Proline132 of Avr3b plays a critical role in activation by cyclophilins.

<p>(<b>A</b>) Avr3b^P132A mutant can not be activated in <i>N</i>. <i>benthamiana</i>. FLAG-GFP, FLAG-Avr3b or FLAG-Avr3b^P132A were expressed in <i>N</i>. <i>benthamiana</i> leaves. Relative Nudix hydrolase activity was analyzed at 48 hpi. Bars represent standard errors from three independent replicates. * representing significantly different than FLAG-GFP (<i>t</i> test <i>P</i><0.05). (<b>B</b>) Avr3b^P132A is impaired in its interaction with GmCYP1. Yeast cells were co-transformed with pGADT7-GmCYP1 and pGBKT7 carrying Avr3b or Avr3b^P132A. Transformants were grown on a selective medium.β-galactosidase activity in yeast cells was measured to determine the Avr3b-GmCYP1 interaction. Bars represent standard errors from three independent replicates. *, <i>t</i> test <i>P</i><0.05. (<b>C</b>) The P132A mutation of Avr3b resulted in loss of virulence activity. FLAG-GFP, FLAG-Avr3b or FLAG-Avr3b^P132A proteins were transiently expressed in <i>N</i>. <i>benthamiana</i> leaves by <i>Agro</i>-infiltration. The leaves were inoculated with <i>P</i>. <i>capsici</i> at 48 hours post <i>Agro</i>-infiltration. Biomass of <i>P</i>. <i>capsici</i> was determined at 36 hours post inoculation by quantitative PCR. Bars represent standard errors from three independent replicates. *, <i>t</i> test <i>P</i><0.05. (<b>D</b>) Avr3b^P132A failed to induce Rps3b-mediated cell death in soybean. Percentage rate of β-glucuronidase (GUS)-positive blue spots following bombardment with Avr3b^P132A compared with the EV. Bars represent standard errors from four independent replicates. **, <i>t</i> test <i>P</i><0.01. (<b>E</b>) Avr3b^P132A could not suppress Avr1b-triggered HR in soybean producing Rps1b. Percentage rate of GUS-positive blue spots following co-bombardment of Avr1b with Avr3b or Avr3b^P132A compared with EV. Data are the means of four independent experiments. Bars represent standard errors from four independent replicates. **, <i>t</i> test <i>P</i><0.01.