1. Introduction
During the growth and development of plants, they are constantly threatened and challenged by various pathogenic microorganisms. To survive, plants have evolved two levels of immune systems [1]. The first level of immune response is initiated by pattern-recognition receptors (PRRs) on the cell surface that recognize pathogen-associated molecular patterns (PAMPs), known as PAMP-triggered immunity (PTI) [2]. Certain pathogens can overcome PTI through secreting effectors. Correspondingly, host plants utilize intracellular resistance (R) proteins to monitor effectors, thereby activating the second level of the immune system, namely effector-triggered immunity (ETI) [3]. ETI is more rapid and intense than PTI, and is often accompanied by visible cell death to limit the invasion of pathogens at the infection site, called hypersensitive response (HR) [4].
Cloning the plant R genes and clarifying its resistance mechanism is of great significance for deepening the understanding of resistance molecular mechanisms and improving agricultural production. In recent years, a large number of R genes have been cloned, with the largest category being R proteins encoding conserved motifs containing nucleotide binding sites and leucine-rich repeats (NBS-LRRs; NLRs). There are about 150 NLR genes in Arabidopsis thaliana [5]. Based on the N-terminal domain, plant NLRs are usually divided into three categories: CC (coiled-coil)-NBS-LRR (CNL), TIR (toll/interleukin-1 receptor)-NBS-LRR (TNL) and RPW8 (resistance to powdery mildew 8)-NBS-LRR (RNL) [6].
The CC domains or TIR domains of multiple NLRs, such as Sr33, Sr50, ZAR1, MLA10, RP1-D21 are enough to induce cell death after transient expression in leaves of Nicotiana benthamiana [7]-[11]. However, there are other NLRs, including RPM1, Rx, and RPS5, whose CC domains do not touch off cell death in N. benthamiana [12]-[14]. The subcellular localization patterns of NLRs are diverse. For example, RPM1 and RPS5 are localized on the plasma membrane [14] [15]. L6 and M proteins are localized on the Golgi and tonoplast, respectively [16]. RGA4 and RGA5 are mainly localized in the cytosol [17]. In addition, SNC1, Rx, and MLA10 exhibit a nucleo-cytoplasmic localization characteristic [7] [18] [19]. Different localizations of NLRs reflect their different activation mechanisms.
To investigate the role of NLR genes, this study cloned a TNL gene SOC3 from wild-type Arabidopsis, conducted bioinformatics analysis on it, and successfully constructed an overexpression vector. Subsequently, the full-length SOC3 and its truncated fragments were transiently expressed in N. benthamiana, providing a basis for further research on the biological function of the SOC3 receptor.
2. Materials and Methods
2.1. Plant Materials and Growth Environment
N. benthamiana and Arabidopsis plants were grown in a growth chamber at 24˚C with a 16 h-light/8 h-dark photoperiod [20].
2.2. Arabidopsis RNA Extraction and cDNA Synthesis
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.
2.3. Construction of the Overexpression Vector
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 Agrobacterium tumefaciens strain GV3101.
2.4. Agrobacterium Transient Expression Assays
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.
2.5. Subcellular Localization Observation
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.
3. Results
3.1. Analysis of Physical and Chemical Properties and Structure
Prediction of SOC3 Protein
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.
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Figure 1. Bioinformatics analysis of SOC3 protein. (A) Hydrophobicity prediction. (B) Protein signal peptide prediction. (C) The tertiary structure prediction of SOC3 protein.
3.2. Construction of the SOC3 Gene Plant Expression Vector
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 cells E. 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.
3.3. SOC3 Was Distributed in the Nucleus, Cytoplasm, and Plasma
Membrane
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)).
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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. benthamiana leaves. Images were taken 3 days after agroinfiltration. SOC3-TIR: bar=50 mm; both SOC3-NBS and SOC3-LRR: bar = 20 mm.
3.4. SOC3-TIR-Induced Cell Death in N. benthamiana Cannot Be
Suppressed by Co-Expression with EXO70B1
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)).
