MAPK cascade positively regulates ToCV infection
To investigate whether ToCV infection can activate the MAPK cascade, we conducted an experiment where N. benthamiana plants were infiltrated with Agrobacterium containing ToCV-infected cDNA, and we analyzed the phosphorylation of the MAPK pathway. Western blot analysis using anti-pTEpY antibody revealed a significant increase in MAPK phosphorylation in systemically infected plants (Fig. 1A). To further assess the impact of the MAPK pathway on ToCV infection, we utilized the MAPK pathway inhibitor U0126 33. Inhibition of the MAPK pathway by U0126 led to alleviation of symptoms in N. benthamiana plants (Fig. 1B). Additionally, total MAPK pathway phosphorylation was reduced in U0126-treated plants (Fig. 1C). Furthermore, U0126 treatment significantly reduced the accumulation of ToCV in plants, as demonstrated by Western blot analysis using anti-CP antibody (Fig. 1C). Both RNA genomic accumulations of ToCV were significantly decreased in U0126-treated N. benthamiana plants (Fig. 1D).
To gain further insights into the role of the MAPK cascade in viral infection, we utilized tobacco rattle virus (TRV)-based gene silencing (VIGS) to silence key components of the MAPK cascade, including NbMKKKα, NbMKK2, NbMPK3, and NbMPK6. The silencing efficiency of these genes was confirmed by quantitative RT-PCR (qRT-PCR) at 7 days post-infiltration (dpi) (Fig. S1). Silencing of NbMKKKα, NbMKK2, NbMPK3, and NbMPK6 resulted in reduced ToCV CP and RNA accumulation compared to control TRV-00 inoculated plants (Fig. 1E-F). Additionally, we overexpressed MAPKKKα after ToCV inoculation, which led to an increase in the phosphorylation level of the MAPK cascade and a increase in the accumulation of ToCV CP (Fig. 1G). ToCV RNA accumulation in plants overexpressing MAPKKKα was higher than in control EV-inoculated plants (Fig. 1H). These results collectively indicate that the MAPK cascade positively regulates ToCV infection.
P7 promotes ToCV infection by activating the MAPK pathway and is associated with membrane localization of P7
Previously, we investigated the role of the P7 protein by conducting RNA-seq analysis in wild-type (WT) and P7-overexpression (OE-P7) plants. Our analysis revealed 508 deferentially expressed genes (DEGs) induced in OE-P7 plants, with 300 genes up-regulated and 208 genes down-regulated using a log2 FC change threshold of > 1 and < − 1 and a t-test ρ value < 0.05 (SI Appendix, Table S1). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed significant enrichment of DEGs in plant-pathogen interaction and the MAPK signaling pathway, with the latter exhibiting the highest number of enriched genes (Fig. 2A). These data suggests P7 protein probably activates plant MAPK cascade.
To further explore the MAPK signaling pathway conducted by P7 protein, we conducted qRT-PCR analysis on the enriched DEGs. Among the 12 DEGs enriched in the plant MAPK signaling pathway, 11 genes were found to be up-regulated (Fig. S2). Notably, key genes such as MPK3, MAPKKK3, MPKKK17, and MPKKK18 in the MAPK pathway showed significant up-regulation, consistent with the RNA-seq data (Fig. 2B). These findings suggest that P7 may activate the MAPK pathway in plants. To corroborate these findings, we assessed the phosphorylation status of proteins in the MAPK pathway using anti-pTEpY antibody in OE-P7 plants. The results indicated a noticeable increase in the phosphorylation levels of proteins related to the MAPK pathway (Fig. 2C).
