Submitted to: Journal of Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/6/1998
Publication Date: N/A
Citation: Interpretive Summary: Plants have a number of mechanisms which serve as defenses against fungal pathogens. Glucanases are produced by plants and are known to inhibit the growth of some pathogenic fungi. We are interested in the role of glucanases in resistance to fungal pathogens of Citrus species. We have purified one of the glucanases from 'Valencia' orange callus. It has properties similar to glucanases from other plant species that are known to have antifungal activity. Antibodies have been raised to the citrus glucanase and these provide us with a tool for quantifying levels of the glucanase in various tissues in response to treatments which may elicit an increase in levels of the glucanase. The antibody was used to isolate a glucanase gene from citrus callus tissue. The glucanase clone makes it possible to study expression of the glucanase gene and develop information about it's regulation. In addition, the cloned glucanase gene will be used to transform citrus plants to overexpress the glucanase protein giving us the opportunity to increase decay resistance. The overall impact of this work will allow us to develop citrus cultivars that are resistant to fungal pathogens and thereby reduce the use of chemical fungicides.
Technical Abstract: We have purified a beta-1,3-endoglucanase (EC 126.96.36.199) from nonembryogenic Citrus sinensis (L.) Osbeck cv. Valencia callus to electrophoretic homogeneity by means of pH precipitation and ion exchange chromatography. The protein has an apparent Mr of 32,000, a pI > pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestans infection. The enzyme hydrolyzes laminarin (Laminaria digitata) optimally at pH 5 and 50øC. The enzyme will hydrolyze pachyman and laminarin extensively and yeast glucan slightly, but does not hydrolyze lichenin, barley glucan, cellulose, or starch. Product characterization by thin-layer chromatography indicates that the enzyme is an endohydrolase. The protein is N-terminal blocked, however, partial internal amino acid sequence analysis revealed that the peptide shared homology with a number of beta-1,3-endoglucanases. Antibody to the purified protein was raised in a rabbit and used to screen an amplified cDNA library prepared from Citrus sinensis (L.) Osbeck cv. Valencia callus. A positive clone (pBGVC-1) containing a 1249 bp insert was isolated. A full length sequence of the clone was obtained and it contained a 1229 bp open reading frame starting at nucleotide 20. Sequence analysis indicated that the clone is homologous to other beta-1,3-endoglucanase genes.