N.benthamiana and Arabidopsis plants were grown in a growth chamber at 24˚C with a 16 h-light/8 h-dark photoperiod [20].

Total RNAs were extracted from four-week-old leaves of Arabidopsis lines and the reverse transcription of RNAs was implemented through using Transcript One-step gDNA Removal and cDNA Synthesis SuperMix. All cDNAs were diluted to 100 ng/moL.

In order to acquire overexpression vectors containing full-length SOC3 and its truncated fragments separately, we first employed cDNA of wild-type Arabidopsis leaves as a template and amplified it by PCR. Conditions for the PCR amplification are: 36 cycles of a 30-second denaturation step at 94˚C, annealing at 56˚C for 30 seconds, and extension at 72˚C for 1 minute. Afterwards, we collected the above fragments separately and cloned them into modified pUC19. After extracting the plasmid, DNA sequencing was conducted, and finally the correct plasmid was ligated to the expression vector pEarleyGate101 fused with the YFP-HA tag utilizing gateway technology. Electro-transfer the expression plasmid into Agrobacteriumtumefaciens strain GV3101.

The Agrobacterium containing different constructs were cultured in LB medium containing kanamycin and rifampicin and grown overnight. Bacteria were collected by centrifugation and resuspended in infiltration medium [10 mM MgCl₂, 10 mM MES (pH 5.6), and 200 muM acetosyringone] at room temperature for 1 hour before injection into 4-week-old N.benthamiana leaves [21]. Each experiment was repeated at least three times and yielded similar results.

The YFP fluorescence was observed using a fluorescence microscope after infiltration for 48 hours. YFP was excited using the 514 nm laser line, and the emission was collected at 520 - 570 nm. All experiments were repeated at least three times to yield comparable results.

The SOC3 belongs to TIR-NBS-LRR protein [22]. The basic physicochemical properties of the proteins encoded by the SOC3 gene were analyzed using ProtParam (https://web.expasy.org/protparam/). The number of amino acid residues of the SOC3 protein was 1049, the formula was C₅₂₇₈H₈₄₇₄N₁₄₄₆O₁₅₃₇S₅₆, the theoretical isoelectric point was 6.9, and the predicted molecular weight was 118.5 kDa, respectively. It is worth noting that the instability coefficient of the SOC3 protein was 42.33, so SOC3 belongs to the unstable proteins. The hydrophilicity/hydrophobicity of SOC3 was analyzed using ExPASy-ProtScale (https://web.expasy.org/protscale/), revealing an uneven distribution of hydrophilic and hydrophobic amino acids across the sequence, with an average hydrophilicity index of -0.490, indicating it is a hydrophilic protein (Figure 1(A)). Signalp-5.0 (https://services.healthtech.dtu.dk/services/SignalP-5.0/) predicted that the SOC3 protein does not possess signal peptides (Figure 1(B)). Using SWISS-MODEL (https://swissmodel.expasy.org/) predicts the three-dimensional structure of the SOC3 protein (Figure 1(C)), and the crystal structure of Arabidopsis RPP1 protein (belonging to TNL protein) (7crc.1.B) was chosen as the template for SOC3 protein with its sequence identity at 35.99%, GMQE (Global Model Quality Estimate) score of 0.51, and QMEANDisCo (Qualitative Model Energy ANalysis DIStance COnstraint) global score of 0.54 ± 0.05.

Figure 1.Bioinformatics analysis of SOC3 protein. (A) Hydrophobicity prediction. (B) Protein signal peptide prediction. (C) The tertiary structure prediction of SOC3 protein.

