Page 13 - Volume 5, Issue 2, July 2012

© Benaki Phytopathological Institute
Phytobacterial type III secretion systems in the era of biotechnology 41
vesicatoria groups in isolations from pep-
per and tomato with the same primers as
Leite et al. (1994), while Park et al. (2010) de-
signed new primers that specifically ampli-
fied a 243-bp product from genomic DNA of
X. arboricola pv. pruni strains, and not from
the 21 other strains of Xanthomonas and two
epiphytic bacterial species. In addition, Zac-
cardelli et al. (2007) proposed a rapid de-
tection method for X. campestris pv. camp-
estris in crucifer seeds and plants, based on
amplification under stringent conditions of
an internal hrcC fragment of 519 bp from a
number of isolates and by means of patho-
gen-specific primers.
Recently, conventional PCR assays were
adapted to real-time PCR to develop more
sensitive and rapid techniques. Even if most
of these studies target gene sequences oth-
er than those involved in T3SS function, as
for example those coding for putative mem-
bers of the ATP-binding cassette (ABC) trans-
porter family in X. arboricola pv. pruni (Pala-
cio-Bielsa et al., 2011), there have been pilot
reports on real-time PCR diagnostic assays
based on hrp genes. Berg et al. (2006) for ex-
ample, used fluorescently labeled probes to
develop a multiplex PCR amplifying a 78-bp
segment of the hrpF gene from different X.
campestris pathovars and a 100-bp segment
of the Brassica spp. 18S–25S internal tran-
scribed spacer. The latter provided an inter-
nal control for the amplification process to
prevent false negatives. Nevertheless, sever-
al real-time PCR protocols have been pub-
lished in the last few years based on T3SS
gene sequences of mammalian pathogens.
Most of these PCR protocols target gene se-
quences such as hilA and ssaN, a T3SS tran-
scriptional regulator and a putative T3SS
ATP synthase gene of Salmonella enterica
serovars, respectively, of pathogens borne
in food and dairy products (McCabe et al.,
2011; Chen et al., 2010). Moreover, similar as-
says target genes coding for T3SS secreted
effectors such as tccP and tccP2 from patho-
genic Escherichia coli (Madic et al., 2011).
Considering the fact that both basic and ap-
plied research on bacterial secretion system
progress since the very beginning hand in
hand for plant and mammalian pathogens,
one should only expect that use of real time
PCR with sensitive and efficient probes (such
as the TaqMan technology), will also ex-
pand rapidly in the next few years for phy-
topathogen detection. This approach offers
the advantage that detection can be com-
pleted within a few hours with a sensitivity
of 102 CFU ml −1, thus surpassing the sensitiv-
ity of the existing conventional PCR.
The microarray technology offers a
promising tool for transcriptional profiling,
which has also been exploited for T3SS ex-
pression profiling and regulation (Ferreira
et al., 2006). Moreover, microarrays have re-
cently been exploited for microbial species
detection, community profiling on specific
niches and/or hosts. High density microar-
rays for example, based on gene sequenc-
es encoding mainly for the small ribosom-
al subunit (16S) rRNA, have been developed
for microbe/pathogen monitoring (Brodie et
al. , 2007). Such tools could complement the
currently available methods for pathogen
detection, which are time consuming, and
specific, but still lack economic feasibility.
The approval of DNA microarray technology
by governmental authorities (US Food and
Drug Administration) has paved the way for
new applications to clinical diagnostics and
pathogen detection in foods and crops.
Microarray applications for the detec-
tion of plant pathogens have already been
developed, mainly targeting a specific host
plant. Probe designing on most cases has
been based on the 16S rRNA gene region
with the complementation of pathoge-
nicity islands containing known virulence
genes (Aittamaa et al. 2008) or housekeep-
ing genes such as rpoB, groEL, ftsZ (Pellu-
dat et al., 2009) and gyrB (Kostic et al.,2007).
The diagnostic ability could extend from ge-
nus and species to pathovar level. However,
such diagnostic tools are still in their infan-
cy with plant pathogens as they have been
applied to pure culture lysates rather than
infected plants and cross hybridization be-
tween probes often occurs. The use of hrp
genes could address the latter problem, as
although they are conserved, they still in-