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Phosphorylated viral protein evades plant immunity through interfering the function of RNA-binding protein

['Juan Li', 'College Of Agriculture', 'Biotechnology', 'Zhejiang University', 'Hangzhou', 'State Key Laboratory For Managing Biotic', 'Chemical Threats To The Quality', 'Safety Of Agro-Products', 'Institute Of Plant Virology', 'Ningbo University']

Date: 2022-05

Successful pathogen infection in plant depends on a proper interaction between the invading pathogen and its host. Post-translational modification (PTM) plays critical role(s) in plant-pathogen interaction. However, how PTM of viral protein regulates plant immunity remains poorly understood. Here, we found that S162 and S165 of Chinese wheat mosaic virus (CWMV) cysteine-rich protein (CRP) are phosphorylated by SAPK7 and play key roles in CWMV infection. Furthermore, the phosphorylation-mimic mutant of CRP (CRP S162/165D ) but not the non-phosphorylatable mutant of CRP (CRP S162/165A ) interacts with RNA-binding protein UBP1-associated protein 2C (TaUBA2C). Silencing of TaUBA2C expression in wheat plants enhanced CWMV infection. In contrast, overexpression of TaUBA2C in wheat plants inhibited CWMV infection. TaUBA2C inhibits CWMV infection through recruiting the pre-mRNA of TaNPR1, TaPR1 and TaRBOHD to induce cell death and H 2 O 2 production. This effect can be supressed by CRP S162/165D through changing TaUBA2C chromatin-bound status and attenuating it’s the RNA- or DNA-binding activities. Taken together, our findings provide new knowledge on how CRP phosphorylation affects CWMV infection as well as the arms race between virus and wheat plants.

Chinese wheat mosaic virus (CWMV) causes a damaging disease in cereal plants. However, CWMV interacts with host factors to facilitate virus infection is not clear yet. Here, we found that S162 and S165 of CWMV cysteine-rich protein (CRP) are phosphorylated by SAPK7 in vivo and in vitro. Mutational analyses have indicated that these two phosphorylation sites of CRP (CRP S162/165D ) promoting CWMV infection in plants, due to the supressed cell death and H 2 O 2 production. Further investigations found the CRP S162/165D can interact with TaUBA2C, while the non-phosphorylatable mutant of CRP (CRP S162/165A ) does not. Futhermore, we have determined that CRP S162/165D and TaUBA2C interaction inhibited the formation of TaUBA2C speckles in nucleus to attenuate its RNA- and DNA-binding activity. We also showed that TaUBA2C recruit the pre-mRNA of TaNPR1, TaPR1 and TaRBOHD to up-regulated these genes expressions and then induce cell death and H 2 O 2 production in plant. This effect can be supressed by the expression of CRP S162/165D , in a dose-dependent manner. Taken together, our discovery may provide a new sight for the arms race between virus and its host plants.

Funding: This work was supported by: China Agriculture Research System from the Ministry of Agriculture of the P.R. China (CARS-03) for J.C., Ningbo Science and Technology Innovation 2025 Major Project, China (Q21C140013) for J.C. and K.C. Wong Magna Funding Ningbo University for J.Y. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, we present evidence that CWMV CRP is phosphorylated at S162 and S165 by a serine/threonine-protein kinase SAPK7 in vivo and in vitro. Using a CWMV mutant encoding a CRP no longer phosphorylatable at S162 and S165 (e.g., CWMV S162/165A ), the inoculated N. benthamiana and wheat plants did not show clear mosaic symptoms compared with CWMV wild-type (WT) infection, due mainly to cell death and H 2 O 2 production. In contrast, using a phosphorylation mimics (e.g., CWMV S162/165D ), the inoculated plants developed stronger disease symptoms than the WT CWMV-inoculated plants, due mainly to the supressed cell death and H 2 O 2 production. In this study, we have also identified a CWMV infection inducible RNA-binding protein gene (TaUBA2C) in wheat that interacts with CRP S162/165D , but not CRP S162/165A . Further analyses showed that silencing of TaUBA2C expression in wheat promoted CWMV infection, while overexpression of TaUBA2C in wheat inhibited CWMV infection. It is noteworthy that TaUBA2C can induce cell death and H 2 O 2 production in wheat plants through up-regulation of TaNPR1, TaPR1 and TaRBOHD expressions. Also, the TaUBA2C-induced cell death and H 2 O 2 production can be supressed by the expression of CRP S162/165D , in a dose-dependent manner. Futhermore, we have determined that CRP S162/165D can bind to TaUBA2C to interfear the formation of TaUBA2C speckles in nuclei to reduce its RNA- and DNA-binding activity. In summary, through this study, we have determined that phosphorylation of CRP promotes CWMV infection in plants. The results described in this paper provide new knowledge of the arms race between CWMV and its host plants.

