Ss‐Rhs1, a secretory Rhs repeat‐containing protein,is required for the virulence of Sclerotinia sclerotiorum

Sclerotinia sclerotiorum is a devastating necrotrophic plant pathogen with a worldwide distribution. Cell wall‐degrading enzymes and oxalic acid are important to the virulence of this pathogen. Here, we report a novel secretory protein, Ss‐Rhs1, which is essential for the virulence of S. sclerotiorum. Ss‐Rhs1 is believed to contain a typical signal peptide at the N‐terminal and eight rearrangement hotspot (Rhs) repeats. Ss‐Rhs1 exhibited a high level of expression at the initial stage of sclerotial development, as well as during the hyphal infection process. Targeted silencing of Ss‐Rhs1 resulted in abnormal colony morphology and reduced virulence on host plants. Microscopic observations indicated that Ss‐Rhs1‐silenced strains exhibited reduced efficiency in compound appressoria formation.     

pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate‐minus mutants of Sclerotinia sclerotiorum

The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV‐induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate‐minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate‐minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate‐minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate‐minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases.     

An atypical forkhead‐containing transcription factor SsFKH1 is involved in sclerotial formation and is essential for pathogenicity in Sclerotinia sclerotiorum

Sclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic plant pathogen with a worldwide distribution. The sclerotia of S. sclerotiorum are pigmented multicellular structures formed from the aggregation of vegetative hyphae. These survival structures play a central role in the life and infection cycles of this pathogen. Here, we characterized an atypical forkhead (FKH)‐box‐containing protein, SsFKH1, involved in sclerotial development and virulence. To investigate the role of SsFkh1 in S. sclerotiorum, the partial sequence of SsFkh1 was cloned and RNA interference (RNAi)‐based gene silencing was employed to alter the expression of SsFkh1. RNA‐silenced mutants with significantly reduced SsFkh1 RNA levels exhibited slow hyphal growth and sclerotial developmental defects. In addition, the expression levels of a set of putative melanin biosynthesis‐related laccase genes and a polyketide synthase‐encoding gene were significantly down‐regulated in silenced strains. Disease assays demonstrated that pathogenicity in RNAi‐silenced strains was significantly compromised with the development of a smaller infection lesion on tomato leaves. Collectively, the results suggest that SsFkh1 is involved in hyphal growth, virulence and sclerotial formation in S. sclerotiorum.     

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.     

The autophagy‐related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea

Autophagy, a ubiquitous intracellular degradation process, is conserved from yeasts to humans. It serves as a major survival function during nutrient depletion stress and is crucial for correct growth and differentiation. In this study, we characterized an atg1 orthologue Bcatg1 in the necrotrophic plant pathogen Botrytis cinerea. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assays showed that the expression of BcATG1 was up‐regulated under carbon or nitrogen starvation conditions. BcATG1 could functionally restore the survival defects of the yeast ATG1 mutant during nitrogen starvation. Deletion of BcATG1 (ΔBcatg1) inhibited autophagosome accumulation in the vacuoles of nitrogen‐starved cells. ΔBcatg1 was dramatically impaired in vegetative growth, conidiation and sclerotial formation. In addition, most conidia of ΔBcatg1 lost the capacity to form the appressorium infection structure and failed to penetrate onion epidermis. Pathogenicity assays showed that the virulence of ΔBcatg1 on different host plant tissues was drastically impaired, which was consistent with its inability to form an appressorium. Moreover, lipid droplet accumulation was significantly reduced in the conidia of ΔBcatg1, but the glycerol content was increased. All of the defects of ΔBcatg1 were complemented by re‐introduction of an intact copy of the wild‐type BcATG1 into the mutant. These results indicate that BcATG1 plays a critical role in numerous developmental processes and is essential to the pathogenesis of B. cinerea.     

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.     

Susceptibility of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> strains different in oxalate production to infection by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g⁻¹ medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.     

