Interaction of Phytophthora sojae Effector Avr1b With E3 Ubiquitin Ligase GmPUB1 Is Required for Recognition by Soybeans Carrying Phytophthora Resistance Rps1-b and Rps1-k Genes

Abstract

<i>Phytophthora sojae</i> is an oomycete that causes stem and root rot disease in soybean. <i>P. sojae</i> delivers many RxLR effector proteins, including Avr1b, into host cells to promote infection. We show here that Avr1b interacts with the soybean U-box protein, GmPUB1-1, in yeast two-hybrid, pull down, and bimolecular fluorescence complementation (BIFC) assays. <i>GmPUB1-1</i>, and a homeologous copy <i>GmPUB1-2</i>, are induced by infection and encode 403 amino acid proteins with U-Box domains at their N-termini. Non-synonymous mutations in the Avr1b C-terminus that abolish suppression of cell death also abolished the interaction of Avr1b with GmPUB1-1, while deletion of the GmPUB1-1 C-terminus, but not the U box, abolished the interaction. BIFC experiments suggested that the GmPUB1-1-Avr1b complex is targeted to the nucleus. <i>In vitro</i> ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the <i>GmPUB1</i> genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by <i>Rps1</i>-b and <i>Rps1</i>-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by <i>Rps1</i>-k plants was not, however, affected following <i>GmPUB1</i>-<i>1</i> silencing. Furthermore, over-expression of GmPUB1-1 in particle bombardment experiments triggered cell death suggesting that GmPUB1 may be a positive regulator of effector-triggered immunity. In a yeast two-hybrid system, GmPUB1-1 also interacted with a number of other RxLR effectors including Avr1d, while Avr1b and Avr1d interacted with a number of other infection-induced GmPUB proteins, suggesting that the pathogen uses a multiplex of interactions of RxLR effectors with GmPUB proteins to modulate host immunity.