The Cytospora chrysosperma Virulence Effector CcCAP1 Mainly Localizes to the Plant Nucleus To Suppress Plant Immune Responses

Abstract

Canker disease is caused by the fungus Cytospora chrysosperma and damages a wide range of woody plants, causing major losses to crops and native plants. Plant pathogens secrete virulence-related effectors into host cells during infection to regulate plant immunity and promote colonization. However, the functions of C. chrysosperma effectors remain largely unknown. In this study, we used Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana and confocal microscopy to investigate the immunoregulation roles and subcellular localization of CcCAP1, a virulence-related effector identified in C. chrysosperma. CcCAP1 was significantly induced in the early stages of infection and contains cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily domain with four cysteines. CcCAP1 suppressed the programmed cell death triggered by Bcl-2-associated X protein (BAX) and the elicitin infestin1 (INF1) in transient expression assays with Nicotiana benthamiana. The CAP superfamily domain was sufficient for its cell death-inhibiting activity and three of the four cysteines in the CAP superfamily domain were indispensable for its activity. Pathogen challenge assays in N. benthamiana demonstrated that transient expression of CcCAP1 promoted Botrytis cinerea infection and restricted reactive oxygen species accumulation, callose deposition, and defense-related gene expression. In addition, expression of green fluorescent protein-labeled CcCAP1 in N. benthamiana showed that it localized to both the plant nucleus and the cytoplasm, but the nuclear localization was essential for its full immune inhibiting activity. These results suggest that this virulence-related effector of C. chrysosperma modulates plant immunity and functions mainly via its nuclear localization and the CAP domain. IMPORTANCE The data presented in this study provide a key resource for understanding the biology and molecular basis of necrotrophic pathogen responses to Nicotiana benthamiana resistance utilizing effector proteins, and CcCAP1 may be used in future studies to understand effector-triggered susceptibility processes in the Cytospora chrysosperma-poplar interaction system.