Purification and characterization of elicitors from the wilt producing fungus, Verticillium dahliae, responsible for the induction of defense responses in plant cells

David Alan Davis, Purdue University

Abstract

Inducible biochemical defense mechanisms play a significant role in the ability of plants to withstand disease causing organisms. The ability of a plant to recognize an invading organism requires specific detection systems for the molecules native to the organism. In this research, plant cell suspension cultures were used as a model system to determine the nature of the eliciting components of phytoalexin production from the wilt producing fungus, Verticillium dahliae. The elicitor activity from the culture fluid was found to be heat stable. Treatment of the crude elicitor with 20 mM periodate did not significantly alter elicitor activity, however, it was sensitive to protease digestion. Treatment of the elicitor with Pronase E or Papain completely abolished elicitor activity. Culture fluid protein was concentrated using acetone precipitation and purification of the elicitor was further carried out using ion exchange, sephacryl S-300 and concanavalin-A lectin affinity chromatography. The elicitor was identified as a 65 kDa glycoprotein. Enzymatic deglycosylation of the conA fraction did not alter elicitor activity indicating that the protein portion was responsible for activity. The 65 kDa glycoprotein elicitor could be enzymatically deglycosylated to a protein of 53 kDa. The elicitor preparation precipitated from the culture fluids of the fungus was also able to elicit an oxidative burst in cotton cells. The oxidative burst activity was not destroyed by protease treatments even though these same treatments destroyed phytoalexin eliciting activity. However, periodate oxidation was able to significantly reduce the elicitation of the oxidative burst indicating that a carbohydrate component was likely to be involved in the stimulation of an oxidative burst in cotton cells. Similar results were obtained when soybean cells were used in place of cotton cells. Oxalate at millimolar levels was found to enhance the accumulation of phytoalexins by the culture fluid elicitor from V. dahliae up to ten fold. The enhancement of phytoalexin elicitation could be reversed by similar levels of the phytoalexin inhibitor, citrate. It was found that the reduction in cell growth induced by high levels of elicitor could be reduced or reversed when oxalate was used to enhance elicitation by low elicitor levels.

Degree

Ph.D.

Advisors

Heinstein, Purdue University.

Subject Area

Biochemistry|Agricultural chemicals|Plant pathology

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