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.

CcPmk1 is a regulator of pathogenicity in Cytospora chrysosperma and can be used as a potential target for disease control

“…In addition, GME7477_g, a small cysteine-rich secretory protein belonging to the CAP superfamily, can inhibit PAMP INF1-triggered cell death and plays a vital role in fungal pathogenicity to the host poplar (Han et al, 2021). Hence, we believe that CcPmk1 may participate in regulating the expression of a subset of downstream components to promote virulence.…”

“…Further analysis revealed that 12 downregulated genes and 7 up-regulated genes met the basic criteria of traditional effectors (Table S3). Among the down-regulated putative effectors, a cysteine-rich secretory protein family gene, GME7477_g, is important for C. chrysosperma pathogenicity and can disturb the plant immunity (Han et al, 2021), while a necrosis-inducing protein (NPP1) was found in the up-regulated putative effectors (Table S3).…”

A Putative Effector CcSp84 of Cytospora chrysosperma Localizes to the Plant Nucleus to Trigger Plant Immunity

“…Cytospora chrysosperma is a necrotrophic fungal pathogen that causes stem canker in a broad range of hosts [1,2]. In nature, the spores of C. chrysosperma germinate and produce the hyphae to invade the host plants with poor conditions through the wound.…”

“…For example, the mitogen-activated protein kinase (CcPmk1) and oxalic acid metabolism were required for the growth and virulence of C. chrysosperma [5,8,9]. The virulence effector CcCAP1, a member of the CAP superfamily (cysteine-rich secretory protein, antigen 5, and pathogenesis-related 1), from C. chrysosperma mainly localized to plant nucleus to suppress plants immune responses [2]. Additionally, the transcription factor CcSge1, belonging to the Gti1/Pac2 family, was essential to the hyphal radial growth, conidiation, and fungal virulence.…”

Phosphoproteomic and Metabolomic Profiling Uncovers the Roles of CcPmk1 in the Pathogenicity of Cytospora chrysosperma

“…Our previous works identified a pathogenicity-related MAPK, CcPmk1 , which acts as a core component to regulate fungal virulence and development by affecting the expression of downstream genes, including transcription factor genes, effector-encoding genes, kinase genes, and so on ( 18 , 26 ). For example, the Gti1/PacII transcription factor CcSge1 and the putative effector CcCAP1 , both of which are required for fungal virulence, are transcriptionally regulated by CcPmk1 ( 42 , 43 ). However, the further mode of action of CcPmk1 is still unclear.…”