VIGS Knockdown of WRKY75 in Tomato

The VIGS vectors pTRV1 and pTRV2^{101 (link)} were stored in our laboratory (Fig. 9). A 199-bp WRKY75 (initially characterized by López-Galiano et al.^{102 (link)} and Chen et al.^{103 (link)}) fragment was amplified from the stem tissue of resistant tomato plants (Hm 2–2) with the specific primers V-F and V-R (listed in Supplementary Table S2) by PCR. The WRKY75 fragment and the vector pTRV2 were then digested with EcoR I and BamH I (Takara, China), and the WRKY75 fragment was ligated into the vector using T4 ligase (Takara, China) to create the TPV::WRKY75 construct; pTRV2 with empty was created to TRV::empty and was used as control. The resulting vector was introduced into Agrobacterium tumefaciens GV3101 (WEIDI, China). The VIGS experiments were carried out as described previously¹⁰⁴ . Three-week-old newly emerged leaves of Hm 2–2 tomato plants were transformed with Agrobacterium containing TPV::WRKY75 (V(pTRV1):V(pTRV2::WRKY75) = 1:1) and TRV::empty (V(pTRV1):V(pTRV2::empty) = 1:1) vectors, respectively, using a 1-mL syringe. Each experiment included three biological replicates. One week later, the plants were inoculated with R. solanacearum by using the root-cutting and root-grafting method. Disease scoring was performed according to that previously described by Lacombe et al^{105 (link)}.Figure 9

The map of pTRV1 and pTRV2 vectors.