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. 2022 Dec 17;24(3):208–220. doi: 10.1111/mpp.13287

Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection

Ya‐Juan Shi 1, Xue Yang 1, Ling‐Ling Yang 1, Qing‐Lun Li 1, Xiao‐Min Liu 2, Xiao‐Yu Han 1, Qin‐Sheng Gu 3, Hong‐Lian Li 1, Lin‐Lin Chen 1, Yiqing Liu 4, Yan Shi 1,✉
PMCID: PMC9923391  PMID: 36528386

Abstract

The movement protein (MP) and coat protein (CP) of tobamoviruses play critical roles in viral cell‐to‐cell and long‐distance movement, respectively. Cucumber green mottle mosaic virus (CGMMV) is a member of the genus Tobamovirus. The functions of CGMMV MP and CP during viral infection remain largely unclear. Here, we show that CGMMV MP can interact with CP in vivo, and the amino acids at positions 79–128 in MP are vital for the MP–CP interaction. To confirm this finding, we mutated five conserved residues within the residue 79–128 region and six other conserved residues flanking this region, followed by in vivo interaction assays. The results showed that the conserved threonine residue at the position 107 in MP (MPT107) is important for the MP–CP interaction. Substitution of T107 with alanine (MPT107A) delayed CGMMV systemic infection in Nicotiana benthamiana plants, but increased CGMMV local accumulation. Substitutions of another 10 conserved residues, not responsible for the MP–CP interaction, with alanine inhibited or abolished CGMMV systemic infection, suggesting that these 10 conserved residues are possibly required for the MP movement function through a CP‐independent manner. Moreover, two movement function‐associated point mutants (MPF17A and MPD97A) failed to cause systemic infection in plants without impacting on the MP–CP interaction. Furthermore, we have found that co‐expression of CGMMV MP and CP increased CP accumulation independent of the interaction. MP and CP interaction inhibits the salicylic acid‐associated defence response at an early infection stage. Taken together, we propose that the suppression of host antiviral defence through the MP–CP interaction facilitates virus systemic infection.

Keywords: accumulation, coat protein, cucumber green mottle mosaic virus, interaction, movement protein

Cucumber green mottle mosaic virus movement protein and coat protein interaction facilitates viral systemic infection, possibly via inhibiting the salicylic acid‐associated defence response at an early stage.

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1. INTRODUCTION

Cucumber green mottle mosaic virus (CGMMV) was first found to cause disease in cucumber in England. Currently, this virus has been reported as a major threat to cucurbit production worldwide (Al‐Shahwan & Abdalla, 1992; Antignus et al., 1990; Dombrovsky et al., 2017; Hilf & Dawson, 1993; Nagendran et al., 2015; Rajamony et al., 1990; Sugiyama et al., 2006; Wang & Chen, 1985).

Cucumber green mottle mosaic virus is a member of the genus Tobamovirus, family Virgaviridae. The genome of CGMMV is a single‐stranded, positive‐sense RNA of about 6.4 kb (Ugaki et al., 1991). CGMMV genomic RNA encodes four proteins, namely the 186, 129, 29, and 17.3 kDa proteins. Both 129 and 186 kDa proteins are known to participate in CGMMV replication and suppression of RNA silencing (Chen et al., 2020). The 29 and 17.3 kDa proteins are known as the movement protein and coat protein (MP and CP), respectively.

Tobamoviruses cause significant economic damage to crop production (Ishikawa et al., 2022; Ruiz et al., 2021; Sofy et al., 2021). The study of tobamovirus has greatly contributed to the development of plant virology (Fraile & García‐Arenal, 2018). The MP of tobacco mosaic virus (TMV) is best‐studied, and is required for TMV cell‐to‐cell movement and plasmodesmata (PD) targeting (Heinlein, 2015). In addition, TMV MP has been found to cause a stress‐like response in plants (Dombrovsky et al., 2017). Apart from encapsidation of viral RNA, tobamovirus CPs are required for viral long‐distance movement (Callaway et al., 2001) as well as suppression of salicylic acid (SA)‐responsive gene expression (Conti et al., 2012; Rodriguez et al., 2014). Down‐regulation of the SA signalling pathway by TMV CP is also required for virus systemic infection and stabilization of DELLA proteins to promote TMV systemic infection (Venturuzzi et al., 2021). Recent studies on CGMMV have found that the amino acid residues at positions 96 and 89 in the CP can affect virus infectivity (Liu et al., 2020; Zhang et al., 2018). TMV CP and MP interaction was observed in cells in the presence of N‐terminus of yellow fluorescent protein and C‐terminus of YFP (Bazzini et al., 2007). The CP of citrus yellow vein clearing virus interacts with all the three movement proteins (TGBp1, TGBp2, and TGBp3), and CP colocalizes with TGBp1 and TGBp3 at the PD of epidermal cells of Nicotiana benthamiana leaves (Ur Rehman et al., 2019). The MP of Sesbania mosaic virus (SeMV) interacts with its CP (Chowdhury & Savithri, 2011).

In contrast to the earlier reports on TMV MP and CP, studies on CGMMV MP and CP and their interaction during virus infection is very limited. In this study we have found that CGMMV MP and CP can interact with each other, and the key MP amino acid residues responsible for the interaction with CP have been determined. We have found that the movement function of MP is dispensable for the MP–CP interaction, and the MP–CP interaction promotes disease symptom development. Furthermore, we have also found that MP and CP co‐expression can increase CP accumulation. The MP–CP interaction inhibits SA‐associated defence response at early infection stage. We propose that the suppression of host antiviral defence through the MP–CP interaction facilitates CGMMV systemic infection.

