Role of the cAMP signaling pathway in the dissemination and development on pepper fruit anthracnose disease caused by Colletotrichum scovillei.

The ascomycete fungus Colletotrichum scovillei causes severe anthracnose disease on the fruit of sweet pepper and chili pepper (Capsicum annuum L.) worldwide. Understanding the biology of C. scovillei would improve the management of fruit anthracnose diseases. The cyclic adenosine monophosphate (cAMP) signaling pathway regulates diverse cellular and physiological processes in several foliar fungal pathogens. We investigated the roles of the cAMP signaling pathway in C. scovillei using pharmaceutical and genetic approaches. Exogenous cAMP was found to increase conidiation, appressorium formation, and anthracnose disease development in C. scovillei. CsAc1, CsCap1, and CsPdeH, which regulate the intracellular cAMP level, were deleted by homology-dependent gene replacement. Expectedly, the intracellular cAMP level was significantly decreased in ΔCsac1 and ΔCscap1 but increased in ΔCspdeh. All three deletion mutants exhibited serious defects in multiple fungal developments and pathogenicity, suggesting regulation of the intracellular cAMP level is important for C. scovillei. Notably, exogenous cAMP recovered the defect of ΔCsac1 in appressorium development, but not penetration, which was further recovered by adding CaCl2. This result suggests that CsAc1 is associated with both the cAMP and Ca2+ signaling pathways in C. scovillei. ΔCscap1 produced morphologically abnormal conidia with reduced tolerance to thermal stress. ΔCspdeh was completely defective in conidiation in C. scovillei, unlike other foliar pathogens. Taken together, these results demonstrate the importance of cAMP signaling in anthracnose disease caused by C. scovillei.

MoRBP9 Encoding a Ran-Binding Protein Microtubule-Organizing Center Is Required for Asexual Reproduction and Infection in the Rice Blast Pathogen Magnaporthe oryzae.

Like many fungal pathogens, the conidium and appressorium play key roles during polycyclic dissemination and infection of Magnaporthe oryzae. Ran-binding protein microtubule-organizing center (RanBPM) is a highly conserved nucleocytoplasmic protein. In animalia, RanBPM has been implicated in apoptosis, cell morphology, and transcription. However, the functional roles of RanBPM, encoded by MGG_00753 (named MoRBP9) in M. oryzae, have not been elucidated. Here, the deletion mutant ΔMorbp9 for MoRBP9 was generated via homologous recombination to investigate the functions of this gene. The ΔMorbp9 exhibited normal conidial germination and vegetative growth but dramatically reduced conidiation compared with the wild type, suggesting that MoRBP9 is involved in conidial production. ΔMorbp9 conidia failed to produce appressoria on hydrophobic surfaces, whereas ΔMorbp9 still developed aberrantly shaped appressorium-like structures at hyphal tips on the same surface, suggesting that MoRBP9 is involved in the morphology of appressorium-like structures from hyphal tips and is critical for development of appressorium from germ tubes. Taken together, our results indicated that MoRBP9 played a pleiotropic role in polycyclic dissemination and infection-related morphogenesis of M. oryzae.

Optimization of Polyethylene Glycol-Mediated Transformation of the Pepper Anthracnose Pathogen Colletotrichum scovillei to Develop an Applied Genomics Approach.

Colletotrichum acutatum is a species complex responsible for anthracnose disease in a wide range of host plants. Strain C. acutatum KC05, which was previously isolated from an infected pepper in Gangwon Province of South Korea, was reidentified as C. scovillei using combined sequence analyses of multiple genes. As a prerequisite for understanding the pathogenic development of the pepper anthracnose pathogen, we optimized the transformation system of C. scovillei KC05. Protoplast generation from young hyphae of KC05 was optimal in an enzymatic digestion using a combined treatment of 2% lysing enzyme and 0.8% driselase in 1 M NH4Cl for 3 h incubation. Prolonged incubation for more than 3 h decreased protoplast yields. Protoplast growth of KC05 was completely inhibited for 4 days on regeneration media containing 200 µg/ml hygromycin B, indicating the viability of this antibiotic as a selection marker. To evaluate transformation efficiency, we tested polyethylene glycol-mediated protoplast transformation of KC05 using 19 different loci found throughout 10 (of 27) scaffolds, covering approximately 84.1% of the entire genome. PCR screening showed that the average transformation efficiency was about 17.1% per 100 colonies. Southern blot analyses revealed that at least one transformant per locus had single copy integration of PCR-screened positive transformants. Our results provide valuable information for a functional genomics approach to the pepper anthracnose pathogen C. scovillei.