Chinese wheat mosaic virus (CWMV), a member in the genus Furovirus, family Virgaviridae, is an RNA virus with two positive-sense single-stranded RNAs (e.g., RNA1 and RNA2). In China, CWMV often infects its host plants, together with Wheat yellow mosaic virus (WYMV), to cause severe disease symptoms and yield losses [ 28 , 29 ]. In the laboratory, CWMV can infect the model plant Nicotiana benthamiana through mechanical inoculation, which has been used as a very common model system for investigating the interaction between CWMV and plants [ 30 ]. CWMV RNA1 encodes three proteins: the replication-associated protein, RNA-dependent RNA polymerase (RdRp), and a movement protein. CWMV RNA2 encodes four proteins: a major capsid protein (CP), two minor CP-related proteins (e.g., N-CP and CP-RT), and a cysteine-rich protein (CRP)[ 28 , 31 ]. The CRP is an RNA silencing suppressor [ 32 ]. Many virus-encoded suppressors of RNA silencing (VSRs) are multifunctional proteins. For example, potyvirus helper component proteinases (HCPro) are not only VSRs, but also important for virus plant-to-plant transmission and viral polyprotein maturation [ 33 ]. Cucumber mosaic virus (CMV) 2b protein is also a VSR and involved in CMV systemic infection [ 34 ]. Barley stripe mosaic virus (BSMV) γb protein can suppress RNA silencing and promote RNA duplex unwinding and chloroplast-associated virus replication as well as cell-to-cell movement. In addition, after phosphorylation, BSMV γb protein can inhibit cell death to benefit virus infection [ 4 , 35 , 36 ]. Although CWMV CRP has been reported as a VSR, how it evades wheat resistance to benefit virus infection is unclear.

During evolution, plants have evolved multiplex defense strategies against pathogen infections. For example, programmed cell death (PCD) can protect plants from pathogen invasion through eliminating damaged or pathogen-infected cells [ 13 , 14 ]. The most well-studied plant PCD is hypersensitive response (HR), which involves activations of PR genes and productions of reactive oxygen species and salicylic acid (SA) to eliminate invading pathogen by dead cells [ 15 , 16 ]. In the process of pathogen infection, a variety of defense-associated genes are induced in plants [ 17 – 19 ]. Regulation of gene expression by post-transcriptional modification in the context of defense is important for PCD and plant immunity [ 20 ]. Previous reports have also shown that plant RNA-binding proteins (RBPs) can regulate gene expressions through post-transcriptional modification [ 21 , 22 ]. Many RBPs contain an RNA recognition motif (RRM), also known as RNA binding domain (RBD), and a domain(s) that are required for protein-protein interactions [ 23 ]. Some RBPs have been demonstrated to regulate plant immune responses. For example, AtRBP-DR1 contains three RRMs and can positively regulate the SA-mediated signaling in Arabidopsis plant [ 24 ]. Arabidopsis Dicer-like 4 (AtDCL4) and Argonaute 2 (AtAGO2) have also been shown to modulate Arabidopsis defense responses to pathogen invasions [ 25 , 26 ]. To counteract plant defense, pathogens have also evolved to encode different proteins to evade host immunity. For instance, HopU1 of Pseudomonas syringae can disrupt the binding between baterial RNA and GRP7, a Glycine-rich RNA binding protein, to regulate the expression of a plant immune receptor [ 27 ]. In this study, we investigated how CWMV evades wheat and N. benthamiana defense response to enhance virus infection.

Post-translational modification (PTM) can greatly modify protein functions, including the functions involved in the responses against abiotic and/or biotic stresses [ 1 , 2 ]. Several PTMs have now been reported. These include protein phosphorylation [ 3 – 5 ], sumoylation [ 6 ], ubiquitination [ 7 ], glycosylation [ 8 ], N-Myristoylation [ 5 ], and S-acylation [ 9 ]. Among these PTMs, protein phosphorylation is considered as one of the most studied PTMs and plays vital roles in many cellular processes [ 10 ]. Protein phosphorylation is a reversible process, and the γ-phosphoryl group of ATP can covalently bind to the serine (S), threonine (T) or tyrosine (Y) residue in the target protein through the actions of protein kinases. Numerous studies have indicated that after phosphorylation, many viral proteins can enhance or suppress virus infection in plants. For instance, the phosphorylated Barley stripe mosaic virus (BSMV) γb protein has been shown to suppress RNA silencing and virus infection-induced cell death in plants [ 4 ]. N. benthamiana NbSKƞ can phosphorylate Tomato leaf curl Yunnan virus (TLCYnV) C4 protein to enhance virus pathogenicity [ 5 , 11 ]. The Casein Kinase 1 (CK1)-mediated phosphorylation of the serine-rich motif in Barley yellow striate mosaic virus (BYSMV) phosphoprotein is essential for viral replication [ 12 ]. However, how protein phosphorylation modulates virus pathogenicity in plants is still largely unknown.

