*3.3. CaHSP18.1a-Silenced Plants Sensitive to Abiotic Stress*

Confirming the VIGS procedure, after about 40–45 days, plants injected with the positive control TRV2: *CaPDS* showed a large area of typical white leaves (Supplementary Figure S1A), while under normal conditions there was no difference between *CaHSP18.1a*silenced (TRV2:*CaHSP18.1a*) and negative control (TRV2:00) pepper plants. The silencing efficiency of *CaHSP18.1a*-silenced and TRV2:00 plants was assessed using q RT-PCR. As shown in Supplementary Figure S1A, the expression level of *CaHSP18.1a* in the silenced pepper plants decreased to less than 20% of that observed in the negative control plants. Thus, the silencing efficiency for *CaHSP18.1a*-silenced plants reached more than 80% (Supplementary Figure S1B). Therefore, control plants (TRV2:00) and silenced plants (TRV2:*CaHSP18.1a*) were used for the follow-up investigation.

HS (42 ◦C) was applied to *CaHSP18.1a*-silenced and control pepper plants for 3 h, and the silenced plants and the control group began to show different degrees of wilting. The heat-stress treatment (42 ◦C) induced significantly different symptoms after 24 h, such that the new growth of *CaHSP18.1a*-silenced plants was seriously wilted with curled leaves and shed lower leaves, while the leaves of the control plants were only slightly curled (Figure 3A). In addition, the MDA content and REL was lower in the control plants compared to *CaHSP18.1a*-silenced pepper plants (Figure 3B,C); however, the total chlorophyll content was higher in the control than in the *CaHSP18.1a*-silenced plants (Supplementary Figure S1C).

**Figure 3.** TRV2:*CaHSP18.1a* and TRV2:00 plant phenotypes under heat, drought, and salt treatments, respectively. (**A**–**C**) TRV2:*CaHSP18.1a* and TRV2:00 plant phenotypes, malonaldehyde (MDA) content, and relative electrolyte leakage (REL) under the 42 ◦C heat treatment for 24 h; (**D**–**F**) TRV2:*CaHSP18.1a* and TRV2:00 plant phenotypes, MDA content, and relative electrolyte leakage (REL) following salt stress by being soaked in 300 mM NaCl solution for 24 h; (**G**–**I**) TRV2:*CaHSP18.1a* and TRV2:00 plant phenotypes, MDA content, and REL following drought stress by being soaked in 300 mM mannitol solution for 24 h. Data are means with standard deviations of three biological replicates. Different letters denote statistical significance (*p* ≤ 0.05).

To study the salt-tolerance of silenced and control plants, we washed their roots and soaked them in 300 mM NaCl solution for 24 h. The leaves of silenced plants showed symptoms of wilting, shriveling, and serious yellowing, with lower leaves that had begun to absciss, while the leaves of the control plants only showed some yellowing and did not exhibit obvious wilting. The leaves of the control plants showed only yellowing and no apparent wilting (Figure 3D). The MDA content of both plants increased significantly, but that of silenced plants was higher than that of control plants (Figure 3E). Relative electrolyte leakage (REL) was higher in silenced plants compared to control plants (0.96 versus 0.65) (Figure 3F). To study the effects of *CaHSP18.1a*-silencing on drought tolerance, the silenced and control plants were soaked in 300 mM mannitol solution for 36 h. The *CaHSP18.1a*-silenced pepper showed severe loss of water and wilting, while control plants showed no obvious change (Figure 3G). Furthermore, the MDA content and REL both exhibited a similar increase in the silenced pepper plants (Figure 3H,I). This indicated that silencing of *CaHSP18.1a* reduced the drought tolerance of pepper plants.
