Page 11 - Volume 5, Issue 2, July 2012

© Benaki Phytopathological Institute
Phytobacterial type III secretion systems in the era of biotechnology 39
and activate the expression of plant genes
that aid in bacterial infection. They recog-
nize plant DNA sequences through a central
domain consisting of a variable number of
tandem, 33–35 amino acid repeats, followed
by a single truncated repeat of 20 amino ac-
ids. The native function of these proteins is
to directly modulate host gene expression.
Upon delivery into host cells via the bacteri-
al T3SS, TAL effectors enter the nucleus, bind
to effector-specific sequences in host gene
promoters and activate transcription (Boch
and Bonas, 2010; Voytas and Bogdanove,
2011). The DNA binding code of TAL effec-
tors is fairly simple: a hypervariable pair
of adjacent residues at positions 12 and 13
in each repeat, the ‘repeat-variable di-resi-
due’ (RVD), specifies the target, one RVD to
one nucleotide, with the four most common
RVDs each preferentially associating with
one of the four bases in the DNA target. In
naturally occurring TAL proteins the recog-
nition sites are uniformly preceded by a T
nucleotide that is required for TAL effector
activity (Voytas and Joung, 2009; Moscou
and Bogdanove, 2009; Boch et al., 2009).
These straightforward sequence rela-
tionships between the variable amino ac-
ids in TAL effector repeats and DNA bases
in their target sites both allow the predic-
tion of TALEs and their target sites and en-
able the redesigning of effectors to selec-
tively bind to DNA targets of choice. These
proteins are interesting to researchers both
for their role in disease of important crop
species and the relative ease of retargeting
them to bind at pre-chosen DNA sequences.
Similar proteins can be found in the patho-
gen Ralstonia solanacearum.
Genome sequencing generates oppor-
tunities to strategically manipulate selected
genes in DNA targeting for a wide range of
applications: understanding gene function
in model organisms, reprogramming the
regulation of selected loci in higher eukary-
otic genomes through novel transcription
factors, site directed mutagenesis and other
techniques. Potential applications are envi-
sioned in treating human genetic disorders,
improving traits in crop plants, genome en-
gineering and synthetic biology. At pres-
ent, a serious technical limitation of such
applications is the difficulty of altering nu-
cleotide sequences and expression of genes
in living cells in a targeted fashion. The TAL
effectors of plant pathogenic Xanthomonas
have provided researchers with a new tool
to meet this challenge. Fusions of these ef-
fectors to rare-cutting restriction endonu-
cleases (called TALENs, TALE nucleases), or
other programmable nucleases, to custom-
izable arrays of polymorphic amino acid re-
peats, direct the nuclease to particular DNA
sites which they subsequently cleave (Bog-
danove and Voytas, 2011; Morbitzer et al.,
2010). In a recent plant-related application
(Li et al. , 2012) a TAL effector of Xanthomonas
oryzae pv. oryzae (Xoo), which transcription-
ally activates a specific rice disease-suscep-
tibility ( S) gene, was fused to the DNA cleav-
age domain of FokI to create a TALEN able to
edit the particular S gene, altering the sus-
ceptibility response and thereby engineer-
ing resistance to bacterial blight.
Harpins
T3SS proteins of phytopathogenic bac-
teria, such as harpins, have been studied for
their use in crop protection. The first exam-
ple was the use of the harpin protein HrpN
from E. amylovora overproduced in a heter-
ologous bacterial system ( Escherichia coli)
(Wei et al., 1992). Harpins elicit a complex set
of metabolic responses in the treated plant,
which result in promotion of plant growth,
induction of defense responses against dif-
ferent types of pathogens and insects, and
tolerance to drought stress (reviewed in
He et al., 2004). Harpins or harpin-like pro-
teins such as HrpZ from P. syringae pv. syrin-
gae (Strobel et al., 1996), PopA from Ralsto-
nia solanacerum (Belbahri et al., 2001) also
elicit similar reactions on non-host plants.
Transgenic cotton lines expressing Harpin Xoo
from X. oryzae pv. oryzae accumulate harpin
along the plant cell wall where it likely
acts as an endogenous elicitor (Miao et al.,
2010), leading the plant to a primed state
which improves resistance against Verticilli-
um wilt. Similarly, transgenically expressed