Characterization of Dicer-like proteins of Fusarium oxysporum f. sp. cubense TR4 for RNAi-mediated gene silencing assessment

Banana fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is a serious epidemic disease worldwide and has recorded significant yield loss in banana farms. The most virulent physiological race of Foc is tropical race 4 (TR4). In commercial cultivations, no effective control measure for fusarium wilt has been realized up to now. Ribonucleic acid interference (RNAi) is a post transcriptional gene silencing mechanism, triggered by small double stranded RNA (dsRNA) which leads to sequence specific messenger RNA (mRNA) degradation. It regulates growth and development of organisms. Dicer like (DCL) proteins are responsible for producing small RNAs from long dsRNAs which is the key step in RNAi mechanism. The overall objective of this study was to assess the effect of Foc TR4 DCL2 gene silencing on pathogenicity of Foc TR4 in banana. The full length DCL1 and DCL2 transcripts were isolated and characterized from Foc TR4 in first time in this study. DCL1 and DCL2 proteins include 1501 and 1441 amino acids respectively. In phylogenetic analysis, all Fusarium species in both DCL1 and DCL2 phylogenetic trees were closely related to each other and classified under nectriaceae family. Foc TR4-DCL2 long dsRNAs were designed in silico, based on identified transcript sequence and synthesized using commercially available MEGAscript RNAi kit. Long dsRNA sequence of DCL2 was 736 base pairs (bp) in length. In RNAi silencing efficiency duration of dsRNA, the relative transcript abundance of Foc TR4-DCL2 was significantly reduced 72 hours post dsRNA application (HPA) by 44% compared to 0 h and thereafter no significant difference with time until 120 HPA. In effective dose of dsRNAs for RNAi silencing, the relative transcript reduction varied between 22-55% compared to control in all doses except 100 ng/mL. The relative transcript abundance was significantly low at 1000-2000 ng/mL of dsRNA levels. At least 1000 ng/mL was required to elicit a significant reduction of relative transcript abundance compared to control. DCL2 gene silencing affected the conidiation of pathogen. In vitro studies were carried out using complete randomized design (CRD) with three replicates. In glasshouse trials, the used dsRNA dose was 2000 ng per injection at 25 ng/μL dilution rate. In vivo dsRNA assessments were carried out CRD with five replicates and relative transcript abundance and diease severity were recorded. Area under disease progress curve (AUDPC) values were significantly less in dsRNA treated roots on 7 DPI compared to control. Relative transcript abundance was reduced in 5 and 7 DPI in roots. Disease severity reduction was similar in pre, post and simultaneous application of dsRNA with pathogen infection. Rhizome injection of dsRNA reduced disease severity and relative transcript abundance on day 7. In stability test, the dsRNA was degraded in 8 h after exposure to ultra violet (UV) rays. In conclusion, in silico designed DCL2 dsRNA molecules could silence Foc TR4 DCL2 gene and it affected conidiation in Foc TR4. Direct exogenous application of Foc TR4 DCL2 dsRNAs significantly decreased fungal infection and has a potential use to control banana fusarium wilt.

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