The P1 Protein of Watermelon mosaic virus Compromises the Activity as RNA Silencing Suppressor of the P25 Protein of Cucurbit yellow stunting disorder virus

Abstract

Mixed viral infections in plants involving a potyvirus and other unrelated virus often result in synergistic effects, with significant increases in accumulation of the non-potyvirus partner, as in the case of melon plants infected by the potyvirus Watermelon mosaic virus (WMV) and the crinivirus Cucurbit yellow stunting disorder virus (CYSDV). To further explore the synergistic interaction between these two viruses, the activity of RNA silencing suppressors (RSSs) was addressed in transiently co-expressed combinations of heterologous viral products in Nicotiana benthamiana leaves. While the strong RSS activity of WMV Helper Component Proteinase (HCPro) was unaltered, including no evident additive effects observed when co-expressed with the weaker CYSDV P25, an unexpected negative effect of WMV P1 was found on the RSS activity of P25. Analysis of protein expression during the assays showed that the amount of P25 was not reduced when co-expressed with P1. The detrimental action of P1 on the activity of P25 was dose-dependent, and the subcellular localization of fluorescently labeled variants of P1 and P25 when transiently co-expressed showed coincidences both in nucleus and cytoplasm. Also, immunoprecipitation experiments showed interaction of tagged versions of the two proteins. This novel interaction, not previously described in other combinations of potyviruses and criniviruses, might play a role in modulating the complexities of the response to multiple viral infections in susceptible plants.

Keywords: RNA silencing suppression; cucurbit yellow stunting disease crinivirus; plant virus mixed infection; virus pathogenesis in plants; watermelon mosaic potyvirus.

Figure 1

Confirmation of RNA silencing suppression (RSS) activity in individually expressed viral gene products of Watermelon mosaic virus (WMV) and Cucurbit yellow stunting disease virus (CYSDV). (A) Genome maps of WMV and CYSDV. Below the size rule in kilobases, the viral ssRNAs are shown as solid horizontal lines (10,035 nucleotides for WMV, and 9,123 and 7,976 for the RNA1 and RNA2 of the bipartite CYSDV, respectively). In the WMV genome, VPg is depicted as a solid circle at the 5' end, and the poly-A tail as An at the 3' end. Viral ORFs are depicted as boxes with the names of the mature gene products. The PIPO region is shown below the polyprotein of the potyvirus leading to the partially out-of-frame product P3N-PIPO, and the protease-specific cleavages sites are indicated by arrows above and matching symbols in the gene products responsible of the proteolytic process. The different frames are shown for the crinivirus gene products. (B) The left part of the panel shows schematically the organization of patches in the Nicotiana benthamiana leaves used to test RSS activity in co-agroinfiltration of the selected gene products with the reporter green fluorescent protein (GFP). The positions for positive and negative controls, corresponding to the P1b of Cucumber vein yellowing virus (CVYV) and an empty vector (delta), respectively, are also shown. Constructs for expression of the individual gene products and the P1-HCPro cis construct are indicated above the pictures of leaves. Pictures were taken at 5 days post agroinfiltration (dpa) under UV light. (C) A representative Western blot analysis of the N-terminus MYC-tagged gene products shown in the diagrams with their expected molecular weights shown in the table. A representative blot revealed after incubation with the indicated anti-MYC specific antibody and the corresponding anti-mouse, is shown with agroinfiltrated samples, collected at 3 and 5 dpa time points, as indicated, and a non-agroinfiltrated N. benthamiana control lane labeled as C. M lane shows the migration of pre-stained molecular weight markers (sizes in KDa on the left side). RbcL corresponds to the Ponceau red-stained blot showing the large subunit of Rubisco protein as loading control.

