Combination of in vivo proximity labeling and co-immunoprecipitation identifies the host target network of a tumor-inducing effector in the fungal maize pathogen Ustilago maydis

Plant pathogens secrete effectors, which target host proteins to facilitate infection. The Ustilago maydis effector UmSee1 is required for tumor formation in the leaf during infection of maize. UmSee1 interacts with maize SGT1 (suppressor of G2 allele of skp1) and blocks its phosphorylation in vivo. In the absence of UmSee1, U. maydis cannot trigger tumor formation in the bundle sheath. However, it remains unclear which host processes are manipulated by UmSee1 and the UmSee1-SGT1 interaction to cause the observed phenotype. Proximity-dependent protein labeling involving the turbo biotin ligase tag (TurboID) for proximal labeling of proteins is a powerful tool for identifying the protein interactome. We have generated transgenic U. maydis that secretes biotin ligase-fused See1 effector (UmSee1-TurboID-3HA) directly into maize cells. This approach, in combination with conventional co-immunoprecipitation, allowed the identification of additional UmSee1 interactors in maize cells. Collectively, our data identified three ubiquitin-proteasome pathway-related proteins (ZmSIP1, ZmSIP2, and ZmSIP3) that either interact with or are close to UmSee1 during host infection of maize with U. maydis. ZmSIP3 represents a cell cycle regulator whose degradation appears to be promoted in the presence of UmSee1. Our data provide a possible explanation of the requirement for UmSee1 in tumor formation during U. maydis-Zea mays interaction. Combining proximity labeling and co-immunoprecipitation identified host targets of a tumor-inducing effector, suggesting that the plant pathogen Ustilago maydismodulates host cell cycle control through the ubiquitin-proteasome pathway.

Automated summary

Plant pathogens secrete effectors to target host proteins for infection. The effector UmSee1 of Ustilago maydis is essential for tumor formation in maize leaves during infection. UmSee1 interacts with maize SGT1 and inhibits its phosphorylation. Using proximity labeling and co-immunoprecipitation, three proteins related to the ubiquitin-proteasome pathway (ZmSIP1, ZmSIP2, and ZmSIP3) were identified as UmSee1 interactors during U. maydis infection of maize. ZmSIP3, a cell cycle regulator, appears to be degraded in the presence of UmSee1, providing a possible explanation for UmSee1's role in tumor formation.