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

Phytophthora sojae is an oomycete that causes stem and root rot disease in soybean. P. sojae 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. GmPUB1-1, and a homeologous copy GmPUB1-2, 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. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activity. Silencing of the GmPUB1 genes in soybean cotyledons resulted in loss of recognition of Avr1b by gene products encoded by Rps1-b and Rps1-k. The recognition of Avr1k (which did not interact with GmPUB1-1) by Rps1-k plants was not, however, affected following GmPUB1-1 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.

How Many Faces Does the Plant U-Box E3 Ligase Have?

“…The mutation in Avr1b C-terminus abolishes the interaction with GmPUB1 and the inhibition of cell death. Silencing of GmPUB1 in soybean cotyledons resulted in loss of recognition by the gene products of Avr1b [72]. Overexpression of GmPUB1 triggers cell death and enhanced resistance to Phytophthora, revealing that GmPUB1 is a positive regulator of effector-triggered immunity, and plays a positive role in the defense against Phytophthora [72].…”

“…Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection, including RxLR effectors Avr1b, Avr1d and Avr1k [72]. Several Phytophthora induced GmPUB proteins can interact with the RxLR effectors.…”

“…Silencing of GmPUB1 in soybean cotyledons resulted in loss of recognition by the gene products of Avr1b [72]. Overexpression of GmPUB1 triggers cell death and enhanced resistance to Phytophthora, revealing that GmPUB1 is a positive regulator of effector-triggered immunity, and plays a positive role in the defense against Phytophthora [72]. Recent data show that Avr1d competes with E2 for GmPUB13 binding to repress the E3 ligase activity and stabilize GmPUB13 to facilitate Phytophthora infection [73].…”

Progress and prospectus in genetics and genomics of Phytophthora root and stem rot resistance in soybean (Glycine max L.)

“…Currently, the management of PRSR is largely dependent on resistant cultivars, having one or more resistance genes. For understanding, pathogen race refers to a pathogen’s ability to cause disease in its host ( Anderson et al, 2010 ); in other words, the pathogen race attacks certain resistance genes ( Dorrance et al, 2016 ), and this kind of resistance is accompanied by several mechanisms including effector-triggered immunity (ETI), where R gene products in the host is recognized (directly/indirectly) by specific pathogen effectors termed avirulence (Avr) proteins ( Li et al, 2021 ). Till now, nine Avr genes of P. sojae have been cloned ( Yang et al, 2019 ).…”

“…During the single dominant gene resistance mechanism against P. sojae , expressed products of Rps genes interact with those of P. sojae through a gene-for-gene interaction and prevent disease development in plants ( Hartwig et al, 1968 ; Schmitthenner, 1999 ; Burnham et al, 2003 ; Fan et al, 2009 ). There are very few reports explaining the detailed expressed products of Rps genes; Gao et al (2005) mentioned the role of coiled-coil–nucleotide-binding–leucine-rich repeat (CC-NB-LRR)-type proteins in the case of Rps1- k locus ,and Li et al (2021) demonstrated that E3 ligase GmPUB1 protein is required for the interaction of P . sojae effector protein Avr1b with the resistance of Rps1b and Rps1k in soybean.…”