Figure 2

Effect on RSS activity of the combination of heterologous selected gene products of WMV and CYSDV. (A) Schematic organization of patches in N. benthamiana leaves and comparison of effects on RSS activity of individual and combined gene products when co-agroinfiltrated with the reporter GFP. The positions for positive and negative controls, corresponding to the P1b of CVYV and an empty vector (delta), respectively, are also shown in the left half of every leaf. In the right side of the pictures and adjacent to the patches are depicted the constructs for expression of the individual gene products of WMV (P1 or HCPro) and of CYSDV (P25 or P22), with their corresponding combinations in the lower row. Pictures were taken at 5 dpa under UV light. (B) Time course evolution of RSS activity at 3, 5, and 7 dpa for the individual gene products WMV P1 (upper right patches), CYSDV P25 (central right patches), and their combination (lower right patches). Positive and negative controls as in A (patches in left side halves). (C) Quantification of GFP mRNA by qRT-PCR, relative to the reference gene ubiquitin, at 3 dpa, in patches agroinfiltrated with the constructs indicated below the bars. Mean values and SDs of three independent replicates are plotted, indicating statistically significant differences after t-test analysis (**indicate p < 0.05, values of p = 0.0013 for P25 vs. P1, and p = 0.0022 for P25 vs. P1+P25). (D) Absence of effect on RSS activity of CYSDV P25 when co-agroinfiltrated with CYSDV P22. Positive and negative controls as in Figure 1B (patches in left side half). Pictures were taken at 3, 5, and 7 dpa under UV light.

Figure 3

Western blot analysis of selected N-terminus tagged proteins after transient expression, individually or combined. (A) Samples of N. benthamiana patches individually agroinfiltrated with MYC-P25 (left panel) and HA-P1 (right panel) constructs collected daily between 1 and 8 dpa are shown, besides a non-agroinfiltrated N. benthamiana control lane labeled as C. The blots are revealed after incubation with the corresponding anti-MYC or anti-HA specific antibodies. (B) Samples of patches co-agroinfiltrated with MYC-P25 and HA-P1 collected daily between 1 and 8 dpa are shown, besides a N. benthamiana control lane labeled as C, and revealed with anti-MYC (left panel) or anti-HA (right panel) specific antibodies. For both (A,B) lanes labeled with M show the migration of pre-stained molecular weight marker (sizes in KDa on the left side), and RbcL correspond to the Ponceau red-stained blots showing the large subunit of Rubisco protein as loading control.

Figure 4

Dose response of the presence of WMV P1 on the RSS activity of CYSDV P25. (A) Schematic organization of patches in N. benthamiana leaves co-agroinfiltrated with the reporter GFP. The same fixed concentration of P25 is used together with different amounts of P1 to reach the indicated ratios as shown in each picture, with positive and negative controls included in the left half of every leaf as in Figure 1B. Pictures were taken at 3 dpa under UV light. (B) Quantitative values showing inverse correlation of the ratio of CYSDV P25: WMV P1 and the relative copies of GFP mRNA measured by qRT-PCR with ubiquitin as reference gene. The graph shows the values for average and SD corresponding to three biological replicates per treatment.

Figure 5

Subcellular localization of fluorescently labeled WMV P1 and CYSDV P25 proteins. Confocal laser microscopy of N. benthamiana leaves agroinfiltrated with the constructs indicated schematically on the left: P1-YFP (upper lane), P25-CFP (central lane) and with both constructs at the same time (bottom lane). The images labeled with YFP correspond to the yellow color field, those labeled with CFP to the blue color field, and in the last column the bright field is shown, the column. In the samples agroinfiltrated with individual constructs observations were also performed with the conditions for the two YFP and CFP, without detecting any cross fluorescence (not shown). Bar size 20 μm.

Figure 6

Interaction of WMV P1 and CYSDV P25. (A) Schematic representation of bimolecular fluorescence complementation (BiFC) constructs with the split YFP fused to WMV P1 and CYSDV P25, and the observation using confocal laser microscopy of N. benthamiana leaves agroinfiltrated simultaneously with the two constructs. The picture YFP corresponds to the yellow color field and the same area is shown under bright field illumination. Control samples agroinfiltrated with individual constructs were analyzed without detecting any fluorescence (not shown). Bar size 20 μm. (B) Immunoprecipitation of viral protein variants tagged with MYC or HA, as indicated. Western blot analysis with anti-MYC and anti-HA antibodies, and Ponceau staining of the large subunit of Rubisco (RbcL) as control of loading, are shown for the input fractions and the corresponding immunoprecipitation samples, as indicated.