Role of Fusarium Graminearum sRNAs in Fusarium Head Blight Pathogenesis on Wheat

Fusarium graminearum causes Fusarium head blight (FHB), a devastating disease on wheat. Mycotoxin deoxynivalenol (DON) acts as a virulent factor for establishment and spread of the disease, though not required for initial infection. DON biosynthesis is highly regulated by Tri genes present on three different clusters. Small RNAs are 20-30 nucleotide non-coding RNA molecules that regulate gene expression by target cleavage, translational repression or RNA directed DNA methylation. In this study, we explored a possible role of siRNAs in DON biosynthesis pathway. When DCL2 (DICER-LIKE 2), one of the major players in sRNA biogenesis, was knocked down through Inverse Repeat Transgene method, fg-siR34 biogenesis was down regulated in the dcl2- mutant compared to the wild-type. The expression assays revealed a significant down-regulation of DON biosynthesis genes Tri4, Tri5, Tri6, Tri10 and Tri14 in the dcl2- mutant, which shows that these genes are regulated by fg-siR34. Though the growth and morphology of the dcl2- mutant remain unaffected in vivo, it was highly affected in planta since the growth of the dcl2- mutant was very less compared to the wild-type. Wheat spikes inoculated with the dcl2- mutant also showed a significant loss of DON production due to the fg-siR34 knockdown. Therefore, our data shows that DON is an important virulent factor that is necessary to F. graminearum colonization in planta. These results indicate that DON biosynthesis may be regulated by siRNAs, though the mechanism of its regulation remains to be elucidated. Furthermore, fg-milR8 a milRNA from Fusarium was also found in Fusarium infected 260-2 and 260-4. As these Near Isogenic Lines differ only for QFhb1 that is known to harbor genes for FHB resistance, this might play a role in FHB pathogenesis. mRNA targets were found only in wheat genome but not Fusarium genome. One of the targets mapped to chromosome 3B and had a similarity with XS domain of Suppressor of Gene Silencing 3 (SGS3) protein. As SGS3 proteins confer resistance to incoming pathogens (viruses) by Post Transcriptional Gene Silencing (PTGS), Fusarium might target this protein by fg-milR8 to increase pathogenesis. Mechanism of target regulation is not clear as the cleavage site could not be identified through 5’RNA Ligase mediated Rapid amplification of cDNA ends (RLM-RACE) experiments. From these results we could conclude that sRNAs play an important role in FHB pathogenesis on wheat.