NIa-Pro of sugarcane mosaic virus targets Corn Cysteine Protease 1 (CCP1) to undermine salicylic acid-mediated defense in maize

Abstract

Papain-like cysteine proteases (PLCPs) play pivotal roles in plant defense against pathogen invasions. While pathogens can secrete effectors to target and inhibit PLCP activities, the roles of PLCPs in plant-virus interactions and the mechanisms through which viruses neutralize PLCP activities remain largely uncharted. Here, we demonstrate that the expression and activity of a maize PLCP CCP1 (Corn Cysteine Protease), is upregulated following sugarcane mosaic virus (SCMV) infection. Transient silencing of CCP1 led to a reduction in PLCP activities, thereby promoting SCMV infection in maize. Furthermore, the knockdown of CCP1 resulted in diminished salicylic acid (SA) levels and suppressed expression of SA-responsive pathogenesis-related genes. This suggests that CCP1 plays a role in modulating the SA signaling pathway. Interestingly, NIa-Pro, the primary protease of SCMV, was found to interact with CCP1, subsequently inhibiting its protease activity. A specific motif within NIa-Pro termed the inhibitor motif was identified as essential for its interaction with CCP1 and the suppression of its activity. We have also discovered that the key amino acids responsible for the interaction between NIa-Pro and CCP1 are crucial for the virulence of SCMV. In conclusion, our findings offer compelling evidence that SCMV undermines maize defense mechanisms through the interaction of NIa-Pro with CCP1. Together, these findings shed a new light on the mechanism(s) controlling the arms races between virus and plant.

Fig 1. SCMV infection up-regulates the expression of CCP1 and alters the activities of maize PLCPs.

A) The expression of CCP1 in maize was determined using quantitative reverse transcription PCR (RT-qPCR) at 5, 7, 10, 12 and 14 days post inoculation (dpi) of SCMV (gray bars). The plants inoculated with phosphate buffer (Mock, black bars) were used as controls. Statistical differences (*, P < 0.05; **, P < 0.01) were evaluated by Student’s t test analysis. B) The accumulation of CCP1 precursor (preCCP1) in maize was determined by immunoblotting assay using antibody specific to CCP1 or SCMV CP at 5, 7, 10 and 12 dpi. C) The relative amount of preCCP1 was quantified by software ImageJ, normalized to actin control. And the lanes of 5 dpi Mock were set to 1.0. Data were presented as means ± SE (n = 4). Statistical differences (***, P < 0.001) were evaluated by two-tailed Student’s t test analysis. D) The protease activities of maize PLCPs were determined by an activity-based protein profiling (ABPP). Only the active forms of PLCPs were labeled with DCG-04 and then detected using a streptavidin-conjugated with horseradish peroxidase (HRP). E) The relative accumulation of active PLCPs was quantified by software ImageJ, normalized to actin control. And the lanes of 5 dpi Mock were set to 1.0. Data were presented as means ± SE (n = 4). Statistical differences (***, P < 0.001) were evaluated by two-tailed Student’s t test analysis. Three independent experiments are conducted.

Fig 2. Knockdown of CCP1 in maize plants enhanced the accumulation level of SCMV.

A) The CCP1-silenced plants showed stronger leaf chlorosis and plant stunting compared with the CMV-GUS control plants at 10 d post challenge inoculation with SCMV. Bar = 5 cm. B) RT-qPCR analysis of the relative accumulation levels of CCP1 and SCMV RNA in the SCMV first systemically infected leaves at 10 dpi. C) Immunoblotting analysis of CCP1 and SCMV CP accumulation levels. Actin was used as gel loading control. Band intensities were measured using software ImageJ. Numbers indicate the accumulation levels of CCP1 and SCMV CP normalized to actin control. D) Activity-based protein profiling (ABPP) analysis of the protease activities of maize PLCPs in the SCMV first systemically infected leaves at 10 dpi. Actin was used as gel loading control. Band intensities were measured using software ImageJ. Numbers indicate the accumulation levels of active PLCPs normalized to actin control. E) The relative accumulations of CCP1 (left), SCMV CP (middle) and active PLCPs (right) were quantified by software ImageJ, and the mean of CMV-GUS+SCMV was set to 1.0. Error bars represent the means ± SE. Statistical differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) were evaluated by two-tailed Student’s t test analysis. Three independent experiments are conducted.

Fig 3. Knockdown of CCP1 in maize plants compromises SA biosynthesis and decreases ZmPR1 and ZmPR5 expressions.

A) Appearance of maize plants silenced for CCP1 gene expression at 7 dpi. B) The contents of SA in the CMV-CCP1-inoculated or the CMV-GUS-inoculated maize plants at 7 d post challenge inoculation with SCMV or Mock. C) and D) Relative expression of ZmPR1 (C) and ZmPR5 (D) in the CMV-CCP1-inoculated or the CMV-GUS-inoculated maize plants. Three independent experiments were conducted with at least three biological replicates per treatment. Error bars represented the means ±SE. Significant differences between CMV-GUS and CMV-CCP1 infected plants are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) were evaluated by Unpaired Student’s t test analysis.

