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RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants. [ABSTRACT FROM AUTHOR]

Mitogen activated protein (MAP) kinases are key components in signalling networks. In Fusarium graminearum, one of the most devastating fungal plant pathogens, two MAP kinases are known to be involved in pathogenicity, Mgv1 and the Gibberella pathogenicity MAP kinase Gpmk1. Δgpmk1 mutants with a disrupted GPMK1 gene are unable to infect wheat spikelets. They exhibit altered secretion of several extracellular enzymes. Among those, the lipase FGL1 is known to be a major virulence factor of F. graminearum. FGL1 gene expression was decreased in Δgpmk1 strains during wheat head infection. To uncouple FGL1 expression from Gpmk1 activity, we generated Δgpmk1 strains that constitutively express FGL1 under control of the Cochliobolus heterostrophus glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter. The level of extracellular lipolytic activity of these strains in culture was comparable to the wild-type. These mutants showed fully restored conidiation and partial complementation of defects in development that were reported from the Δgpmk1 mutant. They also partially complemented the apathogenic disease phenotype of the Δgpmk1 mutants causing lesions in directly inoculated wheat spikelets. But in contrast to wild-type, their growth was restricted to directly inoculated spikelets. A barrier-like formation was observed at the rachis node. Based on these results, we could show that the lipase FGL1 is necessary but not sufficient to restore complete pathogenicity to the apathogenic Δgpmk1 mutant. Hence, we hypothesize that the MAP kinase Gpmk1 is involved in the regulation of additional factors required for complete virulence of F. graminearum. [ABSTRACT FROM AUTHOR]