siRNA quelling of tri4 and tri5 genes related to deoxynivalenol synthesis in Fusarium graminearum and Fusarium culmorum

ABSTRACT

Aim: This study was conducted to investigate the quelling of tri4 and tri5 genes associated with deoxynivalenol, most common class B trichothecene, via siRNA application. Methodology: In this study, one Fusarium graminearum (4F) and two F. culmorum (9F and 20F) isolates, expressing high levels of tri4 and tri5 genes, were targeted for siRNA-based silencing. Full length tri4 and tri5 genes were amplified from all isolates, sequenced and then subjected to CLUSTALW analysis for designing siRNA molecules. Totally designed six siRNAs were separately co-transfected together with helper plasmids (pEGFP75 and pAN7-1) as 36 combinations into protoplast cultures and quelling was analysed via real time polymerase chain reaction, thin layer chromatography and spectrofluorimetric analysis. Results: Alignment of both tri4 and tri5 genes’ sequencing revealed high levels of similarity between isolates (94-99% and 95-100%, respectively). Transfection efficiency ranged from 50±0.00/5x104 to 99.33±10.06/5x104 and relative fluorescence unit values were calculated between 1.37±0.07 and 2.89±0.06. The 0.7 kb length fragments of hph and GFP were amplified from transfectants. The real time PCR revealed that tri4 was completely suppressed in 30 experimental sets whereas down-regulations of tri5, ranging from 74.5 to 99%, was detected in remaining 6 experiment groups. Thin layer chromatography assay also showed that siRNA-transfected Fusarium samples showed no deoxynivalenol spots. Interpretation: This study revealed that tri4 and tri5 genes can be targeted in the development of less deoxynivalenol (DON)-producing strains through RNAi. Moreover, current study has great importance as it demonstrates that quelling can be used as a potential strategy in controlling diseases caused by phytopathogenic species.