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

ORFs on the noncoding strand, a prediction supported by the observation that ORFs
are often visible on the DNA strand opposite to predicted genes [39]. Alternatively,
they may represent read-through from a strong promoter further upstream of the
fusion. While the significance of cryptic fusions remains unclear, they do appear to
be commonly identified from IVET screens [7,17,39,40], and it is of interest to
understand their significance.
Overall, IVET screens have proven to be powerful tools in identifying genes that
are expressed by SBW25 in response to the complex plant environment. This has
provided an insight into revealing the genetic bases underlying SBW25 s ecological
success. In particular, in-depth studies into several plant-induced loci encoding traits
in nutrition acquisition, stress response, physical attachment and potential eukaryotic
signaling systems have provided insight into some of the stresses and obstacles
that are encountered in the plant environment and the physiological responses of
SBW25 to these. While the biological significance of each of the loci investigated has
not always been apparent, investigations into their contributions to the ecological
success of SBW25 have provided clues with regard to the necessity of these loci for
plant colonization.
6.3
Regulatory Networks Controlling Plant-Induced Genes
6.3 Regulatory Networks Controlling Plant-Induced Genesj119
Phenotypic acclimation is the reversible expression of one or more phenotypes in
response to an environmental stimulus. The expression of various phenotypes is the
result of cascades of interactions that are influenced by repressors and activators.
IVETeffectively identifies genes showing elevated levels of expression in a particular
environment, such as the plant, and most of these niche-specific genes will be
involved in bacterial acclimation to the environment to optimize fitness. Genes that
are most likely identified by IVET include structural genes and activators, but not
repressors of plant-induced genes, as these would be active in vitro but downregulated
in vivo. Although IVET systems themselves are inadequate in identifying
repressor genes because they are plant repressed, the auxotrophic basis of the IVET
system is able to provide an efficient framework for identifying repressor genes.
To identify repressors of plant-induced genes in SBW25, a suppressor analysis of
SBW25DdapB strains carrying plant-inducible IVET fusions was undertaken in a
gene discovery method that is referred to as Suppressor-IVET or SPyVET [18]
(Figure 6.4). Two transposons, MiniTn5Km and IS-O-Km/hah, were used to mutagenize
IVET fusion strains. MiniTn5Km was used purely to identify repressors,
but IS-O-Km/hah was used to identify both repressors and activators by virtue of an
outward facing npt promoter located at one end of the transposon [41] (Giddens,
unpublished). Mutants were plated at high density (>10 5 cfu cm 2 ) on minimal
medium containing kanamycin to select for the transposon, but lacking the essential
growth factors DAP and lysine. The strict auxotrophy associated with the dapB
deletion prevented the majority of mutants from growing, thus allowing a large
number of mutants to be efficiently screened. Mutants that contained a transposon