To determine whether other potyvirus NIbs elicit a response from Pvr9, the NIb gene from PVY, Potato CCCP A (PVA), Turnip mosaic virus (TuMV), Soybean mosaic virus (SMV), and Zucchini yellow mosaic virus (ZYMV) were cloned to the modified pPZP212. Co-agroinfiltration of these transformants expressing other potyvirus NIbs with Pvr9 showed that the NIbs from PVY, PVA, and TuMV triggered the hypersensitive response. However, the ZYMV NIb triggered only a weak hypersensitive response, and the SMV NIb failed to trigger a hypersensitive response (Fig. 3A). To explore the phylogenetic relationships among these NIb proteins, we generated an unrooted and scaled phylogenetic tree from the NIb amino acid sequences of potyviruses. As shown in Fig. 3B, the NIbs of PepMoV, PVY, and PVA belonged to a monophyletic group, and the NIbs of ZYMV and SMV belonged to another clade. Branching of the phylogenetic tree which is usually interpreted as a specification event in phylogenetics, especially at the early nodes (Fig. 3B), revealed that the NIb of TuMV shared a common ancestor with the NIb of PepMoV rather than with the NIbs of ZYMV and SMV.
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