Identification Of Pathogenesis Related Protein In Intercellular Washing Fluid Of Rust-infected Wheat Leaves

In this experiment, wheat cultivar Lovrin10,5389 and leaf rust fungus races 260 were defined as study objects. The intercellular washing fluids (IWF) extracted from wheat leaves of combinations(5389×260) after 5 days after inoculation (dai) and incombinations(L10×260) after 3 dai were injected into wheat leaves to determine the activities of PAL, PO and the area of hypersensitive reaction (HR). Then the effects of IWF on hyphal extension and spore germination were observed. The results indicated that both of the IWF from the compatible and incompatible combinations could induce activities of PAL, PO and HR area on leaves of wheat increasing. Furthermore, the defense response induced by IWF from incompatible combination was obviously higher than that of compatible combination. Meanwhile, they could inhibit the hyphal extension and spore germination in the incompatible combination either. This result took a proof of the existence of resistance substances in IWF.In order to isolate pathogenic related protein, the IWF from rust-infected leaves of incompatible combination were used as study objects, and the IWF of healthy wheat leaves served as control, both of them were identified by two-dimensional gel electrophoresis. A suitable test system of two-dimensional electrophoresis for proteomic study on wheat IWF was established in this experiment, which referred a series of optimized effectors as followed: (1) the 50% TCA method was used to precipitate protein of IWF, (2) 800μg of protein was the best loading amount, (3) active hydration was carried out for 12h, (4) the IPG of pI 4-7 was suitable to IWF of wheat, and finally, (5) the best balance time from the one direction to two direction was 15min. This result revealed that high reproducible and resolution two-dimensional gel electrophoresis maps of protein were obtained according to the key effectors mentioned above. A large number of new proteins which were induced by rust fungus in incompatible group were observed. Two significant protein dots in the maps were analyzed by mass spectrometry and then compared with the database in NCBI. The searched information indicated these two proteins had high homology with endo-β-1,3-glucanase andβ-1,3-glucanase precursor. Considered the given result, we supposed thatβ-1,3-glucanase may play a role in wheat resistance to leaf rust fungus. To verify the role ofβ-1,3-glucanase in the process of wheat resistance, firstly,β-1, 3-glucanase activity in IWF from two combinations at different time points after inoculation were detected.The results showed that the activity ofβ-1,3-glucanase from incompatible combination was always higher than that from compatible combination, in addition to that, two peaks were observed at 4hai and 72hai. It indicated thatβ-1,3-glucanase also played a role in wheat resistance to leaf rust fungus. Subsequently, the urediniospores were cultivated in the solution which contained different concentrations of exogenousβ-1,3-glucanase. We found that the germination of spores was not inhibited because of germ tube, which indicated thatβ-1,3-glucanase did not inhibit spore germination in vitro. Thirdly, exogenousβ-1, 3-glucanase were injected into wheat leaves, which inoculated with leaf rust fungus, to measurement its effect on the hyphal extension and the impact of HR. Our results showed thatβ-1,3-glucanase can inhibit the extension of hyphal area in compatible group; and the initiation of HR was getting earlier accompanied with HR area reducing in incompatible group. The experiment results proved thatβ-1, 3-glucanase may be involved in the resistance of wheat to leaf rust fungus infection.In conclusion, the inducing activity to wheat defense response of IWF from incompatible group was obviously higher than compatible group, and there was resistance substances in IWF. Then theβ-1,3-glucanase in IWF was identified by two-dimensional gel electrophoresis analysis, the result indicated thatβ-1,3-glucanase may take part in the wheat resistance to leaf rust through our experiments. The results might lead a way for further studies on the mechanisms of wheat-leaf rust resistance.