The Activation of <i>Phytophthora</i> Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b

<div><p>Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a <i>Phytophthora sojae</i> RXLR effector Avr3b acts as a Nudix hydrolase when expressed <i>in planta</i>; and this enzymatic activity is required for full virulence of <i>P</i>. <i>sojae</i> strain P6497 in soybean (<i>Glycine max</i>). Interestingly, recombinant Avr3b produced by <i>E</i>. <i>coli</i> 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 GmCYP1; as a result, the mutant Avr3b^P132A can no longer be activated by GmCYP1, and is also unable to promote <i>Phytophthora</i> infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3b^P132A lost its ability to trigger HR. Furthermore, silencing of <i>GmCYP1</i> rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of <i>Nicotiana benthamiana</i> can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a “helper” that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.</p>