Silencing of SlDRB1 gene reduces resistance to tomato yellow leaf curl virus (TYLCV) in tomato (Solanum lycopersicum)

Abstract

Double-stranded RNA-binding proteins are small molecules in the RNA interference (RNAi) pathway that form the RNAi machinery together with the Dicer-like protein (DCL) as a cofactor. This machinery cuts double-stranded RNA (dsRNA) to form multiple small interfering RNAs (siRNAs). Our goal was to clarify the function of DRB in tomato resistant to TYLCV. In this experiment, the expression of the SlDRB1 and SlDRB4 genes was analyzed in tomato leaves by qPCR, and the function of SlDRB1 and SlDRB4 in resistance to TYLCV was investigated by virus-induced gene silencing (VIGS). Then, peroxidase activity was determined. The results showed that the expression of SlDRB1 gradually increased after inoculation of 'dwarf tomato' plants with tomato yellow leaf curl virus (TYLCV), but this gene was suppressed after 28 days. Resistance to TYLCV was significantly weakened after silencing of the SlDRB1 gene. However, there were no significant expression differences in SlDRB4 after TYLCV inoculation. Our study showed that silencing SlDRB1 attenuated the ability of tomato plants to resist virus infection; therefore, SlDRB1 may play a key role in the defense against TYLCV in tomato plants, whereas SlDRB4 is likely not involved in this defense response. Taken together, These results suggest that the DRB gene is involved in the mechanism of antiviral activity.

Keywords: TYLCV; Tomato; VIGS; antiviral; double-stranded RNA-binding proteins1(DRB1).

Figure 1.

Structure prediction and phylogenetic analysis of SlDRB1 and SlDRB4.(a) Secondary structure prediction of SlDRB1 and SlDRB4 proteins; (b) Phylogenetic tree of SlDRB1, SlDRB4 and other proteins from Arabidopsis thaliana (ATDRB1:AT1G09700; ATDRB2:AT2G28380; ATDRB3:AT3G26932; ATDRB4:AT3G62800; ATDRB5:AT5G41070) and Solanum lycopersicum (SlDRB2:Solyc11g069460.1.1; SlDRB3:Solyc05g056100.2.1; SlDRB5:Solyc03g118950.1.1; SlDRB6:Solyc01g009190.1.1; SlDRB7:Solyc01g056600.2.1; SlDRB8:Solyc02g091460.2.1); (c) Prediction of the tertiary structure of SlDRB1 and SlDRB4 proteins.

Figure 2.

Relative expression of the SlDRB1 and SlDRB4 genes in leaves under different stress treatments.(a) and (b) 20% PEG-6000 treatment, 0.50 mmol·L⁻ SA treatment, 0.10 mmol·L⁻ BTH treatment, and 300 mmol·L⁻ NaCl treatment.The assays for each treatment consisted of three biological replicates, and different lowercase letters indicate significant differences at the 0.05 level.

Figure 3.

Relative transcript expression of SlDRB1 and SlDRB4 inoculated with TYLCV for different time periods.(a and b) Expression profiles of the SlDRB1 and SlDRB4 genes in response to TYLCV. The reference gene was SlEF1α, and the assays for each treatment consisted of three biological replicates. Data are the means ± standard errors of three independent experiments. Differences between time-course sampling points were assessed using SSPS. *p < .05, n = 3.

Figure 4.

VIGS of SlDRB1 reduced the tolerance of plants to TYLCV.(a) and (b) At the top of the figure, the phenotypes of SlDRB-silenced, negative control pTRV:00 and positive control pTRV:PDS plants are shown. The figure on the left shows the phenotypes of TRV:00, TRV:SlDRB1, and TRV:SlDRB4 at 14 days and 28 days after TYLCV inoculation. The figure on the right shows the relative expression of TYLCV in TRV:SlDRB1, TRV:SlDRB4 and TRV:00 plants at different time periods.

Figure 5.

Changes in antioxidant enzyme activity after virus inoculation.(a)Relative expression levels of POD, SOD, APX and CAT in SlDRB1-silenced plants (* denotes P < .05); (b) Relative expression levels of POD, SOD, APX and CAT in SlDRB4-silenced plants (* denotes P < .05).