H-G WIGGERICH, H KOSTER and A POHLER

Lehrstuhl for Genetik, Fakultit fur Biologie, Universitat Bielefeld, Postfach 1001 31, D-33501 Bielefeld, Germany

Background and objectives
Xanthomonas campestris pv. campestris is the causal agent of black rot disease of crucifers. After infection of a plant with X. c. pv. campestris, two different types of reaction can be observed depending on the susceptibility of the particular plant. If the plant is susceptible, the infection gives rise to blackening of leaf veins, followed by drying and death of the leaf tissue (compatible interaction). In contrast, a hypersensitive reaction (HR) is induced in a resistant plant or a non-host plant (incompatible interaction). The HR is a local defence response characterized by a rapid tissue necrosis in the inoculated area.

Iron is considered to play a crucial role in plant-phytopathogenic interactions. Iron is one of the factors that limits bacterial growth in planta because micromolar concentrations of iron are necessary to support growth and multiplication. The common strategy of bacteria to overcome this problem is the utilization of siderophores with high affinity to ferric iron. Fe(III) is complexed by these chelators which are transported into the cell via an energy-coupled process mediated by the TonB system. In this work, we studied X. c. pv. campestris mutants defective in iron uptake and analysed their ability to induce a hypersensitive response of the non-host plant pepper (Capsicum annuum).

Results and conclusions
The nucleotide sequence of a 3.6-kb HindIII-Smal DNA fragment of X. campestris pv. campestris revealed four open reading frames which, according to sequence homologies, were designated tonB, exbB, exbD1 and exbD2 [1]. Analysis of translational fusions to alkaline phosphatase and P-galactosidase confirmed that the TonB, ExbB, ExbD1 and ExbD2 proteins are anchored in the cytoplasmic membrane. The TonB protein of X. c. pv. campestris lacked the conserved (Glu-Pro)n and (Lys-Pro)m repeats, but harboured a 13-fold repeat of proline residues.

By mutational analysis the tonB, exbB and exbD1 genes were shown to be essential for ferric iron import in X. c. pv. campestris. In contrast, the exbD2 gene was not involved in the uptake of ferric iron. Mutational analysis also revealed that the tonB, exbB, exbD1 and exbD2 genes of X. c. pv. campestris are necessary for the induction of a hypersensitive response on the non-host plant pepper. Whereas the viable bacterial titre of a tonB, an exbB and an exbD1 mutant decreased after leaf infiltration of pepper, the exbD2 mutant multiplied in the pepper leaf tissue and grew even better than the wild-type strain. In further characterizations we found that all mutants mentioned did not produce the extracellular enzyme polygalacturonate lyase, whereas the activities of other extracelluar enzymes, such as amylase, protease and endoglucanase, remained unchanged. These findings indicated that the TonB system of X. c. pv. campestris is likely to be involved in the induction of polygalacturonate lyase production. Interestingly, an exbD2 mutant equipped with the constitutively expressed polygalacturonate lyase 1 gene of X. c. pv. campestris regained the ability to induce a hypersensitive response on pepper. Therefore we propose that polygalacturonate lyase plays an important role in the induction of a hypersensitive response in the interaction of X. c. pv. campestris and the non-host plant pepper.

References
1. Wiggerich H-G, Klauke B et al., 1997. Journal of Bacteriology 179, 7103-7110.