A point mutation in the ethylene-inducing xylanase elicitor inhibits the beta-1-4-endoxylanase activity but not the elicitation activity

Ethylene-inducing xylanase (EIX) elicits plant defense responses in certain tobacco (Nicotiana tabacum) and tomato cultivars in addition to its xylan degradation activity. It is not clear, however, whether elicitation occurs by cell wall fragments released by the enzymatic activity or by the xylanas e protein interacting directly with the plant cells. We cloned the gene enc oding EIX protein and overexpressed it in insect cells. To determine the re lationship between the two activities, substitution of amino acids in the x ylanase active site was performed. Substitution at glutamic acid-86 or -177 with glutamine (Gln), aspartic acid (Asp), or glycine (Cly) inhibited the beta-1-4-endoxylanase activity. Mutants having Asp-86 or Gln-177 also lost the ability to induce the hypersensitive response and ethylene biosynthesis . However, mutants having Gln-86, Gly-86, Asp-177, or Gly-177 retained abil ity to induce ethylene biosynthesis and the hypersensitive response. Our da ta show that the xylanase activity of EIX elicitor can be separated from th e elicitation process, as some of the mutants lack the former but retain th e latter.