Fig 4. SCMV NIa-Pro interacts with the mature protease domain of CCP1 (mCCP1).

A) A schematic diagram showing the domain structure of CCP1. CCP1 contains a signal peptide (SP), an autoinhibitory prodomain (pro-domain) and a mature protease domain. Position of the catalytic triad (Cys, His and Asn) in the mCCP1 is indicated. Different forms of CCP1 are named as preCCP1, iCCP1, and mCCP1, respectively. B) Pair-wise direct Y2H assay was conducted to examine the interaction between mCCP1 and the individual SCMV-encoded proteins. Yeast cells grown on the QDO selective medium are the cells with a positive protein–protein interaction. Yeast cells grown on the DDO medium are the transformed cells. Yeast cells transformed with the empty vector (BD) are used as a negative control. BD, Gal4 Binding Domain; AD, Gal4 activation domain. The AD-T+BD-53 serves as the positive interaction control. C) Co-immunoprecipitation (Co-IP) assay was conducted to verify the interaction between mCCP1 and NIa-Pro in N. benthamiana leaves. Mutant mCCP1_{C161A,H303A,N330A} has three alanine substitutions at its catalytic triad. 3Flag-NIa-Pro was co-expressed with mCCP1-3Myc or mCCP1_{C161A,H303A,N330A}-3Myc in N. benthamiana leaves through agro-infiltration. Leaf tissues were harvested at 48 hours post agroinfiltration (hpai) for Co-IP analysis. Samples of plants co-expressing 3Flag-NIa-Pro and mRFP-3Myc were used as a negative control. Co-IP assay was performed using an anti-Flag affinity agarose gel. Protein samples (Input) and the immunoprecipitated protein samples (Flag-IP) were analyzed through immunoblotting assays using an anti-Flag or an anti-c-Myc antibody. D) Luciferase (Luc) complementation imaging (LCI) analysis of the interaction between NIa-Pro and mCCP1 in N. benthamiana leaves. The indicated plasmid pairs were transiently co-expressed in N. benthamiana. NLuc co-expressed with CLuc or CLuc-NIa -Pro, and mCCP1-NLuc co-expressed with CLuc, were used as negative controls. The luciferase activity was measured using a luminometer at 48 hpai. E) In vitro pull-down assay using a GST-tagged NIa-Pro to immunoprecipitate TF-tagged mCCP1 or mCCP1_{C161A,H303A,N330A}. GST-NIa-Pro was used as a bait in this assay and TF-mCCP1 or TF-mCCP1_{C161A,H303A,N330A} was used as a prey. The input and the pull-down protein samples were analyzed through immunoblotting assays using an anti-GST or an anti-His antibody. Please note that the His Abs did show unspecific binding for the GST and GST-NIa-Pro proteins. *, the protein bands corresponding to the expected TF-mCCP1. Purified GST was used as a negative control.

Fig 5. NIa-Pro inhibits CCP1 activity in vitro and vivo.

A) Relative proteolytic activity of CCP1 was measured by digestion of a fluorescent casein substrate after pre-treatment with 5 μM E-64, 0.5 μM recombinant GST-NIa-Pro or GST (as negative control). In this assay, mutant mCCP1_{C161A, H303A, N330A} with three alanine substitutions at its catalytic triad was used as an inactive control. Fluorescence at 485/530 nm (excitation/emission) was measured. The results are presented as means ± standard deviation (n = 3). Statistical differences between treatments were determined using the unpaired t-test. ****, P < 0.0001. B) ABPP was conducted to estimate the inhibitory effect of NIa-Pro on CCP1 protease activity. preCCP1-3Flag was transiently expressed in N. benthamiana leaves. Then the Flag-enriched proteins were incubated with 5 μM E-64 (as a positive control) or 1.0 μM purified GST-NIa-Pro followed by immunoblotting assay using a streptavidin-conjugated with horseradish peroxidase (HRP). ImageJ software was used to estimate the detection signal intensity. C) Expressing NIa-Pro via FoMV vector exhibited reduced PLCPs activity in maize. Active proteases were enriched using streptavidin beads and detected using streptavidin-HRP conjugates. Numbers indicate the accumulation levels of CCP1 and SCMV CP normalized to actin control. D) The relative accumulations of FoMV-overexpressing proteins (left), and active PLCPs (right) were quantified by software ImageJ, and the mean of FoMV-3Flag-GFP was set to 1.0. Error bars represent the means ± SE. Statistical differences (*, P < 0.05; **, P < 0.01) were evaluated by two-tailed Student’s t test analysis. Three independent experiments are conducted.

Fig 6. Identification of the key amino acids of NIa-Pro for interaction with CCP1.

