1. Introduction
In plants, the two-level innate immune system has evolved to discern potential pathogens and touch off efficacious defense responses [1]. The first type of immune response is triggered by pattern recognition receptors (PRRs) localized at the cell membrane. PRRs identify and response to pathogen-associated molecular pattern (PAMP), known as pattern-triggered immunity (PTI). Certain pathogens secrete virulence effectors to combat PTI. Plants activate the second layer of innate immune system to monitor virulence proteins, named effector-triggered immunity (ETI). ETI is usually sparked by resistance (R) genes and give rise to local necrosis of plants to restrict the sustained spread of pathogens, namely hypersensitive response (HR) [2].
The majority of disease-resistance genes encode nucleotide-binding sites and leucine-rich repeat proteins (NLRs). A typical NLR receptor typically comprises a specific N-terminal domain, a conserved NBS domain, and a highly variable C-terminal LRR domain. According to the differences in its N-terminus, NLRs are predominantly classified into three categories: CC-NBS-LRR (CNL), TIR-NBS-LRR (TNL), and RPW8-NBS-LRR (RNL) [3]. Transient overexpression of the CC or TIR domains of certain NLR receptors is enough to spark off cell death [4]-[9], demonstrating that N-terminal CC and TIR domains are critical for initiating the death signal. In addition, plants also possess a number of truncated NLRs. Such as, a total of 21 TIR-NBS (TN) proteins have been identified in Arabidopsis thaliana (Col-0) [3]. Increasingly studies proclaimed that TN proteins also play a very important role in immunity. For instance, the functional acquisition mutant of TN1, also called CHILLING SENSITIVE 1 (CHS1), emerges cold sensitivity at low temperatures, nevertheless, the cold sensitivity phenotype of the chs1-2 mutant at low temperatures relies on the full-length TNL protein SUPPRESSOR OF chs1-2, 3 (SOC3) [10]. Further experiments demonstrated that SOC3 can associate with TN1 and TN2 respectively to monitor the steady state of Arabidopsis E3 ubiquitin ligase SENESCENCE-ASSOCIATED E3 ubiquitin LIGASE1 (SAUL1) [11]. Additionally, TN13 is reported to be necessary for resistance against Pto DC3000 lacking the effectors AvrPto or AvrptoB [12].
Previous articles reported an Arabidopsis thaliana resistance protein TIR-NBS 2 (TN2) can arise violent cell death in tobacco (Nicotiana benthamiana) leaves [13]. Nevertheless, the specific mechanism by which this protein strikes cell death is generally poor. Here, we preliminarily dissected the activation mechanism of TN2 immune receptor in N. benthamiana. The outcomes of the transient analysis attested that the TN2-TIR domain itself is enough to cell-death induction. The yellow fluorescent protein (YFP)-tagged TN2 and TN2-TIR were major localized to the cytoplasm, which was considered essential to the function of the TN2 and TN2-TIR. Additionally, the cell-death-inducing activity of TN2 can be inhibited by the full-length EXO70B1 instead of its truncated fragments. These research findings provided some clues for understanding the activation mechanism of TN2.
2. Materials and Methods
2.1. Plant Growth Environment
A. thaliana and N. benthamiana plants were cultivated in a greenhouse under a 16 hours light/8 hours dark cycle (24˚C). Plants were used for transient expression assays at 4 - 5 weeks of age.
2.2. Construction of Recombinant Vectors
To acquire cDNA clones of Arabidopsis TN2 genes, total RNA was isolated from 4 - 5 weeks old wild-type Arabidopsis leaves using the Trizol reagent, and cDNA was generated using the cDNA Synthesis Kit following the manufacturers’ instruction. For full-length TN2 gene, PCR product was recombined with the binary vector the modified pUC19 (containing attL1 and attL2 feature sequences) using ClonExpress II One Step Cloning Kit. For truncated domain fragments of TN2 gene, two different domains TIR (1 - 178 aa) and NBS (179 - 380 aa) were identified by TAIR website, and the PCR product was cloned into the modified pUC19 vector. After complete sequencing, they were transferred to the expression vector through Gateway cloning technology (Thermo Fisher Scientific), such as pEarleygate101 for YFP-HA or YFP-HA-Rop or YFP-HA-mutRop tagged protein expression. Electro-transfer the expression plasmid into Agrobacterium tumefaciens strain GV3101.
2.3. Transient Expression
Agrobacterium tumefaciens GV3101 strains carrying recombinant binary vectors were used to infiltrate N. benthamiana leaves. Liquid cultures of all Agrobacterium strains were initially grown at 28˚C with agitation in Luria–Bertani (LB) media supplemented with the appropriate antibiotics. The bacterial cells were pelleted by centrifugation at 11,000 rpm for 1 min, resuspended in infiltration buffer (10 mM MgCl2, 10 mM MES, and 200 μm AS) and adjusted to an appropriate OD600 for infiltration.
