*2.4. Virus-Induced Gene Silencing of CaHSP18.1a*

A 256-bp fragment of the *CaHSP18.1a* ORF was PCR-amplified using a specific primer pair (Supplementary Table S1). The underlined sequences are restriction enzyme cleavage sites (for *Xba*I and *Kpn*I). The resulting *CaHSP18.1a* fragment was inserted into TRV2:00 vectors, with the empty vector TRV2:00 and TRV2: *CaPDS* (phytoene desaturase gene) used

as negative and positive controls. When R9 plants reached the two true leaves stage, we followed the method of Wang et al [38], which involved mixing the pTRV1 bacterial culture with an equal volume of the TRV2:00, TRV2: *CaPDS*, and TRV2: *CaHSP18.1a* cultures; this solution was injected into the leaves of R9 plants. After incubation in the dark at 18 ◦C for 2 days, plants were transferred to incubators under preset normal conditions. After 35 days, when most of the leaves of the TRV2-*CaPDS* pepper plants had become bleached, total RNA was extracted from the leaves of the silenced TRV2: *CaHSP18.1a* plants and the negative control TRV2:00 plants, and qRT-PCR was used to detect the *CaHSP18.1a* expression level, which was used to calculate silencing efficiency.