To investigate the role of P7 during ToCV infection in plants, we constructed a PVX expression vector carrying the ToCV P7 gene (PVX:P7) and inoculated it into N. benthamiana plants. As a control, a PVX expression vector carrying GFP (PVX:GFP) was used. At 4 days post-inoculation (dpi), the phenotype of plants infected with PVX:P7 was similar to that of plants infected with PVX:GFP. However, as the expression of P7 continued, severe mosaic disease, yellowing, and leaf deformities were observed at 8 dpi and 12 dpi in PVX:P7-inoculated plants, while PVX:GFP-inoculated plants exhibited intervein chlorosis and mild mosaic symptoms (Fig. 2D). Furthermore, Western blot analysis showed a significant increase in PVX CP accumulation in the systemic leaves of PVX:P7-inoculated plants compared to PVX:GFP-inoculated plants at 4 dpi, 8 dpi, and 12 dpi (Fig. 2E). The same result was confirmed by qRT-PCR analysis (Fig. 2F). These results strongly indicate that P7 functions as a pathogenic factor that enhances viral infection in N. benthamiana.
Several studies have highlighted the importance of pathogenic factors and their subcellular localization 24, 34. In our study, we analyzed the subcellular localization signal of P7 using TMHMM software (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The prediction revealed a transmembrane region spanning amino acid residues 5 to 27 of P7, suggesting that P7 might be localized in the plasma membrane (Fig. S3A). To confirm this prediction, we fused P7 with GFP at the C-terminal end (P7-GFP) and co-expressed it with Arabidopsis thaliana plasma membrane intrinsic protein 2A (AtPIP2A)-mCherry in N. benthamiana. AtPIP2A-mCherry serves as a marker for plasma membrane localization 35. Laser scanning confocal microscopy (LSCM) confirmed that P7-GFP co-localized with AtPIP2A-mCherry at the plasma membrane (Fig. 2G). Western blot analysis with subcellular fractionation also verified the presence of P7-GFP in the plasma membrane (Fig. 2H). These results provided strong evidence that P7 is indeed a plasma membrane-localized protein.
To further investigate the subcellular localization and significance of the predicted membrane region of P7, we constructed a mutant of P7 (designated as mP7) that could not be localized to the plasma membrane. The expression vector was designed to destroy the transmembrane domain of mP7 through TMHMM software analysis (Fig. S3B). Co-expression of mP7-GFP with AtPIP2A-mCherry in N. benthamiana revealed that mP7-GFP did not co-localize with AtPIP2A-mCherry (Fig. 2I). Western blot analysis with subcellular fractionation confirmed that mP7 was present in the cytoplasm and nucleus (Fig. 2J). These data strongly demonstrated that P7 is indeed a plasma membrane-localized protein.
To test the correlation of the membrane-associated region of P7 with its pathogenicity activation, we constructed a mutant ToCV infectious clone (ToCVmP7), in which the P7 gene was mutated to mP7. Compared with plants infected with wild-type ToCV, plants infected with ToCVmP7 exhibited alleviated symptoms (Fig. 2K). Furthermore, both the viral RNA and CP accumulation levels were reduced in plants infected with ToCVmP7 (Fig. 2L, 2M). And interestingly, the phosphorylation level of the MAPK cascade was similar in ToCVmP7 infected plants with that in ToCV infected plants, suggests mP7 also activates plant MAPK cascades but losses the capacity to enhance virus infection (Fig. 2K-M). Taken together, these findings indicate that P7 promotes ToCV infection through activating the MAPK cascade, and this function is closely associated with the membrane localization of P7.
P7 interacts with NbMPK3 and recruits it to plasma membrane
To elucidate the detailed mechanism of how P7 regulates the MAPK pathway, we conducted Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to screen for potential host factors that interact with P7. The LC-MS/MS results identified 10 proteins from N. benthamiana that probably interact with P7, including NbMPK3, a key protein in the MAPK pathway 12, 36, and a remorin protein NbREM1.1, also named as a plasma membrane nanodomain-associated protein 37, 38, which were found with high frequency in the analysis (SI Appendix, Table S2). As MPK3 is well-documented in the MAPK cascade, we focused on verifying the interactions between P7 and NbMPK3.