Results

CWMV CRP can be phosphorylated in wheat To investigate whether CWMV CRP can be phosphorylated in plant, plasmid expressing CRP-GFP fusion was introduced into wheat protoplasts via PEG 4000-based method. After overnight incubation at room temperature, total protein was extracted from the inoculated protoplasts and CRP-GFP enriched by immunoprecipitation. Comparative western blot analysis using GFP and phosphoserine-specific antibodies revealed in protoplasts expressing the fusion protein a 45 kDa band indicative for phosphorylated CRP-GFP. GFP expressing samples were processed in parallel as a control (Fig 1A). To identify the phosphorylation sites in CRP, the GFP-tagged protein was purified by affinity chromatography from total protein extracts of expressing wheat protoplast (Fig 1B, upper panel). The purified CRP-GFP was then analyzed using Q-Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS) after trypsin treatment. Through 9 independent LC-MS/MS, a total of 8 potential phosphorylation sites were obtained (Fig 1B, lower panel). Considering, S162 and S165 were identified in 6 independent assays (S1 Table), these two sites were chosen for further assays. As previously described by Zhang et al.[4] and Gao et al.[12] with minor modifications, we substituted the two potential phosphorylation sites with alanine in plasmid expressing CRP-GFP fusion to investigate the phosphorylation levels of the WT and S162/165A versions of CRP in plant cells. We transiently expressed CRPS162/165A-GFP in N. benthamiana leaves through agroinfiltration. The leaves expressing CRP-GFP were used as controls. Total protein was extracted from the inoculated leaves, then CRP-GFP and CRPS162/165A-GFP enriched by immunoprecipitation. Western blot analysis showed that the phosphorylation level of CRPS162/165A-GFP was significantly reduced (Fig 1C). Taken together, our results suggest that S162 and S165 phosphorylation sites are important. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Identification of phosphorylation sites in CWMV CRP through LC-MS/MS. A. In vivo phosphorylation of CRP. Total protein was extracted from the protoplasts expressing GFP or CRP-GFP, and then analyzed through immunoprecipitation using an anti-GFP or an anti-phosphoserine antibody. B. Detection of CRP-GFP purified from the inoculated protoplasts using western blot (upper panel). The LC-MS/MS result is shown in the lower panel. The underlined CRP amino acid sequence was identified in this study through LC-MS/MS, and the phosphorylation sites in this protein are shown in red. C. The phosphorylation levels of CRP-GFP and CRPS162/165A-GFP were determined through western blot analysis using a phosphoserine specific antibody. The CBB-stained gel is used to show sample loadings. https://doi.org/10.1371/journal.ppat.1010412.g001

Phosphorylation of CRP at S162 or S165 promoted CWMV infection In order to investigate the role of phosphorylation at S162 and/or S165 in CWMV infection, we generated six mutant CWMV infectious clones (CWMVS162A, CWMVS165A, CWMVS162/165A, CWMVS162D, CWMVS165D, and CWMVS162/165D), through substituting the serine residue (S) at the position 162 and/or 165 with an alanine to mimic the non-phosphorylated state, or with a aspartic acid to mimic the phosphorylated state. The WT and mutant infectious clones were inoculated individually to N. benthamiana plants through agroinfiltration. After 21 days, the CWMVS162A- or CWMVS165A-inoculated plants showed mild mosaic symptoms (Fig 2A). The CWMVS162D- or CWMVS165D-inoculated plants showed moderate mosaic symptoms, while the CWMVS162/165D-inoculated plants showed strong mosaic symptoms. In this study, the CWMVS162/165A-inoculated plants did not show clear mosaic symptoms (Fig 2A). Quantitative RT-PCR (qRT-PCR) result showed that the expression level of CWMV CP in the systemic leaves of the CWMVS162A-, CWMVS165A- or CWMVS162/165A-inoculated plants were significantly reduced, while the expression level of CWMV CP in the systemic leaves of the CWMVS162D-, CWMVS165D- or CWMVS162/165D-inoculated plants were significantly increased compared to the WT CWMV-inoculated plants (Fig 2B). Similar result was also obtained through western blot analysis using a CWMV CP specific antibody (Fig 2C). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Phosphorylation of CRP at S162 or S165 is crucial for CWMV infection. A. Systemic mosaic symptoms in the CWMV-, CWMVS162A-, CWMVS165A-, CWMVS162/165A-, CWMVS162D-, CWMVS165D- or CWMVS162/165D-infected N. benthamiana plants. Photographs were taken at 21 dpi. Scale bar = 5 cm (upper panel), Scale bar = 2 cm (lower panel). B. Relative expression level of CWMV CP in the assayed N. benthamiana plants, determined through qRT-PCR using CWMV CP gene specific primers. The data presented are the means ± standard deviations (SD), calculated using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. C. Accumulation of CWMV CP in the assayed N. benthamiana leaf samples was determined through western blot analysis using a CWMV CP specific antibody. The CBB-stained gel is used to show sample loadings. D. Systemic mosaic symptoms in wheat leaves infected with CWMV, CWMVS162A, CWMVS165A, CWMVS162/165A, CWMVS162D, CWMVS165D or CWMVS162/165D. Photographs were taken at 21 dpi. E. The relative expression level of CWMV CP in the assayed wheat plants was determined through qRT-PCR using CWMV CP gene specific primers. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. F. Accumulation of CWMV CP in the assayed wheat plants was determined through western blot analysis using a CWMV CP specific antibody. The CBB-stained gel is used to show sample loadings. https://doi.org/10.1371/journal.ppat.1010412.g002 We then inoculated wheat seedlings with the WT or mutant CWMV. By 21 dpi, the plants inoculated with CWMVS162/165D showed strong mosaic symptoms in their young developing leaves. The plants inoculated with CWMVS162D or CWMVS165D showed moderate mosaic symptoms, while the plants inoculated with CWMVS162A or CWMVS165A showed mild mosaic symptoms (Fig 2D). In this study, the plants inoculated with CWMVS162/165A again did not show clear mosaic symptoms in their leaves. The qRT-PCR and western blot results revealed that the accumulation levels of CWMV CP and CP were much higher in the plants inoculated with CWMVS162D, CWMVS165D or CWMVS162/165D compared to the WT CWMV-inoculated plants (Fig 2E and 2F). In contrast, the plants inoculated with CWMVS162A, CWMVS165A or CWMVS162/165A accumulated much less CWMV CP compared to the WT CWMV-inoculated plants, indicating that phosphorylation of CRP at S162 and/or S165 can promote CWMV infection in plant. To investigate the effect of phosphorylation of CRP at S162 and S165 on its VSR activity, we expressed CRP, CRPS162/165A and CRPS162/165D in 16c transgenic N. benthamiana leaves as reported [4,37]. By 3 or 6 dpi, the green fluorescence in the 16c plant leaves expressing sGFP was silenced (S1A Fig). Consistent with this result, no GFP protein was detected in these GFP-silenced 16c plant leaves through western blot analysis (S1B Fig). However, the 16c plant leaves co-expressing sGFP and TBSV p19, sGFP and CRP, sGFP and CRPS162/165A or sGFP and CRPS162/165D continued to show strong green fluorescence (S1A Fig). This finding was supported by the result of western blot analysis (S1B Fig), suggesting that phosphorylation of CRP at S162 and S165 does not affect its RNA silencing suppression activity.

