Study On The Changes Of Cytosolic Calcium Dynamics During Wheat-leaf Rust Fungus Interaction

In this paper, wheat (Triticum aestivum L.) cultivars Lovrin 10 (resistant) and 5389 (susceptible) were selected as materials in this system. They make up respectively incompatible combinations and compatible combinations with wheat leaf rust fungus(Puccinia recondita f.sp. tritici)physiological race 162. Distribution of soluble calcium in different phases during leaf rust infect wheat mesophyll cell were showed by subcelluar localization by using potassium pyroantimonate precipitate method and transmission electron microscope technique. Results showed that calcium-containing pyroantimonate precipitate form big granules and amorphous obturation middle empty object in intercellular space of non-inoculated wheat leaves; whereas calcium-containing pyroantimonate deposition form thin granules and a lot of black areas in vacuole. These suggest that there are a lot of soluble calcium in intercellular space and vacuole. There are a few calcium- containing pyroantimonate precipitate in cell wall, chloroplasts and cytosolic, no obvious calcium -containing pyroantimonate precipitate in nucleolus. The distribution trend of calcium is resemble in the samplings taking in different time, suggesting cytosolic calcium concentration maintain stable level under normal state. It is observed from intercellular space mass calcium swarm to cytoplasm at the early stage of wheat-leaf rust interaction in incompatible combinations (4-8h after inoculation). Moreover there are a lot of calcium- containing pyroantimonate deposits in host cell and contacting hypha, and calcium deposits granule distributed in cell wall, chloroplasts and cytoplasm, and chloroplasts disaggregatio- n phenomenon were also observed. In compatible combination (12-24h after inoculation), however, rise of cytosolic calcium concentration was later than incompatible combinations, and the size and quantity of calcium-containing pyroantimona- te deposits granule was less than that in incompatible combination. This result suggests that cytosolic calcium concentration ascend distinctly in incompatible combinations at the early stage of wheat -leaf rust interaction, and the cytosolic calcium rise may mainly depen- ded on extracellar calcium influx.The intercellular washing fluid (IWF) which contains the elicitor from wheat-leaf rust fungus interaction, was used as stimulator to interact with the protoplast of cv. Lovrin 10 or cv. 5389. By using this system, a new method of measuring cytosolic Ca2+ dynamics by incubating with Fluo-3AM in protoplasts was established, Laser Scanning Confocal Micro-scope(LSCM) was used to observe the changes of cytosolic calcium concentration in different wheat (Triticum aestivum) mesophyll protoplast after IWF treatment. The results suggest that the kinetics curve for change of cytosolic calcium concentration is special during investigated period after the mesophyll protoplasts of resistant wheat were treated with IWFi62- In elicitor-treated cv. Lovrin 10 ,a lag phase of 50s preceded a 200s transient and slowly increase of cytosolic calcium concentration, which.peaked at 1.4 after 250s,and last for 100s then decreased.However,it keep at 1.2 until 10 min's treatment.At the same time,the susceptible cultivar protoplast's curve of cytosolic calcium concentration hold the balance on the contrast level all the time. Change of cytosolic calcium concentration of the mesophyll protoplasts of susceptible wheats, however, was not evident after IWF treatment. The pharmacology examination indicated that the cytosolic calcium concentration rise induced by IWF mainly depended on extracellar calcium influx.The results of subcelluar localization and LSCM which were come from different examination system and introduced different means adequately proved that calcium mess-anger was involved in wheat-leaf rust interaction and cytosolic calcium concentration rise mainly depended on extracellar calcium influx.