Based on gene annotation results and the reference sequence of Arabidopsis SOC3 (AT1G17600; https://www.arabidopsis.org/), we designed the specific PCR primers (SOC3-F: 5'-CTCCGCGGCCGCCACCATGGTGTCCTCCTCTGCACC-3', SOC3-R: 5'-GGTCGGCGCGCCCCTTGAAAACACGCAGGAGTAAAATCAC-3'), to amplify the coding sequence of SOC3, which introduces a 15-bp overlap region with the modified cloning vector pUC19 (the gateway entry vector). The results of agarose gel electrophoresis of the PCR amplification product are shown in Figure 2(A), and it was assembled using the ClonExpress II One Step Cloning Kit to construct the pUC19-SOC3 plasmid. Furthermore, pUC19-SOC3 was transformed into competent cellsE. coli, and positive clones were identified through PCR verification using specific primers (501-F: 5'-TAACGCTAGCATGGATGTTTTCC-3', 870-R: 5'-CAGAGCTGCCAGGAAACAGC-3') (Figure 2(B)). Selected clones underwent plasmid extraction, and sequencing results indicated that the amplified product is indeed the coding sequence of the SOC3 gene. Then, the sequenced pUC19-SOC3 plasmid was ligated to the destination binary overexpression vector pEarleyGate101 by LR reaction using the Gateway recombination system following the manufacturer’s instruction, and the product was transformed into competent E. coli cells again. Positive monoclonals were obtained through PCR identification using specific primers (35S-F: 5'-ATGACGCACAATCCCACTATC-3', OCS-R: 5'-GCGCTCTATCATAGATGTCGCTATAAAC-3') (Figure 2(C)), and the extracted plasmid was pEarleyGate101-SOC3-YFP-HA, which was used for subsequent transient expression analysis.

Following the same cloning method, we replaced specific PCR amplification primers and obtained plant overexpression vectors containing three domain fragments of the SOC3 gene (TIR: 1-186 aa; NBS: 187-510 aa; LRR: 511-1049 aa) (COBALT: Multiple Alignment Tool), respectively.

Figure 2.Construction of SOC3 gene plant expression vector. (A) The full-length SOC3 clone was amplified by PCR using cDNA as the template and the gene-specific primer pair. Use 1% agarose gel electrophoresis to check the purity and concentration of the DNA. (B) Fragment of the correct size was cloned into the modified pUC19 plasmid and the insert was verified by PCR and complete sequencing using vector primers and anticipated gene specific primers. (C) Connect the correctly sequenced pUC19-SOC3 plasmid to the pEarletGate101 vector, validate the insert by PCR, and perform complete sequencing using vector primers and expected gene-specific primers.

The subcellular localization of proteins is closely related to their biological function [23]. To investigate the localization pattern of SOC3 receptor, we electroporated the constructed plant overexpression vector into Agrobacterium strain GV3101 and used Agrobacterium-mediated transformation to transiently express the fusion protein in the leaves of N. benthamiana. The yellow fluorescence of the SOC3-YFP-HA fusion proteins was localized to the plasma membrane, cytoplasm and nucleus (Figure 3(A)). Ulteriorly, in N. benthamiana cells expressing SOC3-TIR alone, fluorescence signal was observed in cytoplasm, plasma membrane and nucleus; while in N. benthamiana leaves expressing SOC3-NBS-YFP-HA and SOC3-LRR-YFP-HA, the fluorescence signal was mainly concentrated in the cytoplasm and plasma membrane (Figure 3(B)).

Figure 3.Subcellular localization of the full-length Rx1 and its three domains. (A) Localization pattern of SOC3-YFP-HA. Bar=50 mm. (B-D) Localization pattern of SOC3-TIR, SOC3-NBS, and SOC3-LRR. The YFP-HA-labeled protein was imaged by fluorescence microscopy in epidermal cells of transiently transformed N. benthamianaleaves. Images were taken 3 days after agroinfiltration. SOC3-TIR: bar=50 mm; both SOC3-NBS and SOC3-LRR: bar = 20 mm.

We transiently expressed the full-length SOC3 and its three domains in the leaves of N. benthamiana, respectively, and the results showed that SOC3-TIR alone triggers strong and rapid cell death. We detected that the expression levels of full-length and truncated SOC3 were at comparable levels, ruling out the possibility that their functional differences were caused by changes in protein expression (data not displayed) (Figure 4(A)). Previous studies have shown that expression of TN2 in N. benthamiana induces cell death, which is suppressed by co-expression with EXO70B1 [24]. Considering that SOC3 interacts with TN2 in planta [22], we want to know whether EXO70B1 could also inhibit cell death induced by SOC3-TIR in N. benthamiana. The results indicated that co-expression of EXO70B1 cannot abolish the cell-death-inducing activity of SOC3-TIR (Figure 4(B)).