Mutational analysis of HpaG, a harpin-like protein elicitor, from Xanthomonas axonopodis pv. glycines

Abstract

HpaG is a harpin-like protein elicitor identified from Xanthomonas axonopodis pv. glycines. We compared physical and chemical properties of HpaG with Hpa1Xoo, XopA, and HrpN. These proteins had general characteristics of harpin, but their HR elicitation activity made a difference. Purified HpaG and HrpN elicited an HR at a concentration of 0.1 μM in tobacco plant. On the other hand, XopA did not induce HR in tobacco plant and Hpa1Xoo showed the HR at concentrations above 5 μM. In addition, we compared a xopA gene among Xanthomonas campestris pv. vesicatoria three races, in result, we found that a xopA is identical among three races. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation of HpaG by deletion and site-directed mutagenesis. From the serial deletion analyses, we found that the amino-terminal 67 amino acids were sufficient for inducing HR in tobacco plants. We then mutagenized the plasmid clone carrying hpaG by error-prone PCR and site-directed mutagenesis. We obtained thirty-nine mutants, including three nonsense mutants and thirty-six single missense mutants. Three nonsense mutants and L39A, L43P, L46A substitutions abolished the activity. On the other hand, Q45A, I47A, L50A, Q52A, S54A, G66A, and G67V substitutions showed reduced activity, and the other mutants had the same HR elicitation activity as the wild-type. Through site-directed mutagenesis of XopA, which doesnt possess HR elicitation activity, we obtained two gain of function mutants of XopA. XopA-F48L mutant elicited the typical HR at concentrations above 5 μM and XopA-F48L/M52A mutant exhibited an HR at 1 μM. These results indicate that residues between L39 and G67 have important roles in HR elicitation in tobacco plants.