Analysis Of Recognition Sites And Subcellular Localization Of Avr3A From Phytophthora Sojae

Root and stem rot disease of soybean,which generating great losses to agricultural production annually is caused by the oomycete Phytophthora sojae.Disease outcomes between plants and Phytophthora pathogens often depend on whether plants carry resistance (R) gene-encoded receptors than recognize the presence of pathogen avirulence (Avr) genes. A lot of avirulence genes that trigger plant effector-triggered immunity (ETI) as a result of recognition by plant resistance gene products have been identified in P.sojae, Some isolates of P. sojae evade perception by R genesthrough. sequence mutation in Avr genes and lowered transcript accumulation.Previously, Qutob et al.,have identified a avirulence gene PsAvr3a, which had a gene-for-gene interaction with the soybean resistance gene Rps3a. Avr3a encodes a secreted protein with the RXLR host-targeting motif and C-terminal W motif. Firstly,we confirmed that the signal peptide of20amino acids of Avr3a is not required for recogniton of Avr3a-Rps3a.Sequence analysis of PsAvr3a in different P. sojae strains revealed five Avr3a alleles:Avr3aHN25, Avr3aHN35,Avr3aP6497,Avr3aAcr12and Avr3a P7064. But Avr3a does not transcript in P7064. Transient expression of the Avr3a alleles using a double-barreled bombardment in soybean showed that Avr3aHN25,Avr3aHN35from virulent strains of P. sojae do not cause cell death on Rps3a plants, in contrast to Avr3aP6497and Avr3aACR12from avirulent strains. It is indicated that the the recognition of Avr3a-Rps3a directly affects the Avr3a avirulent function.Avr3a is highly conserved in different P. sojae strains. Analysis of the C-terminal domains of Avr3a proteins have showed that it indeed exposed to strong positive selection, leading to fast evolution and diversifying sequences. Plasmid constructs directing expression of Avr3aP6497deletion mutants and fixed mutants without native signal peptide were introduced into leaves of an Rps3a soybean cultivar together with a reporter gene (beta-glucuronidase, GUS) to measure cell viability. The results indicated that the region of amino acids61to112was the critical to recognition, which contains W motif and its front and rear areas. Either Avr3aP649777G or Avr3aP649782D could trigger Rps3a mediated cell death on soybean leave. Avr3aP649777G82D may affect the regconiton.Expressing different effectors fused GFP protein in plant cells, we found that Avr3aP6497, Avr3aHN25localized to the nuleus and cytoplasma of the plant cells. Meanwhile, the localization nuclear signal (NLS2) and nuclear export signal (NES) fusion to the C-terminal of Avr3aP6497and Avr3aHN25could change localization of Avr3aP6497and Avr3aHN25. Neither Avr3aP6497-NLS2or Avr3aP6497-NES could trigger Rps3a medinated cell death on soybean leave. Avr3aHN25-NLS2and Avr3aHN25-NES showed the same results.We speculated that the recognition of Avr3a and Rps3a was likely to occur in the plant cytoplasm.