Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants.
HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an alpha-helix, as is the HrpW region that is homologous to HpaG. The predicted alpha-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.
Kim
Jung-Gun
JG
School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea.
Jeon
Eunkyung
E
Oh
Jonghee
J
Moon
Jae Sun
JS
Hwang
Ingyu
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology; D001692:Biological Transport; D004252:DNA Mutational Analysis; D004885:Erwinia / Q000235:genetics; D019811:Hydroxylamine / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000494:pharmacology; D020125:Mutation, Missense; D009842:Oligopeptides / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011549:Pseudomonas / Q000235:genetics; D017384:Sequence Deletion; D017385:Sequence Homology; D037521:Virulence Factors / Q000235:genetics* / Q000502:physiology; D014974:Xanthomonas / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D009153:Mutagens; D009842:Oligopeptides; D037521:Virulence Factors; C075462:harpin protein, Erwinia amylovora; D019811:Hydroxylamine