The Virulence-Associated CAP/PR1-Like Proteins From Cytospora chrysosperma Target a Subunit of RNA Polymerase II to...

Plant pathogenesis-related 1 (PR1) proteins are members of the CAP protein superfamily and are widely used immune markers. The precise function and mode of action of CAP proteins, however, remains poorly understood. In Cytospora chrysosperma, the causative agent of poplar canker disease, three CAP family members, CcCAP1-3, have been identified. CcCAP1 is required for fungal virulence and suppresses the plant immune response to facilitate fungal colonisation, in a nuclear-localisation dependent way. In this study, we functionally characterised the other two members of the C. chrysosperma CAP family, CcCAP2 and CcCAP3. Targeted gene deletions revealed that CcCAP2 and CcCAP3 affected vegetative growth and CcCAP2 contributed to fungal virulence during C. chrysosperma- Populus euramericana interaction. While CcCAP3 is predicted to be GPI-anchored, CcCAP2 is a secreted glycoprotein. Ectopic expression of CcCAP2 in Nicotiana benthamiana promoted Botrytis cinerea infection and inhibited plant immune-related gene expression. To elucidate the mode of action of the two virulence-associated proteins, CcCAP1 and CcCAP2, we employed a yeast two-hybrid-based approach and identified interacting proteins. Screening of a poplar library revealed the highest number of hits with RNA polymerase II, subunit 11 (RPB11). The interactions between CcCAP1, 2 and RPB11 were validated by colocalization analysis, FRET-FLIM, and co-immunoprecipitation analysis. Interestingly, virus-induced gene silencing of RPB11 resulted in a dwarf phenotype of the host plant, reduced fungal colonisation, and highly induced PR1 gene expression. These results suggest that the fungal virulence-associated CAP family members, CcCAP1 and CcCAP2, both target plant RNA polymerase II subunit 11 to suppress host immunity and facilitate fungal infection. The dwarf phenotype and induced expression of PR1 upon downregulation of RPB11 suggest that its function strongly affects the plant's growth-defence trade-off. These observations deepen our understanding of the complex functions of CAP family proteins in plant immune resistance and fungal virulence.