| In nature, plants abundantly form beneficial associations with microorganisms, which are important for plant survival and ecosystem functioning. Fungal endophytes can form and maintain a stable symbiotic system with plants without causing plant diseases. Dark septate endophytes (DSEs) are vital components of fungal endophytes and play an important role in plant resistance against biotic and abiotic stress. They colonied plants root and influence phytocoenosium. Harpophora oryzae, a beneficial DSE, was discovered in the wild-rice (Oryza granulata) roots, which strongly promotes rice growth and enhances resistance to pathogens or herbivores. The study in molecular mechanisms of symbiotic interaction can help us take advantage of this symbiotic system.Autophagy is one of the major cellular pathways for the degradation of long-lived proteins and cytoplasmic organisms, which has highly evolutionary conservation from yeast to human. Autophagy plays an important role in plant infection among many plant pathogenic fungi. We are interested in whether autophagy in H. oryzae plays an important role in rice colonization, which have not been studied before. In this study, based on the completed genome sequencing in H. oryzae, we discovered methods to study gene function in H. oryzae, and made a detail research in six autophagy-related genes. The main results acquired are listed below:1) Bioinformatics analysis of autophagy-related genesAutophagy genes and evolutionary relationships between 17 fungal species (including two kinds of Oomycetes) were identified by searching genome database using Blastp against S. cerevisiae and M. oryzae Atgs and Non-Atgs. Autophagy genes in H. oryzae were analyzed in detail.2) Methods of study genes function in H. oryzaeWe used target gene replacement to research genes function in H. oryzae. Optimal Agrobacterium tumefaciens-mediated transformation system was discovered. We successfully got six autophagy genes (HoATG1, HoATG5, HoATG7, HoATG8, HoATG12 and HoATG16) deletion mutants of H. oryzae through this transformation method.3) Six genes are involved in autophagy processSix mutants (△Hoatgl, △Hoatg5, △Hoatg7, AHoatg8, △Hoatgl2 and △Hoatgl6) were all sensitive to starvation. Autophagic body cannot be observed in six deletion mutants through microscopy and electron microscopy. Autophagic process was blocked when HoATG1, HoATG5, HoATG7, HoATG8, HoATG12 and HoATG16 were deleted.4) Conservation verification of various genes in H. oryzae, S. cerevisiae and M. oryzaeComplementation of the M. oryzae mutants (△Moatg5 and △Moatg8) by HoATG5 and HoATG8 gene indicate that these two genes were homologous to MoATG5 of M. oryzae in structure and function; The Y2H assays showed that HoAtg5 interacted with HoAtg3 and HoAtgl6, HoAtg7 interacted with HoAtg8 and HoAtg12, HoAtg8 interacted with HoAtgl. These results were consistent with that in S. cerevisiae, which indicated that two Ubl protein conjugation systems in H. oryzae were conserved.5) Three genes have nothing to do with mutualism system△Hoatg5, AHoatg7 and △Hoatg8 mutants were co-cultured with rice and showed no difference with wild-type, which indicated that these three genes(HoATG5, HoATG7 and HoATG8) have nothing to do with promoting rice growth. Rice roots colonized with △Hoatg5, AHoatg7 and AHoatg8 mutants were observed through microscopy, which showed that they all have the typical structure of infection such as dark septate hyphae, chlamydospore and septate germ tubes and appressorium-like bulges with corresponding infection pegs. This indicated that HoATG5, HoATG7 and HoATG8 cannot influence rice root colonization. |
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