RNAi-mediated gene silencing in Arabidopsis

For RNAi experiments in A. thaliana, a 277-nt Miatrr fragment was amplified from cDNA of M. incognita with two-pair primers using Q5^® High-Fidelity DNA Polymerases (M0419, NEB, MA, USA), and then inserted upstream and downstream of the pSAT5 intron in the forward and reverse orientations [34 (link)]. Then, after digestion with restriction enzymes XbaI and KpnI, the hairpin fragment was acquired and inserted into the pSUPER destination vector to construct the pSUPER-Miatrr-RNAi vector. Via Agrobacterium-mediated transformation, the RNAi vector was then transformed to A. thaliana Col-0 (wild-type) according to the floral dip method, following the description in a previous study [35 (link)]. Lines were verified via PCR and semiquantitative RT-PCR after hygromycin screening. Homozygous T3 plants from three Miatrr-RNAi lines were used for RNAi effect assay. The homozygous GFP-RNAi T3 lines used as control were the same as described in previous study [36 (link)]. The primers (synthesized by Tsingke Biotechnology), restriction enzymes (NEB, Beverly, MA, USA), and T4 ligase enzyme (M0202, NEB, Beverly, MA, USA) used for plasmid construction are listed in Supplementary Table S3.