Construções gênicas para o silenciamento gênico induzido pelo hospedeiro em arabidopsis e trigo para o controle de giberela

Abstract

Gene constructions for host-induced gene silencing in arabidopsis and wheat for Fusarium Head Blight control. [154] f. Thesis (Doctorate in Agronomy) – University of Passo Fundo, Passo Fundo, 2021. Fusarium Head Blight caused by the ascomycetus fungus Gibberella zeae (Schwein.) Petch, asexual form Fusarium graminearum Schwabe stands out as an important wheat disease, for reducing the productivity and quality of grains, in addition to causing contamination by mycotoxins. The available control strategies, such as genetic resistance, chemical and cultural controls, are insufficient to contain epidemics in years when environmental conditions are favorable to infection. Certain factors, it is necessary to seek new strategies for the control of this disease. The Host Induced Gene Silencing (HIGS) technology, which is based on interference RNA (RNAi) and, in the case of plants, aims at silencing important genes for the growth, development or pathogenicity of pathogens. This strategy can be used to control phytopathogenic fungi, and an increase in resistance to F. graminearum in wheat and barley has already been developed. The major objective was to verify the ability of RNAi gene constructs for HIGS of target genes important for pathogenicity or virulence without F. graminearum control, and the specific objectives i) To evaluate the potential of multiple RNAi-Chs3b, RNAi-Cyp51-Tri multiple gene constructs in arabidopsis and wheat and the RNAi-Synchit2 gene construct, with part of the Fusarium verticillioides Chitin synthase gene, in arabidopsis and ii) Evaluate the potential of the unique RNAi gene constructs (RNAi-Lae1, -Hog1, -Snf1, -Sid1, -Top1 and -Fgl1 in arabidopsis for F. graminearum HIGS aiming at silencing the corresponding genes in the fungus. Arabidopsis plants were transformed with Agrobacterium tumefaciens containing these gene constructs, correlated with the number of insertions, and the response to F. graminearum was verified via inoculation in detached leaves and inflorescences. In wheat, spikes of transgenic lines in the T2 and T3 generations containing the RNAi-Chs3b and RNAi-Cyp51-Tri gene constructs were inoculated with F. graminearum and evaluated for severity, Fusarium-damaged kernels, DON levels and relative expression of target genes from the phatogen. Lines of arabidopsis containing the RNAi-Chs3b, -Cyp51-Tri and -Synchit2 gene constructs did not show difference in severity in detached leaves and inflorescences, two transgenic RNAi-Cyp51-Tri lines show reduction in deoxynivalenol (DON) levels. The transgenic BRS Guamirim wheat lines containing the RNAi-Chs3b and RNAi-Cyp51-Tri gene constructions showed no reductions related to severity, AACPD, Fusarium-damaged kernels and grain weight. A wheat line with RNAi-Cyp51-Tri showed a reduction in DON level. No reduction in the expression of the Chs3b, Cyp51A, Cyp51B and Tri5 genes was detected in F. graminearum exposed to transgenic wheat lines. In transgenic lines of arabidopsis targeting the genes Lae1, Hog1, Snf1, Sid1 and Top1 of F. graminearum for HIGS, dissipated response regarding the severity of the disease in detached leaves. Additional studies are added in order to verify whether siRNAs are being found in plants or in the fungus, analyzing the expression levels of target genes not yet verified, in addition to testing silencing in more events, new sequences and target genes.