To confirm the interaction between P7 and NbMPK3, we employed pull-down assays, luciferase complementation assay (LCI), and bimolecular fluorescence complementation (BiFC). These experiments demonstrated that P7 interacted with NbMPK3 both in vitro and in vivo (Fig. 3A-C). Furthermore, in the subcellular fractionation experiments using BiFC assays, Western blot analysis revealed that NbMPK3 was located in both the nucleus and cytoplasm when co-expressing GUS-nYFP and NbMPK3-cYFP (GUS-nYFP + NbMPK3-cYFP). On the other hand, P7 was localized in the plasma membrane when co-expressing P7-nYFP and GUS-cYFP (P7-nYFP + GUS-cYFP). When co-expressing P7-nYFP and NbMPK3-cYFP (P7-nYFP + NbMPK3-cYFP), NbMPK3 was recruited by P7 and transferred to the plasma membrane, disappearing from the nucleus (Fig. 3D).
In addition, we examined the subcellular localization of NbMPK3 fused with GFP at the C-terminal end (NbMPK3-GFP) in N. benthamiana plants using LSCM at 48 hours post-infiltration (hpi). We observed that NbMPK3-GFP was present in both the cytoplasm and nucleus (Fig. 3E). Western blot analysis further confirmed the presence of NbMPK3-GFP in protein extracts from both the cytoplasm and nucleus (Fig. 3F). These results collectively indicate that NbMPK3 is recruited by P7 and transferred to the plasma membrane during their interaction.
NbMPK3 phosphorylates the Ser59 residue of P7 and enhances virus infection
To investigate the role of P7 in regulating the MAPK pathway, we hypothesized that P7 might act as a substrate of NbMPK3, leading to its phosphorylation. To validate this hypothesis, we conducted several experiments. Firstly, we transiently expressed P7-Flag in wild-type (WT) or NbMPK3-overexpressing (OE-NbMPK3) plants. Immunoprecipitation of P7-Flag followed by phosphoprotein detection using pIMAGO reagent revealed that overexpression of NbMPK3 enhanced the phosphorylation level of P7 (Fig. 4A). Conversely, when P7-Flag was expressed in plants with silenced NbMPK3 (TRV:NbMPK3), the phosphorylation level of P7 was impaired (Fig. 4B).
To further confirm P7 phosphorylation by NbMPK3, we purified recombinant NbMPK3-GST and P7-GST proteins from E. coli and performed in vitro phosphorylation assays using pIMAGO. The results showed that P7-GST alone did not show any phosphorylation signal, but when incubated with pre-activated NbMPK3-GST, a clear phosphorylation signal was detected (Fig. 4C). To identify the precise amino acid residues of P7 phosphorylated by NbMPK3, LC-MS/MS analysis was performed on in vitro phosphorylated P7. The analysis revealed serine residues at positions 39, 54, and 59 (designated as S39, S54, and S59) and a tyrosine residue at position 45 (designated as Y45) as potential phosphorylation sites (Fig. 4D, upper panel). Among them, S59 was consistently identified in four independent assays out of nine independent LC-MS/MS analyses (Fig. 4D, lower panel; SI Appendix, Table S3). To verify the phosphorylation site, we overexpressed P7-Flag in TRV:00- or TRV:NbMPK3-inoculated plants, and the phosphorylation of P7 at S39, Y45, S54, and S59 was monitored using a parallel reaction monitoring (PRM) system. The results showed that phosphorylation of P7 at S59 was significantly reduced in TRV:NbMPK3-inoculated plants compared to TRV:00-inoculated plants (Fig. 4E). However, there was no difference in phosphorylation of P7 at S39, Y45, and S54 between TRV:00- and TRV:NbMPK3-inoculated plants (Fig. 4E). These results indicated that Ser59 is the critical residue phosphorylated by NbMPK3.
