Discontinuous and repeatable peaks of small interfering RNAs mapping to TRI6 associated with deoxynivalenol reductions by RNA interference in Fusarium graminearum

Authors: Baldwin, Thomas; Bregitzer, Paul; Esvelt Klos, Kathy; ISLAMOVIC, EMIR - Basf Corporation North America; SCHWARTZ, PAUL - North Dakota State University; GILLESPIE, JAMES - North Dakota State University

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/9/2018
Publication Date: 8/30/2018
Citation: Baldwin, T.T., Bregitzer, P.P., Esvelt Klos, K.L., Islamovic, E., Schwartz, P., Gillespie, J. 2018. Discontinuous and repeatable peaks of small interfering RNAs mapping to TRI6 associated with deoxynivalenol reductions by RNA interference in Fusarium graminearum. Fungal Genetics and Biology. 13(8):Article 0202798. https://doi.org/10.1371/journal.pone.0202798.
DOI: https://doi.org/10.1371/journal.pone.0202798

Interpretive Summary: Fusarium graminearum is a fungal pathogen causing Fusarium head blight of small grains and can result in dangerous levels of mycotoxin accumulation, most prominently the mycotoxin deoxynivalenol (DON). A new breeding method call host-induced gene silencing (HIGS) is a potential solution to control this disease. However, the underlying mechanisms of HIGS are not fully understood. HIGS is based on RNA interference (RNAi) that ‘silence’ (i.e. inactivate) genes via creation of small interfering RNA (siRNA). In this study, an RNAi construct was transformed into Fusarium graminearum to silence the gene TRI6 that regulates the production of DON. We found patterns of siRNA to be constant and discontinuously abundant in “hot spots” in different regions of TRI6 associated with reduction in DON. These results may enable optimization of RNAi constructs to be used in HIGS resistance against Fusarium head blight.

Technical Abstract: Deoxynivalenol (DON) contamination of cereal grains caused by Fusarium head blight may be addressed by future RNA interference (RNAi)-based gene silencing approaches. However, utilizing these approaches will require a greater understanding of the principles that govern RNAi effectiveness in the pathogen Fusarium graminearum. RNAi in higher eukaryotes, including fungi, involves processing double stranded RNA (dsRNA) into small interfering RNA (siRNA) that silence gene expression based on base pair complementarity. This study examined virulence, DON production, and the small RNA (sRNA) populations in response to RNAi-based silencing of TRI6, a transcription factor that positively regulates DON synthesis via control of TRI5 expression. Silencing was accomplished via the expression of transgenes encoding inverted repeats targeting various regions of TRI6 (RNAi vectors). Transgene expression was associated with novel, TRI6-specific siRNAs. For RNAi vectors targeting the majority of TRI6 sequence (~600 bp), a discontinuous, repeatable pattern was observed in which most siRNAs mapped to specific regions of TRI6. Targeting shorter regions (250±350 bp) did not alter the siRNA populations corresponding to that region of TRI6. No phased processing was observed. The 5' base of ~83% of siRNAs was uracil, consistent with DICER processing and ARGONAUTE binding preferences for siRNA. Mutant lines showed TRI6 siRNA-associated reductions of TRI5 expression on toxin inducing media and DON in infected wheat and barley spikes. Shorter RNAi vectors resulted in variable levels of silencing that were less than for the ~600 bp RNAi vector, with a 343 bp RNAi vector targeting the 5' end of TRI6 having the best silencing efficiency. This work identifies efficient shorter region for silencing of TRI6 and describes the patterns of siRNA corresponding to those regions.