Genome Organization and Comparative Genomics - Wheat Science and Trade - page 346

Virus-induced gene silencing was then used
to assess the functional identity of the Lr21 gene
candidate. A 174-bp fragment from the 3′ UTR
of the Lr21 candidate was cloned into the BSMV
genome. Inoculation of a wheat line carrying the
Lr21 resistance gene fi rst with the BSMV: Lr21
construct and then with a leaf rust isolate that is
avirulent to Lr21 led to virulent interactions in
some areas of the leaf. Similar results were obtained
with three other genes, RAR1 , SGT1 , and cyto-
solic HSP90 , which have been shown to function
in many of the NBS-LRR resistance pathways
(Scofi eld et al., 2005). The products of all four
genes are thus required for Lr21 - mediated resis-
tance. This study demonstrated the use of VIGS
for functional analysis of genes that are expressed
in the leaves of seedlings. Since then, VIGS pro-
tocols have also been developed for silencing genes
in the spikes of wheat, making VIGS a tool that
can be employed for the rapid screening of gene
candidates (S. Scofi eld, pers. comm.).
Research is also being conducted to facilitate
wheat transformation technologies. Wheat trans-
formation using biolistics is robust and can reach
effi ciencies of over 60% with select genotypes
(Pellegrineschi et al., 2002). The drawback of
biolistic DNA delivery is that it often results
in complex integration patterns. This has led to
the development of Agrobacterium -mediated gene
transfer, which generally results in the insertion
of single or few copies of the transgene in a
cost-effi cient manner. Transformation effi cien-
cies obtained in wheat with Agrobacterium are
generally lower than the best published values for
biolistic transfers, but are likely to increase as
protocols are further developed and optimized
(Jones 2005). The main drawback of both systems
is that fertile plants need to be regenerated from
transformed somatic cells via somatic embryogen-
esis, a process that is time-consuming, is highly
genotype-dependent, and can create somaclonal
variants. These disadvantages can be overcome
by using an in planta transformation system,
modeled on the “dipping” protocol that exists in
Arabidopsis (Bechtold et al., 1993; Clough and
Bent 1998). Dipping wheat infl orescences into
Agrobacterium infi ltration medium has yielded a
small number of successful transformation events

Reverse genetics, whereby the gene is the start-
ing point and elucidation of its function is the aim,
is in its infancy. Technologies and resources that
have been developed in wheat to aid this endeavor
are targeted induced local lesions in genomes
(TILLING) populations and VIGS. In TILLING,
chemical mutagenesis is used to create a popula-
tion of mutants. Primers, made against the gene
that is the target of the functional analysis, are
used to screen the DNA of the M2 mutant popula-
tion using a pooled approach (McCallum et al.,
2000). Key to the success of TILLING is the high
density of mutations that can be achieved. This
density is much higher in polyploids compared
with diploids because most genes are redundant
and the organism is thus buffered against muta-
tions that would be lethal in a diploid. In tetraploid
and hexaploid wheat, respectively, mutation rates
of 1 per 40 bp and 24 bp have been obtained by
Slade et al. (2005) and of 1 per 45 and 60 kb by
Dubcovsky and colleagues (J. Dubcovsky, pers.
comm.). Considering the high mutation rate, it is
feasible to fi nd mutations in the orthologous genes
on the A, B, and D genomes. These genes then
have to be combined into a single plant to assess
the phenotype. This has been successfully dem-
onstrated in the combination of EMS mutations
in waxy alleles, which has led to the creation of a
waxy wheat in which the synthesis of amylose was
greatly reduced (Slade et al., 2005).
A second reverse-genetics tool available in
wheat is VIGS using Barley stripe mosaic virus
(BSMV) (Scofi eld et al., 2005), which is a positive-
sense single-strand RNA virus. Double-stranded
RNA, which occurs as an intermediate in viral
replication, triggers sequence-specifi c degrada-
tion. When transcribed fragments corresponding
to a wheat gene are cloned into the viral genome,
the mRNA produced by the endogenous target
gene will be degraded, leading to gene silencing.
The potential of VIGS in wheat was fi rst demon-
strated by silencing phytoene desaturase (PDS)
expression. Suppression of PDS, an enzyme in the
carotenoid pigment biosynthetic pathway, results
in photolysis of chlorophyll in the affected tissues.
Photobleaching was observed 10 days after infec-
tion with the BSMV:PDS construct (Scofi eld
et al., 2005; Color Plate 31).