*4.4. Expression Analysis of LcCLA1 and LcPDS Using qRT-PCR*

Leaf chlorosis was observed after 2 weeks, and the leaves of *L. chinensis* that were not injected with infection solution, injected with pTRV1+pTRV2 and injected with pTRV1+pTRV2- *LcCLA1*, pTRV1+pTRV2-*LcPDS* were collected. The collected leaves were immediately frozen in liquid nitrogen and stored at −80 ◦C for qRT-PCR experiments. The *EXP1* gene was used as an endogenous control to normalize the results. To determine the relative levels of endogenous *LcCLA1* and *LcPDS* transcripts in infected leaves, qRT-PCR was performed using the primer pair EXP1-RT-F, EXP1-RT-R, *LcCLA1*-RT-F, *LcCLA1*-RT-R and *LcPDS*-RT-F, *LcPDS*-RT-R (Table 1). The steps of extracting total RNA, detecting RNA concentration, purity, integrity and reverse transcription are the same as in Section 4.2. qRT-PCR was performed using a StepOnePlus real-time PCR system (Applied Biosystems, Beijing, China) and a 20 μL reaction mixture. Each 20 μL reaction mixture contained 10 μL SYBR Premix Ex TaqTMII (TaKaRa, Dalian, China), 5 μL diluted cDNA, 0.8 μL forward and reverse primer (10 μM), 0.4 μL ROX Reference Dye and 3 μL ddH2O. The experiment was repeated three times, and the data were analyzed using the 2−ΔΔCt method to calculate relative gene expression levels [55].
