Comprehensive analysis of full genome sequence and Bd-milRNA/target mRNAs to discover the mechanism of hypovirulence in Botryosphaeria dothidea strains on pear infection with BdCV1 and BdPV1

Abstract

Pear ring rot disease, mainly caused by <i>Botryosphaeria dothidea</i>, is widespread in most pear and apple-growing regions. Mycoviruses are used for biocontrol, especially in fruit tree disease. BdCV1 (<i>Botryosphaeria dothidea</i> chrysovirus 1) and BdPV1 (<i>Botryosphaeria dothidea</i> partitivirus 1) influence the biological characteristics of <i>B. dothidea</i> strains. BdCV1 is a potential candidate for the control of fungal disease. Therefore, it is vital to explore interactions between <i>B. dothidea</i> and mycovirus to clarify the pathogenic mechanisms of <i>B. dothidea</i> and hypovirulence of <i>B. dothidea</i> in pear. A high-quality full-length genome sequence of the <i>B. dothidea</i> LW-Hubei isolate was obtained using Single Molecule Real-Time sequencing. It has high repeat sequence with 9.3% and DNA methylation existence in the genome. The 46.34 Mb genomes contained 14,091 predicted genes, which of 13,135 were annotated. <i>B. dothidea</i> was predicted to express 3833 secreted proteins. In bioinformatics analysis, 351 CAZy members, 552 transporters, 128 kinases, and 1096 proteins associated with plant-host interaction (PHI) were identified. RNA-silencing components including two endoribonuclease Dicer, four argonaute (Ago) and three RNA-dependent RNA polymerase (RdRp) molecules were identified and expressed in response to mycovirus infection<i>.</i> Horizontal transfer of the LW-C and LW-P strains indicated that BdCV1 induced host gene silencing in LW-C to suppress BdPV1 transmission. To investigate the role of RNA-silencing in <i>B. dothidea</i> defense, we constructed four small RNA libraries and sequenced <i>B. dothidea</i> micro-like RNAs (<i>Bd</i>-milRNAs) produced in response to BdCV1 and BdPV1 infection. Among these, 167 conserved and 68 candidate novel <i>Bd</i>-milRNAs were identified, of which 161 conserved and 20 novel <i>Bd</i>-milRNA were differentially expressed. WEGO analysis revealed involvement of the differentially expressed <i>Bd</i>-milRNA-targeted genes in metabolic process, catalytic activity, cell process and response to stress or stimulus. BdCV1 had a greater effect on the phenotype, virulence, conidiomata, vertical and horizontal transmission ability, and mycelia cellular structure biological characteristics of <i>B. dothidea</i> strains than BdPV1 and virus-free strains. The results obtained in this study indicate that mycovirus regulates biological processes in <i>B. dothidea</i> through the combined interaction of antiviral defense mediated by RNA-silencing and milRNA-mediated regulation of target gene mRNA expression.