SAPK7 kinase is responsible for the phosphorylation at S162 or S165 To identify which kinase(s) phosphorylates CRP at S162 or S165, we screened a wheat cDNA library through yeast two hybrid (YTH) assays using CRP as the bait. Through three independent screenings, a total of 15 positive clones were obtained (S2 Table). After sequence analysis, six of these clones were found to encode a polypeptide of 355 amino acids (aa). Blast search result showed that this polypeptide shared 95.8% sequence identity with the serine/threonine-protein kinase SAPK7 (TraesCS2A02G303900.1, named as TaSAPK7 thereafter), and this polypeptide represents the full-length of TaSAPK7. Further analysis revealed that wheat has three TaSAPK7 homology sequences (TraesCS2A02G303900.1, TraesCS2B02G320500.1, and TraesCS2D02G302500.1), and these three sequences share 99.91% sequence identity (S2A Fig). In addition, phylogenetic analysis showed that SAPK7 is a member of the SnRK2 subfamily of plant protein kinase (S2B Fig). For convenience, we selected TraesCS2A02G303900.1 for our further analysis. To validate the function of this protein, we constructed a pAD-TaSAPK7 plasmid and used it in further YTH assays. The result showed that AD-TaSAPK7 interacted with BD-CRP in the yeast cells (Fig 3A). To confirm this finding, we conducted bimolecular fluorescence complementation (BiFC) and firefly luciferase (LUC) complementation imaging assays, and confirmed that TaSAPK7 interacted with CRP (Fig 3B and 3C). PPT PowerPoint slide

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TIFF original image Download: Fig 3. CWMV CRP can be phosphorylated by TaSAPK7. A. Yeast two-hybrid (YTH) assay was first used to determine the interaction between TaSAPK7 and CRP (TaSAPK7+CRP). AD-TaSAPK7 and BD-CRP were co-expressed in yeast cells, and the transformed cells were grown on the SD/-Leu/-Trp medium and then on the SD/-Trp/-Leu/-His/-Ade medium to determine the protein-protein interaction. Yeast cells co-transformed with AD-T+BD-Lam, AD+BD-CRP and AD- TaSAPK7+BD were used as the negative controls, yeast cells co-transformed with AD-T+BD-53 were used as the positive controls. B. BiFC assay was used to confirm the interaction between TaSAPK7-cYFP and CRP-nYFP in N. benthamiana leaves. These two proteins were co-expressed in N. benthamiana leaves followed by Confocal Microscopy at 60 h post agroinfiltration (hpi). Scale bar = 20 μm. C. LCI assay was used to confirm the interaction between cLuc-TaSAPK7 and CRP-nLuc in N. benthamiana leaves. Agrobacterium cultures carrying pCRP-nLuc or pcLuc-TaSAPK7 were mixed (cLuc-TaSAPK7+CRP-nLuc), and infiltrated into N. benthamiana leaves. The leaf areas infiltrated with Agrobacterium cultures expressing sGF-nLuc+cLuc-SAR were used as the positive controls, CRP-nLuc+cLuc and nLuc+cLuc-TaSRK7 were used as the negative controls, respectively. Luciferase activity was captured using a low-light cooled CCD imaging apparatus at 3dpi. D. Detection of kinase activity after CRP and TaSAPK7 interaction in vitro. In this study, recombinant CRP-6×His and TaSAPK7-GST were separately expressed and purified from E. coli, and then incubated together. The reactions lacking CRP-6×His or TaSAPK7-GST were used as controls. The CBB-stained gel is used to show sample loadings. E. Detection of the effect of TaSAPK7 on CRP phosphorylation in vitro. The radioactive protein bands indicate the levels of CRP phosphorylation. The CBB-stained gel is used to show sample loadings. F. Analysis of phosphorylations of CRP and its mutants by TaSAPK7. The radioactive protein bands indicate the levels of phosphorylation of various proteins. The CBB-stained gel is used to show sample loadings. https://doi.org/10.1371/journal.ppat.1010412.g003 To investigate whether TaSAPK7 phosphorylates CRP, we purified GST-TaSAPK7 and CRP-6×His from E. coli, and subjected them to in vitro kinase assay in 1× kinase reaction buffer supplemented with [γ-32P] ATP. The result showed that TaSAPK7 was responsible for phosphorylating CRP (Fig 3D). In this study, we have also found that phosphorylation of CRP was TaSAPK7 dose-dependent (Fig 3E). Because CRP has several consensus phosphorylation sites, we substituted S162 and/or S165 with A or D, respectively, and performed further in vitro phosphorylation assays. The result showed that CRPS162A and CRPS165A were phosphorylated by TaSAPK7, but not CRPS162/165A (Fig 3F), further confirming that SAPK7 kinase is responsible for the phosphorylation of CRP at S162 and S165. However, we unexpectedly observed that the CRPS162/165D mutant showed a strong phosphorylation level (Fig 3F), but aspartic acid should not be phosphorylated by TaSAPK7. It suggested that either some sites other than S162 and S165 could be phosphorylated by TaSAPK7 and their phosphorylation is dependent on the phosphorylation of S162 and/or S165 or their Asp substitutes, or the Ser-to-Asp mutations resulted in new phosphorylation sites for TaSPAK7. To clarify the secondary phosphorylation sites of CRP, we analyzed the phosphorylation status of CRPS162/165D by LC-MS/MS. The results showed that S156 and T159 are secondary phosphorylation sites of CRP (S3A Fig). To investigate the role of phosphorylation at S156 and T159 in CWMV infection, we generated two mutant CWMV infectious clones CWMVS156/T159A to mimic the non-phosphorylated state, and CWMVS156/T159D to mimic the phosphorylated state. The WT and mutant infectious clones were inoculated individually to N. benthamiana plants through agroinfiltration. After 21 days, the CWMVS156/T159A- or CWMVS156/T159D-inoculated plants showed mosaic symptoms similar to WT CWMV (S3B Fig). The qRT-PCR and western blot results revealed that the accumulation levels of CWMV CP and CP in plants inoculated with CWMVS156/T159A or CWMVS156/T159D were similar to those in plants inoculated with WT CWMV (S3C and S3D Fig). These results suggested that phosphorylation of CRP at S156 and T159 does not affect the pathogenicity of CWMV.