2.4. Subcellular Localization
Subcellular localization was executed following a previously mentioned method, with minor modifications [9]. In one word, a fluorescence microscope (Olympus BX53) was used to observe photographs of live cells on the abaxial sides of N. benthamiana leaves at 28 or 40 h post target protein expression.
3. Results
3.1. Membrane and Cytoplasmic Localization of TN2 Triggers Cell Death in N. benthamiana
We previously cloned a range of TIR-NBS (TN) genes from Arabidopsis, and transiently expressed them in N. benthamiana leaves to plumb the subcellular localization and function of these receptors. We found that several TN proteins, including TIR-NBS 2 (TN2), can trigger cell death N. benthamiana (Figure 1(A)), a characteristic function of activating a TN protein. This discovery is consistent with the previous findings published in scientific papers [13]. Subcellular localization of proteins is an important aspect of protein function study. Therefore, we employed a fluorescence microscope based on C-terminal YFP tagging to investigate the subcellular localization of TN2, and observed that the cellular localization of TN2-YFP seemed to exhibit a dynamic process. Specifically, in the early stage of transient expression, the fluorescence signal of TN2-YFP appears in the plasma membrane and cytoplasm, and over time, a large amount of aggregated fluorescence signals appears in the cytoplasm (Figure 1(B)).
Thus, the membrane and cytoplasmic localization of TN2 receptor was auto-active in the leaves of N. benthamiana.
Figure 1. Membrane and cytoplasmic localization of TN2 triggers cell death in N. benthamiana. (A) Transiently expressed TN2 induces cell death. 35S::TN2-YFP-HA was transiently expressed in N. benthamiana by Agrobacterium infiltration (OD600 = 0.8). 35S::YFP-HA was used as a negative control. Picture was photographed at 72 h post infiltration (hpi). The experiment was repeated independently three times, and similar results were obtained in each replicate; (B) Observation the subcellular localization of TN2 with fluorescence microscope. TN2-YFP-HA was transiently expressed in N. benthamiana (OD600 = 0.8). Images were observed at 24 hpi and 40 hpi, respectively. Bar = 20 μm. The experiments were repeated at least three times with minimum three technical replicates and similar results were obtained.
3.2. The TIR Domain of TN2 Is Sufficient for Cell-Death Induction
The results of sequence alignment showed that TN2 encoded a potentially resistant protein harboring two domains. Consequently, two truncated fragments were generated [14], and both fused with the YFP-HA tag to determine whether these two domains possess cell-death-inducing activity (Figure 2(A)). On overexpressing two segments in N. benthamiana, the TN2-TIR domain caused sever cell death, and NBS domain was defective in causing macroscopic cell death, and we detected that the expression levels of different truncated proteins were at comparable levels, ruling out the possibility that their functional differences were caused by changes in protein expression (data not displayed) (Figure 2(B)). Fluorescence microscope results showed that TN2-TIR domain was also membrane and cytoplasmic-localized, but NBS module exhibited apparent cytoplasmic and nuclear distribution (Figure 2(C)).
In summary, TN2-TIR domain exhibited the almost identical localization pattern as full-length TN2, and the former is enough to trigger cell death in N. benthamiana leaves.
Figure 2. The TIR domain of TN2 can induce cell death in N. benthamiana. (A) Schematic diagram of TN2 receptor domain structure and the derived fragments. The two domains of TN2 are rendered in different colors. The numbers in the picture indicate the length of the protein; (B) Analysis of cell-death-inducing ability of TN2 fragments (OD600 = 0.8). The presented data are the average of three technical replicates, and experiments were biologically repeated at least three times with similar results; (C) Observation the subcellular localization of TN2 fragments with fluorescence microscope. Images were observed at 40 hpi. Bar = 20 μm. The experiment was repeated at least three times, each time minimum three technical replicates were analyzed.
3.3. Cytoplasmic Localization Is Crucial for the Function of
Full-Length TN2 and Its Tir Domain
Figure 3. Cytoplasmic localization is crucial for the function of full-length TN2 and its TIR domain. (A, C) Rop tag can indeed anchor TN2 and TN2-TIR proteins separately to the PM. (B, D) Analysis of cell-death-inducing activity of TN2-YFP-HA-Rop and TIR-YFP-HA-Rop, respectively (OD600 = 0.8). Each experiment was performed at least three times with similar results and three technical replicates were performed for each sample. Fluorescent images were observed at 40 hpi. Bar = 20 μm.
