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.

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.

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.

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.

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.

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.

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.

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.