2. RESULTS

2.1. Interaction between CGMMV MP and CP in vivo

The interaction between CGMMV P2 (the 57 kDa readthrough part between 129 and 186 kDa), MP, and CP was tested in N. benthamiana leaf cells using bimolecular fluorescence complementation (BiFC) assays. Agrobacteria harbouring constructs pMP‐nYFP (N‐terminus of YFP), pMP‐cYFP (C‐terminus of YFP), pCP‐nYFP, pCP‐cYFP, and pP2‐cYFP were co‐infiltrated, in various combinations, into N. benthamiana leaves. The combinations of pMP‐nYFP and pP2‐cYFP, pCP‐nYFP and pP2‐cYFP were used as negative controls. By 2 days post‐agro‐infiltration (dpai), strong YFP fluorescence was observed in the cells in N. benthamiana leaves co‐infiltrated with pMP‐nYFP and pCP‐cYFP, pCP‐cYFP and pCP‐nYFP, or pCP‐nYFP and pMP‐cYFP. The strong YFP fluorescence appeared as punctate signals in different parts of the cytoplasm (Figure 1a). Furthermore, co‐immunoprecipitation (Co‐IP) assays indicated that MP interacted with CP in vivo (Figure 1b).

FIGURE 1.

FIGURE 1

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Cucumber green mottle mosaic virus (CGMMV) movement protein (MP) can interact with coat protein (CP) in vivo. (a) CGMMV MP and CP were fused to the N‐terminus of YFP (MP‐nYFP and CP‐nYFP) and transiently co‐expressed with CP‐cYFP, P2‐cYFP or MP‐cYFP in Nicotiana benthamiana leaf cells. The infiltrated leaves were harvested at 2 days post‐agro‐infiltration (dpai), and examined and imaged under a LSM710 laser scanning microscope. Bar = 20 μm. (b) The interaction analysis of MP and CP in vivo by co‐immunoprecipitation (Co‐IP). N. benthamiana leaves were co‐infiltrated with Agrobacterium tumefaciens cells harbouring expression vectors to express CP‐YFP and Myc‐MP, CP‐YFP and Myc‐GUS. Leaf protein extracts were incubated with GFP beads. Samples before (input) and after (IP) immunoprecipitation were analysed by immunoblotting using anti‐GFP or anti‐Myc antibody

To further confirm the interaction between CGMMV MP and CP, we co‐expressed YFP fused to the C‐terminus of CP (CP‐YFP) and red fluorescent protein [RFP] fused to the C‐terminus of MP (MP‐RFP) fusions (CP‐YFP + MP‐RFP) in cells of N. benthamiana leaves. As shown in Figure 2a,b, the MP‐RFP was indeed colocalized with CP‐YFP in the cytoplasm. The colocalization of CP‐YFP and MP‐RFP was further verified through coefficient analysis (Figure 2c) as previously reported (Zinchuk & Grossenbacher‐Zinchuk, 2009). When MP‐RFP or CP‐YFP was expressed individually in N. benthamiana cells, the MP‐RFP fluorescence appeared as punctate signals in the cytoplasm, while the CP‐YFP fluorescence appeared as free signals in the cytoplasm and nucleus (Figure 2d,e).

FIGURE 2.

FIGURE 2

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Colocalization of MP‐RFP and CP‐YFP in Nicotiana benthamiana leaf cells. N. benthamiana leaves were agro‐infiltrated with Agrobacterium cultures carrying plasmid pMP‐RFP, pCP‐YFP, or both. (a) Confocal images showing colocalization of MP‐RFP and CP‐YFP in cells. Merged image shows fluorescent punctate signals near the cell walls. Images were taken at 2 days postinfiltration under a LSM710 laser scanning microscope. Bar = 20 μm. (b) Enlarged images from (a) show the colocalization of MP‐RFP and CP‐YFP. Bar = 5 μm. (c) Colocalization of MP‐RFP and CP‐YFP was confirmed through coefficient value analysis. (d) Images show CP‐YFP localization in cell periphery and nucleus of H2B‐RFP transgenic N. benthamiana leaf cells. Bar = 10 μm. (e) Images show MP‐RFP localization in N. benthamiana leaf cells. Bar represents 10 μm

2.2. Amino acid residues 79–128 in the MP are responsible for the interaction with CP

To determine which domain in the MP is necessary for the interaction with CP, we first fused the MP N terminal 50 amino acid residues to the N terminus of YFP to produce pMP1‐50‐nYFP and the amino acid residues 51–264 of the MP to the C terminus of YFP to produce pMP51‐264‐cYFP, based on a report by Yuan et al. (2016). Plasmid combinations of pMP1‐50‐nYFP/CP‐cYFP and pMP51‐264‐cYFP/CP‐nYFP were co‐expressed in N. benthamiana leaves (Figure 3a). Co‐infiltration of MP1‐50‐nYFP and CP‐cYFP into N. benthamiana leaves yielded YFP fluorescence in cells by 2 dpi (Figure 3b). Co‐expression of MP51‐264‐cYFP and CP‐nYFP in N. benthamiana leaf cells produced fluorescent punctate signals near the cell walls. Further analyses of the interactions between MP51‐182‐cYFP and CP‐nYFP, and MP183‐264‐cYFP and CP‐nYFP indicated that the residues 51–182 of the MP were responsible for formation of the fluorescent punctate signals near the cell walls (Figure 3b). Analysis of the MP amino acid sequence using MEME online software (https://meme‐suite.org/meme/tools/meme) identified three motifs in the region encompassing amino acid residues 51–182 (Figure 3c). These three fragments were cloned into the p35S‐nYFP vector to produce plasmids pMP50‐78‐nYFP, pMP79‐128‐nYFP, and pMP130‐163‐nYFP, and were then co‐expressed, individually, with CP‐cYFP in N. benthamiana leaves through agro‐infiltration. By 2 dpi, the leaves co‐infiltrated with pMP79‐128‐nYFP and pCP‐cYFP produced fluorescent punctate signals near the cell walls, while the leaves co‐infiltrated with pMP50‐78‐nYFP and pCP‐cYFP or pMP130‐163‐nYFP and pCP‐cYFP did not (Figure 3d), indicating that the motif encompassing residues 79–128 is crucial for the interaction between MP and CP.

