Cloning of Genes Affecting Polygalacturonase Production in Pseudomonas solanacearum. Caitilyn Allen. Department of Plant Pathology, University of Wisconsin, Madison, WI 53706 U.S.A. Yong Huang, and Luis Sequeira. Department of Plant Pathology, University of Wisconsin, Madison, WI 53706 U.S.A. MPMI 4:147-154. Accepted 5 November 1990. Copyright 1991 The American Phytopathological Society.

An extracellular polygalacturonase (PG) produced by the bacterial wilt pathogen Pseudomonas solanacearum induced rapid browning of potato tissue culture callus and was identical to a previously described browning factor. The structural gene for this enzyme was cloned from a genomic library of P. solanacearum K60 and mutagenized with Tn3 carrying a Β-glucuronidase reporter gene. The PG gene (called pehA) was located at the edge of the Vir2 cluster of hrp genes previously described by Christian Boucher and co-workers in strain GMI1000 of P. solanacearum. The mutagenized PG gene was marker-exchanged into the wild-type chromosome of strain K60; the resulting mutant strain no longer caused browning on callus but was still pathogenic on tobacco and eggplant and retained the ability to elicit a hypersensitive response on nonhost species. The mutant no longer produced the pI 9.0 enzyme encoded by pehA. In the wild-type strain, total extracellular PG activity increased about fivefold when bacteria were grown in the presence of tobacco leaf intracellular fluids and about 100-fold when bacteria were inoculated into tobacco leaves. Three Tn5 mutants, one from strain B1 and two from strain K60, no longer browned callus or produced detectable PG activity in culture, but Southern blot analysis showed that in all three mutants the transposon was inserted into a common 4.5-kilobase region which is not in or near pehA. When these mutants were complemented with the corresponding wild-type fragment, the resulting trans-merodiploid strains produced about eight times the wild-type level of PG activity. This locus, called pehR, may encode a trans-acting positive regulator of PG production.