GoKawiilPlant materials and growth conditions
The Arabidopsis thaliana ecotype Col-0 was used as the WT in this study. The mekk1 (salk_052557), mpk4, summ2 (summ2-8) and mekk2 mutants have been reported previously18,61, and the T-DNA insertion mutant (salk_022911C, sag101) was obtained from the Arabidopsis Biological Resource Center. The adr1 triple (adr1/L1/L2)62, nrg1 double (nrg1.1/1.2)63, adr1 triple/nrg1 double (adr1/L1/L2/nrg1.1/1.2)53, mekk1/summ2, mpk4/mekk2 ref. 32, pad4-1 ref. 64 and eds1-2 ref. 46 were previously described. The adr1/L1/L2/mekk1 or adr1/L1/L2/mpk4 mutants were obtained by crossing adr1/L1/L2 with mekk1 or mpk4 heterozygous mutants, respectively. The genotypes of the mutants were confirmed by PCR using the primers listed in Supplementary Table 1. The N. benthamiana eds1 ref. 65, pad4 ref. 65, epss (eds1a/pad4/sag101a/sag101b)66, nrg1-1 ref. 67 and nrc2/3/4 ref. 68 mutants were previously described.
The Arabidopsis and N. benthamiana plants were grown in soil (Metro Mix 366 for Arabidopsis and LP5 for N. benthamiana) in a growth room at 23 °C, 50% relative humidity, 75–100 µE m−2 s−1 light with a photoperiod of 12-h light–12-h dark. Seedlings were grown on plates containing half-strength Murashige and Skoog medium (1/2 MS) with 0.5% sucrose, 0.8% agar and 2.5 mM MES at pH 5.7 in a growth room with the same conditions as above.
Construction of plasmids
The virus-induced gene silencing (VIGS) constructs of pTRV-RNA1 and pTRV-RNA2 of pYL156-GFP (vector control) and pYL156-MEKK1 and plant expression constructs of MEKK2, HopAI1, SUMM2 and SUMM2ac have been previously reported18,61. The cDNA coding regions of ADR1-L2 (AT5G04720), EDS1 (AT3G48090), SAG101 (AT5G14930), PAD4 (AT3G52430) and RBA1 (AT1G47370) were amplified from Col-0 cDNA using the primers containing BamHI at the 5′ end and StuI at the 3′ end and ligated into a plant gene expression pHBT vector under the control of the CaMV 35S promoter with a HA, FLAG, GFP or mCherry tag at the C terminus. The genomic DNA fragment of gADR1-L1 (AT4G33300, 2,844 bp) was amplified from Col-0 genomic DNA and subcloned into the pHBT vector under the CaMV 35S promoter with a FLAG, GFP or mCherry tag at the C terminus using a one-step cloning kit (Vazyme Biotech). SUMM2G2A, SUMM2ac, SUMM2ac/G2A, SUMM2ac/L22A, SUMM2ac/D12N, SUMM2ac/E13Q and SUMM2ac/D12N/E13Q were generated by site-directed mutagenesis using Platinum Pfx DNA polymerase-mediated PCR.
To make binary vector constructs, gADR1 (AT1G33560, 2,787 bp), gADR1-L1, ADR1-L2, EDS1, RBA1 or SUMM2 mutant variants were subcloned into the binary vectors pMDC32-p2x35S::HA, pMDC32-p2x35S::FLAG or pMDC32-p2x35S::GFP by BamHI and StuI digestions or pTA7002-HA and pTA7002-GFP by XhoI and StuI digestions. To generate the binary vector pCB302-pSUMM2::SUMM2-GFP, the promoter of SUMM2 (2,685 bp upstream of ATG) was subcloned into the binary vector pCB302::GFP by XhoI and BamHI digestions, and SUMM2 was inserted into pCB302-pSUMM2::GFP by BamHI and StuI digestions. The fragment of SUMM2CC (1–480 bp) was subcloned into the binary vector pMDC32-p2x35S::GFP by BamHI and StuI digestions. To generate pCAMBIA2300-p35S::gADR1-L1-nYFP (1–174 amino acids) and pCAMBIA2300-p35S::gADR1-L1-cYFP (175–239 amino acids) plasmids, gADR1-L1 was amplified from pHBT-p35S::gADR1-L1-FLAG and subcloned into pCAMBIA2300-p35S::nYFP and pCAMBIA2300-p35S::cYFP using a one-step cloning kit by BamHI and SmaI digestions. To generate pCAMBIA2300-p35S::EDS1-nYFP and pCAMBIA2300-p35S::PAD4-cYFP plasmids, EDS1 or PAD4 was amplified from pHBT-p35S::EDS1-FLAG or pHBT-p35S::PAD4-FLAG and subcloned into pCAMBIA2300-p35S::nYFP and pCAMBIA2300-p35S::cYFP using a one-step cloning kit by BamHI and SmaI digestions. To generate pCAMBIA1300-p35S::gADR1-L1-TagRFP-HA, gADR1-L1 was amplified from pHBT-p35S::gADR1-L1-FLAG and subcloned into pCAMBIA1300-p35S::TagRFP-HA69 using a one-step cloning kit by BamHI or SpeI digestion. To construct the pCAMBIA1300 binary vector containing the native promoter-driven gADR1-L1 with a GFP tag at the C terminus, the promoter (2,036 bp upstream of the start codon) and genomic sequence were amplified from Col-0 genomic DNA using primers containing XbaI at the 5′ end and StuI at the 3′ end, and ligated into pCAMBIA1300-GFP to obtain the pCAMBIA1300-pADR1-L1::gADR1-L1-GFP construct. HopAI1 was subcloned into the binary vector pMDC32-p2x35S::HA by BamHI and StuI digestions. The SlNRC3 gene from tomato was synthesized into the pUC57 vector (GenScript). To generate pMDC32-p2x35S::SlNRC3-GFP, SlNRC3 was amplified from pUC57-SlNRC3 and ligated into the pMDC32-p2x35S::GFP vector using a one-step cloning kit by BamHI and StuI digestions. The construct of pBRI1::BRI1-GFP has been previously described70.
All the primer sequences have been listed in Supplementary Table 1. The sequences of all genes and mutations were verified by Sanger sequencing.
Agrobacterium-mediated VIGS assays
Agrobacterium tumefaciens strain GV3101 carrying pTRV-RNA1 or pTRV-RNA2 was first cultured in 2 ml LB liquid medium containing 50 µg ml−1 kanamycin and 25 µg ml−1 gentamicin in the 20-ml glass culture tubes overnight at 28 °C in a roller drum and then transferred to 200 ml fresh LB liquid medium containing 50 µg ml−1 kanamycin, 25 µg ml−1 gentamicin, 10 mM MES and 20 µM acetosyringone for overnight at 28 °C with 200 rpm shaking. Cells were pelleted by 1,300g centrifugation, resuspended in buffer containing 10 mM MgCl 2 , 10 mM MES and 200 μM acetosyringone, adjusted to optical density at 600 nm of 1.5, and incubated at 25 °C for at least 3 h. Bacterial cultures containing pTRV-RNA1 and pTRV-RNA2 derivatives were mixed at a ratio of 1:1 and inoculated into the first pair of true leaves of 2-week-old soil-grown plants using a needleless syringe.
Generation of transgenic plants and growth phenotype analyses
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