FIGURE 3.

FIGURE 3

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Identification of the movement protein–coat protein (MP–CP) interacting domain in cucumber green mottle mosaic virus MP. (a) Schematic representations of the four MP regions. (b) Determination of MP–CP interaction through bimolecular fluorescence complementation (BiFC) using truncated MP mutants. Nicotiana benthamiana leaves were infiltrated with Agrobacterium cultures carrying plasmids pCP‐cYFP and pMP1‐50‐nYFP, pCP‐nYFP and pMP51‐264‐cYFP, pCP‐nYFP and pMP51‐182‐cYFP, pCP‐nYFP and pMP183‐264‐cYFP. The infiltrated leaves were harvested at 2 days post‐agro‐infiltration (dpai), and examined and imaged under a LSM710 laser scanning microscope. Bar = 10 μm. (c) Schematic representation of the three conserved motifs (positions 50–78, 79–128, and 130–163) was prepared using the MEME online software (https://meme‐suite.org/meme/tools/meme). (d) BiFC analysis of the interaction between CP and MP conserved motifs. N. benthamiana leaves were infiltrated with Agrobacterium cultures carrying plasmids pCP‐cYFP and pMP50‐78‐nYFP, pCP‐cYFP and pMP79‐128‐nYFP, or pCP‐cYFP and pMP130‐163‐nYFP. The infiltrated leaves were harvested at 2 dpai, and examined and imaged under a LSM710 laser scanning microscope. Bar = 10 μm

2.3. Residue T107 in the MP is important for the interaction with CP

To determine the key amino acid in the MP that is necessary for the interaction with CP, we performed an alignment using the amino acid sequence of CGMMV MP and that of other tobamovirus MPs with the online PROMALS3D software (http://prodata.swmed.edu/promals3d/promals3d.php) (Figure S1). Five conserved amino acids (C87, R99, E105, T107, and Y128) in the MPs were found to harbour hydroxyl groups and were substituted with alanine. The mutated sequences were cloned individually into the p35S‐nYFP vector (Figure 4a). After co‐infiltration of individual mutant MP constructs with pCP‐cYFP into N. benthamiana leaves, YFP fluorescence was examined in the leaves co‐expressing one of the five mutant MPs and the CP‐cYFP (Figures 4b and S2). The result showed that co‐expression of MPT107A‐nYFP and CP‐cYFP in N. benthamiana leaf cells failed to produce YFP fluorescence (Figure 4b). To further confirm the above findings, we constructed plasmid MPT107A‐cYFP and co‐infiltrated it with pCP‐nYFP into N. benthamiana leaves. As expected, no YFP fluorescence was observed (Figure S2). We then substituted the other six conserved amino acid residues (E50, S51, Y69, S78, D134, E161) with alanine to produce pMPES5051AA‐nYFP, pMPY69A‐nYFP, pMPS78A‐nYFP, pMPD134A‐nYFP, and pMPE161A‐nYFP. These mutant fusions were then individually co‐expressed with CP‐cYFP in N. benthamiana leaf cells. In this experiment, YFP fluorescence was observed in cells of all the assayed leaves (Figures 4c and S2). Furthermore, Co‐IP assays indicated that MP interacted with CP in N. benthamiana leaves, while a very weak band was observed for the combination of MPT107A and CP. These results indicated that residue 107 in the MP is important for the interaction with CP (Figure 4d).

FIGURE 4.

FIGURE 4

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The amino acid residue at the position 107 in movement protein (MP) is important for the MP–coat protein (CP) interaction. (a) Amino acid sequence of cucumber green mottle mosaic virus MP (GenBank accession no. KY753929.1). Conserved residues are shown in bold and red letters. Substitutions of the conserved amino acids with alanine in different MP mutants. (b, c) Bimolecular fluorescence complementation analysis of interactions between CP‐cYFP and one of the 10 MP‐nYFP mutants. Nicotiana benthamiana leaves infiltrated with Agrobacterium cultures carrying plasmid pCP‐cYFP and one of the 10 pMP‐nYFP mutants were harvested at 2 days postinfiltration, and examined and imaged under a LSM710 laser scanning microscope. Bar = 10 μm. (d) The interaction analysis of MP–CP and MPT107A–CP in vivo by co‐immunoprecipitation. N. benthamiana leaves were co‐infiltrated with Agrobacterium tumefaciens cells harbouring expression vectors to express CP‐YFP and Myc‐GUS (lane 1 for IP, lane 3 for input), CP‐YFP and Myc‐MP (lane 2), CP‐YFP and Myc‐MPT107A (lane 3 for IP, lane 1 for input). Leaf protein extracts were incubated with Myc beads. Samples before (input) and after (IP) immunoprecipitation were analysed by immunoblotting using anti‐GFP or anti‐Myc antibody

