Purification Of An Elicitor From Intercellular Washing Fluid Of Rust-infected Wheat Leaves And Researching Into Its Origin

In this study , an elicitor which exists in the intercellular washing fluid(IWF) in the progress of the interaction between leaf rust and wheat was analyzed and purified.. At first. The germ tube extracts of wheat leaf rust race 165 or its germination medium was injected into health wheat leaves in order to find out whether they can induce the increase of PAL and POD activities or not. The results showed that there were not any elicitor activities exist in the extracts of wheat leaf rust race 165 and its germination medium. Subsequently, we inoculated wheat seedings of cultivar lovrin 10 with leaf rust race 165 to form compatible combination and prepared intercellular washing fluid(IWF) after 5 days, named IWF₁₆₅. Cells suspension of wheat were treated with IWF₁₆₅ and found that IWF₁₆₅ can induce PCD in these cells. These results suggest that there is an elicitor in the IWF₁₆₅ and the elicitor activities can be checked by PCD in the progress of purification. The IWF₁₆₅ was purified by (NH₄)₂SO₄ precipitation fraction, and then applied to Sephadex G-100 column. The column eluates were collected separately according to ΔA280nm, and the healthy wheat leaves and cells suspension of wheat were treated with the eluate fractions in order that elicitor activities were determined by PAL, POD and PCD assays. After the IEF-PAGE analysis, the Sephadex G-100 elution peak 1(F1) of IWF₁₆₅ was further purified by DEAE anion exchange chromatography. And the elution peaks were treated as: (1) Elicitor activities were checked by PAL, POD and PCD assasys.(2) SDS-PAGE analysis . This paper is to search out the origin of the elicitor and purify it. And it can also throw light on further studies about the elicitor's characteristics, receptor and infectious signal transduction system.The results suggested: elicitor may not exist in the extracts of wheat leaf rust uredosPODre race 165 and its culture medium. The IWF₁₆₅ not only can induce the obvious increase of the activities of PAL and POD in wheat leaves, but also can induce cells suspension of wheat occurring PCD. Therefore, we believe that the elicitor may produce in the interaction between the leaf rust and wheat, so, we purified the elicitor from IWF₁₆₅. The eluates were collected from gel-filtration of the IWF₁₆₅ which showed different inductive effect. The eluate peak 1(F1) of the IWF₁₆₅ had the highest activities determined by PAL, POD and PCD assays. So, further purified F1 using the DEAE anion exchange chromatography were conducted.In the DEAE anion exchange chromatography two elution peak 1(LF1) and peak 2(LF2) all showed certain inductive activities for PAL and POD. But LF1's inductive activity is higher than LF2 and only LF1 can induce the cells suspension of wheat occurring PCD. So, the LF1 contain elicitor. SDS-PAGE analysis indicated that eluate LF1 from DEAE anion exchange chromatography contained the protein of Mr1=28 kD and the...