Isolation And Purification Of Elicitor(s) From Wheat-leaf Rust Interaction System And CDNA-AFLP Display For Isolation Of Lr19 Expressed During Infection

Recently, many studies suggested that the pathotypes in plant-pathogen interactions, compatible or incompatible, are determined by not only the presence of the defence genes, but also when, where and how strong those genes are expressed. The defence response is induced by host-parasite interaction firstly. The studies of original signal- elicitors, were payed increasing attention. To the system of interaction between wheat and leaf rust, elicitors were found in intercellular washing fluids (IWF) of both compatible and incompatible interactions. Thatcher and its near-isogenic lines carrying ThLrl9 resistance gene against leaf rust were inoculated with leaf rust race 99-5-49-4. Thatcher X 99-5-49-4 is compatible combination and ThLrl9 X 99-5-49-4 is incompatible combination. Intercellular washing fluids were prepared five days after inoculation(Thatcher X 99-5-49-4) or three days (ThLrl9X99-5-49-4). The IWFs were purified by (NH4)2SO4 precipitation fractionation, and then applied to Sephadex G-75 column. The column eluates were collected separately according to A280 nm and injected into the health wheat leaves to examine the eliciting activities by PAL, PO assays.It was shown that the eluates collected from gel-filtration of the two combination IWFs showed different eliciting effect. For the compatible combination IWF, the eluate peakl(Pl) and peak2 (P2) contain the higher active constituent(s), but the eluate peak3(P3) showed the inhibiting effect determined by PAL and PO assays; and for the incompatible combination IWF, the eluate peak 1 (PI) shows higher active effect on PAL activity.The interaction of host-pathogen can be analyzed at mRNA level. We applied the same system (Thatcher X 99-5-49-4 and ThLrl9X99-5-49-4) for cDNA-AFLP analysis. Wheat leaf samples were harvested in 12, 24, 36, 48, 60, 72, 120 hours after inoculated with leaf rust race 99-5-49-4 and control. We use the extracted total RNA of wheat leaves to replace the mRNA to analysis the difference of resistance gene induced expression.The results could be divided into two aspects. One side, some differential cDNA bands could be examined from 12 to 120 hours, and on the other side, the differentialcDNA bands could only be examined from 12 to 36 hours after inoculated. To collect the differential cDNA fragment from the PAGE and reamplifyit at the same conditionare expected to be helpful for further research, such as Northern blotting, molecular hybrid, cloning, sequencing and so on.