Knockdown of Bmp1 and Pls1 Virulence Genes by Exogenous Application of RNAi-Inducing dsRNA in Botrytis cinerea

Abstract

Botrytis cinerea is a pathogen of wide agronomic and scientific importance partly due to its tendency to develop fungicide resistance. Recently, there has been great interest in the use of RNA interference as a control strategy against B. cinerea. In order to reduce the possible effects on non-target species, the sequence-dependent nature of RNAi can be used as an advantage to customize the design of dsRNA molecules. We selected two genes related to virulence: BcBmp1 (a MAP kinase essential for fungal pathogenesis) and BcPls1 (a tetraspanin related to appressorium penetration). After performing a prediction analysis of small interfering RNAs, dsRNAs of 344 (BcBmp1) and 413 (BcPls1) nucleotides were synthesized in vitro. We tested the effect of topical applications of dsRNAs, both in vitro by a fungal growth assay in microtiter plates and in vivo on artificially inoculated detached lettuce leaves. In both cases, topical applications of dsRNA led to gene knockdown with a delay in conidial germination for BcBmp1, an evident growth retardation for BcPls1, and a strong reduction in necrotic lesions on lettuce leaves for both genes. Furthermore, a strongly reduced expression of the BcBmp1 and BcPls1 genes was observed in both in vitro and in vivo experiments, suggesting that these genes could be promising targets for the development of RNAi-based fungicides against B. cinerea.