Considering that both full-length TN2 and its TIR domain represent partial membrane localization, and both proteins can strike cell death in N. benthamiana. We want to validate whether these two proteins appear in large quantities in the cytoplasm after initiating the death signal on the plasma membrane (PM), or whether they need to dissociate from the PM into the cytoplasm before starting signal in the cytoplasm. In other words, are TN2 and TN2-TIR activated on the PM or in the cytoplasm? Full-length TN2 and its TIR domain were fused with the Rop tag, respectively, which could tether fusion protein on PM [15], to further assess whether the TN2 and its TIR domain worked on the PM. The TN2-YFP-Rop was found to be successfully anchored to PM and could not mediate cell death in N. benthamiana (Figure 3(A) and Figure 3(B)). Not surprising, TN2-TIR domain forcibly anchored to the PM has also completely lost its ability to trigger cell death (Figure 3(C) and Figure 3(D)). In conclusion, cytoplasmic localization is important for the function of full-length TN2 and its TIR domain.
3.4. Cell-Death-Inducing Activity of TN2 Can Be Inhibited by
Full-Length EXO70B1 Instead of Its Truncated Form
Figure 4. Cell-death-inducing activity of TN2 can be inhibited by full-length EXO70B1 instead of its truncated form. (A) Observation the subcellular localization of EXO70B1 and its truncated fragments with fluorescence microscope (OD600 = 0.8). Images were observed at 40 hpi. Bar = 20 μm; (B) Cell-death-inducing activity of TN2 can be suppressed by full-length EXO70B1 rather than its truncated form. Each experiment was performed at least three times with similar results and three technical replicates were performed for each sample.
Wang et al. demonstrated that EXO70B1 can suppresses TN2- or TN2-TIR-triggered cell death in tobacco (N. tabacum and N. benthamiana) [13]. We constructed recombinant plasmids expressing full-length EXO70B1 and two truncated forms, EXO70B1-N and EXO70B1-C, which represent the N- and C-terminal halves of the protein, respectively. Fluorescence microscopy manifested that EXO70B1-YFP exhibited accumulation in PM, cytoplasm, and nucleus, whereas, EXO70B1-N and EXO70B1-C only exhibited GFP signals in PM and cytoplasm (Figure 4(A)).
Furthermore, we co-expressed full-length EXO70B1, EXO70B1-N or EXO70B1-C with EXO70E2 in N. benthamiana, respectively. The results indicated that the cell-death-inducing ability of TN2 was abrogated when co-expressed with full-length EXO70B1, while the other fragments of EXO70B1 did not affect the cell-death-inducing activity of TN2 (Figure 4(B)). These results suggested that the cell-death-inducing ability of TN2 can be abolished by full-length EXO70B1 rather than its truncated form.
4. Discussion
In the absence of pathogens, ectopic expression of full-length or truncated TIR domains of some NLR proteins can trigger cell death in tobacco [16]. In this study, we demonstrate that transient expression of TN2 or TN2-TIR alone triggers strong and rapid cell death in N. benthamiana, proclaiming its act as a pro-cell death signaling domain. However, such a role differed from the N-termini of several TNs, such as TN10, suggesting that TNs exploited diverse domains for mediating downstream cell death signaling [16]. Our results confirmed that cytoplasmic localization was crucial for the functioning of TN2, but we cannot rule out the possibility that TN2 needs to enter the nucleus to initiate cell death response, that is to say, activated TN2 was likely to undergo re-localization, which was confirmed in NLR receptors including MLA10, RPS4, etc. [17] [18]. Additionally, Zhao et al. confirmed that EXO70B1 can interact with TN2 [19], we observed that cell-death-inducing activity of TN2 was suppressed by co-expression of full-length EXO70B1, not its truncated form (Figure 4(B)), indicating that TN2 is likely to only interact with the full-length EXO70B1.
The genome of Arabidopsis (Col-0) encodes approximately 20 TN proteins, some of which have been reported to play a role in plant immunity. For example, TN13 is a modifier of SNC1 and a 6 (MOS6) interaction partner required to resist Pto DC3000 lacking AvrPto and AvrPtoB [12]. Recent studies have shown that TN13 also interact with the CNL protein RPS5 and contribute to RPS5 mediated disease resistance. Nevertheless, it is still unclear how truncated NLR proteins activate disease resistance. Future studies are needed to determine whether TN2-activated resistance depends on full-length NLR proteins.
Previous studies have shown that the TIR domains of certain plant NLR receptors, such as SNC1-TIR (1-226), and RPS4-TIR (1-236), can induce cell death in N. benthamiana, and these TIR domains all possess NAD+ cleavage activity [20]. In this study, we found that the TN2-TIR domain also has cell-death-inducing activity. Future work should focus on exploring the impact of highly conserved sites in TN2-TIR on its function, and further analysis is needed to determine whether this domain also possesses NAD+ cleavage activity similar to SNC1-TIR and RPS4-TIR.
Funding
This study received funding support from the Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ2403304 and GJJ218112), and the school-level Science and Technology Project of Fuzhou Medical University (fykj202201).
NOTES
*Corresponding author.