crassa. The BRRG-1 gene deletion mutant ΔBrrg1-62 was unable to produce conidia. The mutant showed increased sensitivity to osmotic stress mediated by NaCl and KCl, and to oxidative stress generated by H₂O₂. Additionally, the mutant was more sensitive to the fungicides iprodione, fludioxonil, and triadimefon than the parental strain. Western-blot analysis showed that the Bos-2 protein, the putative downstream component of Brrg-1, was not phosphorylated in the ΔBrrg1-62. Real-time polymerase chain reaction assays showed that expression of BOS-2 also decreased significantly in the mutant. All of the defects were restored by genetic complementation of the ΔBrrg1-62 with the wild-type BRRG-1 gene. Plant inoculation tests showed that the mutant did not show changes in pathogenicity on rapeseed leaves. These results indicated that Brrg-1 is involved in the regulation of asexual development, sensitivity to iprodione, fludioxonil, and triadimefon fungicides, and adaptation to osmotic and oxidative stresses in B. cinerea.     

Involvement of BcStr2 in methionine biosynthesis,vegetative differentiation,multiple stress tolerance and virulence in Botrytis cinerea

The Str2 gene encodes a cystathionine γ‐synthase that is a key enzyme in methionine (Met) biosynthesis in Saccharomyces cerevisiae. Met plays a critical role in protein synthesis and diverse cellular processes in both eukaryotes and prokaryotes. In this study, we characterized the Str2 orthologue gene BcStr2 in Botrytis cinerea. The BcStr2 mutant was unable to grow on minimal medium (MM). In addition, conidia of the mutant were unable to germinate in water–agar medium within 15 h of incubation. Supplementation with 1 mm Met or 0.5 mg/mL homocysteine, but not 1 mm cysteine or 0.5 mg/mL glutathione, rescued the defect in mycelial growth of the BcStr2 deletion mutant. These results indicate that the enzyme encoded by BcStr2 is involved in the conversion of cysteine into homocysteine. The mutant exhibited decreased conidiation and impaired sclerotium development. In addition, the BcStr2 mutant exhibited increased sensitivity to osmotic and oxidative stresses, cell wall‐damaging agents and thermal stress. The mutant demonstrated dramatically decreased virulence on host plant tissues. All of the defects were restored by genetic complementation of the mutant with wild‐type BcStr2. Taken together, the results of this study indicate that BcStr2 plays a critical role in the regulation of various cellular processes in B. cinerea.