*4.10. VIGS Assay of CaChiIV1*

The VIGS approach was used for the knock-down of the *CaChiIV1* gene in the pepper plant cultivar AA3, and the VIGS assay was performed as described by Liu et al. (2016) [93]. For *pTRV2:CaChiIV1*, a 232-bp cDNA part of *CaChiIV1* gene was PCR-amplified. Briefly, the *CaChiIV1* gene was cloned into a pTRV2 vector to construct the recombinant plasmid *pTRV2:CaChiIV1*, which was further used in the subsequent research to confirm the exact silencing of *CaChiIV1* (primer pairs used for vector construction are given in Table S5. Afterwards, the freeze–thaw method was used to transform pTRV1, pTRV2 (negative control), and *pTRV2:CaPDS* (positive control) along with the combined vector *pTRV2:CaChiIV1* into an *Agrobacterium tumefaciens* strain (GV3101). *A. tumefaciens* harboring pTRV1 was mixed at a 1:1 ratio with pTRV2, pTRV2-CaPDS and pTRV2-*CaChiIV1*. The agrobacterium inocula suspensions harboring pTRV1, pTRV2:00, *pTRV2:CaPDS* or *pTRV2:CaChiIV1* (OD600 = 1.0) were infiltrated into the full extended cotyledons leaves of pepper plants using a 1.0 mL clean needleless syringe [94]. Then, these infiltrated plants were conserved at 18–22 ◦C in a plant growth chamber with a 16/8 h light/dark period as defined by Wang et al. (2013) [62,95]. Forty-five days post-infiltration, leaf samples from the control and *CaChiIV1*-silenced plants were collected to measure the silencing efficiency by RT-PCR. The triphenyltetrazolium chloride (TTC) method was used to measure the root activity [62,95]. Before the TTC test, root tips (approximately 0.2 g) from the control (TRV:00) and *CaChiIV1*-silenced (*pTRV2*:*CaChiIV1*) plants were collected at various time points after NaCl stress as described by Khan et al. (2018) [77]. These experiments were executed with three biological repeats.
