A Phytophthora sojae CRN effector mediates phosphorylation and degradation of plant aquaporin proteins to suppress host immune signaling

Author summary CRN effectors are conserved in diverse pathogens of plants, animals, and insects, and highly expanded in Phytophthora species. Nevertheless, little is known about their functions, targets, and action mechanisms. Here, we characterized a kinase-domain-containing CRN effector (CRN78) in a notorious oomycete pathogen, P. sojae. CRN78 is a virulence-essential effector of P. sojae infection, and acts via suppression of plant H2O2 accumulation and defense gene expressions. We demonstrated that CRN78 might interact with plant PIP2-family aquaporin proteins, including N. benthamiana NbPIP2;2 and soybean GmPIP2-13, and regulate their phosphorylation, resulting in subsequent 26S-dependent protein degradation. Furthermore, we revealed that NbPIP2;2 was an apoplast-to-cytoplast H2O2 transporter and positively regulated plant immunity and ROS accumulation. Importantly, this phosphorylation may be highly conserved in many plant aquaporin proteins. Thus, this study identifies a virulence-related effector from P. sojae and a novel plant immunity-related gene, and reveals a detailed mechanism of effector-mediated phosphorylation and degradation of plant aquaporin proteins. Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

Automated summary

The study identifies a virulence-related effector, CRN78, from the oomycete pathogen Phytophthora sojae, which promotes infection by inhibiting H2O2 accumulation in plants. Furthermore, CRN78 interacts with plant aquaporin proteins and regulates their phosphorylation for subsequent protein degradation. The phosphorylation mechanism of CRN78 on aquaporin proteins is highly conserved among higher plants and oomycete pathogens, suggesting a novel pathway for effector proteins to inhibit host cellular defenses.