VIEW ARTICLE | DOI: 10.1094/MPMI-1-275
Cloning of Two Endoglucanase Genes of Xanthomonas campestris pv. campestris: Analysis of the Role of the Major Endoglucanase in Pathogenesis. Clare L. Gough. John Innes Institute, Colney Lane, Norwich, NR4 7UH, U.K.. J. Maxwell Dow, Christine E. Barber, and Michael J. Daniels. John Innes Institute, Colney Lane, Norwich, NR4 7UH, U.K.. MPMI 1:275-281. Accepted 29 September 1988. Copyright 1988 The American Phytopathological Society.
Additional Keywords: marker-exchange mutagenesis, pathogenicity testing, Brassica.
A genomic library of Xanthomonas campestris pv. campestris was mobilized into the noncellulolytic Xanthomonas campestris pv. translucens, and transconjugants were screened for endoglucanase activity on carboxymethylcellulose. pIJ308I and pIJ3082, two positive clones whose inserts shared no homology, were identified. pIJ3082 encoded an endoglucanase activity that had the same pH optimum as the major endoglucanase of X. c. pv. campestris, and like the major endoglucanase of X. c. pv. campestris, was mainly extracellular. Conversely, pIJ3081 directed the synthesis of an endoglucanase with a different pH optimum that was largely intracellular in X. c. pv. translucens. Viscometric and reducing sugar analysis confirmed that the enzyme activities encoded by pIJ3081 and pIJ3082 and the major enzyme activity of X. c. pv. campestris were all endoglucanases. The major endoglucanase activity of X. c. pv. campestris was purified and shown to be a protein of 53 kD. This protein was absent from culture filtrates of endoglucanase-minus mutants made by marker exchange of endoglucanase-minus Tn5 insertions in pIJ3082, implying that pIJ3082 contains the gene for the major endoglucanase of X. c. pv. campestris. Subcloning and Tn5 mutagenesis located the gene to a 1.5-kb region of pIJ3082 DNA. Several different pathogenicity tests with these endoglucanase-minus mutants showed that the major endoglucanase of X. c. pv. campestris plays a minor role in the early stages of pathogenicity of X. c. pv. campestris on turnip and radish.