Plasmid construction and genetic transformation
To study the expression patterns of miR398b, a 1233 bp promoter region of miR398b was cloned into the pGWB433 vector and used to drive expression of the GUS reporter gene in Arabidopsis . To localize the CSD family proteins, cDNA sequences lacking stop codons corresponding toGhCSD1 , GhCSD2 , GhCCS and GhCSD3 were inserted into the N-terminal GFP-fusion expression vector PMDC84 (Curtis and Grossniklaus, 2003) by Gateway Cloning (Invitrogen, USA). To express VdNLP1:GFP protein, the cDNA sequence of VdNLP1 lacking the stop codon was inserted into the N-terminal GFP -fusion expression vector PMDC84 by Gateway Cloning. To examine whether miR398b represses the expression of the NBS-LRR gene, the 35S promoter with duplicated enhancers was cloned into the reporter vector pGreenII 0800-LUC to generate the vector pGreenII 0800-35s:LUC . A 100 bp fragment of Gh_A04G0380 and Gh_D05G3257containing the predicted binding sites was amplified from cotton genomic DNA and ligated into pGreenII 0800-35s:LUCat the Not Ι site. The 21 bp target sites of Gh_D05G3257(5NBts) and the mutated target sites of Gh_D05G3257 (5NBmts) were amplified using the primers 5NBts-F/R and 5NBmts-F/R, respectively, according to the method described previously (Li et al., 2019). The isolated fragments were ligated into the N-terminal GFP-fusion expression vector pMDC84-35s:GFP at the Kpn Ι site. For VIGS vector construction, fragments of GhCSD1 , GhCSD2 ,GhCCS , and NB-ARC were amplified from root samples ofG. hirsutum . PCR products were digested by two restriction endonucleases, Bam HI and Kpn I, and ligated to the TRV vector as reported previously (Gao et al., 2013). To study the function of miR398b, a 313 bp genomic sequence containing the miR398b precursor was cloned and ligated into a pK2GW7.0 vector to overexpress the miR398b precursor. The miR157 overexpression vector was generated previously (Liu et al., 2017). A miR398b-resistant version of GhCSD2 (rGhCSD2 ) was generated by introducing six mutations into the target sites of GhCSD2 through recombinant PCR and cloned into the vector pK2GW7.0. The primer sequences used in vector construction are listed in Table S2. All vectors were transferred to Agrobacterium tumefacien (GV3101).
For genetic transformation, Agrobacterium tumefaciens (strain GV3101)-mediated transformation was used to transform hypocotyl sections of G. hirsutum cv. J668 according to a previously described method (Jin et al., 2006). Null plants containing no transgenes were separated from self-pollinated35s:rCSD2 hemizygotes TN and self-pollinated 35s:miR398bhemizygotes ON.