Nicotiana benthamiana and Arabidopsis thaliana plants were grown on soil in a greenhouse with a 16 h light/8 h darkness photoperiod and temperatures of 25˚C.

To create the constructs, cDNA fragments of EXO70B1 or EXO70B2 were amplified and cloned into pEarleygate101-YFP-HA vectors to generate 35S:: EXO70B1-YFP-HA and 35S:: EXO70B2-YFP-HA vectors.

Agrobacterium tumefaciens GV3101 cultures containing the relevant binary constructs were centrifuged and suspended in 10 mM MES (pH 5.6), 10 mM MgCl₂, 150 mM acetosyringone, and the OD₆₀₀ was adjusted to 0.8. Transient expression was performed by agroinfiltration into N. benthamiana leaves. After 2 days, fluorescence was analyzed in infiltrated leaves using a fluorescence microscope (Olympus BX53). YFP-derived fluorescence was monitored through excitation with a 488 nm argon laser.

In the BiFC assays, the EXO70B1 coding fragment was amplified and cloned into the pEarleygate 201-Yn vector to obtain EXO70B1-YFPn, and the EXO70B2 protein was fused into the pEarleygate 202-Yc vector to acquire EXO70B2-YFPc. Bacterial suspensions containing the BiFC constructs and p19 silencing plasmid were infiltrated into N. benthamiana leaves. After the infiltration, fluorescence was observed by a fluorescence microscope after 2-3 days.

The evolutionary history was inferred employing the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site. This analysis involved 23 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Evolutionary analyses were conducted in MEGA-X.

Sequence information from this paper can be observed in the GenBank data libraries under accession numbers EXO70A1 (AT5G03540), EXO7OA2 (AT5G52340), EXO7OA3 (AT5G52350), EXO7OB1 (AT5G58430), EXO7OB2 (AT1G07000), EXO7OC1 (AT5G13150), EXO7OC2 (AT5G13990), EXO7OD1 (AT1G72470), EXO7OD2 (AT1G54090), EXO7OD3 (AT3G14090), EXO7OE1 (AT3G29400), EXO7OE2 (AT5G61010), EXO7OF1 (AT5G50380), EXO7OG1 (AT4G31540), EXO7OG2 (AT1G51640), EXO7OH1 (AT3G55150), EXO7OH2 (AT2G39380), EXO7OH3 (AT3G09530), EXO7OH4 (AT3G09520), EXO7OH5 (AT2G28640), EXO7OH6 (AT1G07725), EXO7OH7 (AT5G59730), and EXO7OH8 (AT2G28650).

The structural models were retrieved from the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). AlphaFold is a new machine learning approach that incorporates previous knowledge about protein structure and that, by leveraging multi-sequence alignments into the design of a deep learning algorithm, can predict protein structures with great accuracy [18] [19] . The protein structure from this article can be obtained in the AlphaFold Protein Structure Database under the accession codes EXO70A1 (AF-Q9LZD3-F1), EXO70A2 (AF-F4KG57-F1), EXO70A3 (AF-F4KG58-F1), EXO70B1 (AF-Q9FGH9-F1), EXO70B2 (AF-Q9LMJ4-F1), EXO70C1 (AF-Q9FY95-F1), EXO70C2 (AF-Q9FFX6-F1), EXO70D1 (AF-Q9C9E5-F1), EXO70D2 (AF-Q9SYG5-F1), EXO70D3 (AF-Q9LJH9-F1), EXO70E1 (AF-Q9LIA2-F1), EXO70E2 (AF-Q9FNR3-F1), EXO70F1 (AF-F4K8Y6-F1), EXO70G1 (AF-Q7XYW9-F1), EXO70G2 (AF-Q9C8H6-F1), EXO70H1 (AF-Q8VY27-F1), EXO70H2 (AF-O80625-F1), EXO70H3 (AF-Q9SF50-F1), EXO70H4 (AF-Q9SF51-F1), EXO70H5 (AF-F4IIS8-F1), EXO70H6 (AF-Q9LQP9-F1), EXO70H7 (AF-F4KJ98-F1), and EXO70H8 (AF-Q9SIB0-F1).

