*3.2. Diversification of Subcellular Localization Pattern of the EXO70 in H. villosa*

Protein subcellular localization analysis provided important clues to their specialized biological functions [58]. The diversification of EXO70-V subcellular localization patterns implies functional differentiation. Except for EXO70I1-V, all *EXO70* genes showed plasma membrane (PM) signals. The EXO70D1/F2-V-GFP locates to the PM merged with some small, discrete punctate. At the same time, EXO70I1-V-GFP only gave rise to smaller fluorescent discrete punctate along with PM, which are similar to AtEXO70E2 in *Arabidopsis* protoplasts, which was a maker of a novel double-membraned structure termed EXPO (exocyst-positive organelles). AtEXO70E2 was involved in unconventional protein secretion for cytosolic proteins that lack a signal peptide, because of its ability to recruit several other exocyst complex subunits [8,9,59,60]. Therefore, *EXO70D1*, *F2* and *I1-V* might have the ability to recruit different partners, then form various complexes to execute different biological functions. AtEXO70A1 was distributed in different patterns in different systems' cytosol; it showed up in the nucleus and numerous small punctate structures in the BY-2 cell [36], at the apex of growing tobacco pollen tubes [61] and is strongly present in the cell plate [62]. In the study, EXO70A1/A3-V showed a weak PM signal, while EXO70A2-V was characterized by mis-localizations. This was probably because *AtEXO70A1* took part in a different vesicular transport process. Moreover, despite both AtEXO70B2 and AtEXO70H1 participating in the interaction between plants and pathogens, the signal of EXO70B2-GFP was mainly found in the cytoplasm, while EXO70H1-GFP was in vesicle-like structures in *Nicotiana benthamiana* leaf [31]. In our analysis, EXO70B1-V was present in the PM and nucleus (Figure 7g). It is likely that they went through different action sites to take part in the process of disease resistance. An exocyst is a tethering factor that mediates secretory vesicles to the plasma membrane before SNARE-mediated fusion [63,64]. EXPOs deliver cytosolic proteins to the cell surface [65,66] and therefore all of those were related to PM. The results explained why most genes had the PM location pattern.
