Abstract
ABSTRACTCanker 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.
Highlights
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
Nearly 300 candidate effector genes were identified in the C. chrysosperma genome based on general criteria, including their small sizes, the presence of a signal peptide in the N terminus, the lack of transmembrane domains, and being rich in cysteines, as described previously [70]
The expression of hemagglutinin (HA)-tagged CcCAP1, green fluorescent protein (GFP), Bax, and INF1 were confirmed by Western blotting (Fig. 3B). These results suggested that CcCAP1 is an important virulence-related effector of C. chrysosperma that is involved in manipulating plant immunity by suppressing cell death
Summary
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. 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. PAMPs are often recognized by plants via pattern recognition receptors on the plasma membrane, including receptor-like kinases, receptor-like proteins, and receptor-like cytoplasmic kinases [21,22,23] This basal defense response can restrict the proliferation of most pathogens via callose deposition in the cell walls, reactive oxygen species (ROS) accumulation, and transcriptional upregulation of immune-related genes [23]. When effectors are recognized by corresponding resistance (R) proteins in the host plants, effector-triggered immunity is induced, which is a qualitatively swifter and more vigorous immune response than PTI and induces localized programmed cell death (PCD) in the host, called the hypersensitive response (HR) [30]
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