Transcriptome analyses suggest a disturbance of iron homeostasis in soybean leaves during white mould disease establishment

Sclerotinia sclerotiorum is a serious pathogen of numerous crops around the world. The major virulence factor of this pathogen is oxalic acid (OA). Mutants that cannot produce OA do not cause disease, and plants that express enzymes that degrade OA, such as oxalate oxidase (OxO), are very resistant to S. sclerotiorum. To examine the effect of OA on plants, we infiltrated soybean leaves with 5 mm OA and examined the gene expression changes at 2 h post‐infiltration. By comparing the gene expression levels between leaves of a transgenic soybean carrying an OxO gene (OxO) and its parent AC Colibri (AC) infiltrated with OA (pH 2.4) or water (pH 2.4 or 5.5), we were able to compare the effects of OA dependent or independent of its pH. Gene expression by microarray analysis identified 2390 genes that showed changes in expression, as determined using an overall F‐test P‐value cut‐off of 0.001. The additional requirement that at least one pairwise t‐test false discovery rate (FDR)‐corrected P value should be less than 0.001 reduced the list of the most highly significant differentially expressed genes to 1054. Independent of pH, OA altered the expression levels of 78 genes, with ferritin showing the strongest induction by OA. The combination of OA plus its low pH caused 1045 genes (99% of all significant genes) to be differentially expressed, with many of the up‐regulated genes being related to basal defence, such as genes of the phenylpropanoid pathway and various cytochrome P450s. RNA‐seq was also conducted on four samples: OxO and AC genotypes infiltrated with either OA pH 2.4 or water pH 2.4. The RNA‐seq analysis also identified ferritin paralogues as being strongly induced by OA. As the expression of ferritin, a gene that encodes for an iron storage protein, is induced by free iron, these results suggest that S. sclerotiorum benefits from the ability of OA to free iron from plant proteins, as this induces host cell death, and also allows the uptake and assimilation of the iron for its own metabolic needs.     

The septins FaCdc3 and FaCdc12 are required for cytokinesis and affect asexual and sexual development,lipid metabolism and virulence in Fusarium asiaticum

Septins are a highly conserved family of GTP‐binding proteins that contribute to many cellular and metabolic functions, including cell polarity, cytokinesis, cell morphogenesis and pathogenesis. In this study, we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus Fusarium asiaticum. The functions of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12, designated ΔFaCdc3‐5 and ΔFaCdc12‐71, respectively. The deletion mutants exhibited a reduced rate of mycelial growth, increased aerial hyphae formation, irregularly shaped hyphae, reduced conidiation and a lack of sexual reproduction in wheat kernels. Histochemical analysis revealed that the conidia and hyphae of ΔFaCdc3‐5 and ΔFaCdc12‐71 formed large lipid droplets (LDs). ΔFaCdc3‐5 and ΔFaCdc12‐71 also exhibited increased resistance to agents that induce osmotic stress and damage the cell membrane and cell wall. In addition, the hyphae and conidia of the two mutants formed fewer septa than those of the wild‐type and exhibited aberrant nuclear distribution. Pathogenicity assays showed that ΔFaCdc3‐5 and ΔFaCdc12‐71 exhibited reduced virulence on wheat spikelets, which was indirectly correlated with a reduced level of deoxynivalenol accumulation. All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12. These results indicate that FaCdc3 and FaCdc12 play a critical role in various cellular processes in F. asiaticum.     

Degradation of oxalic acid by the mycoparasite Coniothyrium minitans plays an important role in interacting with Sclerotinia sclerotiorum