2.4. MP and CP interaction affects disease symptom development

To determine the biological function caused by the MP and CP interaction during CGMMV infection, we constructed mutant CGMMVMPT107A and inoculated it to N. benthamiana plants through agro‐infiltration. The CGMMV‐inoculated N. benthamiana plants showed mosaic symptoms by 11 days postinoculation (dpi), while the CGMMVMPT107A‐inoculated plants did not show virus symptoms by 11 dpi (Figure 5a). Western blot and reverse transcription‐quantitative PCR (RT‐qPCR) analysis results showed that CGMMV viral RNA and CP accumulations were significantly reduced in the upper leaves of the CGMMVMPT107A‐inoculated plants compared to that in CGMMV‐inoculated plants, indicating that disruption of MP and CP interaction inhibited the systemic infection of CGMMV (Figure 5b,c). Time‐course analysis showed that the disease symptoms and CP systemic accumulation were inhibited in CGMMVMPT107A‐inoculated N. benthamiana plants compared with CGMMV‐inoculated plants (Figure S3). We analysed the CP accumulation on the inoculated leaves. Time‐course analysis showed that the accumulation levels of CGMMV CP in the CGMMVMPT107A‐inoculated leaves were higher than that in the CGMMMV‐inoculated leaves from 4 to 7 dpi (Figure 5d). This finding indicates that the MP–CP interaction promotes CGMMV systemic infection.

FIGURE 5.

FIGURE 5

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Interaction between cucumber green mottle mosaic virus (CGMMV) movement protein (MP) and coat protein (CP) affects viral accumulation and symptom induction. (a) Observations of Nicotiana benthamiana plants infected with the wildtype CGMMV and the CGMMVMPT107A mutant at 11 days postinoculation (dpi). (b, c) Detection of CGMMV RNA and CP accumulations in the systemically infected leaves. Upper leaves were harvested from the CGMMV‐ or CGMMVMPT107A‐inoculated plants at 11 dpi. The accumulation of CGMMV RNA was determined through reverse transcription‐quantitative PCR (RT‐qPCR) using CGMMV‐specific primers. The expression level of NbUBC was used as an internal control in this experiment. The accumulation of viral CP in these samples was determined through western blot assay using a CP‐specific antibody. The Coomassie brilliant blue (CBB)‐stained RuBisCO large subunit protein gel was used to show sample loadings. The RT‐qPCR result is presented as the means ± SD from three biological replicates per treatment. **Significant difference at p < 0.01. (d) Time‐course analysis of CGMMV CP accumulation in inoculated leaves through western blot assay. The Coomassie brilliant blue‐stained RuBisCO large subunit protein gel is used to show sample loadings

2.5. Conserved amino acids in MP are crucial for viral accumulation

To determine the roles of the other 10 conserved amino acids in MP, which have no impact on MP–CP interaction, on CGMMV accumulation, we constructed nine CGMMV MP mutants. By 14 dpi, CGMMVMPY69A, CGMMVMPS78A, CGMMVMPC87A, and CGMMVMPE161A mutants caused milder mosaic symptoms in N. benthamiana plants compared to the wildtype CGMMV. The other five mutant viruses (CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A) caused no disease symptoms in the inoculated N. benthamiana plants by 14 dpi (Figure 6a). Then we tested CGMMV accumulation in the upper leaves of the plants inoculated with wildtype and CGMMV mutants through western blot and RT‐qPCR. Consistent with the disease symptom observation result, mutants CGMMVMPY69A, CGMMVMPS78A, CGMMVMPC87A, and CGMMVMPE161A accumulated to much lower levels than the wildtype CGMMV. CGMMV viral RNA and CP were not detected in the upper leaves of the plants inoculated with CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A (Figure 6b,c). By 21 dpi, symptoms on N. benthamiana plants inoculated with CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A were still not obvious (Figure S4). We further analysed the viral RNA accumulation of the five mutants on the inoculated leaves at 6 dpi. The viral accumulation in the leaves inoculated with mutants CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A was significantly lower than that in the CGMMMV‐inoculated leaves (Figure S5), indicating these mutants were possibly impaired in MP movement activity.

FIGURE 6.

FIGURE 6

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Conserved amino acids in cucumber green mottle mosaic virus (CGMMV) movement protein (MP) are crucial for viral accumulation. (a) Observations of Nicotiana benthamiana plants inoculated with the wildtype CGMMV and one of the nine CGMMV MP mutants at 14 days postinoculation (dpi). (b, c) Determination of CGMMV RNA and coat protein (CP) accumulation in the upper leaves. Upper leaves of the plants inoculated with the wildtype CGMMV or nine mutants were harvested and analysed at 14 dpi. The accumulation of CGMMV RNA was determined through reverse transcription‐quantitative PCR (RT‐qPCR) using CGMMV‐specific primers. The expression of NbUBC was used as an internal control. The accumulation of CGMMV CP in these samples was determined through western blot assay using a CP‐specific antibody. The Coomassie brilliant blue (CBB)‐stained RuBisCO large subunit protein gel is used to show sample loadings. The RT‐qPCR results are presented as the means ± SD from three biological replicates per treatment. The statistical significance between the treatments was determined using Student's t test (p < 0.05)