SRK promotes CWMV infection Considering CWMV can infect the model plant Nicotiana benthamiana, which has been used as a very common model system for investigating the interaction between virus and plants. Firstly, we search the homologous gene of TaSAPK7 through NCBI database. The blast search result showed that TaSAPK7 have 80.39% amino acid identity to the serine/threonine-protein kinase SRK2A-like (LOC107817827, named as NbSRK thereafter) (S2C Fig). Through YTH assays, we have found that AD-NbSRK could also interact with BD-CRP (Fig 4A). This finding was then validated through BiFC and LCI assays (Fig 4B and 4C). Next, in order to investigate the role of SRK in CWMV infection, we first silenced NbSRK expression in N. benthamiana plants using a TRV-based VIGS technology. qRT-PCR result showed that the expression level of NbSRK was knocked down by about 70% in the TRV:NbSRK-infected plants compared to the TRV:00-infected plants (S4A Fig). These plants were then inoculated again with CWMV. At 21 days post CWMV inoculation, the TRV:NbSRK- and CWMV-inoculated (referred to as TRV:NbSRK+CWMV thereafter) plants showed milder mosaic symptoms than the plants inoculated with TRV:00 and CWMV (TRV:00+CWMV) (Fig 4D). qRT-PCR result showed that the accumulation level of CWMV CP in the TRV:NbSRK+CWMV-inoculated plants was decreased by 0.5 fold compared to the TRV:00+CWMV-inoculated plants (Fig 4E). Western blot result showed that the accumulation level of CWMV CP in the TRV:NbSRK+CWMV-inoculated plants was reduced by about 50% compared to the TRV:00+CWMV-inoculated plants (Fig 4F). To investigate that the effect of CRP phosphorylation on CWMV infection, we inoculated the NbSRK-silenced plants with CWMVS162/165A (TRV:NbSRK+CWMVS162/165A) or CWMVS162/165D (TRV:NbSRK+CWMVS162/165D). By 21 dpi, the TRV:NbSRK+CWMVS162/165A-inoculated plants showed the similar mosaic symptoms as the TRV:NbSRK+CWMV-inoculated plants, while the TRV:NbSRK+CWMVS162/165D-inoculated plants showed stronger mosaic symptoms than the TRV:NbSRK+CWMV-inoculated plants (Fig 4D). qRT-PCR result showed that the accumulation level of CWMV CP in the TRV:NbSRK+CWMVS162/165A-inoculated plants was similar to that in the TRV:NbSRK+CWMV-inoculated plants. In contrast, the accumulation level of CWMV CP in the TRV:NbSRK+CWMVS162/165D-inoculated plants were about 2.2 fold higher than that in the TRV:NbSRK+CWMV-inoculated plants (Fig 4G). Western blot result agreed with the qRT-PCR result and showed that the infection of CWMVS162/165D, but not CWMVS162/165A, in the NbSRK-silenced plants was enhanced (Fig 4H). PPT PowerPoint slide