Coniothyrium minitans (Cm) is a mycoparasite of the phytopathogenic fungus Sclerotinia sclerotiorum (Ss). Ss produces a virulence factor oxalic acid (OA) which is toxic to plants and also to Cm, and Cm detoxifies OA by degradation. In this study, two oxalate decarboxylase genes, Cmoxdc1 and Cmoxdc2, were cloned from Cm strain Chy‐1. OA and low pH induced expression of Cmoxdc1, but not Cmoxdc2. Cmoxdc1 was partially responsible for OA degradation, whereas Cmoxdc2 had no effect on OA degradation. Disruption of Cmoxdc1 in Cm reduced its ability to infect Ss in dual cultures where OA accumulated. Compared with Chy‐1, the Cmoxdc1‐disrupted mutants had reduced expression levels of two mycoparasitism‐related genes chitinase (Cmch1) and β‐1,3‐glucanase (Cmg1), and had no detectable activity of extracellular proteases in the presence of OA. On the other hand, the cultural filtrates of the Cmoxdc1‐disrupted mutants in OA‐amended media showed enhanced antifungal activity, possibly because of increased production of antifungal substances under acidic pH condition resulted from reduced Cmoxdc1‐mediated OA degradation. This study provides direct genetic evidence of OA degradation regulating mycoparasitism and antibiosis of Cm against Ss, and sheds light on the sophisticated strategies of Cm in interacting with metabolically active mycelia and dormant sclerotia of Ss.     

The transmembrane protein Sho1 cooperates with the mucin Msb2 to regulate invasive growth and plant infection in Fusarium oxysporum

In the vascular wilt pathogen Fusarium oxysporum, the mitogen‐activated protein kinase (MAPK) Fmk1 is essential for plant infection. The mucin‐like membrane protein Msb2 regulates a subset of Fmk1‐dependent functions. Here, we examined the role of the tetraspan transmembrane protein Sho1 as an additional regulator of the Fmk1 pathway and determined its genetic interaction with Msb2. Targeted Δsho1 mutants were generated in wild‐type and Δmsb2 backgrounds to test possible interactions between the two genes. The mutants were examined for hyphal growth under different stress conditions, phosphorylation of the MAPK Fmk1 and an array of Fmk1‐dependent virulence functions. Similar to Msb2, Sho1 was required for the activation of Fmk1 phosphorylation, as well as Fmk1‐dependent gene expression and invasive growth functions, including extracellular pectinolytic activity, cellophane penetration, plant tissue colonization and virulence on tomato plants. Δsho1 mutants were hypersensitive to the cell wall‐perturbing compound Calcofluor White, and this phenotype was exacerbated in the Δmsb2 Δsho1 double mutant. These results highlight that Sho1 and Msb2 have partially overlapping functions upstream of the Fmk1 MAPK cascade, to promote invasive growth and plant infection, as well as cell wall integrity, in F. oxysporum.     

A two‐component histidine kinase Shk1 controls stress response,sclerotial formation and fungicide resistance in Sclerotinia sclerotiorum

Fungal histidine kinases (HKs) are involved in osmotic and oxidative stress responses, hyphal development, fungicide sensitivity and virulence. Members of HK class III are known to signal through the high‐osmolarity glycerol mitogen‐activated protein kinase (HOG MAPK). In this study, we characterized the Shk1 gene (SS1G_12694.3), which encodes a putative class III HK, from the plant pathogen Sclerotinia sclerotiorum. Disruption of Shk1 resulted in resistance to phenylpyrrole and dicarboximide fungicides and increased sensitivity to hyperosmotic stress and H₂O₂‐induced oxidative stress. The Shk1 mutant showed a significant reduction in vegetative hyphal growth and was unable to produce sclerotia. Quantitative real‐time polymerase chain reaction (qRT‐PCR and glycerol determination assays showed that the expression of SsHOG1 (the last kinase of the Hog pathway) and glycerol accumulation were regulated by the Shk1 gene, but PAK (p21‐activated kinase) was not. In addition, the Shk1 mutant showed no change in virulence. All the defects were restored by genetic complementation of the Shk1 deletion mutant with the wild‐type Shk1 gene. These findings indicate that Shk1 is involved in vegetative differentiation, sclerotial formation, glycerol accumulation and adaption to hyperosmotic and oxidative stresses, and to fungicides, in S. sclerotiorum. Taken together, our results demonstrate, for the first time, the role of two‐component HKs in Sclerotinia.     

Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase

Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β‐N‐oxalyl‐L ‐α,β‐diaminopropionic acid (β‐ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed‐specific expression of an oxalate‐degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β‐ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC‐expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate‐degrading enzyme.     

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.