Single amino acid in V2 encoded by TYLCV is responsible for its self-interaction, aggregates and pathogenicity

Abstract

The V2 protein encoded by Begomovirus is essential for virus infection and is involved in multiple functions, such as virus movement and suppression of the host defence response. In this study, we reported that V2 encoded by the Tomato yellow leaf curl virus (TYLCV), which is one of the most devastating tomato-infecting begomoviruses, could interact with itself and a S71A mutation of V2 (V2^S71A) abolished its self-interaction. Fluorescence results showed that V2 localized primarily in the cytoplasm and around the nucleus. Site-directed mutagenesis V2^S71A had the similar subcellular localization, but V2^S71A formed fewer large aggregates in the cytoplasm compared to wild-type V2, whereas the level of aggregates came to a similar after treatment with MG132, which indicates that the S71A mutation might affect 26S proteasome-mediated degradation of V2 aggregates. Meanwhile, heterologous expression of V2^S71A from a Potato virus X vector induced mild symptoms compared to wild-type V2, delay of virus infection associated with mild symptoms was observed in plants inoculated with TYLCV-S71A, which indicates that the amino acid on position 71 is also involved in the pathogenicity of V2. To the best of our knowledge, this report is the first to state that the S71A mutation of V2 encoded by TYLCV affects the self-interaction, aggregate formation and pathogenicity of V2.

Figure 1

Identification of the interaction of V2 encoded by TYLCV with itself and mutants. (a) The self-interaction of TYLCV V2 in a yeast two-hybrid system. Co-transformed yeast strain AH109 grew on SD/-Ade/-His/-Leu/-Trp medium with X-α-gal. (b) BiFC visualization of the interaction between V2 and itself/mutants. Fluorescence was observed 48 hours post-infiltration. Bars: 100 μm. (c) Western blot analyses of BiFC construct combinations from the same experiments as in (b). All combinations were detected with anti-GFP polyclonal antibody (d) Co-Immunoprecipitation assay to show the in vitro self-interaction of V2 and the interaction of V2^S71A and V2.

Figure 2

Identification the essential motif and site responsible for the self-interaction of V2. (a) Schematic representation of the motif and sites of TYLCV V2 based online prediction. (b) Schematic representation of the truncated mutants of V2 and yeast-two-hybrid analysis of the interactions with V2. (c) Identification of the interaction between the wide-type V2 and the site-directed mutants V2^G70A, V2^S71A, V2^K73A in a yeast two-hybrid assay.

Figure 3

Effects of site-directed mutation of V2 on its large aggregates. (a) Diagram of the plant expression vector containing unfused yellow fluorescent protein (YFP) or YFP fused at the N-terminus of V2 and V2^S71A. L-Bonder is the left border and R-Bonder is the right border of the T-DNA; p35S is the 35S transcript termination signal. (b) Expression of V2-YFP and V2^S71A-YFP in epidermal cells of N. benthamiana leaves. The aggregates are indicated with arrows. Bar: 50 μm. Western blot analyses of construct combinations from the same experiment. (c) Comparative analysis of large aggregates between V2-YFP and V2^S71A-YFP in N. benthamiana leaves. Bars: 500 μm. (d) The statistical number of large aggregates. Bars and numbers indicate the mean number of aggregates as measured for leaf areas of 4 mm² in 10 independent experiments for each treatment. Different lowercase letters above the bars denote significant differences (Fisher’s LSD method; P < 0.05).

Figure 4

Inhibition of the cytoskeleton and the 26S proteasome lead to changes in the number of V2-YFP and V2^S71A-YFP large aggregates in N. benthamiana. (a) Number of large aggregates in latB-treated cells and oryzalin-treated cells. Bars and numbers indicate the mean sizes of aggregates as measured for leaf areas of 4 mm² in 10 independent experiments for each treatment. Different lowercase letters above the bars denote significant differences (Fisher’s LSD method; P < 0.05). (b) Number of large aggregates in MG132-treated cells. The average number of aggregates as measured for leaf areas of 4 mm² in 10 independent experiments for each treatment. (c) Western blot analysis V2 and V2^S71A in large aggregates with or without MG132.

Figure 5

Symptoms exhibited by N. benthamiana plants infected with PVX expressing V2 and V2^S71A. (a) The symptoms of N. benthamiana plants at 16 dpi. (b) Western blot analyses of V2 and V2^S71A expression with pGR-V2, pGR-V2^S71A and pGR. (c) The virus accumulation in plants infiltrated with pGR-V2, pGR-V2^S71A and pGR. (d) Sequencing analysis of V2 in plants infiltrated with pGR-V2 and pGR-V2^S71A.

Figure 6

Effects of site mutation S71A on virus infection and relative viral DNA accumulation levels in agroinoculated solanum lycopersicum plants. (a) Symptoms of plants agroinoculated with TYLCV or TYLCV-S71A at 14 dpi. CK indicates the mock-inoculated plant. (b) Infection course of TYLCV or TYLCV-S71A. Values represent percentages of systemically infected plants at different dpi and are given as means ± SD of triplicate experiments. In each experiment, 12 plants were inoculated. (c) Accumulation of virus in TYLCV and TYLCV-S71A inoculated plants.