*4.2. Cloning of the LcCLA1 and LcPDS Genes and VIGS Vector Construction*

The gene sequences of *LcCLA1* and *LcPDS* were obtained from transcriptome data of *L. chinensis* [6]. Raw reads were deposited in the NCBI database (https://www.ncbi. nlm.nih.gov/, accessed on 8 June 2022) under BioProject number PRJNA847051. The fresh leaves of *L. chinensis* were collected and put into liquid nitrogen for RNA isolation. Total RNA was extracted using the Plant RNA Extraction Kit (Huayueyang, Beijing, China) according to the manufacturer's instructions. The RNA concentration and purity were detected using the OneDrop OD1000 system (Wuyi Technology, Nanjing, China) and gel electrophoresis. The cDNA was synthesized using the PrimeScriptTM RT kit with the gDNA Eraser kit (TaKaRa, Dalian, China) according to the manufacturer's instructions. Primers of *LcCLA1* and *LcPDS* genes (Table 1) were designed using Primier 5 software. The fragments of *LcCLA1* and *LcPDS* were amplified using the designed primers *LcCLA1*-F/R and *LcPDS*-F/R, respectively. The Xba I restriction site was added to the 5 end of the upstream primer, and the Kpn I restriction site was added to the 5 end of the downstream primer. The PCR reaction was performed in a 50 μL volume containing 4 μL cDNA, 1 μL dNTP Mix (10 mM), 25 μL 2 × Phanta Max Buffer, 2 μL each of the forward and reverse primers (10 μM), 1 μL Phanta Max Super-Fidelity DNA Polymerase and 15 μL ddH2O. The PCR reaction was performed in the following conditions: denaturation at 95 ◦C for 3 min, followed by 35 cycles of 95 ◦C for 15 s, 56 ◦C for 15 s, 72 ◦C for 1 min, and finally extension at 72 ◦C for 5 min.


**Table 1.** List of primers used in this study.

The TRV-VIGS vectors pTRV1 and pTRV2 were used in this study as reported in previous studies [52]. AxyPrep DNA Gel Extraction Kit (Axygen, Nanjing, China) was used to purify PCR products. After purification, *LcCLA1* and *LcPDS* fragments were assembled into the pTRV2 vector using ClonExpress II one-step cloning kit (Vazyme, Nanjing, China). The specific fragments were respectively attached to pTRV2, and then the silent vectors pTRV2-*LcCLA1* and pTRV2-*LcPDS* were obtained (Figure 5).

**Figure 5.** TRV-based VIGS vectors and construction. LB and RB: left and right borders of T-DNA; 2 × 35 S: the duplicated CaMV 35 S promoter; RdRp: RNA-dependent RNA polymerase; MP: movement protein; CP: coat protein; 16 K: 16 kDa cysteine-rich protein; MCS: multiple cloning sites; Rz: self-cleaving ribozyme; NOSt: nopaline synthase terminator.

## *4.3. Preparation of Agroinfiltration Infection Solution*

The plasmids of TRV2-*LcCLA1*, TRV2-*LcPDS*, TRV2 and TRV1 were transformed into *Agrobacterium tumefaciens* strain GV3101 using the freeze-thaw method [53], separately. The transformed plasmids of TRV1, TRV2, TRV2-*LcCLA1* and TRV2-*LcPDS* were cultured in LB solid medium (with 50 μg/mL kanamycin; 25 μg/mL rifampicin). Freshly cultured *A. tumefaciens* GV3101 colonies containing pTRV1, pTRV2, pTRV2-*LcCLA1* and pTRV2- *LcPDS* were selected and added to 3 mL YEB liquid medium (with 50 μg/mL kanamycin; 25 μg/mL rifampicin). Then, they were cultured in a shaker at 28 ◦C and 170 rpm/min for 16 h. Add 1 mL of the cultured solution to 100 mL of YEB liquid medium (with

50 μg/mL kanamycin; 25 μg/mL rifampicin), and incubate at 28 ◦C and 170 rpm/min for 20–24 h. When the OD600 of the detected solution is about 1.0, take 40 mL and centrifuge at 4000 rpm for 10 min to collect the cells, and add an appropriate volume of infection buffer to resuspend until the final concentration of OD600 is 2.0. The configuration of the infection buffer was 100 mL of permeate containing 1 mL of MgCl2 (1 mol/L), 1 mL of MES (1 mol/L), and 200 μL of acetosyringone (1 mol/L), and the pH was adjusted to 5.6. Make up to volume with sterilized distilled water, which needs to be prepared and used immediately. Two kinds of infection solutions were prepared by mixing pTRV1 with pTRV2 and pTRV2-*LcCLA1* resuspension at a volume ratio of 1:1 [54], respectively. Another infection solution also mixed pTRV1 and pTRV2-*LcPDS* at the same ratio. The mixed infecting solution was placed in a 25 ◦C shaker at 100 rpm/min and cultured in the dark for 4 h to infect the leaves.
