RNA-Mediated Gene Silencing of Superoxide Dismutase (bcsod1) in Botrytis cinerea

Abstract

Gene silencing is a powerful tool utilized for identification of gene function and analysis in plants, animals, and fungi. Here, we report the silencing of superoxide dismutase (bcsod1) in Botrytis cinerea through sense and antisense-mediated silencing mechanisms. Because superoxide dismutase (SOD) is a virulence factor, transformants were tested for phenotypic silencing in vitro and reduction in pathogenicity in planta. Plate-based assays with and without paraquat were performed to screen initial silencing efficiency, and a subset of transformants was used for in planta studies of virulence. Transformants exhibiting strongly decreased transcripts levels were recovered with both constructs but none of those exhibited a reduction in virulence in planta. Our investigations may help optimize a high-throughput gene silencing system useful for identifying potential gene targets for future fungal control.

Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

“…Whether virulence attenuation in these mutants arises from failure to defend against host-produced oxidative stress awaits testing in hosts unable to produce superoxide. Data for the involvement of superoxide dismutases in fungal infection of plants is based primarily on correlations with SOD gene transcription during fungus-plant interaction [48]–[51], but genetic evidence of the role of superoxide dismutase is largely lacking or contradictory in the case of Botrytis Sod1 [52], [53]. The results of our study using Histoplasma strains deficient in Sod1 and Sod3 demonstrate the spatial specificity of the Sod1 and Sod3 superoxide dismutases for internal and external (i.e., host-derived) superoxide, respectively.…”

Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches

“…An important aspect of molecular genetics of B. cinerea is the high efficiency of targeted gene inactivation by replacement approaches, which allows a rapid functional analysis of putative pathogenicity-related genes. RNA-mediated gene silencing approaches were also implemented as an alternative strategy for gene function characterization especially for essential genes (Patel et al, 2008;Schumacher et al, 2008b). In addition, random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) has been shown to be a useful tool for the identification of new virulence-associated genes (Giesbert et al, 2012).…”

The pOT and pLOB vector systems: Improving ease of transgene expression in Botrytis cinerea

“…For instance, one may consider the utilization of a vector that contains an intron sequence in the 5 -UTR, which is believed to provide a greater degree of transcript stability and thereby increases the heterologous protein production level (Burns et al, 2005). Even so, the pOT vector system may be used as a tool to improve ease of transgene expression and increase previously observed gene silencing frequencies in B. cinerea and other ascomycetes (Patel et al, 2008). Activity is measured in fl uorometric (FLU) and relative light (RLU) units per microgram protein (μg 1 ), respectively.…”