Plants rely on two levels of innate immune system to ward off pathogen attacks [1] [2] . The first layer is basal immunity triggered by membrane-localized pattern recognition receptors (PRRs), which can recognize highly conserved microbial molecules such as bacterial flagellin or fungal chitin, known as pattern-triggered immunity (PTI) [3] . In order to overcome PTI, pathogens secrete effector proteins into plant cells through a type III secretion system to inhibit plant immunity. Once pathogens suppress PTI and enter host cells, the second layer of immunity is then activated. The intracellular nucleotide-binding leucine rich repeat receptors (NB-LRR/NLRs) activate the second layer of immunity by directly or indirectly recognizing effectors released by pathogens, known as effector-triggered immunity (ETI) [4] . ETI typically leads to rapid and intense reactions, commonly referred to as hypersensitivity reactions (HR), displaying local host cell death [5] .

Based on this N-terminus motif NLRs are classified into three subclasses CC (coiled-coil)-NLRs (CNLs) or TIR (Toll/interleukin-1 receptor)-NLRs (TNLs) and RPW8-NLRs (RNLs) [6] . Many NLR receptors function on the plasma membrane [7] [8] . In addition, some NB-LRRs have been described in plant genomes, which lack some NB-LRR specific domains and only contain TIR, TIR-NB, CC, and CC-NB domains [9] . Studies using overexpression of plant resistance genes, implying that the CC, TIR and NBS domains themselves might be sufficient to cause HR and to initiate plant defense responses [10] - [19] .

The genome of Arabidopsis contains 21 genes encoding TN proteins [20] , the functions of most TN proteins, including TIR-NBS 2 (TN2), are not particularly clear. Yeast two-hybrid system and BIFC technology are widely used for screening target protein interactors and verifying protein interactions in planta, respectively. The model plant N. benthamiana combined with Agrobacterium-mediated transient expression system is frequently applied to clone plant NLRs genes or plumb the function of plant NLRs receptors [21] . In this study, we utilized TN2 TIR domain (TN2-TIR) as a bait to screen for interacting proteins in the Arabidopsis leaf cDNA library, and demonstrated through BIFC technology that EXO70B1, SOC3, and CPK5-VK all interact with TN2 on the plasma membrane. Further transient expression results indicate that EXO70B1 can inhibit cell death of TN2 and TN2-TIR in N. benthamiana. In summary, our research findings provide valuable reference clues for understanding the activation mechanism of TN2.

TN2 is one of the 21 TIR-NBS receptors in the Arabidopsis thaliana genome [22] . Although the specific function of TN receptors is not yet clear, some evidence suggests that they can positively regulate the immune system of plants. For example, ectopic expression of the full-length or truncated TIR domain of some NLR proteins in the absence of pathogens can trigger an HR in N. benthamiana [19] [23] [24] . Previous studies have shown that the deficiency of TN2 receptor can partially inhibit cell death caused by the bon1/bon3 double mutant [25] . In this study, we demonstrated that transient expression of TN2 or TN2-TIR alone induces strong and rapid cell death in N. benthamiana, which is suppressed by co-expression of EXO70B1, not SOC3 and CPK5-VK ( Figure 2 ). This suggests that EXO70B1 specifically suppresses the activation of TN2, indicating that EXO70B1 associates with TN2 and may maintain TN2 in an inactive status.

EXO70B1 belongs to the EXO70 protein family and encodes a component of the vesicle transport machinery, playing a critical role in anchoring and binding vesicles to specific sites on the plasma membrane [26] . In vesicle trafficking, exocytosis plays an important role in maintaining membrane integrity and promoting membrane remodeling to cope with changes in environmental conditions [27] . In this study, we found that TN2 interacts with EXO70B1 on the plasma membrane ( Figure 1 ). Previous studies have found that RIN4 recruits EXO70B1 into the plasma membrane, and AvrRpt2 can release RIN4 and EXO70B1 into the cytoplasm [28] , declaring that the localization of EXO70B1 on the plasma membrane may be essential for plant immunity.

SOC3 encodes a typical TIR-type NLR (TNL) receptor. Liang et al. found that SOC3 can guard the homeostasis of the E3 ligase SAUL1 by interacting with CHS1 (TN1) and TN2, respectively. Overaccumulation of SAUL1 is monitored by the SOC3-TN2 pair, while SAUL1’s disappearance is guarded by the SOC3-CHS1 pair. SOC3 forms a head-to-head genomic arrangement with CHS1 and TN2, indicative of transcriptional co-regulation [29] . This complex cooperative interaction may expand the recognition range of NLR and increase its functional flexibility, which can partially explain the overwhelming occurrence of NLR gene clustering in higher plants.

Generally speaking, functional redundancy in multi-gene families is an unavoidable challenge for studying specific genes within them. CPK5 belongs to the CPK family in plants, and previous studies have shown that in the absence of functional CPK5 instead of CPK4, CPK6, and CPK11, EXO70B1-mediated cell death and enhanced resistance to the Golovinomyces cichoracearum phenotype are lost [30] , indicating that the specific function of CPK5 cannot be achieved through its functionally redundant homologs. In addition to CPK5, EXO70B1-mediated autoimmunity also requires the atypical truncated NLR resistance protein TN2 [26] . Therefore, the tripartite interaction between EXO70B1, CPK5, and TN2 controls cell death. The activity of CPK5 kinase and its membrane binding are both essential, although the exact mechanism is not fully understood. Whether other CPK family members are also involved in similar interactions and whether this mechanism represents innovation that only exists in Arabidopsis remains an unresolved question.

The research was funded by the Science and Technology Research Project of Jiangxi Provincial Department of Education (GJJ2403304 and GJJ218112).

*Corresponding author.