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TIFF original image Download: Fig 4. SRK is required for CWMV infection in plant. A, B and C. YTH, BiFC and LCI assay were used to confirm the interaction between NbSRK and CRP. D. Systemic mosaic symptoms on the TRV:00+CWMV-, TRV:SRK+CWMV-, TRV:SRK+CWMVS162/165A-, or TRV:SRK+CWMVS162/165D-inoculated N. benthamiana plants. Photographs were taken at 21 days post CWMV inoculation. Scale bar = 5 cm (upper panel), Scale bar = 2 cm (lower panel). E. Quantitative RT-PCR analysis of relative expression level of CWMV CP in the TRV:00+CWMV- or TRV:SRK+CWMV-inoculated N. benthamiana plants. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. F. The accumulation of CWMV CP in the assayed N. benthamiana plants was determined through western blot analysis using a CWMV CP specific antibody at 21 dpi. The CBB-stained gel is used to show sample loadings. G. Relative expression levels of CWMV CP in the TRV:SRK+CWMV-, TRV:SRK+CWMVS162/165A-, or TRV:SRK+CWMVS162/165D-inoculated N. benthamiana plants were determined through qRT-PCR. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05; n.s, no significant difference. H. The accumulation of CWMV CP in the assayed N. benthamiana plants was determined through western blot analysis at 21 dpi. The CBB-stained gel is used to show sample loadings. I. Relative expression levels of CWMV CP in the EV+CWMV-, NbSRK+CWMV-, NbSRK+CWMVS162/165A- or NbSRK+CWMVS162/165D-inoculated N. benthamiana plants were determined through qRT-PCR. The data presented are the means ± SD, determined through the Tukey’s test (P < 0.05). Each treatment had three biological replicates. J. Accumulation of CWMV CP in the assayed N. benthamiana plants was determined through western blot analysis using a CWMV CP specific antibody at 21 dpi. The CBB was used as loading controls. K and L. Quantifications of phosphorylation at S162 and S165. CRP-GFP fusion was expressed in the leaves of the TRV:00- or TRV:SRK-inoculated N. benthamiana plants followed by immunoprecipitation using an anti-GFP antibody. The phosphorylated peptides were identified through LC-MS/MS using the PRM method. Peptide phosphorylation ratios (e.g., phosphorylated/non-phosphorylated) were then determined. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. https://doi.org/10.1371/journal.ppat.1010412.g004 To further elucidate the role of NbSRK in CWMV infection, we constructed a p35S:NbSRK-His expression vector and co-inoculated this vector with CWMV (NbSRK+CWMV), CWMVS162/165A (NbSRK+CWMVS162/165A) or CWMVS162/165D (NbSRK+CWMVS162/165D) into N. benthamiana leaves through agroinfiltration. The plants co-inoculated with the empty vector and CWMV (EV+CWMV) were used as controls. At 3 dpi, the inoculated leaves were harvested and analyzed for NbSRK-His expression through western blot assays (S4B Fig). These plants were then analyzed through qRT-PCR at 7 dpi and the result showed that the accumulation level of CWMV CP was significantly increased in the NbSRK+CWMV- or the NbSRK+CWMVS162/165D-inoculated plants compared to the control plants, while the accumulation level of CWMV CP in the NbSRK+CWMVS162/165A-inoculated plants was similar to that in the EV+CWMV-inoculated plants (Fig 4I). Further analysis of CWMV CP accumulation in these samples through western blot assays yielded a similar result (Fig 4J), indicating that overexpression of SRK can promote CWMV infection through phosphorylation of CRP at S162 and S165. To elucidate the importance of NbSRK in CRP phosphorylation, CRP-GFP was transiently over-expressed in the TRV:00- or TRV:NbSRK-inoculated plants. At 3 dpi, CRP-GFP fusion was isolated through immunoprecipitation and then subjected to LC-MS/MS analysis. Phosphorylation of CRP at S162 and S165 was monitored through a parallel reaction monitoring (PRM) system. The result showed that the phosphorylation of CRP at S162 and S165 in the TRV:NbSRK-inoculated plants was significantly reduced compared to the TRV:00-inoculated control plants (Fig 4K and 4L), indicating that silencing of NbSRK expression affected the phosphorylation of CRP.

CRPS162/165D can interact with TaUBA2C In order to clarify the molecular mechanism of how S162 and S165 of CRP phosphorylation promotes CWMV infection, we screened a wheat cDNA library through YTH using CRPS162/165D as the bait. The results from three independent screenings showed that wheat UBP1-associated protein 2C (TraesCS3A02G220400.1, named as TaUBA2C thereafter) interacted with CRPS162/165D. Blast searching T. aestivum database (http://plants.ensembl.org/) identified three TaUBA2C sequences (TraesCS3A02G220400.1, TraesCS3B02G250700.1, and TraesCS3D02G232300.1) (S5A Fig). Because these three sequences shear 98.06% sequence identity, we decided to use TraesCS3A02G220400.1 in further studies. Through YTH, we have found that CRPS162D, CRPS165D and CRPS162/165D, but not CRPS162/165A, interacted with TaUBA2C, indicating that phosphorylation of CRP at S162 and/or S165 is crucial for the interaction (Fig 5A). This finding is supported by the results from the microscale thermophoresis (MST) and pull-down assays (Fig 5B and 5C). While MST assays show an interaction of TaUBAC with CRPS162/165A, the different results obtained in different interaction experiments may be due to different experimental conditions. Because TaUBA2C contains two RRMs (Fig 5D), we analyzed the roles of these two RRMs via YTH using CRPS162/165D as the bait. The result showed that the second RRM (TaUBA2CD2) was responsible for the interaction (Fig 5E). PPT PowerPoint slide

