Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens intoNicotiana benthamianaleaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector ofPhytophthora sojae. Plants infected with PVX carryingAvh148showed strong viral symptoms and high-levelAvh148and viral RNA accumulation. Analysis ofP. sojae Avh148deletion mutants and soybean hairy roots overexpressingAvh148revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized inN. benthamianaplants at different developmental stages across a range ofAgrobacteriumcell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.