2.6. MP movement activity‐associated residue is dispensable for MP and CP interaction

PD localization is a typical MP function. A previous study by Yuan and colleagues indicated that the phenylalanine residue at position 14 in TMV MP is responsible for PD localization (Yuan et al., 2018). In this study, we found the corresponding phenylalanine residue in the CGMMV MP is located at amino acid position 17. We also found the specific motif LXD/X50‐70G (Mann et al., 2016) in the CGMMV MP (Figure S6). Inoculation of CGMMVMPF17A and CGMMVMPD97A mutants to leaves of N. benthamiana plants did not cause systemic symptoms in the plants by 30 dpi, while the plants inoculated with the wildtype CGMMV showed systemic mosaic symptoms by 10 dpi (Figure 7a). Analyses of systemically infected leaf tissues harvested at 14 dpi from the assayed plants through semiquantitative RT‐PCR (sqRT‐PCR) and western blot showed that substitution of D97 with alanine significantly reduced the accumulation of CGMMV, and substitution of F17 with alanine abolished the systemic accumulation of CGMMV in upper leaves (Figure 7b,c), indicating the importance of MP movement activity in viral systemic infection. We analysed the viral RNA accumulation on the inoculated leaves at 6 dpi. The viral accumulation levels in leaves inoculated with the CGMMVMPF17A and CGMMVMPD97A mutantswere lower than that in the CGMMMV‐inoculated leaves (Figure S7). To investigate the role of MP–CP interaction in MP movement activity, we tested the interaction between one of the two MP mutants and CP through BiFC. The results showed that co‐expression of CP‐nYFP and MPD97A‐cYFP or CP‐nYFP and MPF17A‐cYFP produced fluorescent punctate signals, similar to that found in the leaves co‐expressing CP‐nYFP and MP‐cYFP, indicating that the residues responsible for MP movement are dispensable for the interaction between CGMMV MP and CP (Figure 7d).

FIGURE 7.

FIGURE 7

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Movement protein (MP) movement function was dispensable for the MP–coat protein (CP) interaction. (a) Phenotypes of Nicotiana benthamiana plants inoculated with the wildtype cucumber green mottle mosaic virus (CGMMV) and one of the two MP mutants (CGMMVMPF17A and CGMMVMPD97A) at 14 days postinoculation (dpi). (b, c) Detection of CGMMV CP and RNA accumulation in systemically infected leaves. Upper leaves were harvested from the plants inoculated with the wildtype CGMMV and one of the two MP mutants at 14 dpi. The accumulation of CGMMV CP was detected through western blot assay using a CGMMV CP‐specific antibody. The Coomassie brilliant blue (CBB)‐stained RuBisCO large subunit protein gel was used to show sample loadings. The expression of NbUBC in these samples was used as an internal control. The accumulation of CGMMV RNA was detected through semiquantitative RT‐PCR using CGMMV‐specific primers. (d) Bimolecular fluorescence complementation analysis of the interaction between CP‐nYFP and one of the two MP mutants. N. benthamiana leaves were co‐infiltrated with Agrobacterium cultures carrying plasmid pMP17‐cYFP and pCP‐nYFP, pMP97‐cYFP and pCP‐nYFP, pMP17‐cYFP and pnYFP, or pMP97‐cYFP and pnYFP. The infiltrated leaves were harvested and imaged at 2 days post‐agro‐infiltration under a LSM710 laser scanning microscope. Bar = 10 μm

2.7. Co‐expression of MP and CP increases CP accumulation and interaction between MP and CP inhibits SA‐related gene expression at an early infection stage

To investigate the role of MP and CP interaction on CGMMV infection, we tested the stabilities of MP and CP after co‐expression. FLAG‐MP and CP‐YFP, FLAG‐MP and YFP or Myc‐GUS and CP‐YFP, Myc‐MP and CP‐YFP were transiently co‐expressed in N. benthamiana leaves, and the infiltrated leaves were harvested and analysed for protein expression through western blot at 36 h post‐agro‐infiltration (hpai). The results showed that the expression of MP significantly increased the accumulation of CP (Figure 8a), while the expression of CP slightly suppressed the expression of MP (Figure 8b), suggesting that MP and CP co‐expression could affect the accumulation of MP and CP. We further tested the effect of co‐expression of MPT107A and CP on the accumulation of MP and CP (Figure 8a,c). The results indicated that MPT107A mutant expression showed a similar effect on CP accumulation as MP, indicating that the regulation of CP accumulation by MP was possibly not directly correlated with the interaction. A previous study using TMV showed that the CP of TMV can regulate the expression of SA‐responsive genes, which are required for TMV long‐distance movement (Conti et al., 2012; Venturuzzi et al., 2021). To evaluate the interaction on the expression of SA‐related genes in the N. benthamiana leaves, we analysed the expression levels of NPR1and PR1a. The results showed that the expression levels of NPR1 and PR1a were significantly up‐regulated in the CGMMVMPT107A‐inoculated systemic leaves compared with CGMMV and healthy control at 4 dpi. At 5 dpi, the expression levels of PR1a in both CGMMV‐inoculated and systemic leaves were significantly lower than in CGMMVMPT107A and control. This finding indicates that CGMMV MP–CP interaction could inhibit the SA‐associated defence response at an early infection stage (Figure S8).

FIGURE 8.

