Preliminary Study On Molecular Mechanisms And Omics Of Lasiodiplodia Theobromae Infecting Grapevine

Grapevine Botryosphaeria dieback caused by species belonging to family Botryosphaeriaceae have become an economically important disease in grapevine growing countries worldwide in the last few decades.However,there are still lack of effective disease control measures.Species of this family are mostly known as opportunistic pathogens and there are few studies on their molecular infection mechanism.In the present study,we attempted to reveal the omics basis of Lasiodiplodia theobromae infecting grapevine and the preliminary molecular mechanism using genomic and transcriptional sequencing.This study will provide evidence for the pathogenesis of opportunistic plant pathogens which will contribute to develop new control strategies for grapevine Botryosphaeria dieback and for resistance breeding.To reveal the adaptation mechanism of Botiyosphaeriaceae to the grapevine,genomic and transcriptional sequencing were performed for three main Botryosphaeria dieback pathogens,Botryosphaeria dothidea,L.theobromae and Neofusicoccum parvum,with two isolates form each species.DNA sequencing results showed that the three Botryosphaeriaceae species have similar genome sizes(42.1Mb to 43.3Mb),and have less repetitive sequences(<5%).Phylogenetic analysis which included common phytopathogenic fungi,plant opportunistic pathogens and non-pathogenic fungi revealed that the species belonging to Botryosphaeriaceae are closely related to plant opportunistic fungi.Compared to the all other fungal genomes analysed in this study,five gene families which are related to cell wall degradation,nutrient absorption,secondary metabolism and membrane transport have significantly expanded in Botryosphaeriaceae species.We speculated that the expansion of these gene families facilitates the fungi in plant cell wall degradation,thereafter,complete its colonization and expansion,which ultimately resulted in disease symptom development.Furthermore,RNA-seq analysis was conducted with grapevine samples infected with L.theobromae,which is the most pathogenic species among these three species.The results showed that when L.theobromae infected grapes,the genes involved in carbohydrate catabolism,pectin,starch and sucrose metabolism,pentose and glucuronate conversion were up-regulated,suggesting that these pathways play an important role in the process of the pathogen infecting grapes.Based on the genomic and RNA-seq data,the author indicated that the cell wall degradation enzymes and secondary metabolites secreted by L.theobromae contributed to its infection process.RNA-seq analysis was conducted to profile the transcriptome of grapevine green shoots infected with L.theobromae over a time periods of 4,8 and 12 hr of post-infection.A total of 5181 genes were identified as differentially expressed genes(DEGs).GO annotation and KEGG enrichment analysis showed that more than half of the commonly shared DEGs were up-regulated and these DEGs were mainly involved in the primary and secondary metabolic pathways.Additionally,some pathways which are related to plant defence responses,such as Ca2+,MAPK.salicylic acid(SA)and Jasmonic acid(JA)signal transduction pathways and phenylpropanoid biosynthesis pathway were also enriched in grapevine after infection.Moreover,a total of 616 DEGs which were uniquely expressed in the grapevine during L.theobromae infection were identified,these genes may play important roles in grapevine-L.theobromae interaction.To further reveal the molecular mechanism of Botryosphaeriaceae infection based on the genomic and transcriptomic data,the author optimized the genetic transformation system of L.theobromae combined with the separation technology of the hyphal tip.A putative effector gene LtLysMl,which was up-regulated during L.theobromae infection,was selected for further analysis.Overexpressed transformants of LtLysMl were constructed.Pathogenicity results showed that the over expressed transformants were more virulent than the wild type strain.LtLysM1 can slightly inhibit the HR induced by B.glumae in N.benthamiana.Further,the protein secretion characteristic of LtLysM1 was proved in the yeast signal peptide trap system,indicated that LtLysM1 may function outside the fungal cells.In addition,we generated TO generation transgenic tobacco of LtLysMl,providing materials for a further study on the function of this gene.In conclusion,this study provided a comprehensive knowledge of the grapevine-L.theobromae interaction by using next generation sequencing.And the genetic transformation system of L.theobromae was optimized.In addition to that a putative effector protein was proved to have an important role in the infection process of L.theobromae.These results will provide useful evidence for analyzing the pathogenic mechanism of opportunistic pathogenic fungi,and then provide a new theoretical basis for the prevention and control of woody plant diseases caused by these pathogens.