Characterization and gene expression analysis of dicer-like genes of Fusarium oxysporum f. sp. cubense tropical race 4 for RNAi-Mediated Crop Protection

Fusarium wilt is the most devastating banana disease caused by a soilborne pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Non-coding small RNAs (sRNAs) are key components in regulating gene expression via RNA interference (RNAi).Dicer-like (DCL) genes are responsible for digesting double-stranded RNAs into sNA duolexes. Understanding the biological function of DCL genes is important for RNAi-mediated suppression of pathogen infection against the host. This study identified the full length of two DCL transcripts in Foc TR4 using Rapid Amplification of cDNA Ends (RACE) technique, bioinformatic predictions and quantitative polymerase chain reactions (qPCR). Full-length mRNA sequences comprised of 1501 amino acids and 1441 amino acids in DCL1 and DCL2 (DCL1/2) genes, respectively. Domain prediction using SMART program showed that Foc TR4-DCLi protein comprises four domains; DEAD-like helicase superfamily domain, helicase superfamily c-terminal domain, and dual ribonuclease 3 family domains whereas Foc TR4-DCL2 protein has similar domains plus double-stranded RNA binding motif. Foc TR4-DCL1 genes were expressed in spores and mycelia, DCL2 gene was expressed in rhizome from 3 days after infection (DAI) while DCL1 gene expression started 14 DAI. Phyre2 software predicted an 'L' shaped DCL protein tertiary structure, equivalent to the human endoribonuclease dicer with 100% confidence. Phylogenetic analysis of the Foc TR4-DCLs with other plant pathogenic ascomycetes showed that DCL1/2 primary clades were divided into four subgroups of Sodariomycetes, Leotiomycetes, Dothideomycetes, and Eurotiomycetes.Fusarium species formed a separate clade comprising proteins from the Nectriaceae family and their sequence similarity range from 94.99-98.63% and 98.07-99.50% in DCL1 and DCL2, respectively. These results demonstrated that dicer-dependent RNAi pathway exists in Foc TR4 which may regulate the pathogenicity of the pathogen. In addition, this study generates important information for the development of RNAi-mediated gene silencing against fusarium wilt in banana.