To study the importance of Ser59 in P7 during ToCV infection, we generated two mutant ToCV infectious clones (ToCVS59A and ToCVS59D), where the serine residue at position 59 was substituted with an alanine to mimic the non-phosphorylated state, or with an aspartic acid to mimic the phosphorylated state. In comparison to the plants infected with wild-type ToCV, plants infected with ToCVS59D displayed the most severe yellowing of leaves (Fig. 4G). Moreover, both the accumulation of CP and RNA were highest in plants infected with ToCVS59D (Fig. 4H-I). These findings collectively demonstrate that P7 is phosphorylated at Ser59 to enhance virus infection.
Furthermore, we investigated the role of NbMPK3 in ToCV infection by silencing NbMPK3 through tobacco rattle virus (TRV)-mediated gene silencing (VIGS) in N. benthamiana. We then inoculated ToCV into NbMPK3-silenced plants (ToCV + TRV:NbMPK3) or TRV:00-infected plants (ToCV + TRV:00). Plants infected with ToCV + TRV:NbMPK3 exhibited milder mosaic symptoms and significantly decreased levels of ToCV RNA compared to ToCV + TRV:00-infected plants (Fig. 4J, 4K, and 4L). Additionally, we inoculated ToCVS59D into NbMPK3-silenced plants (ToCVS59D + TRV:NbMPK3) and found that these plants exhibited severe mosaic symptoms with increased ToCV RNA levels compared to ToCV + TRV:00 or ToCV + TRV:NbMPK3-infected plants (Fig. 4J, 4K, and 4L). Similarly, upon ToCV infection, overexpressing NbMPK3 (OE-NbMPK3) plants showed more severe yellowing of leaves and higher ToCV RNA levels compared to control wild-type (WT) plants (Fig. 4M, 4N, and 4O). However, when ToCVS59A was inoculated into OE-NbMPK3 plants, the symptoms and ToCV RNA levels were reduced compared to ToCVWT-infected plants (Fig. 4M, 4N, and 4O). These results further confirm that NbMPK3 phosphorylates the Ser59 residue of P7, enhancing virus infection.
Phosphorylated P7 interacts with NbREM1 to reduce callose deposition
Considering that P7 is phosphorylated on the plasma membrane and enhances viral infection, we sought to identify host factors involved in P7 functions on the plasma membrane. Among the candidates screened in the LC-MS/MS analysis (SI Appendix, Table S2), we focused on NbREM1, which has been implicated in callose deposition processes 37, 38. Through LCI and pull-down assays, we confirmed the interaction between P7 and NbREM1 both in vivo and in vitro (Fig. 5A-B). Interestingly, we observed that only the phosphorylated form of P7 at S59 (designated as P7S59D) was capable of interacting with NbREM1 in the LCI and pull-down analyses (Fig. 5A-B). This finding suggested that the phosphorylation status of P7 at S59 might regulate callose deposition and, consequently, enhance virus transmission.
To investigate the effect of different P7 phosphorylation states on callose deposition, we individually inoculated ToCV and its mutant infectious clones (ToCVS59D and ToCVS59A) into N. benthamiana plants through agro-infiltration. After 15 days, we observed a significant reduction in callose deposition in plants inoculated with ToCVS59D compared to those inoculated with ToCV or ToCVS59A (Fig. 5C-D, upper panel). Similarly, when we inoculated ToCV and its mutants in OE-NbMPK3 plants, callose deposition was significantly lower in plants inoculated with ToCVS59D and ToCV than in plants inoculated with ToCVS59A (Fig. 5C-D, lower panel).
