1.6.3
CLONING AND CHARACTERIZATION OF hrp OR hrc GENES FROM BURKHOLDERIA GLUMAE, ACIDOVORAX AVENAE, PANTOEA AGGLOMERANS PV. MILLETTIAE AND PSEUDOMONAS FLUORESCENS

Y TAKIKAWA, M KOJIMA, S ADACHI, M KOBAYASHI, T KUMAKURA and Y INOUE

Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422, Japan

Background and objectives
Genes conferring both pathogenicity and ability to induce hypersensitive reaction (HR) of plant pathogenic bacteria have been identified and named hrp genes. The similarity of hrp genes to some pathogenicity genes of animal pathogens led to the finding of a common type III secretion pathway in both pathogens. Some of the well conserved hrp genes are now named hrc genes. Although they have been demonstrated in a wide variety of plant pathogens, it is still not known whether having hrp/hrc genes is a prerequisite for all plant pathogens. Therefore, we have begun the detection of genes homologous to known hrp/hrc genes in various plant pathogenic and saprophytic bacteria, and their cloning and functional analysis.

Materials and methods
Probes were derived from well known hrp/hrc clones of Pseudomonas syringae pv. phaseolicola (pPL6) and Ralstonia solanacearum (pVir2) or from PCR products of Erwinia herbicola pv. gypsophilae hrp genes. Various plant pathogenic and saprophytic bacteria were subjected to genomic Southern hybridization. Genomic libraries of some bacteria were constructed using the cosmid vector pLAFR3. Homologous cosmid clones were screened by hybridization, subcloned and sequenced. Marker-exchange mutations were introduced into hrp/hrc genes of some bacteria with Tn3-Spice insertions.

Results and conclusions
Among the bacteria tested, many plant pathogenic Burkholderia and Acidovorax species showed positive signals against the probe derived from hrpO(hrcV) of R. solanacearum. They include B. andropogonis, B. caryophylli, B. gladioli, B. glumae, A. avenae and A. konjaci. B. cepacia gave uncertain signals. Clones containing deduced hrp genes were screened from genomic libraries of B. glumae and A. avenae. They were sequenced and shown to possess the ORF corresponding to hrcV. Their deduced amino-acid sequences retain a membrane-spanning motif. The nucleotide sequence of the hrcV gene of B. glumae has 69 and 62% homologies against hrpO of R. solanacearum and hrpC2 of X. campestris pv. vesicatoria, respectively. A. avenae has less similarity to both. A marker-exchange mutant of hrcV negative B. glumae was constructed. This mutant lost the ability to incite HR on tobacco plants. The inability was complemented by introducing an intact cosmid clone of hrcV of B. glumae into the mutant. A clone of hrcV of A. avenae also complemented HR induction, however pVir2, the clone from R. solanacearum, did not. The marker-exchange mutant retained the ability to induce seedling rot of rice, which is one of the major symptoms in bacterial glume rot disease caused by B. glumae, probably because the mutant keeps producing toxins. The ability of B. glumae to cause rotting of onion was reduced by marker-exchange mutation. These results suggest that the deduced hrcV gene of B. glumae was functionally active in its pathogenesis.

Pantoea agglomerans pv. millettiae (=E. herbicola pv. millettiae) was also shown to have hrp-homologous genes on its plasmid. They were sequenced and revealed to correspond to hrcQ, R, S and T of E. herbicola pv. gypsophilae and E. amylovora. The distribution of those genes was further investigated. Many, but not all, pathogenic isolates of pv. millettiae and a few saprophytic Pantoea strains had hrp-homologous genes.

We have previously demonstrated that a strain of Pseudomonas fluorescens which is non-pathogenic and a biological control agent shows homology to hrp genes [1]. We cloned and sequenced the hrp-homologous genes, which were identified as hrcC and hrpL genes, and others.

The above results show that hrp/hrc genes are widely distributed among plant pathogenic bacteria, but are not considered useful as determinative markers for pathogenic ability.

Reference
1. Mulya K, Takikawa Y, Tsuyumu S, 1996. Annals of the Phytopathological Society of Japan 62, 355-359.