Exogenous siRNAs against chitin synthase gene suppress the growth of the pathogenic fungus Macrophomina phaseolina

Abstract

Macrophomina phaseolina, causative agent of the charcoal rot disease, impacts over 500 plant species, causing devastating crop failures worldwide. Due to the lack of effective management approach, charcoal rot can result in serious loss, especially during years with hot and dry summers. This study aims to evaluate the effectiveness of using the RNA interference (RNAi) mechanism to suppress the growth of M. phaseolina, through exogenous application of small interfering RNA (siRNA) targeting one of the chitin synthase (MpCHS) genes in the M. phaseolina genome. Since chitin is one of the main components in the fungal cell wall, we hypothesize that suppressing the expression of MpCHS through RNAi will negatively affect the growth of the fungus, which may result in reduced virulence toward the host. Of the seven candidate MpCHS genes assessed, MpCHS6 showed consistent expression in plate-grown fungus and in infected plant tissue and was chosen as the target for RNAi knockdown. Two siRNAs were designed to target MpCHS6 and tested through in vitro application in M. phaseolina culture. Both siRNAs suppressed growth of the fungus at multiple time points and under varied environmental conditions. More importantly, the growth suppression was well correlated with down-regulation of the MpCHS6 gene. These results indicate that exogenous siRNAs can suppress gene expression in M. phaseolina even when applied in vitro. In addition, targeting a MpCHS gene may provide an effective way to control the fungal growth. Our study provides firsthand knowledge on RNAi in M. phaseolina, which benefits not only basic research that focuses on understanding gene function in this species but also application of RNAi-based technology in disease control in the future.

Keywords: Charcoal rot; RNAi; RT-qPCR; chitin.