Agriculture Reference
In-Depth Information
native gene promoters or by rearrangements in
the integrating DNA that result in reversal of
the coding sequence or promoter orientations
(Fig. 18.2). In effect, the sense construct is con-
verted into an antisense construct. Overexpres-
sion sense constructs can also suppress
homologous native genes at the DNA level in
some transgenic events, precluding their tran-
scription, but the molecular mechanisms under-
lying these interactions are not understood
(Matzke and Matzke 1995).
In recent years it has become clear that in
eukaryotic cells, dsRNAs have much wider
impacts than prevention of translation. It is now
known that dsRNAs are perceived by plant cells
as viral replicons, triggering a multicomponent
response called RNA interference (RNAi), which
results in the degradation of the dsRNAs as well
as all closely related single-stranded mRNAs
(Ossowski et al., 2008). The dsRNAs are cut into
small fragments by the dicer RNAse, and these
fragments are incorporated as targeting guides
into complexes that degrade all homologous
RNAs. As understanding of this phenomenon has
grown from studies in model systems such as
tobacco (
Nicotiana tabacum
L.), it has become
clear that more effective than antisense transcripts
for triggering the RNAi response are hairpin
RNAs. These single RNA molecules include
complementary sequences in reverse orientation
so that they can fold back on themselves to form
regions of dsRNA (Fig. 18.2). Such constructions
reliably suppress the expression of related
sequences at the RNA level. For a detailed review
of the application of RNAi in wheat functional
genomics, see Fu et al. (2007).
RNA interference is a powerful tool for func-
tional genomics in wheat, because the suppression
effects of the interfering construct are dominant,
making it reliable for generating losses of gene
function even though the polyploid genome con-
tains multiple alleles of each gene. Moreover,
experimenters can target either individual genes
or entire gene families, simply by including
unique or shared gene sequences, respectively, in
the region of the construct that becomes double-
stranded. Travella et al. (2006) used RNAi to
obtain phenocopies in hexaploid wheat of mutants
that had been previously characterized in diploid
model plants. Their RNAi construct for the phy-
toene desaturase gene down-regulated all three
homoeologous genome copies, resulting in seed-
lings with photobleached leaves. Introduction of
an RNAi construct for the
EIN2
gene, which
encodes an ethylene signal transduction factor,
resulted in wheat insensitive to ethylene. In both
cases, different transgenic lines showed different
levels of reductions of the targeted transcripts,
which was refl ected in the range of severity of the
phenotypes in the different transformed lines.
The RNAi-induced phenotypes were stable for at
least two selfi ng generations.
Overexpression, antisense RNA, RNAi, and
complementation constructs have all been suc-
cessfully employed to confi rm the identity of
wheat gene sequences isolated either by map-
based cloning or by homology to genes with
proven functions in model plant systems with
sequenced genomes such as Arabidopsis (
Arabi-
dopsis thaliana
), rice, or Brachypodium
(Brachy-
podium distachyon
L.) (www.brachypodium.org).
An interesting wheat gene whose identity and
pleiotropic effects were confi rmed by genetic
transformation is the
Q
gene, a key gene in the
domestication of wheat that had been linked to
the square spike and free-threshing (fragile glume
combined with tough rachis) characteristics, with
minor pleiotropic effects on glume shape and
tenacity, rachis fragility, spike length, plant
height, and spike-emergence time. Simons et al.
(2006) used map-based cloning to isolate a candi-
date sequence for the Q gene that encoded a
transcription factor. To verify its identity, they
transformed wheat with an overexpression con-
struct and obtained some plants with increased
expression and some showing cosuppression. The
range of phenotypes of the transgenic plants was
correlated with expression levels of the candidate
transgene, confi rming that a single gene controls
both spike compactness and the free-threshing
character, the major traits that had been attrib-
uted to the
Q
locus.
To understand the molecular basis for leaf rust
resistance, candidate sequences for the
Lr1
,
Lr10
,
