Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

6.2 Identification of Plant-Induced Genes of SBW25 Using IVETj115
egf deletion; silent genes do not express egf and the number of these bacterial strains
decreases. Harvested bacteria were subsequently screened in vitro for constitutive
promoter activity by virtue of the lacZY reporter, thus enabling the identification of
gene fusions that are active in the plant environment and not in the culture. Positive
IVET fusion activities were confirmed by inoculating IVET strains (purified monocultures)
back onto the sugar beet seedlings and testing for colonization relative to
wild-type SBW25. The IVET fusion points were identified by recovering integrated
IVET constructs by conjugation into Escherichia coli [23] and sequencing across the
junction of the reporter gene to the genomic fragment insertion. The availability of the
SBW25 genome sequence (http://www.sanger.ac.uk/Projects/P_fluorescens/) has
greatly facilitated the mapping of insertion points, providing a detailed context of the
candidate plant-induced genes upstream of the reporter and insight into the promoters
driving their activities.
In the preliminary IVET screening for rhizosphere-induced SBW25 genes employing
the panB reporter, a panel of IVETclones representing 10% of the coverage
needed to comprehensively survey the SBW25 genome were screened, revealing the
specific upregulation of 20 loci in the rhizosphere environment [7]. These results
provided the first major insights into how SBW25 perceives and responds to this
environment, as identified rhizosphere-induced genes had expected roles in nutrient
acquisition, secretion, stress response and a number of other unidentified
(novel) traits.
One of the most intriguing findings of this screening was the expression of a gene
with 51% sequence identity to the plant pathogen P. syringae gene hrcC, which
encodes a putative pore-forming outer membrane component of a type III protein
secretion system (TTSS) [7]. Further analysis of this locus revealed a 20-kb cluster of
TTSS-related genes (designated the Rsp cluster), which is closely related to the TTSS
gene cluster of P. syringae, though SBW25 appears to lack a number of P. syringae
TTSS gene homologues including an EBP gene, harpin gene and parts of the hrpJ
cluster [6]. TTSSs are commonly associated with pathogenic bacteria and function in
the delivery of virulence/modulating proteins into host cells that may result in
parasitism or elicitation of host defense responses. Screening of other P. fluorescens
strains showed the gene cluster to be widely distributed in nonpathogens. Thus, the
discovery that SBW25 possesses TTSS gene homologues led to further investigations
into the functionality and ecological role of this locus in SBW25. While wildtype
SBW25 does not cause disease symptoms in plants or elicit host defense
responses in test host organisms, ectopic expression of a key sigma factor gene,
rspL, that directs the expression of rsp genes, resulted in the elicitation of a hypersensitive
response (HR) in Nicotiana clevelandii. Furthermore, expression of rspL in
combination with the heterologous P. syringae avirulence protein, AvrB, resulted in a
gene-for-gene specific HR reaction in Arabidopsis thaliana Col-0, albeit by using very
high inoculum loads [6]. Although the SBW25 TTSS genes appear to function in
protein secretion under contrived conditions, the ecological role and function of
these genes in their natural environment remains to be elucidated. Evidence from
plant colonization experiments with rsp mutants suggests that the rsp genes are
involved in the active colonization of root surfaces and also affect growth in vitro.