FIGURE 8

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Effects of co‐expression of movement protein (MP) and coat protein (CP), MPT107A and CP on the accumulation of CP or MP. Agrobacterium cultures harbouring Myc‐GUS and CP‐YFP, Myc‐MP and CP‐YFP, Myc‐MPT107A and CP‐YFP, FLAG‐MP and CP‐YFP, FLAG‐MP and YFP were infiltrated individually into Nicotiana benthamiana leaves. The accumulation of Myc‐GUS, Myc‐MP, Myc‐MPT107A, FLAG‐MP, CP‐YFP, and YFP in these infiltrated leaf tissues was determined through western blot assay using a GFP‐, Myc‐, or a FLAG‐specific antibody at 36 h postinfiltration. The Coomassie brilliant blue (CBB)‐stained RuBisCO large subunit protein gel was used to show sample loadings. (a) The effect of MP and MPT107A expression on the CP accumulation. The accumulation of CP‐YFP in these samples was detected through western blot assay using a GFP‐specific antibody. The accumulation of Myc‐MP, Myc‐MPT107A and Myc‐GUS in these samples were detected through western blot assay using an anti‐Myc antibody. (b) The effect of CP expression on the MP accumulation. The accumulation of CP‐YFP and YFP in these samples was detected through western blot assay using a GFP‐specific antibody. The accumulation of FLAG‐MP in these samples was detected through western blot assay using a FLAG‐specific antibody. (c) The effect of CP expression on the MPT107A accumulation. The accumulation of CP‐YFP and YFP in these samples was detected through western blot assay using a GFP‐specific antibody. The accumulation of Myc‐MPT107A in these samples was detected through western blot assay using a Myc‐specific antibody

3. DISCUSSION

Plant viruses have small genomes, and therefore encode only a few proteins with multiple functions, important for different steps of viral infections. Here we present evidence to show that CGMMV MP can interact with CP, and this interaction is possibly not linked to the movement ability of MP. The interaction is, however, involved in systemic infection, possibly through suppression of host antiviral defence.

The TMV CPT42W mutant virus is known to inhibit MP production or accumulation, while the wildtype CP shows the opposite effect (Bendahmane et al., 2002). TMV CP and MP interaction was observed in cells in the presence of CP‐nYFP and MP‐cYFP, but not in the presence of CP‐cYFP and MP‐nYFP (Bazzini et al., 2007). In our study, both combinations (i.e., CP‐cYFP and MP‐nYFP, and CP‐nYFP and MP‐cYFP) produced strong YFP fluorescence. In addition, we have found that the CGMMV MP and CP interaction site appeared as punctate signals in the cells, which is different from the interaction of TMV MP and CP.

According to previous reports on the MP of TMV, we have found two corresponding amino acid residues in the CGMMV MP, and these two conserved amino acid residues are phenylalanine (F) and aspartic acid (D) at positions 17 and 97, respectively (Yuan et al., 2018). As expected, substitution of F17 or D97 with alanine inhibited CGMMV systemic infection, but not the interaction between the mutant MPs and the CP. This finding indicates that the MP–CP interaction probably exerts other functions without impact on the movement activity of MP. Infection assays using non‐CP‐interacting MP mutant virus showed that the MP–CP interaction is possibly independent of MP movement activity. It is noteworthy that all the conserved amino acids of MP selected for mutational analyses without impact on the MP–CP interaction are crucial for CGMMV infection.

As reported previously, TMV MP is the second protein to accumulate during virus infection and is required for cell‐to‐cell movement through interaction with PD (Heinlein, 2015). After cell‐to‐cell movement to reach vascular tissues, TMV systemic spread is initiated with the participation of viral CP (Conti et al., 2017). How MP–CP interaction coordinates the switch from cell‐to‐cell movement to systemic infection is unknown. In our study, we further confirmed that the movement function of MP is indispensable for viral infection via two CGMMV MP‐specific mutants. We also found that the movement function of MP was not linked to its interaction with CP. Analysis of this interaction using MP deletion mutants further confirmed the above finding. The N‐terminal 50 amino acids in TMV MP have been reported as the TMV MP PD localization signal (Yuan et al., 2016) but are not essential for the MP and CP interaction.

In this study, we analysed the effect of CGMMV MP and CP interaction on their accumulation and CGMMV infection. Our results showed that the expression of both MP and MPT107A can positively regulate the expression of CP, indicating that the regulation of CP accumulation by MP was possibly not directly correlated with the interaction. Previous studies had shown that TMV CP can repress the host defence response (Callaway et al., 2001; Conti et al., 2017; Venturuzzi et al., 2021). Negative regulation of the SA signalling pathway by the CP of TMV is required for viral systemic movement (Venturuzzi et al., 2021). Based on the inhibition of the SA‐associated defence response at an early infection stage by MP–CP interaction, we speculate that the suppression of host antiviral defence through the MP–CP interaction possibly facilitates viral systemic movement.

4. EXPERIMENTAL PROCEDURES

4.1. Plant growth and virus inoculation

Wildtype and H2B‐transgenic N. benthamiana plants were grown in pots inside a growth room maintained at 25°С, 16 h light/8 h dark photoperiod, and 60% humidity. CGMMV was from a previously reported source (Liu et al., 2017). For agro‐infiltration, Agrobacterium tumefaciens GV3101 carrying individual infectious viral clones was suspended in an infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 μM acetosyringone, pH 5.6) to OD600 = 1, incubated at room temperature for 2–4 h, and then infiltrated into leaves of N. benthamiana using 1‐ml needleless syringes.

