Molecular characterisation of RNA interference in Fusarium circinatum

Abstract

RNA interference is an important biological pathway that can be induced to control plant pathogens. Methods of application include host-, virus- and spray-induced gene silencing. While earlier research concentrated on host-induced gene silencing (HIGS), recent studies indicate that spray-induced gene silencing is now considered the more effective approach. This study focused on using spray-induced gene silencing (SIGS) to induce silencing in Fusarium circinatum which causes pitch canker on Pinus species. This work first determined whether RNAi pathways are functional in F. circinatum. The protein sequences of Neurospora crassa RNAi genes were used to identify the 7 RNAi-related genes in 21 Fusarium species including F. circinatum and 13 other fungal species. All the species had the core RNAi genes including Argonaute, Dicer and RNA-dependent RNA polymerase (RdRP). An additional Argonaute gene was found, forming a sister clade to SMS-2 (suppressor of meiotic silencing 2, an argonaute protein in N. crassa) containing clade. A total of 99 Argonaute, 70 Dicer, 120 RdRP, 38 RecQ helicase, 68 RPA, 34 QIP and MRPL3 genes were found in the 34 species. Most species possessed 3 Argonaute, 2 Dicer, 4 RdRP, 2 RPA, 1 QIP, RecQ helicase and MRPL3 genes. The greatest variation in gene copy number was observed with regards to Argonaute and RdRP genes. Gene phylogenies were also generated for comparison to the species tree. The congruency between the gene trees and species tree suggests that the RNAi genes are conserved. InterProScan was used to determine the protein domain organisation in each gene. All the identified putative proteins had the necessary domains associated with RNAi proteins. In a subsequent pilot study, application of double-stranded RNA (dsRNA) to F. circinatum GFP (green fluorescent protein) mutants reduced the expression of GFP. This was reflected by a significant difference in GFP fluorescence compared to the control. In a bigger study, this was not observed, potentially due to use of growth medium containing rifampicin, which have been shown to affect eukaryotic RNA dependent RNase activity. Additional studies are thus needed to decipher the impact of rifampicin on RNAi, and the original experiment should be repeated without rifampicin. Furthermore, Reverse Transcriptase-quantitative PCR (RT-qPCR) and small RNA-Seq analyses should be performed to validate the efficacy of the applied dsRNA in gene silencing. Overall, the work presented in this dissertation presents a valuable first step towards the development of SIGS or some other RNAi-based approach for controlling F. circinatum.