T. Gregory McCollum

PURIFICATION AND CHARACTERIZATION OF AN ENDO-POLYGALACTURONASE FROM THE GUT OF WEST INDIES SUGARCANE ROOTSTALK BORER WEEVIL (DIAPREPES ABBREVIATUS L.) LARVAE

Abstract An endo-polygalacturonase (PG) (EC:3.2.1.15) with a pI of 9.4 and an Mr of 44,500 was purified to electrophoretic homogeneity from the gut of West Indies sugarcane rootstalk borer weevil (Diaprepes abbreviatus L.) larvae. Hydrolytic activity was maximal in 150 mM sodium acetate, pH 5.5, at 30°C. Kinetic determinations yielded an apparent Km of 3.68 mg polygalacturonic acid (PGA)/ml and a Vmax of 283 μmol galacturonic acid/min/mg protein for PGA. Enzymatic activity was inhibited by a polygalacturonase inhibitor protein from “Hamlin” orange flavedo. The purified protein does not appear to be glycosylated, and its N-terminal sequence showed no homology to any PG protein sequences in data banks.

Isolation of a citrus chitinase cDNA and characterization of its expression in response to elicitation of fruit pathogen resistance

Summary Chitinases are well-known antifungal proteins and belong to the pathogenesis-related (PR) group of proteins. In the present study, we screened a Valencia orange flavedo cDNA expression library with an antibody raised against a purified Valencia basic chitinase polypeptide and isolated its corresponding cDNA. The Valencia flavedo chitinase cDNA, designated chi1, is 875 bp in length, with an open reading frame of 693 bp. The chi1 gene encodes a predicted polypeptide of 231 amino acids with a predicted molecular mass of 25.1 kDa and a pI of 9. 15. The CHI1 protein shares 60, 58, and 56 % identity with the basic chitinase proteins of rice, grape and maize, respectively. Southern blot analysis indicated that chi1 is present as a low-copy gene. RNA gel blot hybridizations revealed that chi1 gene expression was markedly induced by various treatments that induce fruit resistance against the green mould pathogen Penicillium digitatum (Pers.:Fr.) Sacc. These treatments included elicitation of fruit pathogen resistance by UV irradiation, hot water brushing, and application of β-aminobutyric acid (BABA) and Candida oleophila antagonist yeast cells.

Biochemical, Molecular Genetic, and Immunological Characterization of a β-1,3-Endoglucanase from ‘Valencia’ Orange Callus

Summary We have purified a β-1,3-endoglucanase (EC 3.2.1.39) 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 pl > pH 10 and is serologically similar to a potato leaf glucanase induced by Phytophthora infestans infection. The enzyme hydrolyzes laminarin (Lam- inaria digitata) optimally at pH 5 and 50 °C. 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 β-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. Valen- cia callus. A positive clone (pBGVC-1) containing a 1,249 by insert was isolated. A full length sequence of the clone was obtained and it contained a 1,229 by open reading frame starting at nucleotide 20. Sequence analysis indicated that the clone is homologous to other 0-1,3-endoglucanase genes. The predicted amino acid sequence was homologous with other 0-1,3-glucanases, contained both N- and C-terminal signal sequences, the glycosyl hydrolase family 17 signature sequence, and the sequence identical to the peptide that was sequenced from the purified protein.