Biocenter Helsinki, Department of Biosciences, Division of General Microbiology, PO Box 56, FIN-0001 4 University of Helsinki, Finland

Background and objectives
Foliar plant pathogenic bacteria such as Pseudomonas syringae and Xanthomonas campestris are opportunistic pathogens that may colonize leaves epi- and endophytically without causing disease symptoms. Outbreak of disease occurs in suitable conditions, and is correlated to the leaf-associated population density. Bacteria in the non-symptom-generating growth phase express partially different sets of genes from those in the pathogenic phase. The micro-environment on the leaf surface differs significantly from that inside the leaf tissue. On the surface the bacteria are exposed to rapid and extensive variations in humidity, higher levels of UV radiation and presumably lower nutrient concentrations. In this growth phase the bacteria have to be able to sustain the killing effect of high-UV doses and drought, or alternatively the flushing, cell dislocating effect of running water. The objective of the studies in my laboratory is to map the behaviour of plant pathogenic and/or epiphytic bacteria before and during the onset of plant disease.

Results and conclusions
Even on the surface of dry plant leaves where the bacterial cell numbers are stable or decrease, P. syringae cells are metabolically active and, for example, exchange DNA by conjugation very efficiently. In leaf surface conditions where no population growth occurs, the metabolically active bacterial cells become very short. In the epiphytic growth phase a role for type IV pili has been postulated in the case of both P. syringae and X. campestris , in high-radiation (dry) conditions and/or in wet conditions. The type IV pili mediated bacterial aggregate formation, i.e. attachment of bacteria to each other, increases their UV tolerance. In addition to an effect of bacteria shielding each other, also the local concentration of extracellular polysaccharide and UV-absorbing pigments is likely to be higher in a cluster or micro-colony. On the other hand, in conditions mimicking rainy and wet conditions, the type IV pili of certain P. syringae and X. campestris pv. hyacinthi strains promote leaf surface attachment and enable the bacterial cells to resist the flushing effect of running water. Whether this effect of the pilus is a fitness determinant more generally among epiphytic colonizers remains to be seen.

In certain in vitro conditions, and apparently at the onset of the pathogenic phase, the expression of the hrp gene cluster is induced. At this point also the hrp pilus is produced. In P. syringae pv. tomato DC3000 a functional hrpa -gene, encoding the major structural component of the hrp pilus, is required for pathogenicity, and therefore it is likely that the pilus itself plays an important role in disease development.

The HrpA protein is found in the extracellular medium as four different-sized proteins. In each case a successively larger amino-terminal fragment has been removed, apparently by specific protease cleavage. Each of these size-class proteins is able to re-assemble into filamentous structures. The biological significance of the HrpA-cleavage is under investigation. Also, in X. campestris pv. campestris hrp pili are produced. Isolated pili attach efficiently to tobacco cells in liquid culture. Whether it is the major pilin or a putative minor subunit that is the active adhesin is not presently known. Although the hrp cluster forms a type III protein-secretion pathway with homology to the pathogenicity related gene clusters in animal pathogens such as Yersinia, mechanistically and morphologically the hrp pilus resembles more closely the Agrobacterium (type IV protein secretion pathway) pilus involved in transfer of T-DNA into plant cells. This resemblance may reflect the need for a plant cell wall-traversing structure, which is met by the same structural solution by each of the plant pathogenic bacteria, despite the differences in specific function of the pili.

1. Roine E, Wei W, Yuan J et al., 1997. Proceedings of the National Academy of Sciences, USA 94, 3459-3464.
2. Roine E, Saarinen J, Kalkkinen N, Romantschuk M, 1997. FEBS Letters 417, 168-172.