Updated project metadata. Extracellular vesicles (EVs) have been extensively studied in animal cells, and play an important role in cell-to-cell communications. Emerging evidence shows that EVs also act as important agents in plant-microbe interactions. However, the mechanisms by which EVs mediate cross-kingdom interactions between plants and microbial pathogens remain largely elusive. Here, proteomic analyses of soybean root rot pathogen Phytophthora sojae EVs identified tetraspanin family proteins, PsTET1 and PsTET3, that can be recognized by Nicotiana benthamiana to trigger plant immune responses. PsTET1 and PsTET3 were redundantly required for the full virulence of P. sojae. Further analyses revealed that the large extracellular loop (EC2) of PsTET3 is the key region recognized by N. benthamiana and also by Glycine max, and that recognition depends on the plant receptor-like kinase BAK1. TET proteins from oomycete and fungal plant pathogens could be recognized by N. benthamiana and induce immune responses. However, plant-derived TET proteins failed to do so, due to the divergent sequences of the final 16 amino acids of EC2, which ultimately makes plants distinguish self and non-self EVs, triggering active defenses against pathogenic eukaryotes.