*2.2. Silencing Efficiency of LcCLA1 and LcPDS in L. chinensis after Infection*

Two weeks after injection, an obvious yellow leaf phenotype was observed in the leaves of *L. chinensis* after injection of pTRV2-*LcCLA1*. The difference is that the leaves injected with pTRV2-*LcPDS* infection solution had less yellow leaf phenotype. Injection efficiency was detected using PCR, and leaves not injected with *Agrobacterium* (CK), pTRV1+pTRV2 (Mock), pTRV1+pTRV2-*LcCLA1* and pTRV1+pTRV2-*LcPDS* were selected for PCR detection. The results showed that no band was seen in CK, while pTRV1+pTRV2 and pTRV1+pTRV2- *LcCLA1* had a clear band at 260 bp (Figure 2a). Similarly, for the injection of *LcPDS*, no band was observed in the CK region, but about 240 bp bands were observed in pTRV1+pTRV2 and pTRV1+pTRV2-*LcPDS* (Figure 2b). This indicates that the tip needle injection method was successful in achieving infection of the leaves of *L. chinensis*.

**Figure 2.** Testing of *LcCLA1* and *LcPDS* injection efficiency. M: marker; 1–3: leaves without injection; 4–6: leaves injected with pTRV1 and pTRV2; 7–9 (**a**): leaves injected with pTRV1 and pTRV2-*LcCLA1*: 7–9 (**b**): leaves injected with pTRV1 and pTRV2-*LcPDS*.

From the perspective of leaf phenotype, plants inoculated with pTRV1 and pTRV2 showed no significant difference in leaf morphology compared to CK (Figure 3a,b). In comparison with CK, leaves injected with pTRV1+pTRV2-*LcCLA1* were found to show a distinct etiolation phenotype (Figure 3c). However, leaves injected with pTRV1+pTRV2- *LcPDS* showed a weaker etiolation phenotype, which was less pronounced than that of leaves injected with pTRV1+pTRV2-*LcCLA1*. (Figure 3d). Phenotypic differences suggest that the expression of the gene *LcCLA1* may be repressed in leaves infiltrated by pTRV2- *LcCLA1*. The expression level of *LcPDS* might not be completely suppressed because the leaves showed incomplete chlorotic phenotype. The reason for this discrepancy is unclear,

but it is speculated that *LcPDS* may have a lower density of *Agrobacterium* and less function in leaves.

**Figure 3.** Silencing of *LcCLA1* and *LcPDS* in *L. chinensis* using TRV VIGS vector. (**a**) Leaf of *L. chinensis* without *Agrobacterium* injected as a control (CK). (**b**) Leaf of *L. chinensis* injected with pTRV1 and pTRV2. (**c**) Leaves of *L. chinensis* injected with pTRV1 and pTRV2-*LcCLA1* showed etiolation phenotype after two weeks. (**d**) Leaves of *L. chinensis* injected with pTRV1 and pTRV2-*LcPDS* showed less etiolation phenotype after two weeks.
