Function And Mechanism Of Action Analysis Of Avirulence Gene Avr3b From Phytophthora Sojae

Soybean root rot caused by Phytophthora sojae is one of destructive diseases to soybean production, causing losse of $1-2 billion per year worldwide. P. sojae deploys a large number of effector proteins that enter plant cells to disturb plant immunity and promote infection. One type of these effectors contains conserved Arg-X-Lys-Arg (RXLR) motif, termed RXLR effector. There are more than 300 genes coding RXLReffectors in the genome of P. sojae, among them, 9 RXLR effectors from P. sojae were identified as Avirulence (Avr) effector, such as Avr1b?Avr3a?Avr4/6.Products coding by these genes can be recognized by soybean resistance proteins and induce soybean resistance response. However the biological functions and molecular machemisms in plant-pathogen interaction of these effectors are unclear. To investigate this problem, our study focus on Phytophthora sojae avirulence gene Avr3b, which is indentified by our lab, and found that Avr3b is a NADH and ADP-ribose pyrophosphorylase that modulates plant immunity by a series of genetic and biochemical experiment. In addition, Avr3b also requires plant cyclophilin to activate the Nudix activity.P. sojae avirulence effector Avr3b is a secreted NADH and ADP-ribose pyrophosphorylase that modulates plant immunity. In this study, we found that transient expression of Avr3b in Nicotiana benthamiana increased susceptibility to P. capsici and P.parasitica, with significantly reduced accumulation of reactive oxygen species (ROS) around invasion sites. Avr3b encodes a secreted protein with the RXLR host-targeting motif and C-terminal W and Nudix hydrolase motifs. Biochemical assays confirmed that Avr3b is an NADH/ADP-ribose pyrophosphorylase, as predicted from the Nudix motif. Deletion of the Nudix motif or mutation of key residues of this motif significantly impaired enzyme activity and virulence functions of Avr3b.In addition, Avr3b also suppresses ETI triggered by P. sojae effector Avrlb. Considering that some plant Nudix hydrolases act as negative regulators of plant immunity, and this study suggested that pathogen might deliver Nudix effector, such as Avr3b, into host cells to impair plant immunity.The activation of Phytophthora effector Avr3b by plant cyclophilin is required for the nudix hydrolase activity of Avr3b. Previous study demonstrated that a P. sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta. Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase),cyclophilin,in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1,which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYPl; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1,and is also unable to promote Phytophthora infection. Finally, cyclophilins of N. benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, this study demonstrates the molecular machenism that Avr3b recruits plant cyclophilin activates the enzymatic activity of itself.GmCYPl associates with the recognition of Avr3b by Rps3b. Avr3b elicits specific ETI in soybean cultivars containing the resistance gene Rps3b. To test the role of GmCYPl in Avr3b-induced ETI in resistant cultivars of soybean, we delivered the constructs for GmCYPl silencing and Avr3b expression into soybean using bombardment, then analyzed the cell death.Quantitative RT-PCR data showed that GmCYP1 transcript level was significantly reduced in the delivery region, and the cell death assay indicated that Avr3b triggered cell death was weakened in soybean when GmCYPl was silenced. Pharmacological experiments showed Avr3b-triggered cell death was suppressed,in the presence of PPIase inhibitor CsA (20 ?M) on soybean leaves,indicating the PPIase activity is required for the activation of Avr3b avirulence function. In addition,transient expression of Avr3bP132A by bombardment could not induce Rps3b-mediated cell death on soybean leaves. Taken all the data together, our experiments demonstrate that GmCYPl is required for the recognition of Avr3b by Rps3b.RXLR-Nudix proteins are effectors which exist widely in Phytophthora species.In general,avirulence genes are not conserved in Phytophthora species, then, we examine the distribution of Avr3b homologs in other Phytophthora species. Searches of the P. sojae, P. infestans, P. capsici and P. ramorum, Avr3b homologs (RXLR-Nudix effector) were identified in all of these Phytophthora species (one from P. sojae, five from P. infestan, one from P. capsici and three from P. ramorum) ,the conserved Nudix hydrolase motif is located at the C-terminus of each of the RXLR-Nudix effectors, and the N-terminus of RXLR-Nudix effectors showed more polymorphism, suggesting that Phytophthora pathogens might share similar strategies to suppress plant immunity. Secreted Nudix proteins were also found in fungus. Unlike RXLR-Nudix effectors, these Nudix proteins don't contain RXLR motif or W motif. Sequence analysis found that Phytophthora RXLR-Nudix effector and other Nudix effector belong to different group and evolve separately. These results suggest two type of Nudix effector undergo different evolution, indicating they possess diverse functions for different pathogens.