Next, we investigated the impact of P7 phosphorylation status on the cell-to-cell transmission of PVX-GFP 41. We co-inoculated P7-Flag, P7S59D-Flag, or P7S59A-Flag with PVX-GFP and observed the transmission efficiency. After 3 days, 25% (6 of 24) of PVX-GFP fluorescence was removed from more than 40 cells in the presence of P7-Flag, and approximately 12.5% of fluorescence was removed from more than 50 cells (Fig. 5E-F, upper panel). In contrast, with the presence of P7S59D-Flag, 56% (17 of 30) of fluorescence moved out of more than 40 cells, and about 26% of fluorescence moved out of more than 50 cells (Fig. 5E-F, upper panel). These results indicated that the mimic-phosphorylated state of P7 (P7S59D) exhibited a higher cell-to-cell transmission efficiency for PVX-GFP compared to the non-phosphorylated state of P7 (P7S59A).
Additionally, we explored the effect of NbMPK3-phosphorylated P7 on viral spread. We co-inoculated NbMPK3-Flag into N. benthamiana with PVX-GFP and P7 or its mutants. After 3 days, 51% (17 of 33) of PVX-GFP fluorescence was removed from more than 40 cells in the presence of P7-Flag, and approximately 21% of fluorescence was removed from more than 50 cells (Fig. 5E-F, lower panel). In contrast, when P7S59D-Flag was present, 50% (16 of 32) of fluorescence moved out of more than 40 cells, and about 22% of fluorescence moved out of more than 50 cells (Fig. 5E-F, lower panel). These findings provided further evidence that NbMPK3-phosphorylated P7 interacts with NbREM1, thereby reducing callose deposition and promoting viral transmission. Overall, these findings demonstrate the importance of P7 phosphorylation at Ser59, mediated by NbMPK3, in regulating callose deposition and facilitating virus transmission during the interaction of ToCV with the host plant.
NbREM1 negatively regulates ToCV infection
To investigate the role of NbREM1 in ToCV infection, we utilized NbREM1-overexpression (OE-REM1) plants and NbREM1 knockout mutant plant lines (ΔNbrem1) 42. After 15 days of ToCV infection, we observed that the accumulation levels of ToCV CP and RNA were significantly reduced in the OE-REM1 plants compared to the WT plants, as demonstrated by Western blot and qRT-PCR analyses (Fig. 6A-B). Conversely, in the ΔNbrem1 plants, the accumulation of ToCV RNA and CP was significantly increased compared to the WT plants (Fig. 6A-B). These results suggest that NbREM1 negatively regulates ToCV infection. Additionally, to assess callose deposition in these plants, we employed aniline blue staining. The callose deposition was significantly increased in OE-REM1 plants, whereas it was reduced in ΔNbrem1 plants compared to WT plants (Fig. 6C-D).
To further investigate the correlation of NbREM1 with virus transmission, we inoculated PVX-GFP into OE-REM1 and ΔNbrem1 plants. In OE-REM1 leaves, the area of the green fluorescent infection foci was significantly smaller than that in the wild-type plants, with only 3% (1/30) of PVX-GFP spread beyond 40 cells. Conversely, in ΔNbrem1 leaves, the infection foci were significantly larger than those in the wild-type leaves, with 24% (6/25) of PVX-GFP spread beyond 40 cells (Fig. 6E-F, left panel). Furthermore, when co-inoculating P7-Flag, P7S59D-Flag, or P7S59A-Flag with PVX-GFP into OE-REM1 and ΔNbrem1 plants, we observed that in both OE-REM1 and ΔNbrem1 plants, P7-Flag and P7S59D-Flag increased the transmission efficiency of PVX-GFP compared to P7S59A-Flag (Fig. 6E-F, right panels). These findings indicate that phosphorylated P7 influences the function of NbREM1 to facilitate viral spread.
In summary, our results suggest that NbREM1 plays a negative regulatory role in ToCV infection, as demonstrated by reduced viral accumulation and increased callose deposition in OE-REM1 plants, and increased viral accumulation and reduced callose deposition in ΔNbrem1 plants. Additionally, phosphorylated P7 disrupts the function of NbREM1 in facilitating viral spread, further highlighting the interplay between P7, NbMPK3, and NbREM1 in the context of ToCV infection.