4.2. Amino acid sequence alignment

Sequences of 20 tobamovirus MPs were from the database at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) (O'Leary et al., 2016). Multiple sequence alignment was performed using DNAMAN software. Motifs in MP partial sequences were predicted using MEME software (https://meme‐suite.org/meme/tools/meme).

4.3. Plasmid construction

Detailed information about the constructions of plasmids is listed in the Table S1. All the resulting constructs were sequenced before use.

For BiFC analysis, coding sequences of CGMMV MP, CP, and P2 genes were individually amplified and cloned into the entry vector pGWC via the in‐fusion method (Zhou et al., 2018). The sequence representing the MP amino acids 1–50 (referred to as MP1–50), 51–182 (MP51–182), 50–78 (MP50–78), 51–264 (MP51–264), 79–128 (MP79–128), 130–163 (MP130–163), 183–264 (MP183–264), MPF17A, MPES50,51AA, MPY69A, MPS78A, MPC87A, MPD97A, MPR99A, MPE105A, MPT107A, MPY128A, MPD134A, and MPE161A were amplified individually and cloned into the Gateway Entry vector pGWC using ClonExpress II One Step Cloning Kit as instructed (Vazyme). The resulting clones were then used to clone into the Gateway vector p35S‐nYFP and p35S‐cYFP vectors through the LR reaction.

For subcellular localization and transient expression assays, MP and CP were cloned into the Gateway vector RFP and pEarleyGate101 to produce plasmid pMP‐RFP and pCP‐YFP, respectively. The MP coding region was also PCR amplified and cloned into the expression vector pGDFlag to produce pGDFlagMP.

To generate CGMMV MP mutants, the codons of E50, S51, Y69, S78, C87, R99, E105, T107, Y128, D134, and E161 were substituted with alanine (A) through overlapping PCR to produce plasmids pCGMMVMP ES50,51AA, pCGMMVMPY69A, pCGMMVMPS78A, pCGMMVMPC87A, pCGMMVMPR99A, pCGMMVMPE105A, pCGMMVMPT107A, pCGMMVMPY128A, pCGMMVMPD134A, and pCGMMVMPE161A.

4.4. Confocal laser scanning microscopy

For BiFC and colocalization assays, A grobacterium cultures carrying the corresponding plasmids were individually infiltrated into the leaves of N. benthamiana plants as described in the figure legends. The infiltrated leaves were detached at 48 hpai. Fluorescence signals were examined under an inverted confocal laser scanning microscope (LSM 710; Zeiss). The YFP and RFP were excited at 514 and 561 nm wavelengths, respectively.

4.5. Western blot analysis

The agro‐infiltrated leaves were harvested, and total protein was extracted from these leaves (0.2 g per sample) using an extraction buffer (20% glycerol, 20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM PMSF, and 1× protease inhibitor cocktail; Sigma‐Aldrich). The extracted protein samples were separated in SDS‐polyacrylamide gels through electrophoresis, followed by transferring them to nitrocellulose membranes. The membranes were then probed with a polyclonal anti‐CP antibody, an anti‐GFP antibody (TransGen), an anti‐Myc antibody (Sigma‐Aldrich) or an anti‐FLAG antibody (Sigma‐Aldrich), followed by a horseradish peroxidase‐conjugated secondary antibody. The detection signals were developed by addition of an ECL reagent (Vazyme) as instructed.

4.6. Quantitative and semiquantitative RT‐PCR

Total RNA was extracted from the harvested N. benthamiana leaf samples using TRIzol reagent (Invitrogen) and treated with RNase‐free DNase I. First‐strand cDNA was synthesized using 1 μg of total RNA (per 20 μl reaction), random primers, and M‐MLV reverse transcriptase. Ten‐fold diluted cDNA products and the SYBR Green master mix (Takara) were used for qPCR on an Eppendorf Real‐Time PCR system. The expression of NbUBC was used as an internal control. All the RT‐qPCR primers are listed in Table S1. The relative RNA level in each sample was calculated using the 2−ΔΔCt method (Livak & Schmittgen, 2001).

For sqRT‐PCR, a 307 bp fragment representing a partial sequence NbUBC and a 795 bp fragment representing the CGMMV MP gene was amplified. The expression level of NbUBC was used as an internal control. The sqRT‐PCR was performed as follows: predenaturation at 94°C for 5 min; 35 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s; followed by the final extension at 72°C for 10 min. The resulting products were analysed in 1% agarose gels through electrophoresis.

CONFLICT OF INTEREST

The authors declare no conflict of interests.

Supporting information

Figure S1 Multiple alignment using movement protein (MP) sequences of CGMMV and other tobamoviruses. Conservative residues were circled in red. The alignment was performed using the DNAMAN software

Click here for additional data file. (648.1KB, docx)

Figure S2 Bimolecular fluorescence complementation analysis of the interactions between individual movement protein (MP) mutants and cYFP, or MPT 107A‐cYFP and CP‐nYFP in Nicotiana benthamiana leaf cells. Images were taken at 2 days post‐agro‐infiltration under a LSM710 laser scanning microscope. Bar = 10 μm

Click here for additional data file. (189.5KB, docx)

Figure S3 Time‐course observation of Nicotiana benthamiana plants infected by the wildtype CGMMV and CGMMVMPT107A. (a) Observations of N. benthamiana plants infected with the wildtype CGMMV and the CGMMVMPT107A mutant at 14 days postinoculation (dpi), 17 dpi and 19 dpi. (b) Time‐course analysis of CGMMV coat protein (CP) accumulation in systemically infected leaves through western blot assay. The Coomassie brilliant blue‐stained RuBisCO large subunit protein gel was used to show sample loadings

