Interaction with host SGS3 is required for suppression of RNA silencing by tomato yellow leaf curl virus V2 protein

Abstract

The V2 protein of tomato yellow leaf curl geminivirus (TYLCV) functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. The host-cell target of V2, however, remains unknown. Here we show that V2 interacts directly with SlSGS3, the tomato homolog of the Arabidopsis SGS3 protein (AtSGS3), which is known to be involved in the RNA-silencing pathway. SlSGS3 genetically complemented an AtSGS3 mutation and restored RNA silencing, indicating that SlSGS3 is indeed a functional homolog of AtSGS3. A point mutant of V2 that is unable to bind SlSGS3 also lost its ability to suppress RNA silencing, suggesting a correlation between the V2-SlSGS3 interaction in planta and the suppressor activity of V2.

Fig. 1.

Identification of SlSGS3 and its interaction with TYLCV V2 in yeast. (A) Interaction in yeast two-hybrid system. The indicated cell inocula were plated on growth media without leucine, tryptophan, histidine, or uracil. Because growth on leucine-deficient medium represents selective conditions for protein–protein interactions, efficient growth of cells coexpressing SlSGS3 and V2 indicated interaction between these proteins, whereas the absence of cell growth in two negative controls indicated the specificity of this interaction. (B) Alignment of the SlSGS3 with AtSGS3. Regions of identity and similarity are indicated by black and shaded boxes, respectively; gaps introduced for alignment are indicated by dashes. The XS domain is denoted by a horizontal bar under its sequence. Alignment was performed by using the ClustalW algorithm (www.genebee.msu.su/clustal/advanced.html).

Fig. 2.

Subcellular colocalization of V2 with SlSGS3 and AtSGS3. (A and B) V2-CFP coexpressed with YFP-SlSGS3 or YFP-AtSGS3, respectively, in N. tabacum protoplasts. (C and D) V2-CFP coexpressed with YFP-SlSGS3 in agroinfiltrated tomato leaf epidermis. Note that, adjacent to the coexpressing cell (cell 1), C and D also show a cell (cell 2) that expresses either YFP-AtSGS3 or V2-CFP, respectively. CFP signal is shown in green, YFP signal is shown in red, and signal produced by comparable levels of the colocalizing proteins is shown in yellow. Plastid autofluorescence was filtered out in A and B and is shown in blue in C. All images are projections of several confocal sections.

Fig. 3.

Interaction between V2 and SlSGS3 or AtSGS3 in planta. (A and B) Protein–protein interaction was monitored by FRET microscopy of living N. tabacum protoplasts coexpressing V2-CFP and either YFP-SlSGS3 (A) or YFP-AtSGS3 (B). Representative acceptor photobleaching images show CFP (donor) and YFP (acceptor) channels before and after bleaching. After bleaching, CFP fluorescence increases and YFP fluorescence decreases in the bleached areas indicated by rectangles. All images are projections of several confocal sections. (C) Quantification of donor fluorescent intensity in representative samples. Data represent average values of three independent experiments, with 10 protoplasts each, with indicated standard deviation values.

Fig. 4.

Genetic complementation of the Arabidopsis L1/sgs3-1 phenotype by SlSGS3 and restoration of RNA silencing. (A) PCR-based identification of L1/sgs3-1/SlSGS3 and L1/sgs3-1/AtSGS3 transgenic plants by detection of the SlSGS3 (Left) and AtSGS3 (Right) transgenes, respectively. Lane M, molecular size markers [indicated in kilobase pairs (kbp)]; lane 1, L1/sgs3-1 parental line showing no SlSGS3 cDNA-specific PCR product; lane 2, L1/sgs3-1/SlSGS3 line producing the SlSGS3 cDNA-specific 786-bp PCR product; lane 3, L1/sgs3-1 parental line showing a 1,191-bp PCR product specific for the endogenous AtSGS3 gene with its intron, but no AtSGS3 cDNA-specific 888-bp band; lane 4, L1/sgs3-1/AtSGS3 plants yielding 1,191- and 888-bp PCR products specific for the intron-containing endogenous AtSGS3 gene and the AtSGS3 cDNA transgene, respectively. (B) RT-PCR-based detection of transcripts derived from the SlSGS3 and AtSGS3 cDNA transgenes. Lane M, molecular size markers (kbp); lane 1, L1/sgs3-1 parental line; lane 2, L1/sgs3-1/SlSGS3 plants; lane 3, L1/sgs3-1/AtSGS3 plants. L1/sgs3-1/SlSGS3 and L1/sgs3-1/AtSGS3 plants yielded RT-PCR products specific for the SlSGS3 and AtSGS3 transcripts, respectively, whereas all plants contained transcripts of the constitutively expressed Actin gene. (C) Silencing of the GUS reporter in the indicated plant lines. Note that expression of SlSGS3 or AtSGS3 transgenes restored RNA silencing of GUS in L1/sgs3-1 plants to levels comparable to those of the original silenced L1 line. Data represent average values of three independent experiments with indicated standard deviations.

Fig. 5.

Correlation between V2-induced suppression of RNA silencing and V2-SlSGS3 binding. V2 and its C84S/C86S mutants were tested for their ability to suppress RNA silencing, subcellular localization, and interaction with SlSGS3. (A) RNA-silencing suppression. N. benthamiana leaves were agroinfiltrated with a mixture of a GFP-expressing construct and an empty expression vector (GFP), a V2-expressing vector (GFP+V2), or a C84S/C86S-expressing vector (GFP+C84S/C86S). (Left) Leaves 7 days after infiltration. (Right) GFP fluorescence in the same leaves. (B) Subcellular localization in N. tabacum protoplasts coexpressing C84S/C86S-CFP and YFP-SlSGS3. CFP signal is shown in green, YFP signal is shown in red, comparable levels of colocalizing green and red signals produce yellow color, and plastid autofluorescence is shown in blue. Images are projections of several confocal sections. (C) Interaction with SlSGS3. By using FRET microscopy, donor fluorescent intensity was quantified in representative samples. Data represent average values of three independent experiments, with 10 protoplasts each, with indicated standard deviation values.