N Protein of Tomato Spotted Wilt Virus Proven to Be Antagonistic Against Tomato Yellow Leaf Curl Virus in Nicotiana benthamiana

Abstract

Two phylogenetically unrelated viruses transmitted by different insect vectors, tomato spotted wilt virus (TSWV) and tomato yellow leaf curl virus (TYLCV), are major threats to tomato and other vegetable production. Although co-infections of TSWV and TYLCV on the same host plant have been reported on numerous occasions, there is still lack of research attempting to elucidate the mechanisms underlying the relationship between two viruses when they coexist in the same tomato or other plants. After assessing the effect of four TSWV-coded proteins on suppressing TYLCV in TSWV N transgenic Nicotiana benthamiana seedlings, the TSWV N protein proved to be effective in reducing TYLCV quantity and viral symptoms. Western blot analysis indicated that TSWV N was involved in down-regulating the expression level of the V1, C3, and C4 proteins of TYLCV, among which V1 was the most significantly suppressed one. Moreover, TSWV N was confirmed to reduce TYLCV V1 within both nucleus and cytoplasm, but a greater suppression was observed in cytoplasm. The co-immunoprecipitation and mass spectrometry identified 244 differential proteins from the TYLCV-infected TSWV N transgenic N. benthamiana seedling. These proteins pertaining to energy metabolism pathways were enriched, suggesting that TSWV N could inhibit TYLCV through competing for energy or regulating energy-related metabolism. The evidence presented here offers a novel perspective that will facilitate a comprehensive understanding of virus-virus and virus-host interactions, as well as a potential strategy for plant virus control through using TSWV N in the near future.

Keywords: N protein; antagonism; tomato spotted wilt virus; tomato yellow leaf curl virus; viral interactions.

FIGURE 1

Identification of TSWV‐encoded proteins affecting the pathogenicity of TYLCV. (a) A schematic diagram of the construction of each protein gene of TSWV (except RdRp) within the vector pCB301. (b, c) The reverse transcription‐quantitative PCR and western blot analysis of the relative expression of TYLCV co‐inoculated with an individual protein of TSWV in Nicotiana benthamiana 3 days post‐inoculation, respectively. The data were subjected to multiple comparisons using the Duncan's new complex polarity method. The error line represents the standard deviation of three biological replicates for each treatment, and different letters represent significant differences (p < 0.05). The symbol @ represents the antibody for TYLCV V1. Coomassie Brilliant Blue R‐250 (CBB) staining gel is used to indicate the sample loading quantity.

FIGURE 2

Interactions between TSWV N and each protein of TYLCV in Nicotiana benthamiana. (a) Western blot analysis of the relative expression of each protein with the FLAG tag of TYLCV in N. benthamiana co‐inoculated with the TSWV N or β‐glucuronidase (GUS) control at 3 days post‐inoculation. A quantitative analysis of the western blot bands was conducted using ImageJ software, and the resulting values are presented in italics above the image. (b) Western blot analysis of the expression of TSWV N‐Myc and GUS‐Myc proteins in each treatment. M: Protein marker. The symbol @ represents the antibody for the corresponding proteins. A Coomassie Brilliant Blue R‐250 (CBB) staining gel is used to indicate the sample loading quantity.

FIGURE 3

The subcellular localisation of TSWV N and V1, C3, C4 of TYLCV following transient infection in Nicotiana benthamiana (bar = 50 μm). (a) The subcellular localisation of TSWV N and V1, C3, C4 of TYLCV after single‐ or co‐transfection in N. benthamiana. (b) The subcellular localisation of TYLCV V1 in N. benthamiana is altered by TSWV N. Yellow arrows indicate the localisation in cytoplasm and white arrows indicate the localisation in nucleus.

FIGURE 4

Test results for genetically modified Nicotiana benthamiana. (a) Reverse transcription‐quantitative PCR results for the relative expression levels of each genetically modified (GM) N. benthamiana line. The data were subjected to multiple comparisons using the Duncan's new complex polarity method. The error line represents the standard deviation of three biological replicates for each treatment, and different letters represent significant differences (p < 0.05). (b) PCR results for each of the three selected transgenic lines. The PCR products were sequenced separately. (c) Western blot results for each of the three selected transgenic lines with GFP antibody. M, marker; non‐GM, non‐GM N. benthamiana; GM‐CK, GM N. benthamiana with empty‐GFP (the total length of the gene fragment was 1034 bp containing 726 bp of full‐length sequence of GFP gene, and the molecular weight of GFP protein was about 27 kDa); GM‐N, GM N. benthamiana with TSWV N (the total length of the gene fragment was 1796 bp containing 774 bp of TSWV N gene and 726 bp of GFP gene, and the molecular weight of TSWV N+GFP protein was about 57 kDa). The symbol @ represents the antibody for GFP. (d) The subcellular localisation of GM‐CK (#7) and GM‐N (#1) (bar = 50 μm).

FIGURE 5

Analysis of TYLCV pathogenicity in genetically modified (GM) Nicotiana benthamiana inoculated with TYLCV. (a) Symptoms of GM N. benthamiana inoculated with TYLCV and non‐GM N. benthamiana inoculated with TYLCV or TSWV+TYLCV at 7 and 14 days post‐inoculation (dpi). The quantitative PCR (qPCR) (b) and western blot (d) analysis of the relative expression of TYLCV V1 at 7 dpi for each treatment. The reverse transcription‐qPCR (c) and western blot (e) analysis of the relative expression of TYLCV V1 at 14 dpi for each treatment. The data were subjected to multiple comparisons using the Duncan's new complex polarity method. The error line represents the standard deviation of three biological replicates for each treatment, and different letters represent significant differences (p < 0.05). The symbol @ represents the antibody for TYLCV V1. Coomassie Brilliant Blue R‐250 (CBB) staining gel is used to indicate the sample loading amount.

FIGURE 6

Results of western blot detection of proteins with different treatments in co‐immunoprecipitation (Co‐IP) assays and the Venn diagram of host proteins obtained by mass spectrometry. (a) Western blot analysis of proteins obtained from Co‐IP of genetically modified (GM) Nicotiana benthamiana plants with the GFP antibody. M: protein marker; 1: negative control (non‐GM); 2, 3: total plant protein and protein obtained by Co‐IP from GM‐CK plants inoculated with TYLCV, respectively; 4, 5: total plant protein and protein obtained by Co‐IP from GM‐N plants inoculated with TYLCV, respectively; 6, 7: total plant proteins and protein obtained by Co‐IP from GM‐N plants inoculated with an empty vector, respectively. Samples from lanes 3, 5, and 7 were also selected for mass spectrometry analysis. (b) Venn diagram of host proteins obtained by mass spectrometry for the different treatments described above.

FIGURE 7

Bubble plots of GO terms and KEGG pathways enriched in differential proteins. (a) Bubble plots of top 15 GO terms enriched for differential proteins (p < 0.05). The GO terms belong to biological processes, cellular components, and molecular functions. (b) Bubble plots of top 20 KEGG pathways to which the differential proteins were enriched (p < 0.05). The enrichment score is used to indicate the degree of enrichment of the given GO term or KEGG pathway. The size of the bubbles (ListHits) represents the number of differential proteins enriched, with larger bubbles indicating a greater number of differential proteins included. The colour change of the bubbles represents the size of the p value.