Exploring The Potential Function Of Non-Coding RNA Of Fusarium Graminearum Based On RNA Sequencing

Fusarium graminearum is the main pathogen of wheat head blight,and it seriously endanger the wheat fields all over the world.At present,the prevention of head blight mainly rely on chemical pesticides,but the problem of drug resistance could not be ignored.Ascospores play an important role in the infection of wheat head blight.Indeed,the further studies on the development of F.graminearum ascospores and the regulators of discharge will provide new ideas or theories for the control measure of head blight.Non-coding RNA plays an important role in the growth and development of plants and animals.Dicer and Argonaute are widely existed in various organisms such as plants,animals,fungi,etc.,and are key factors in the processing of non-coding small RNAs.To explore the biological function of Dicer and Argonaute in F.graminearum,single knockout and double knockout mutants of Fgdcl and Fgago were obtained according to split-marker.At the asexual stage,no significant differences were observed between mutants and the wild-type stain PH-1 in mycelium growth,colony morphology,spore morphology,sporulation,pathogenicity,and abiotic stress.However,in the sexual stage,the numbers of mature and normal asci were reduced in ?Fgdcl1,?Fgdcl1/2,?Fgago2,and ?Fgago1/2.In addition,the ascospore discharge was decreased in ?Fgdcl1,and absolutely blocked in ?Fgdcl1/2,?Fgago2 and ?Fgago1/2.Indeed,comparing with the asexual stage,Fgdcl1 and Fgago2 have more important biological function in the sexual stage,and Fgdcl1 and Fgdcl2 genes were partly functionally redundant in biological function.To explore the mechanism of Fgdcl and Fgago affecting sexual reproduction,transcriptome sequencing and analysis of ?Fgdcl1,?Fgdcl1/2,?Fgago2 and ?Fgdcl2were performed in this study.Correlation analysis showed that ?Fgdcl1 have a strongly correlation with ?Fgdcl1/2,?Fgdcl1/2 and ?Fgago2,and the up-regulated genes in?Fgdcl1 were also induced in ?Fgdcl1/2 and ?Fgago2.The functional annotations of differentially expressed genes(DEGs)indicated that the down-regulated DEGs in?Fgdcl1,?Fgdcl1/2 and ?Fgago2 regulated the development of the asci,and up-regulated DEGs affected the release of ascospores by interfering with the transport of substances in the asci.In order to explore the potential function of small RNA in F.graminearum,we analyzed the mil RNA at different development stages of F.graminearum and found that there are many differences between mil RNA in sexual stage and asexual stage,including the number,location,preference for 5' first base and biogenesis pathway.During sexual phase,the prediction of mil RNA targets showed that the genes,which associated with ascospore dicharge were predicted to be candidate targets,and the expression of these candidate targets increased significantly in ?Fgdcl1/2 and ?Fgdcl1.On the other hand,in the sexual development stage,the exon-specific ex-si RNAs were similar to mil RNAs in sequence characteristics and synthetic pathways.Indeed,we speculated that sexual stage mil RNA and ex-si RNA co-regulate sexual development.In addition,we selected two mil RNAs that have high expression in asexual stages to explore their functions.However,compared with the wild type PH-1 strain,these two mil RNA deletion mutants showed no significant differences in mycelial growth,sporulation,pathogenicity,and perithecia.Meanwhile,the conidia-related lncRNAs in F.graminearum were predicted and analyzed in this study.The results showed that a majority of lncRNAs located on the upstream or downstream of gene.In this study,five lncRNAs and nine NATs,which have different expression before and after sporulation,were selected to verify by q RT-PCR,Furthermore,11 lncRNAs were knockout and preliminary functional study were performed on these mutatnts.The results confirmed that ?In R15174 may affect conidiation,perithecia development and ascospore discharge by negatively regulating the expression of adjacent genes;FG05658 collaborating with NAT05658 play a role in pigmentation,conidiation,virulence and sexual reproduction.This study laid the foundation for further study of the role and mechanism of non-coding RNA in F.graminearum asexual reproduction and sexual reproduction.