A) Schematic representation of NIa-Pro in SCMV genome. SCMV protein: P1: the first protein; HC-Pro: Helper Component-proteinase; P3: the third protein; 6K1: the first 6K protein; CI: cytoplasmic inclusion protein, 6K2: the second 6K protein, VPg: viral genome-linked protein, NIa-Pro: nuclear inclusion protein a-proteinase, NIb: nuclear inclusion protein b, CP: coat protein, P3N-PIPO: the N-terminal half of P3 fused to Pretty Interesting Potyvirus Open Reading Frame. B) The predicted 3D structure of SCMV NIa-Pro shows a protease domain (Cartoon shape) and an inhibitor motif (the blue line indicated). C) LCI assay of the interaction between mCCP1 and pdNIa-Pro or imNIa-Pro in N. benthamiana leaves. The Agrobacterium strains carrying the indicated constructs were infiltrated into N. benthamiana leaves. D) LCI assay for identification of the amino acid within the imNIa-Pro essential for interaction with mCCP1. The Agrobacterium strains carrying the indicated constructs were infiltrated into N. benthamiana leaves. NLuc co-expressed with CLuc or CLuc-NIa-Pro-, and mCCP1-NLuc co-expressed with CLuc, were used as negative controls. The luciferase activity was measured using a luminometer at 48 hpai. E) ABPP analysis to estimate the inhibitory effect of different form NIa-Pro on CCP1 protease activity. preCCP1-3Flag or preCCP1_{C161A,H303A,N330A} (preCCP1_m)-3Flag was transiently expressed in N. benthamiana leaves. Then the Flag-enriched proteins were incubated with 5 μM E-64 (as a positive control) or 1.0 μM purified GST-NIa-Pro, GST-NIa-Pro_{K230A, D234A}(NIa-Pro_m), GST-imNIa-Pro and GST (as a negative control), followed by immunoblotting assay using a streptavidin-conjugated with horseradish peroxidase (HRP). PreCCP1_m-3Flag was used as an inactive control. ImageJ software was used to estimate the detection signal intensity.

Fig 7. Truncated NIa-Pro or key amino acids mutant NIa-Pro_{K230A, D234A} inhibited SCMV infection in maize plants.

A) GFP analysis of the N. benthamiana leaves agro-infiltrated with the wild type virus SCMV-GFP, the inhibitor motif of NIa-Pro-defective mutant SCMV-NIa-Pro_IMD-GFP, and substitutions mutant SCMV-NIa-Pro_{K230A, D234A}-GFP at 5 dpai. Under UV light, strong GFP fluorescence was evident in pSCMV-GFP and pSCMV-NIa-Pro_K230A,D234A-GFP infected areas. No GFP fluorescence was observed on pSCMV-NIa-Pro_IMD-GFP infected areas. B) RT-qPCR analysis of the relative accumulation levels of SCMV RNA in the SCMV agro-infiltrated leaves at 5 dpi. Error bars represent the means ± SE. Statistical differences (**, P < 0.01) were evaluated by Student’s t test analysis. Three independent experiments are conducted. C) Statistical analysis of the incidence of symptoms appearance on maize plants. D) Symptoms of maize plants infected with SCMV-GFP, and its NIa-Pro mutants SCMV-NIa-Pro_IMD-GFP or SCMV-NIa-Pro_{K230A, D234A}-GFP at 8 dpi. Bar = 5 cm.

Fig 8. The double mutant virus SCMV-NIa-Pro_{K230A, D234A}-GFP fails to induce CCP1 gene expression and activates PLCP activity.

A) RT-qPCR analysis of the relative accumulation levels of SCMV RNA in the SCMV-GFP- or SCMV-NIa-Pro_{K230A, D234A}-GFP- infected first systemically leaves at 8 dpi. B) Immunoblotting analysis of SCMV CP accumulation in the first systemically infected maize leaves. C) RT-qPCR analysis of the relative accumulation levels of CCP1 in the mock, SCMV-GFP- or SCMV-NIa-Pro_{K230A, D234A}-GFP- infected first systemically leaves. D) Immunoblotting analysis of CCP1 accumulation and activity-based protein profiling (ABPP) analysis of the protease activities of maize PLCPs in the first systemically infected leaves at 8 dpi. E) The contents of SA in the mock, SCMV-GFP- or SCMV-NIa-Pro_{K230A, D234A}-GFP- infected first systemically leaves. F) RT-qPCR analysis of the relative accumulation levels of PR1 and PR5 in the mock, SCMV-GFP- or SCMV-NIa-Pro_{K230A, D234A}-GFP- infected first systemically leaves. Error bars represent the means ± SE. Statistical differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001) were evaluated by two-tailed Student’s t test analysis. Three independent experiments are conducted.

Fig 9. Proposed model of NIa-Pro targeting Corn Cysteine Protease 1 (CCP1) for efficient infection of SCMV in maize.

CCP1 is a maize PLCP which confers resistance to SCMV infection via activation of SA signaling pathway. NIa-Pro is the main viral protease of SCMV. NIa-Pro can interact with CCP1 and inhibit its activity to counteract CCP1-mediated resistance and facilitate SCMV infection in maize plants.