1.7.4S
ROLE(S) OF PATHOGENICITY-ASSOCIATED GENES IN INTERACTIONS OF POPULATIONS OF PSEUDOMONAS SYRINGAE PV. SYRINGAE B728A WITH SNAP BEAN (PHASEOLUS VULGARIS) PLANTS IN THE FIELD

SS HIRANO1, AO CHARKOWSKI2, A COLLMER2, DK WILLIS1,3 and CD UPPER1,3

1Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wl 53706, USA; 2Department of Plant Pathology, Cornell University, lthaca, NY 14853, USA; 3US Department of Agriculture, Agricultural Research Service, Plant Disease Resistance Research Unit, University of Wisconsin-Madison, Madison, Wl 53706, USA

Background and objectives
A number of genes required for pathogenicity have been identified in Pseudomonas syringae pv. syringae (Pss). Among these are genes in the hrp and lemA/gacA regulons. Understanding the role of these genes is based largely on laboratory assays which have demonstrated, for example, that defects in hrp genes result in an inability of Pss to cause disease and inability to grow in planta. Such assays, however, circumvent interactions that occur between bacteria and plants in the field. We have used Pss-snap bean-bacterial brown spot disease as a model system to examine pathogen-host interactions at the population level in the field.

Materials and methods
Field behaviour was examined of three hrp mutants (deltahrpZ::nptll, deltahrcC::nptll, hrpJ::omegaIspc operon), all derived from the highly virulent Pss strain B728a. hrpZ encodes for a protein (harpinPss) that is secreted via the Hrp type-III secretory system. hrcC is thought to encode for an outer membrane component of the Hrp secretory system. The third mutant (i.e. hrpJo) carries a polar mutation in hrpJ. In laboratory assays, the hrpJo and hrcC mutants have phenotypes typical of 'hrp' mutants - they are both HR and path minus. The strains were inoculated onto bean seeds at the time of planting in the field. Population sizes of the strains were estimated by dilution plating of samples collected at frequent intervals following planting.

Results and conclusions
No significant differences were found in the population sizes of the deltahrpZ mutant relative to B728a on emerging seedlings and leaf samples. The deltahrpZ mutant was isolated from brown spot lesions as was the wild-type. Hence, hrpZ in Pss B728a appears neither to be required for growth/survival nor for disease causation. Growth of the hrpJo and hrcC mutants was similar to that of the wild-type on germinating bean seeds. At 12 days after planting (DAP) relatively large numbers (>105 to 106 of both mutants were found on some of the developing primary leaves. At subsequent sampling times, leaf-associated population sizes of the mutants diminished, even during periods when numbers of B728a increased. Hence, mutations in hrp genes that affect the Hrp secretion system substantially affect growth and survival of Pss in association with bean leaves but not with seedlings before emergence.

Population sizes greater than roughly 105 to 106 c.f.u./leaf are sufficiently large to be predictive of disease for strains capable of causing brown spot disease. By 14 DAP, lesions were detected in plots inoculated with the hrpJo and hrcC mutants. The only occupants of the lesions examined from these plots were either the hrcC or hrpJo mutant. Isolates purified from these lesions were non-pathogenic when inoculated into growth-chamber bean plants - the expected reaction for hrp mutants. Thus, under some conditions, hrp mutants are able to grow and cause disease in the field; hrp genes are not absolutely required for lesion formation. Co-inoculation of the hrpJo mutant with B728a in the field and in growth-chamber experiments substantially increased growth of the mutant relative to the mutant inoculated alone. When the hrpJo mutant and a non-pathogenic B728a lemA mutant were co-infiltrated into bean leaves in the growth chamber, growth of the hrpJo mutant was similarly elevated and a pathogenic reaction was observed.

Findings from the field and growth-chamber experiments demonstrate that growth and survival of hrp mutants are substantially reduced, but when tricked into growing in association with plants, these mutants are able to cause disease. The data strongly suggest that hrp genes are fundamental plant-associated growth/survival-enabling genes but they are not absolutely required for lesion formation in Pss B728a.