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TIFF original image Download: Fig 5. CWMV CRPS162/165D can interact with TaUBA2C. A. YTH assay was used to detect the interaction between TaUBA2C and CRP, CRPS162/165A, CRPS162/165D, CRPS162D or CRPS165D. AD-TaUBA2C and BD-CRP or one of its derivatives were co-expressed in yeast cells. The transformed yeast cells were grown on the SD/-Leu/-Trp medium and then on the SD/-Trp/-Leu/-His/-Ade medium. Yeast cells co-expressing AD-T+BD-Lam or AD-T+BD-53 were used as controls. B. Microscale thermophoresis assay was used to detect the binding affinity of TaUBA2C to CRP, CRPS162/165A or CRPS162/165D. Three independent experiments were conducted in this study and yielded similar results. Bars represent standard errors. C. Pull-down assay was used to confirm the in vitro interaction between TaUBA2C-GST and CRP-His, CRPS162/165A-His or CRPS162/165D-His. In this study, GST was used as a control. Black arrowheads indicate the positions of TaUBA2C-GST, CRP-His, CRPS162/165A-His, and CRPS162/165D-His, respectively. D. A schematic shows the arrangement of domains in TaUBA2C. The numbers above the schematic indicate the amino acid positions of different domains. RRM, RNA-recognition motif; aa, amino acids. E. YTH assay was used to detect the interaction between CRP and TaUBA2C, TaUBA2CD1 or TaUBA2CD2. https://doi.org/10.1371/journal.ppat.1010412.g005

UBA2C can regulate CWMV infection in wheat qRT-PCR analysis using tissues from the CWMV-infected or non-infected wheat plants showed that the expression of TaUBA2C was significantly up-regulated by CWMV infection (Fig 6A). To investigate the role of TaUBA2C on CWMV infection in wheat plants, we first silenced TaUBA2C expression using a BSMV-based VIGS vector. qRT-PCR at 10 dpi showed that the expression of TaUBA2C in the BSMV:TaUBA2C-inoculated plants was reduced by about 50% (S6A Fig). We then inoculated the BSMV:TaUBA2C- or BSMV:00-inoculated plants with CWMV (BSMV:TaUBA2C+CWMV or BSMV:00+CWMV). After 21 days post CWMV inoculation, stronger mosaic symptoms were observed on the BSMV:TaUBA2C+CWMV-inoculated plants than the BSMV:00+CWMV-inoculated plants (Fig 6B). The result of qRT-PCR showed that the accumulation level of CWMV CP in the BSMV:TaUBA2C+CWMV-inoculated plants was much higher than that in the BSMV:00+CWMV-inoculated plants (Fig 6C). Western blot result showed that the accumulation level of CWMV CP in the BSMV:TaUBA2C+CWMV-inoculated plants was much higher than that in BSMV:00+CWMV-inoculated plants (Fig 6D), indicating that TaUBA2C has a key role in CWMV infection in wheat. PPT PowerPoint slide

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TIFF original image Download: Fig 6. TaUBA2C can facilitate CWMV infection. A. Analysis of TaUBA2C expression in the mock- or the CWMV-inoculated wheat plants through qRT-PCR. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. B. Mosaic symptoms in the wheat leaves infected with CWMV, BSMV:00, BSMV:PDS, BSMV:TaUBA2C, BSMV:00+CWMV or BSMV:TaUBA2C+CWMV. Mock indicates that the leaf was from a plant inoculated with the FES buffer. Photographs were taken at 21 dpi. C. Quantitative RT-PCR analysis of CWMV CP accumulation in the BSMV:00+CWMV- or BSMV:TaUBA2C+CWMV-inoculated wheat plants at 21 dpi. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. D. Detection of CWMV CP in the assayed wheat plants through western blot analysis using a CWMV CP specific antibody. The data presented are the means ± SD. Each treatment had three biological replicates. The CBB-stained gel is used to show sample loadings. E. Disease symptoms on the CWMV-inoculated wild type (WT) or the two TaUBA2C overexpression transgenic lines (L3 and L5). Photographs were taken at 21 days post CWMV inoculated. Scale bar = 5 cm. F. Detection of CWMV CP accumulation in the CWMV-inoculated WT, L3, and L5 plants, respectively, through qRT-PCR at 21 dpi. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. G. Detection of CWMV CP accumulation in the assayed plants through western blot analysis using a CWMV CP specific antibody. The data presented are the means ± SD. Each treatment had three biological replicates. CBB-stained gel is used to show sample loadings. https://doi.org/10.1371/journal.ppat.1010412.g006 To further validate this finding, we generated several transgenic wheat lines overexpressing TaUBA2C-His. Analysis these lines through western blot assay showed that line 3 and line 5 (L3 and L5) plants produced more TaUBA2C-His than other line plants (S6B Fig). We then inoculated the seedlings of L3 and L5 with in vitro transcribed CWMV RNAs. After 21 days, the CWMV-inoculated L3 and L5 plants showed milder mosaic symptoms than the CWMV-inoculated wild type (WT) wheat plants (Fig 6E). The results of qRT-PCR and western blot analysis revealed that the accumulation level of CWMV CP were significantly decreased in the two CWMV-inoculated transgenic lines compared to that in the CWMV-inoculated WT control plants (Fig 6F and 6G), indicating that TaUBA2C is regulator of CWMV infection.

