Generation of an expression cassette for gene silencing the tRNA ISOPENTENYL TRANSFERASE (tRNA IPT) gene of Moniliophtora perniciosa by RNA interference (RNAi).

Abstract

"Witches' broom" is an important disease that affects cacao (Theobroma cacao) and is caused by the basidiomycete fungus Moniliophtora perniciosa. The fungus is endemic to the Amazon region, and its introduction in southern Bahia led to the collapse of cocoa plantations and caused important economic and social changes in the region. Moniliophtora perniciosa is characterized as a hemibiotrophic fungus, but unlike other pathogens with this lifestyle, M. perniciosa has a symptomatic and long biotrophic phase. The main symptoms of the infection are swelling and loss of apical dominance of branches, with the induction of lateral shoots. The study of the disease is difficult because the interaction occurs between two non-model organisms. Therefore, since there is a biotype that infects members of the Solanaceae family (Biotype-S) and that, when inoculated, infects the tomato plant (Solanum lycopersicum), it is possible to study the defense mechanisms and pathogenicity using the tomato plant cv. 'Micro-Tone' (MT). Using MT, our group demonstrated that at least part of the plant's response to infection is related to cytokinin signaling. The transcriptomic atlas of witches' broom indicated the expression of a candidate gene for an alternative route of cytokinin biosynthesis, annotated as tRNA ISOPENTENYL TRANSFERASE (tRNA IPT) in monokaryotic, dikaryotic mycelium and during the biotrophic phase of infection in cacao. Thus, this gene needs to be functionally validated as associated with the induction of symptoms related to cytokinin imbalance. As it is not possible to genetically manipulate M. perniciosa, we intend to generate MT transgenic plants expressing interfering RNAs for IPT tRNA (p35S:RNAiMptRNA) to evaluate this virulence mechanism and the development of symptoms in the biotrophic phase of M. perniciosa infection. To this end, this project aims to develop an expression cassette for silencing the gene tRNA ISOPENTENYL TRANSFERASE (tRNA IPT) from M. perniciosa to be introduced into tomato.