Upgrading of the genetic engineering toolkit accelerated the discovery process of the virulence effect of PsGH7d on Phytophthora sojae invasion.

The genus of Phytophthora includes numerous phytopathogens that have devastating impacts on agricultural production. However, the limited availability of selection markers for numerous pathogenicity pathogens of the genus Phytophthora genetic transformation hinders further research on their pathogenic functional genes. Here we report a gene of NAT I, which serves as a novel selection marker for the Phytophthora sojae transformation. Additionally, we developed a new genetic manipulation toolkit based on vectors containing NAT I, which facilitates gene editing in P. sojae. With the toolkit, the gene PsGH7d of P. sojae, which encodes a glycosyl hydrolase, was edited consecutively via the CRISPR/Cas9 system to obtain gene knockout and enzymatic active site mutation strains. The pathogenicity analysis of these transformants revealed that PsGH7d is a virulence factor dependent on its bifunctional glucanase-xylanase activities. This study develops an updated toolkit for the genus Phytophthora genetic transformation and provides initial insights into the virulence of the bifunctional enzyme PsGH7d.