Identification of virulence associated milRNAs and their bidirectional targets in Rhizoctonia solani and maize during infection

Abstract

Background Anastomosis group 1 IA (AG1-IA) of Rhizoctonia solani is the major agent of banded leaf and sheath blight (BLSB) disease that causes severe yield loss in many worldwide crops. MicroRNAs (miRNAs) are ~ 22 nt non-coding RNAs that negatively regulate gene expression levels by mRNA degradation or translation inhibition. A better understanding of miRNA function during AG1-IA infection can expedite to elucidate the molecular mechanisms of fungi-host interactions. Results In this study, we sequenced three small RNA libraries obtained from the mycelium of AG1-IA isolate, non-infected maize sheath and mixed maize sheath 3 days after inoculation. In total, 137 conserved and 34 novel microRNA-like small RNAs (milRNAs) were identified from the pathogen. Among these, one novel and 17 conserved milRNAs were identified as potential virulence-associated (VA) milRNAs. Subsequently, the prediction of target genes for these milRNAs was performed in both AG1-IA and maize, while functional annotation of these targets suggested a link to pathogenesis-related biological processes. Further, expression patterns of these virulence-associated milRNAs demonstrated that theyparticipate in the virulence of AG1-IA. Finally, regulation of one maize targeting gene, GRMZM2G412674 for Rhi-milRNA-9829-5p, was validated by dual-luciferase assay and identified to play a positive role in BLSB resistance in two maize mutants. These results suggest the global differentially expressed milRNAs of R. solani AG1-IA that participate in the regulation of target genes in both AG1-IA and maize to reinforce its pathogenicity. Conclusions Our data have provided a comprehensive overview of the VA-milRNAs of R. solani and identified that they are probably the virulence factors by directly interfered in host targeting genes. These results offer new insights on the molecular mechanisms of R.solani-maize interactions during the process of infection.

Genome-Wide Expression Profiling of Small RNAs in Indian Strain of Rhizoctonia solani AG1-1A Reveals Differential Regulation of milRNAs during Pathogenesis and Crosstalk of Gene Regulation

“…Interestingly, R. solani milRNAs showed their targets in the host (rice) genome also. A similar observation was made on R. solani targeting maize genes [ 25 ]. Recently, cross kingdom regulation of genes by miRNAs during host–pathogen interaction has been demonstrated in several pathosystems [ 41 ].…”

“…The novel milRNA scaffold13_22000 showed >6000 read count which emphasizes that several novel players identified in this study might regulate fungal genome. Earlier studies suggested that such novel milRNAs may have crucial roles in pathogenesis and virulence of R. solani [ 21 , 25 ]. 1725 target genes of 128 known and 22 novel milRNAs were identified that are involved in various metabolic and developmental processes of fungi indicating that milRNAs are major regulators of the R. solani genome.…”

“…The milRNAs-Rhi-milR-141, Rhi-milR-120, Rhi-milR-41, and Rhi-milR-91 target pectinesterase and pectate lyase domain-containing protein genes signifying their crucial role in plant cell wall degradation and pathogenesis. milRNAs of R. solani targeting carbohydrate active enzymes and other fungal genes including those involved in virulence and pathogen–host interaction were reported recently [ 25 ]. The genes encoding plant cell wall degrading enzymes of R. solani play significant role in disease initiation and establishment [ 4 ].…”

“…Other host target genes of fungal milRNAs included isoflavone reductase, OsFBX438-F-box domain containing protein, NB-ARC domain containing protein, serine/threonine-protein kinase receptor, and rabGAP/TBC domain-containing protein which are involved in plant defense against pathogens [ 50 , 51 , 52 , 53 , 54 ]. R. solani -encoded milRNAs target maize defensegenesduring infection and negatively regulate resistance of maize [ 25 ]. Notably, the regulons such as Osa-KANADI1:Rhi-milR-124, Osa-vacuolar-sorting receptor precursor:Rhi-milR-13, Osa-plastocyanin-like domain containing protein:Rhi-milR-22, and Osa-serine/threonine-protein kinase receptor precursor: Rhi-milR-51 were verified by analysing their expression through qRT-PCR during host–fungal interaction and disease development.…”

“…To date, very few milRNAs have been reported in plant pathogenic fungi, which could be due to the low accumulation of milRNAs or the small number of cells at certain infection stages, and also due to lesser attention of researchers. Recently, Meng et al (2021) [25] identified virulence-associated milRNAs from R. solani AG1-IA infecting maize crop. With this perspective, our current study focussed on discovering the milRNAs in R. solani causing sheath blight disease in rice.…”