Role of the Hrp type III protein secretion system in growth of Pseudomonas syringae pv. syringae B728a on host plants in the field

Abstract

hrp genes are reportedly required for pathogenicity in Pseudomonas syringae pv. syringae (Pss) and other phytopathogenic bacterial species. A subset of these genes encodes a type III secretion system through which virulence factors are thought to be delivered to plant cells. In this study, we sought to better understand the role that hrp genes play in interactions of Pss with its host as they occur naturally under field conditions. Population sizes of hrp mutants with defects in genes that encode components of the Hrp secretion system (DeltahrcC::nptII and hrpJ:: OmegaSpc) and a protein secreted via the system (DeltahrpZ::nptII) were similar to B728a on germinating seeds. However, phyllosphere (i.e., leaf) population sizes of the hrcC and hrpJ secretion mutants, but not the hrpZ mutant, were significantly reduced relative to B728a. Thus, the Hrp type III secretion system, but not HrpZ, plays an important role in enabling Pss to flourish in the phyllosphere, but not the spermosphere. The hrcC and hrpJ mutants caused brown spot lesions on primary leaves at a low frequency when they were inoculated onto seeds at the time of planting. Pathogenic reactions also were found when the hrp secretion mutants were co-infiltrated into bean leaves with a non-lesion-forming gacS mutant of B728a. In both cases, the occurrence of disease was associated with elevated population sizes of the hrp secretion mutants. The role of the Hrp type III secretion system in pathogenicity appears to be largely mediated by its requirement for growth of Pss in the phyllosphere. Without growth, disease does not occur.

Figure 1

Population sizes of Pss B728a hrp mutants (ΔhrpZ∷nptII, hrpJ∷ΩSpc^r, ΔhrcC∷nptII) in association with field-grown bean plants. The bacterial strains were inoculated onto seeds immediately before planting. Samples were seeds or germinating seedlings collected between 0 and 3 DAP (A) and 0 and 7 DAP (B); cotyledons and primary leaves were collected at 7 (A) and 9 DAP (B); primary leaves were collected between 9 and 14 (A) and 12 and 20 DAP (B); and single leaflet from trifoliolate leaves were collected at all other times. Each datum point represents the mean log cfu/sample and SE based on three (A) or four (B) replicate plots with six or eight individual samples per plot. Inverted solid triangles indicate rain events with sustained rates >1 mm/min.

Figure 2

Distribution of population sizes of B728aCm and the hrp mutants in association with individual asymptomatic primary leaves collected at 12 DAP in 1997. Mean bacterial population sizes for the sets of individual leaves are shown in Fig. 1B.

Figure 3

Brown spot lesions caused by the B728a ΔhrcC∷nptII mutant under field conditions. Lesions were detected 14 DAP in 1997.

Figure 4

Rescued growth of the B728a hrpJ∷ΩSpc mutant by a non-lesion-forming B728a gacS1∷Tn5 mutant. The mutants were infiltrated into primary leaves of growth chamber-grown bean plants either alone or in a 1:1 mixture. Approximately 3 × 10³ cfu were deposited initially in each of the 6-mm-diameter disks removed for sampling. Samples inoculated with the mixture were dilution-plated onto both KBRSpc and KBRKan to enumerate population sizes of the hrpJ and gacS mutants, respectively. Each datum point represents the mean log cfu/sample and SE based on dilution plating of five individual samples.

Figure 5

Leaf infiltration assay for pathogenicity. Necrosis indicative of brown spot disease developed at sites infiltrated with either B728a or a 1:1 mixture of the hrpJ and gacS mutants. Neither mutant caused the pathogenic reaction when inoculated alone. The dose was ≈10³ cfu for B728a and the mutants when inoculated alone. In the co-inoculation treatment, the dose for each mutant was ≈5 × 10² cfu.