Phosphorylation of CRP suppresses cell death induced by TaUBA2C Transient expression of AtUBA2s in Arabidopsis leaves caused cell death [38]. To investigate whether TaUBA2C is also an inducer of cell death, we transiently expressed TaUBA2C-Flag in N. benthamiana leaves through agroinfiltration (S7A Fig). The leaves expressing PVX-bax were used as positive controls. Trypan blue staining result showed that at 5 dpi, the TaUBA2C-Flag or PVX-bax expressing N. benthamiana leaves developed cell death symptom (Fig 7A). To investigate the role of CRP and TaUBA2C interaction on cell death induction, we transiently co-expressed CRP-HA, CRPS162/165A-HA or CRPS162/165D-HA with TaUBA2C-Flag in N. benthamiana leaves through agroinfiltration (S7A Fig). Trypan blue staining result showed that the N. benthamiana leaves co-expressing TaUBA2C-Flag and CRP-HA (TaUBA2C-Flag+CRP-HA) or TaUBA2C-Flag and CRPS162/165D-HA (TaUBA2C-Flag+CRPS162/165D-HA) showed less cell death than that in the leaves expressing TaUBA2C-Flag alone (Fig 7A and 7B). In this study, the leaves co-expressing TaUBA2C-Flag and CRPS162/165A-HA (TaUBA2C-Flag+CRPS162/165A-HA) showed moderate cell death (Fig 7A and 7B), indicating that phosphorylation of CRP can suppress the TaUBA2C-induced cell death in plant. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Phosphorylation of CRP at S162 and S165 suppresses cell death induced by TaUBA2C. A. Cell death in N. benthamiana leaf tissues expressing TaUBA2C, TaUBA2C+CRP, TaUBA2C+CRPS162/165A or TaUBA2C+CRPS162/165D. The infiltrated leaves were harvested and photographed at 5 days post agroinfiltration (upper panel), and then stained with a Trypan blue solution (lower panel). An N. benthamiana leaf inoculated with PVX-bax was used as a positive control. B. Measurement of the relative Trypan blue staining intensity shown in Fig 7A. Each treatment had three biological replicates, the data presented are the means ± SD. Different letters show statistically significant differences (P < 0.05, Tukey’s test). C. Assayed N. benthamiana leaves were stained with a DAB solution at 5 dpi. The N. benthamiana leaf inoculated with the wild type Agrobacterium DC3000 was used as a control. D. Measurement of the relative DAB staining intensity shown in Fig 7C. Each treatment had three biological replicates, the data presented are the means ± SD. Different letters show statistically significant differences (P < 0.05, Tukey’s test). E. N. benthamiana leaves co-inoculated with TaUBA2C and different concentrations of CRPS162/165D were harvested and photographed at 5 dpi (upper panel), and then stained with a Trypan blue solution (lower panel). F. Measurement of the relative Trypan blue staining intensity shown in Fig 7E. Each treatment had three biological replicates, the data presented are the means ± SD. Different letters show statistically significant differences (P < 0.05, Tukey’s test). G. Quantitative RT-PCR analyses of TaNPR1, TaPR1 and TaRBOHD expressions in the wild type (WT), L5 transgenic and BSMV:TaUBA2C-inoculated plants. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. H. Quantitative RT-PCR analyses of TaNPR1, TaPR1 and TaRBOHD expressions in the CWMV-inoculated, CWMVS162/165A-inoculated or CWMVS162/165D-inoculated plants. The data presented are the means ± SD, determined using the Student’s t-test. Each treatment had three biological replicates. *, P <0.05. https://doi.org/10.1371/journal.ppat.1010412.g007 Because cell death is related to H 2 O 2 production, we analyzed the accumulation level of H 2 O 2 in various N. benthamiana leaves through DAB staining. The result showed that less H 2 O 2 had accumulated in the leaves co-expressing TaUBA2C-Flag+CRP-HA or TaUBA2C-Flag+CRPS162/165D-HA compared to the leaves expressing TaUBA2C-Flag alone (Fig 7C and 7D). The leaves co-expressing TaUBA2C-Flag+CRPS162/165A-HA accumulated moderate level of H 2 O 2 (Fig 7C and 7D). In the next experiment, we co-expressed TaUBA2C-Flag and different concentrations of CRPS162/165D-HA in N. benthamiana leaves (S7C Fig). The result of Trypan blue staining showed that the effect of CRPS162/165D-HA on TaUBA2C-induced cell death was indeed dose-dependent (Fig 7E and 7F). In addition, we analyzed the effect of TaUBA2C on the expressions of defense-related genes and H 2 O 2 biogenesis-related genes. Total RNA was isolated from the WT, TaUBA2C transgenic and BSMV:TaUBA2C-inoculated plant tissues, respectively, and analyzed for the expressions of TaNPR1, TaPR1 and TaRBOHD through qRT-PCR. The results showed that the expressions of these three genes were significantly up-regulated in the TaUBA2C transgenic plants, but significantly down-regulated in the BSMV:TaUBA2C-inoculated plants (Fig 7G), suggesting that TaUBA2C may regulate the expressions of TaNPR1, TaPR1 and TaRBOHD. Moreover, we studied the expressions of these three target genes in CWMV-inoculated, CWMVS162/165A-inoculated and CWMVS162/165D-inoculated plants. The results showed that the expressions of these three genes were significantly up-regulated in the CWMVS162/165A-inoculated plants, but significantly down-regulated in the CWMVS162/165D-inoculated plants (Fig 7H), indicating that the pathogenicity of CWMV is closely related to the expression levels of three target genes. Based on the above findings we conclude that phosphorylation of CRP at S162 and S165 can attenuate the TaUBA2C-induced cell death.

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