Preparation Of Antiserum Against Wheat Mlo IL2 Polypeptide And Its Cytolocalization Specificity

Recessive mutations in the barley Mlo gene confer durable, broad-spectrum resistance to almost all known isolates of the powdery mildew pathogen, Blumeria graminis f. sp. hordei. The race-specific resistance always causes the hypersensitive host cell death response at the site of attempted cell. But the mlo gene confers papilla-associated resistance to restrict the pathogen invasion. In this paper, the polyclonal antiserum against wheat Mlo protein was prepared by molecular biological and immunological techniques. Furthermore, the cytolocalization of Mlo protein during the interaction between host and pathogen was studied. The main results obtained in this paper were as follows.1. According to wheat TaMlo3 cDNA sequence, the DNA fragment encoding the intracellular loop 2 of wheat Mlo protein was cloned into the vector pET-30a, and the fusion polypeptide was expressed in E.coli BL21. The fusion protein purified by Ni2+-NTA agarose column was used to immunize the rabbits. The titre of the antiserum obtained is 1:10000 measured by indirect ELISA. The western blotting showed that this antiserum can specifically react with the expressed protein.2. The leaves of wheat, tobacco and barley cultivars with different resistance were labeled with two antisera against the intracellular loop 2 and C-terminus of wheat Mlo protein, respectively. The results revealed that there was no Mlo protein to be detectable in the healthy epidermal cells. After inoculation with powdery mildew fungi, the gold particles were specifically bound in the extrahaustorial matrix in both barley and wheat haustorium-containing cells. These indicated that Mlo protein is a low abundant protein and specifically accumulated in the extrahaustorial matrix.3. The gold particles labeled with the two wheat antisera were also specifically bound in the plant chloroplasts. The control experiments revealed that the labeling was caused by crossing interaction.4. Wheat or barley powdery mildew fungi were also labeled with the two antisera against the intracellular loop 2 and C-terminus of wheat Mlo protein, respectively.