S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Food and Animal Biotechnology (식품·동물생명공학부) Theses (Master's Degree_식품·동물생명공학부)
Mutational analysis of HpaG, a harpin-like protein elicitor, from Xanthomonas axonopodis pv. glycines
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- 서울대학교 대학원
- Thesis (master`s)--서울대학교 대학원 :농생명공학부,2003.
- 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.
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