Click here for additional data file. (161.4KB, docx)

Figure S4 Observations of Nicotiana benthamiana plants infected by CGMMV mutants at 21 days postinoculation

Click here for additional data file. (177.1KB, docx)

Figure S5 The accumulation levels of viral genomic RNA in CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A in inoculated leaves were determined through reverse transcription‐quantitative PCR using CGMMV‐specific primers. The expression level of NbUBC was used as an internal control in this experiment

Click here for additional data file. (62.6KB, docx)

Figure S6 Alignment of CGMMV movement protein (MP) and TMV MP amino acid sequences using the PROMALS3D software. Red circles indicate the key motifs and amino acids responsible for the MP movement function

Click here for additional data file. (222.3KB, docx)

Figure S7 The accumulation levels of CGMMV RNA in leaves inoculated with CGMMVMPF17A and CGMMVMPD97Awere determined through reverse transcription‐quantitative PCR using CGMMV‐specific primers. The expression level of NbUBC was used as an internal control in this experiment

Click here for additional data file. (47.6KB, docx)

Figure S8 Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the expression of salicylic acid‐responsive genes in the Nicotiana benthamiana plants infected by the wildtype CGMMV or CGMMVMPT107A. The expression of NbUBC was used as an internal control. The RT‐qPCR results are presented as the means ± SD from three biological replicates per treatment. The statistical significance between the treatments was determined using the Student’s t test (p < 0.05)

Click here for additional data file. (176.5KB, docx)

Table S1 Primers used in this study

Click here for additional data file. (20.5KB, docx)

ACKNOWLEDGEMENTS

This work was funded by the Henan Science Fund for Excellent Young Scholars (grant 202300410194), the National Natural Science Foundation of China (3210170372), and the Key Specialized Research and Development Breakthrough Project in Henan province (212102110451). We would like to thank Dr Xinshun Ding for critical reading of the manuscript.

Shi, Y.‐J. , Yang, X. , Yang, L.‐L. , Li, Q.‐L. , Liu, X.‐M. & Han, X.‐Y. et al. (2023) Interaction between cucumber green mottle mosaic virus MP and CP promotes virus systemic infection Molecular Plant Pathology, 24, 208–220. Available from: 10.1111/mpp.13287

Ya‐Juan Shi and Xue Yang contributed equally to this work.

DATA AVAILABILITY STATEMENT

The data that support the finding of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Multiple alignment using movement protein (MP) sequences of CGMMV and other tobamoviruses. Conservative residues were circled in red. The alignment was performed using the DNAMAN software

Click here for additional data file. (648.1KB, docx)

Figure S2 Bimolecular fluorescence complementation analysis of the interactions between individual movement protein (MP) mutants and cYFP, or MPT 107A‐cYFP and CP‐nYFP in Nicotiana benthamiana leaf cells. Images were taken at 2 days post‐agro‐infiltration under a LSM710 laser scanning microscope. Bar = 10 μm

Click here for additional data file. (189.5KB, docx)

Figure S3 Time‐course observation of Nicotiana benthamiana plants infected by the wildtype CGMMV and CGMMVMPT107A. (a) Observations of N. benthamiana plants infected with the wildtype CGMMV and the CGMMVMPT107A mutant at 14 days postinoculation (dpi), 17 dpi and 19 dpi. (b) Time‐course analysis of CGMMV coat protein (CP) accumulation in systemically infected leaves through western blot assay. The Coomassie brilliant blue‐stained RuBisCO large subunit protein gel was used to show sample loadings

Click here for additional data file. (161.4KB, docx)

Figure S4 Observations of Nicotiana benthamiana plants infected by CGMMV mutants at 21 days postinoculation

Click here for additional data file. (177.1KB, docx)

Figure S5 The accumulation levels of viral genomic RNA in CGMMVMPES50,51AA, CGMMVMPR99A, CGMMVMPE105A, CGMMVMPY128A, and CGMMVMPD134A in inoculated leaves were determined through reverse transcription‐quantitative PCR using CGMMV‐specific primers. The expression level of NbUBC was used as an internal control in this experiment

Click here for additional data file. (62.6KB, docx)

Figure S6 Alignment of CGMMV movement protein (MP) and TMV MP amino acid sequences using the PROMALS3D software. Red circles indicate the key motifs and amino acids responsible for the MP movement function

Click here for additional data file. (222.3KB, docx)

Figure S7 The accumulation levels of CGMMV RNA in leaves inoculated with CGMMVMPF17A and CGMMVMPD97Awere determined through reverse transcription‐quantitative PCR using CGMMV‐specific primers. The expression level of NbUBC was used as an internal control in this experiment

Click here for additional data file. (47.6KB, docx)

Figure S8 Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the expression of salicylic acid‐responsive genes in the Nicotiana benthamiana plants infected by the wildtype CGMMV or CGMMVMPT107A. The expression of NbUBC was used as an internal control. The RT‐qPCR results are presented as the means ± SD from three biological replicates per treatment. The statistical significance between the treatments was determined using the Student’s t test (p < 0.05)

Click here for additional data file. (176.5KB, docx)

Table S1 Primers used in this study

Click here for additional data file. (20.5KB, docx)

Data Availability Statement

The data that support the finding of this study are available from the corresponding author upon reasonable request.

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