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.

Keywords: Cytospora chrysosperma; plant immunity; subcellular localization; virulence effector.

FIG 1

Significant upregulation of CcCAP1 at early infection stages. (A) Relative expression levels of the candidate effector CcCAP1 were detected at 0, 1, 2, 3, 6, and 12 dpi with the WT strain of Cytospora chrysosperma on poplar twigs, with CcActin as a reference gene. This experiment was performed three times. The statistical analyses were conducted by SPSS v16.0, and Duncan’s test at P = 0.05 was used to determine the differences. Bars indicate ± the standard errors (SE). Different letters indicate significant differences at P ≤ 0.01. (B) A schematic diagram of putative CcCAP1 architecture structure. SP, signal peptide, indicated in gray; CAP, the CAP domain, indicated in green; C, cysteine, indicated in yellow. (C) Sequence alignment of CcCAP1 CAP domain with six homologs. The cysteine residues conserved in these homologs are indicated in red.

FIG 2

Indispensable role of CcCAP1 in ROS defense and pathogenicity. (A) Vegetative growth and morphological development of C. chrysosperma WT strain, ΔCcCAP1-4, ΔCcCAP1-8, and ΔCcCAP1/C-1 on PDA at 25°C for 3 days. (C) Colonial morphology of C. chrysosperma WT strain, ΔCcCAP1-4, ΔCcCAP1-8, and ΔCcCAP1/C-1 on PDA added with 5 and 7% H₂O₂ for 3 days, respectively. (E) Infection symptoms of C. chrysosperma WT strain, ΔCcCAP1-4, ΔCcCAP1-8, and ΔCcCAP1/C-1 on poplar twigs for 4 days. (B, D, and F) Quantification of colony diameter, halo size, and lesion area on the WT strain and on ΔCcCAP1-4-, ΔCcCAP1-8-, and ΔCcCAP1/C-1-treated media or twigs. This experiment was performed three times with similar results. Each assay was performed on at least three independent biological repeats. The statistical analyses were conducted by SPSS v16.0, and Duncan’s test at P ≤ 0.05 or P ≤ 0.01 was used for determining the differences between mutants and WT strain. Bars indicate ± the SE. The letters above the error bars indicate the different groups with statistical significance (P ≤ 0.01 or P ≤ 0.05).

FIG 3

Inhibition of BAX- and INF1-induced cell death by transient expression of CcCAP1 in N. benthamiana leaves. (A) Representative symptoms on leaves of N. benthamiana were assessed at 5 dpa of CcCAP1-pGR106, with GFP-pGR106, BAX-pGR106, and INF1-pGR106 as a control. This experiment was performed at least three times with similar results. Each assay was performed on at least three plants. (B) Western blot analysis of proteins in N. benthamiana transiently expressing HA-tagged CcCAP1, GFP, Bax, and INF1. White asterisks indicate protein bands of interest.

FIG 4

Significance of CAP domain in suppression of INF1-induced cell death. (A) Deletion mutants of CcCAP1. L, linker motif, indicated in orange; T, terminal motif, indicated in blue. (B) Deletion mutants were transient expressed by agroinfiltration in N. benthamiana to assay the suppression of INF1-induced cell death. Representative symptoms on leaves of N. benthamiana were photographed at 5 dpa. This experiment was performed at least three times with similar results. Each assay was performed on at least three plants. (C) Western blot analysis of proteins in N. benthamiana transiently expressing CcCAP1 and its variants fused with an HA tag. White asterisks indicate protein bands of interest.

FIG 5

Determination role of the 154th, 238th, and 259th cysteine residues of CAP domain in suppression of INF1-induced cell death. (A) Representative symptoms on leaves of transient expressed CAP mutants CcCAP1-C^C154S-pGR106 (C¹⁵⁴S), CcCAP1-C^C238S-pGR106 (C²³⁸S), CcCAP1-C^C243S-pGR106 (C²⁴³S), and CcCAP1-C^C259S-pGR106 (C²⁵⁹S) at 5 dpa. This experiment was performed at least three times with similar results. Each assay was performed on at least three plants. (B) Western blot analysis of proteins in N. benthamiana transiently expressing HA-tagged cysteine substitution mutants. White asterisks indicate protein bands of interest.

