Identification Of Virulence Associated MilRNAs And Their Bidirectional Targets In Rhizoctonia Solani And Maize During Infection

Maize(Zea mays L.)is an important food crop,energy crop and forage crop in China,and its stable and high yield is very important to maintain food security.The banded leaf and sheath blight(BLSB)caused by Rhizoctonia solani is one of the most important fungal diseases affecting the yield of maize,so it is urgent to further study the sheath blight.MicroRNAs(miRNAs)are a class of non-coding endogenous smallRNAs with a length of21-25 bases,which play important regulatory roles in both animals and plants.Currently,a large number of microRNA-like smallRNAs(milRNAs)have been identified in fungi,and milRNAs have been shown to be directly involved in fungal-plant interactions.In this study,three smallRNA libraries were constructed,namely Maize leaf sheath(Maize),a normal culture of bacterial sheath(IA),and a mixed sample of bacterial sheath infected with Maize(IA-3d).Through high-throughput sequencing of smallRNA,milRNAs related to the virulence of Rhizoctonia solani AG1-IA were identified,and their interactions with Maize genes were analyzed.The following results were obtained:1.A total of 137 conserved milRNAs and 34 noval milRNAs were obtained through analysis of the sequencing results,and the reliability of the sequencing data was verified by Real-Time PCR.2.Through the Differential analysis of the expression levels of milRNAs between IA and IA-3d,17 conserved milRNAs and novel-milRNA-108 were found to be the virulence associated milRNAs.Among these milRNAs,seven conserved milRNAs were up-regulated in IA-3d compared with IA,while ten conserved milRNAs and novel-milRNA-108 were down-regulated.3.The target genes of 18 milRNAs related to the pathogenicity of sheath blight in maize and fungi were predicted,and their expression patterns were analyzed.Correlation analysis showed that some target genes were negatively correlated with the expression of milRNAs.In addition,the target genes were annotated functionally and the biological pathways involved in them were analyzed.4.We validated that the maize gene GRMZM2G412674 was decreased when cotransformed with Rhi-mil R-9829-5p utilizing a dual-luciferase assay and it was a positive regulator of BLSB resistance confirmed by the inoculation of R.solani in the EMS mutants.5.The cis-elements of the promoters of virulency-related milRNAs were analyzed,and the results showed that the expression of these milRNAs might be regulated by plant hormones such as ABA,GA,Me JA and transcription factors such as MYB.In this study,we identified differentially expressed milRNAs related to virulence of BLSB,analyzed the regulation of these milRNAs on maize target genes,verified the interaction between maize gene GRMZM2G412674 and Rhizoctonia solani Rhi-milRNA-9829-5P,and provided new evidence that Rhizoctonia solani may cause disease through its own milRNAs interaction with maize target genes.