4. Discussion
The TIR domain of SOC3 initiated cell death in N. benthamiana, but the TIR-NBS fragment cannot (Figure 4(A)), declaring that the NBS domain of SOC3 suppressed the activation of TIR, and the intra-molecular interaction can regulate the activation of full-length SOC3. Previous studies have suggested that the TIR modules of some plant NLRs, such as L6TIR, SNC1TIR, and RPS4TIR, can trigger cell death in N. benthamiana leaves, and these TIR domains exhibit NAD+ cleavage activity. In NADases, a catalytic glutamate typically interacts with the C-2 and C-3 hydroxyl groups of the Namri bose in NAD+ [25]. Mutation of the conserved catalytic glutamate in L6TIR-YFP, SNC1TIR-YFP, and RPS4TIR-YFP abrogated effector-independent HR detected by transient expression [26]. In addition, Li et al. found that TIR immune signaling is blocked by phosphorylation to maintain growth in plants and animals [27]. In the SOC3TIR structure, E86 forms hydrogen bonds with the C-2 and C-3 hydroxyl groups of glycerol and also contains conserved serine residues. In this article, we demonstrated that the SOC3-TIR also has cell-death-inducing activity in plants. Future research should focus on determining whether the SOC3-TIR domain also has NAD+ cleavage activity and whether mutated conserved serine residues can abolish its cell-death-inducing activity.
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Figure 4. SOC3-TIR-triggered cell death in N. benthamiana cannot be abolished by co-expression with EXO70B1. (A) SOC3-TIR overexpression rapidly induced cell death (OD600 = 0.1). The image was recorded 48 h post infiltration (hpi). White solid line circles show cell death, while the white dashed line circles indicate no apparent cell death in the infiltrated area. (B) Co-expression of EXO70B1 inhibited the hypersensitive response triggered by TN2-TIR instead of SOC3-TIR in N. benthamiana leaves (both TN2-TIR and SOC3-TIR: OD600 = 0.6). The image was recorded 72 hpi.
We observed that SOC3 was distributed in the cell membrane, nucleus, and cytoplasm and was therefore a nucleoplasmic protein (Figure 3(A)). Due to the lack of a typical nuclear localization signal (NLS) sequence, NBS-LRR protein was previously believed to function in the cytoplasm. However, a number of studies have demonstrated that coordinated nucleo-cytoplasmic trafficking of plant NLRs is required for the full activation of defense responses, suggesting that a single NLR protein may activate distinct signaling pathways in the cytoplasm and nucleus. For example, Arabidopsis TNL protein RRS1-R interacts with the effector PopP2 of Ralstonia solanacearum in the nucleus, and forms a complex with the nuclear-expressed protein cysteine protease RD19 (Responsive Dehydration 19), initiating resistance to pathogens [28]. In addition, the recognition of barley disease resistance protein MLA10 and powdery mildew effector factor A10 induces MLA10 to bind to the transcription factor WRKY in the nucleus, initiating the disease resistance response. The co-expression of MLA10 fusion protein containing a nuclear export signal (NES) leads to the export of R protein to the extracellular space, resulting in the disappearance of resistance [29]. The potato Rx1, which confers extreme resistance to Potato virus X, is located both in the nucleus and cytoplasm. Manipulating the nucleocytoplasmic distribution of Rx1 or its elicitor revealed that Rx1 is activated in the cytoplasm and cannot be activated in the nucleus [18].
5. Conclusion
SOC3 and SOC-TIR are both located in the plasma membrane, cytoplasm, and nucleus, and SOC-TIR domain exhibits cell-death-inducing ability in N. benthamiana, which cannot be suppressed by co-expression of EXO70B1.
Acknowledgements
Our functional data are limited to the transient system of N. benthamiana, and confirmation in Arabidopsis remains a future objective.
Funding
This study received grant from the Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ2403304 and GJJ218112).
NOTES
*Corresponding author.