The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae.

growth ehamber at 25°C (day) and 20°C (night) with a 14-h photoperiod. The isolates were eultured on PDA at 25°C for 7 days. Stems were wounded at 10 em height with a drill. Eaeh isolate was inoeulated to 25 replieates per eultivar by plaeing a myeelia agar plug (4 to 5 mm diameter) in the hole and wrapping the stem with Parafilm. Plants treated identieally with sterile agar plugs were used as eontrols. Plants were then maintained at 100% relative humidity for 2 h. Both isolates indueed diseases symptoms and death of seedlings of both cultivars at a mean time of 37.5 days after inoeulation. No signifieant differenees between isolates or between cultivars were deteeted. Twenty eontrol plants similarly treated with sterile PDA dises did not display symptoms. C. parasítica was re-isolated from lesions, eonfirming Koeh's postulates. Andalusia has 14,000 ha of chestnut crops with high eommereial value due to their preeoeity. Dispersion of ehestnut blight in this zone ean reduee crop produetivity. To our knowledge, this is the first report of C. parasítica eausing chestnut blight in Andalusia (southern Spain), one of the few areas left in southwestern Europe free of ehestnut blight. . Plant Dis. 98:284, 2014; published online as http://dx.

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Protein O-mannosylation is a type of O-glycosylation that is characterized by the addition of mannose residues to target proteins, and is initially catalyzed by evolutionarily conserved protein O-mannosyltransferases (PMTs). In this study, three members of PMT were identified in Magnaporthe oryzae, and the pathogenic roles of MoPmt2, a member of PMT2 subfamily, were analyzed. We found that MoPmt2 is a homolog of Saccharomyces cerevisiae Pmt2 and could complement yeast Pmt2 function in resistance to CFW. Quantitative RT–PCR revealed that MoPmt2 is highly expressed during conidiation, and targeted disruption of MoPmt2 resulted in defects in conidiation and conidia morphology. The MoPmt2 mutants also showed a distinct reduction in fungal growth, which was associated with severe alterations in hyphal polarity. In addition, we found that the MoPmt2 mutants severely reduced virulence on both rice plants and barley leaves. The subsequent examination revealed that the fungal adhesion, conidial germination, CWI and invasive hyphae growth in host cells are responsible for defects on appressorium mediated penetration, and thus attenuated the pathogenicity of MoPmt2 mutants. Taken together, our results suggest that protein O-mannosyltransferase MoPmt2 plays essential roles in fungal growth and development, and is required for the full pathogenicity of M. oryzae.

In fungi, O-mannosylation is one type of conserved protein modifications that add the carbohydrate residues to specific residues of target proteins by protein O-mannosyltransferases. Previously, three members of O-mannosyltransferases were identified in Magnaporthe oryzae, with MoPmt2 playing important roles in fungal growth and pathogenicity. However, the biological roles of the rest Pmt proteins remain unclear. In this study, to understand if O-mannosyltransferases are crucial for fungal pathogenicity of M. oryzae, the Pmt-coding genes MoPmt1 and MoPmt4 were separately disrupted and their roles in pathogenesis were analyzed. Of the two genes, only MoPmt4 is specifically required for full virulence of M. oryzae. Deletion of MoPmt4 resulted in defects on radial growth, with more branching hyphae and septa as compared to Guy11. The MoPmt4 mutant was severely impaired not only in conidiation, but also in both penetration and biotrophic invasion in susceptible rice plants. This mutant also had defects in suppression of host-derived ROS-mediated plant defense responses that might be ascribed from the reduced activities of extracellular enzymes. Furthermore, like their fungi counterparts, MoPmt4 localized in the ER and had O-mannosyltransferase activity. Domain disruption analysis indicated that mannosyltransferase activity regulated by PMT domain of MoPmt4 is crucial for fungal development and pathogenicity of M. oryzae. Taken together, these data suggest that MoPmt4 is a protein O-mannosyltransferase essential for fungal development and full virulence of M. oryzae.

The accurate manipulation of genomic integration of mutant alleles or fluorescent fusion-protein constructs is necessary for understanding of pathogenic mechanism of Magnaporthe oryzae. Recently, this can be achieved by integrating of exogenous DNA randomly into genome of this pathogen, but ectopic integration may result in alteration of gene expression or gene disruption due to unpredictable position effects and/or disruption of protein-coding regions. In this study, we establish a novel strategy for locus-specific integration of exogenous DNA via carboxin-resistance reconstitution by a point-mutation (H245L) on succinate dehydrogenase subunit Mosdi1. Independent transformants derived from the same reconstitution construct showed consistent fluorescent signal and undiversified phenotypes, including hyphae growth, conidiation and pathogenicity, in M. oryzae. Meanwhile, 96 % of all transformants integrate correctly into the Mosdi1 locus as a single copy. Furthermore, we provide a vector carrying yeast recombination cassette and thus allow assembly of multiple overlapping DNA fragments by yeast in vivo recombination for gene complementation and protein localization assay.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0232-x) contains supplementary material, which is available to authorized users.

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