Plants rely on two-levels of innate immune systems to defend against various pathogens in nature [1] . The first level of the immune system is activated by pattern recognition receptors (PRRs) located on the plasma membrane, which can recognize conserved pathogen associated molecular patterns (PAMPs), known as pattern triggered immunity (PTI). However, some host-adapted pathogens can enter plant cells by secreting effectors that contribute to pathogen virulence, thereby interfering with plant immunity [2] . Correspondingly, plants have evolved intracellular nucleotide-binding sites (NBS) and leucine-rich repeat (LRR) domain receptors (NLRs), also known as resistance (R) proteins, which activate the second level of the immune system called effector triggered immunity (ETI) by directly or indirectly recognizing these effectors. Compared to PTI, ETI typically responds stronger and faster, accompanied by hypersensitive responses (HR), which is programmed cell death at the site of infection [3] .

Generally speaking, a typical plant NLR protein contains a specific N-terminal domain, a conserved NBS domain, and a highly variable C-terminal LRR domain. The N-terminal domain are typically coiled-coil (CC) domain, Toll/interleukin receptor (TIR) domain, or RESISTANCE TO POWDERY MILDEW 8 (RPW8)-like domain. Therefore, according to different N-terminal domains, NLRs can be roughly divided into three subfamilies: CC-NBS-LRR (CNL), TIR-NBS-LRR (TNL), and RPW8-NBS-LRR (RNL) [4] . Besides, plants also contain some truncated NLR receptors. For example, 21 truncated TIR-NBS (TN) proteins lacking the C-terminal LRR domain have been identified in the Arabidopsis thaliana ecological type Col-0 [4] . An increasing number of studies indicated that TN proteins also play a crucial role in plant immunity. Such as, the gain-of-function mutant of TN1, also known as CHILLING SENSITIVE 1 (CHS1), exhibits cold sensitivity at low temperatures, yet the cold sensitivity phenotype of chs1-2 mutant at low temperatures counts on the full-length TNL protein SUPPRESSOR OF chs1-2, 3 (SOC3) [5] . Further experiments revealed that SOC3 can interact with TN1 and TN2 respectively to monitor the homeostasis of Arabidopsis E3 ubiquitin ligase SENESCENCE-ASSOCIATED E3 UBIQUITIN LIGASE1 (SAUL1). Specifically, the excessive accumulation of SAUL1 is monitored by the SOC3-TN2 pair, meanwhile the disappearance of SAUL1 is guarded by the SOC3-CHS1 pair [6] . Ectopic expression of the TN2-TIR domain causes cell death in N. benthamiana. The autoimmune responses in exo70B1 mutants are caused by the activation of TN2, and co-expression of EXO70B1 and TN2 can inhibit the TN2-triggered cell death in N. benthamiana, suggesting that TN2 may monitor the presence or absence of EXO70B1 [7] [8] . Furthermore, TN2 associates with CALCIUM-DEPENDENT PROTEIN KINASE 5 (CPK5) and stabilizes its kinase activity [9] . The loss of function of TN8 and TN11 genes results in increased susceptibility to Pseudomonas syringae pv. tomato (Pto) strain DC3000 [10] . Additionally, TN13 is necessary to resistance against Pto DC3000 lacking the effectors AvrPto or AvrptoB [11] .

However, there have been no systematic studies published on the subcellular localization and function of TN receptors. In this article, we first conducted a brief analysis of the proteins and evolutionary tree expressed by these 20 TN genes. Furthermore, we focused on the TN genes on chromosome 1, which included 12 TN genes, TN1 to TN12, and observed different subcellular localization patterns of these proteins through fluorescence microscopy, including membrane, cytoplasmic, and/or nuclear localization, suggesting diverse roles for these proteins. Additionally, we identified four receptors, including TN2, TN9, TN11, and TN12, that can trigger visible cell death in N. benthamiana. Taken together, our study provides valuable clues for further investigating the activation mechanisms of TN family members.

In this article, we focused on analyzing the subcellular localization and function of 12 TN receptors (TN1-TN12) derived from Arabidopsis, confirming that TN receptors have diverse cellular localization, including cytoplasm, cell membrane, nucleus, etc. Additionally, we observed that transient expression of four TN proteins, including TN2, TN9, TN11, and TN12, can trigger significant cell death in Nicotiana benthamiana leaves. Subsequent focus would be on screening the interacting proteins of these receptors, especially full-length TNL or CNL receptors, to observe whether these TNs need to interact with full-length NLRs receptors to mediate defense responses.

We have demonstrated that the localization of 12 designed TNs from Arabidopsis is not entirely identical ( Figure 2 ), but we cannot rule out the possibility that TNs may have other subcellular localization. According to reports, some NBS-LRR proteins, such as MLA10, N, and RPS4, can relocate to the nucleus after activation [13] [15] [16] . Therefore, studying whether TNs receptors can be repositioned would help analyze the activation mechanism of these proteins. Coincidentally, animal NBS-LRR has also been observed in different subcellular locations, requiring similar partners to maintain pre-activation ability and can be repositioned during activation to participate in various signaling complexes. Mammalian CIITA NBS-LRR receptors function in the nucleus [17] . NOD2 exists on the plasma membrane and recruits downstream partners to the membrane; the mutation of its LRR causes NOD2 to appear cytoplasmic and non-functional [18] . NOD1 and NOD2 recruit autophagic components locally at the plasma membrane site of bacterial infection, while common NOD2 variants associated with Crohn’s disease fail to do so [19] . These studies indicate that mammalian NBS-LRRs are located at different positions in the cell before activation and can dynamically relocate, and their cellular localization affects downstream functions. The sum of data on intracellular immune receptors of NBS-LRR in plants and animals is consistent with the conceptual role of NBS-LRR protein as a cell homeostasis monitor, which studies a wide range of cellular defense mechanisms through various subcellular addresses.

Out of 12 TN genes tested for transient overexpression in N. benthamiana, four of the TN genes induced macroscopic cell death responses ( Figure 3 ). Induction of a localized cell death has been demonstrated previously in N. benthamiana when an R gene is transiently overexpressed in the absence of a corresponding Avr protein [12] . Cell death induced in response to the transient overexpression of TNL genes was shown in the cases of RPP1A and RPS4 [13] [14] . Therefore, the TIR domain is shared between TN and TNL proteins and may have a common role in triggering cell death.

This study was funded by Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ2403304 and GJJ218112), and School-level Science and Technology Project of Fuzhou Medical University (fykj202201).

*Corresponding author.