Characterization of soybean GmPUB1 proteins that interact with the Phytophthora sojae effector Avr1b protein

Phytophthora sojae is an oomycete which causes stem and root rot disease in soybean. Upon infection, P. sojae could deliver a series of virulent effector proteins into host cell to promote disease on susceptible plants (Tyler et al. 2008, Göhre and Robatzek 2008). Avr1b is one such protein (Tyler et al. 2008). In this report, a soybean U-box protein, GmPUB1-1, was isolated by interaction with P. sojae effector Avr1b in yeast. It has a homologous copy which was named GmPUB1-2. These two GmPUB1 proteins share 94% identities. Pull-down assay confirmed the interaction between GmPUB1s and Avr1b in vitro. Mutations in Avr1b C-terminal conserved residuals abolished the interaction between Avr1b and GmPUB1 proteins. These residuals are also known involved in Avr1b virulence activity. By linking GmPUB1 genes with the GFP tag, GmPUB1-GFP fusion proteins was localized throughout soybean hypocotyl and onion epidermal cells. Bimolecular fluorescence complementation (BIFC) experiment suggests the interaction of GmPUB1 and Avr1b, most likely predominantly occurs in nuclei. In vitro ubiquitination assay demonstrated that GmPUB1-1 and GmPUB1-2 possess E3 ligase activities and mediated self-monoubiquitination. Gene silencing of the GmPUB1 genes in cotyledons of the soybean cultivar Williams 82 showed loss of race-specific Phytophthora resistance encoded by Rps1-k. GmPUB1-1 mRNA level was up-regulated following P. sojae infection. Eight candidate interactors of GmPUB1-2 are identified from a yeast two-hybrid library screening. Overall, this study implicates that finctional E3 ligase activities of GmPUB1 proteins may be involved in Rps1-k mediated Phytophthora resistance in soybean.