We identified the whole EXO70 gene in Arabidopsis. The number of amino acid residues of EXO70 protein ranged from 573 aa (EXO70H8) to 695 aa (EXO70C2), and their molecular weight ranged from 64799 Da (EXO70H8) to 79765.6 Da (EXO70C2). Theoretical isoelectric points are located between 4.64 (EXO70E1) and 8.94 (EXO70A1), which can be roughly divided into two categories: basic proteins (EXO70A1, EXO70A2, EXO70G1, EXO70G2, EXO70H2, EXO70H3, EXO70H6) and acidic proteins (EXO70A3, EXO70B1, EXO70B2, EXO70D1-EXO70D3, EXO70E1, EXO70E2, EXO70F1, EXO70H1, EXO70H4, EXO70H5, EXO70H7, EXO70H8) ( Table 1 ).

To further clarify the relationship among the EXO70 genes in Arabidopsis, a phylogenetic tree was constructed based on the protein sequences encoded by these EXO70 genes. And the results showed that homologous genes belonging to the same subclass are generally distributed on the same main trunk. There are 9 pairs of paralogs on the side branches of the phylogenetic tree, including ( Figure 1 )

EXO70H1/EXO70H2; EXO70H3/EXO70H4;

EXO70H5/EXO70H6; EXO70E1/EXO70E2;

EXO70D2/EXO70D3; EXO70B1/EXO70B2;

EXO70C1/EXO70C2; EXO70G1/EXO70G2;

EXO70A2/EXO70A3.

The 3D structures of proteins encoded by the EXO70 gene family were predicted using the online AlphaFold3 platform. The results revealed that the proteins encoded by the EXO70 family all contained alpha-helix, extended strand, and random coil ( Figure 2 ). Moreover, the majority of members within the same subclass exhibited similar structures, such as EXO70B1/EXO70B2, EXO70C1/EXO70C2, providing additional insights into the evolutionary relationship of the EXO70 gene. Significantly, EXO70A3 seems to be an exception, although in terms of evolution, this protein belongs to the same branch as EXO70A2 and EXO70A1. However, its protein structure differs significantly from that of EXO70A1 and EXO70A2, while the latter two exhibit a high degree of structural similarity.

EXO70B1 is a component of the exocyst complex and belongs to the EXO70 protein family, and previous studies indicated that the exocyst subunit EXO70B1 is involved in the immune response to different pathogens in Arabidopsis [20] [21] . In this study, we used EXO70B1 in an experimental study on Arabidopsis EXO70 genes. In order to examine the localization of EXO70B1, the EXO70B1 fused with the yellow fluorescent protein (YFP) was expressed under the control of the 35S promoter in N. benthamiana leaves. We observed clear fluorescence signals in the cytoplasm, nucleus, and plasma membrane ( Figure 3(A) ). This discovery was consistent with the roles of EXO70s in vesicle transport.

Considering that some plasma membrane localization proteins appear as membrane components of transport vesicles. In Arabidopsis, FLS2 (flagellin receptor), a PRR protein, is localized on the plasma membrane. We would like to know if EXO70B1 directly interacts with FLS2. We then performed BiFC assays and observed strong YFP fluorescence signals on the plasma membrane in N. benthamiana leaves only co-transformed with EXO70B1-Yc and FLS2-Yn constructs, but not in the negative controls ( Figure 3(B) ), indicating that EXO70B1 and FLS2 indeed interact with each other. This discovery is consistent with the previous research results of Wang et al. [16] .

A previous study had demonstrated that EXO70B1, co-localized with TN2 at the plasma membrane and interacted with TN2 [21] , and EXO70B1 could inhibit cell death caused by TN2 in N. benthamiana [22] . Considering that the sequences of EXO70B2 and EXO70B1 share the highest similarity, EXO70B1 and EXO70B2 were found to directly interact with FLS2 and hetero-oligomerize [16] . Thus, we further investigate whether EXO70B2 could also abolish the cell-death-inducing ability of TN2. We first observed and discovered that the fluorescence distribution of YFP-labeled EXO70B2 was in the nucleus, cytoplasm, and plasma membrane ( Figure 4(A) ), suggesting that EXO70B2 exhibited a subcellular localization pattern similar to EXO70B1. Using the Agrobacterium tumefaciens-mediated transient expression system, it was confirmed that EXO70B1, rather than EXO70B2, could suppress TN2-triggered cell death in N. benthamiana ( Figure 4(B) ).