Study On The Formation Of Specific Cytosolic Calcium Signal During Wheat-leaf Rust Fungus Interaction

The incompatible combination included wheat cultivar Lovrin10 and leaf rust fungus races 260 in this experiment. First, the pharmacology experiment was carried out to test the effect of stabilizing agent taxol on hypersensitive reaction (HR) when it was injected into wheat leaves before inoculation of rust fungus. The results indicated that the injection could obviously reduce the area of dying cells caused by HR, and the higher the concentration of taxol, the smaller the area of dying cells. Combined with our early results that cystoskeleton involved in the influx of extracelluar calcium, it could be deduced that the dynamics of microtube may participate in the formation of calcium signalling during wheat interact with leaf rust fungus to effect on provocation level of HR.The results of prior pharmacology experiment showed that the concentration of cytosolic calcium rising may mainly depend on extracelluar calcium influx. In order to verify this hypothesis, potassium pyroantimonate precipitate method and transmission electron microscope technique were used to observe subcelluar distribution of calcium in wheat mesophyll cells after plasma membrane calcium channel blocker LaCl₃ was injected into in different infection phases, the information about provocation status of HR after prohibition of influx of extracelluar calcium, and the variation of cytosolic calcium and intracellular calcium were obtained meanwhile. Results showed that it could inhibit entry of extracellular calcium when LaCl₃ was injected into wheat leaves before inoculation, and there were similar tendencies of calcium concentration between the treatment and control except at the early stage of infection (8～12h), compared with the results of normal inoculation, there were no obviously differences in distribution and quantity of calcium at later stage, and tendency changes of the structures of cells looks same. According to the results, the host cells can activate HR though LaCl₃ block up the influx of extracellular calcium, so intracellular calcium may also be involved in the formation of the signal in the process of wheat-leaf rust fungus interaction.To verify weather intracellular calcium involved in induction of HR during interaction between wheat leave rust fugus and host, pharmacology experiment were carried out. The intercellular calcium chelators (BAPTA-AM) at different concentration were injected into wheat leaves before inoculation with wheat leave rust race 260. The results show that injecting BAPTA-AM could obviously reduce the area of dying cells caused by HR. The higher the concentration was, the smaller the area of dying cells was. While intercellular calcium activator (caffeine) was injected into wheat leaves, HR could also be detected. The HR area increased with caffeine concentration. Calcium channel blocker (Heparin,RR,and 8-Br-cADPR) were separately injected into wheat leaves before inoculation. The results show that there is an concentration-depended effect on herapin, and no obviously effect with RR and 8-Br-cADPR. So we deduce that intercellular calcium might involve in the formation of calcium signal transduction during infection of wheat leaf rust fungus, which used IP3 passway..Considering all results mentioned above, we suggested that the formation of calcium signal transduction was caused by the co-working of extracellular calcium influx and intercellular calcium releasing in HR reaction during interaction of wheat leave rust fugus and its host. Thus these results provided more powerful proof for calcium involving in HR signal transduction in wheat-leaf rust fungus interaction.