Application of Double-Strand RNAs Targeting Chitin Synthase, Glucan Synthase, and Protein Kinase Reduces Fusarium graminearum Spreading in Wheat.

Controlling the devastating fungal pathogen Fusarium graminearum (Fg) is a challenge due to inadequate resistance in nature. Here, we report on the identification of RNAi molecules and their applications for controlling Fg in wheat through silencing chitin synthase 7 (Chs7), glucan synthase (Gls) and protein kinase C (Pkc). From transgenic Fg strains four RNAi constructs from Chs7 (Chs7RNAi-1, -2, -3, and -4), three RNAi constructs from Gls (GlsRNAi-2, -3, and -6), and one RNAi construct from Pkc (PkcRNAi-5) were identified that displayed effective silencing effects on mycelium growth in medium and pathogenicity in wheat spikes. Transcript levels of Chs7, Gls and Pkc were markedly reduced in those strains. Double-strand RNAs (dsRNAs) of three selected RNAi constructs (Chs7RNAi-4, GlsRNAi-6 and PkcRNA-5) strongly inhibited mycelium growth in vitro. Spray of those dsRNAs on detached wheat leaves significantly reduced lesion sizes; the independent dsRNAs showed comparable effects on lesions with combination of two or three dsRNAs. Expression of three targets Chs7, Gls, and Pkc was substantially down-regulated in Fg-infected wheat leaves. Further application of dsRNAs on wheat spikes in greenhouse significantly reduced infected spikelets. The identified RNAi constructs may be directly used for spray-induced gene silencing and stable expression in plants to control Fusarium pathogens in agriculture.

Expression of microRNA-like RNA-2 (Fgmil-2) and bioH1 from a single transcript in Fusarium graminearum are inversely correlated to regulate biotin synthesis during vegetative growth and host infection.

MicroRNA-like RNAs (milRNAs) post-transcriptionally down-regulate target genes. We investigated Fusarium graminearum (Fg) milRNA expression during fungal vegetative growth and infection of wheat. Small RNA sequencing identified 36 milRNAs from Fg, one of which, Fgmil-2, had >100 transcripts per million in conidia, mycelia and infected wheat, with the highest expression in conidia and the lowest expression in colonized wheat tissue. Fgmil-2 displays perfect homology to the 3'-untranslated region (3'-UTR) of an FgbioH1 messenger RNA that is involved in biotin biosynthesis. Poly(A) polymerase-mediated rapid amplification of cDNA ends combined with sequencing analysis demonstrated that cleavage at a specific site by FgDicer2 in the 3'-UTR of FgbioH1 transcripts generated the Fgmil-2 precursor with a typical hairpin structure. Deletion of FgbioH1 or FgDicer2 genes abolished Fgmil-2 biogenesis. FgbioH1 had an inversely correlated pattern of expression to that of Fgmil-2 and FgDicer2. Deletion of FgbioH1 also showed that it is required for mycelial growth, virulence, mycotoxin biosynthesis and expression of biotin-dependent carboxylase genes. This study reveals in Fg a novel mode of inversely correlated post-transcriptional regulation in which Fgmil-2 originates from its own target transcript, FgbioH, to govern biotin biosynthesis.