FIG 6

Localization of CcCAP1 in both the nucleus and cytoplasm in N. benthamiana. (A) Subcellular localization was observed 3 h after nucleus being stained with DAPI at 2 dpa of CcCAP1-pBinGFP2. n, nucleus. The white arrow indicates the region of interest, and the line chart indicates the fluorescence intensity of the region of interest. (B) Western blot analysis of proteins in N. benthamiana transiently expressing GFP control and CcCAP1 fused with an N-terminal GFP.

FIG 7

Suppression of the immune responses and enhancement of susceptibility to pathogen of N. benthamiana by overexpression of CcCAP1. (A) Representative infection symptoms, ROS accumulation, and callose deposition on leaves of CcCAP1-pBinGFP2 or EV agroinfiltrated N. benthamiana at 2 dpi with B. cinerea. (B) Quantification of lesion area, ROS, and callose intensity with ImageJ. (C) Transcriptional levels of defense-related genes were detected at 2 dpi with B. cinerea on leaves of CcCAP1-pBinGFP2 or EV agroinfiltrated N. benthamiana. This experiment was performed three times with similar results. Each assay was performed on at least six independent biological repeats. The statistical analyses were conducted by SPSS v16.0, which was used to analyze the experimental data, and Duncan’s test at P = 0.05 was used to determine the differences in the expression level of defense-related genes. Bars indicate ± SE. The letters above the error bars indicate the different groups with statistical significance (P ≤ 0.01).

FIG 8

Artificial alteration of the subcellular localization of CcCAP1 with NLS and NES sequence. (A) Schematic diagram of CcCAP1 that was artificially added with NLS or NES sequence at the C terminus. NLS, nuclear localization signal, indicated in brown; NES, nuclear export signal, indicated in pink. (B) Confocal microscopy images showing the subcellular localization of CcCAP1 and its modified mutants. Alteration of the subcellular localization of CcCAP1 was observed at 2 dpa, with the nucleus being stained with DAPI 3 h before confocal observation. n, nucleus. (C) Western blot analysis of proteins in N. benthamiana transiently expressing GFP control and GFP-tagged CcCAP1, CcCAP1-NLS, and CcCAP1-NES.

FIG 9

Significant role of the nuclear localization in immunoinhibiting activity of CcCAP1. (A) Representative infection symptoms, ROS accumulation, and callose deposition on leaves of EV, CcCAP1-pBinGFP2, CcCAP1-NLS-pBinGFP2, and CcCAP1-NES-pBinGFP2 agroinfiltrated N. benthamiana at 2 dpi with B. cinerea. (B) Quantification of lesion area, ROS, and callose intensity with ImageJ. (C) Relative transcriptional levels of defense-related genes were detected at 2 dpi with B. cinerea on leaves of EV, CcCAP1-pBinGFP2, CcCAP1-NLS-pBinGFP2, or CcCAP1-NES-pBinGFP2 agroinfiltrated N. benthamiana. This experiment was performed three times with similar results. Each assay was performed on at least six independent biological repeats. SPSS v16.0 was used to analyze the experimental data, and Duncan’s test at P = 0.05 was used to determine the differences. Bars indicate ± SE. The letters above the error bars indicate the different groups with statistical significance (P ≤ 0.01, P ≤ 0.05).

FIG 10

Hypothesis of CcCAP1 functions during C. chrysosperma-host interaction. Inductively expressed CcCAP1 proteins were first transported to the apoplastic space; they then translocate to the cytoplasmic space and finally to the nucleus to inhibit the plant immunity.