Recruiting Genetic Engineering and Gene Silencing Technology to ...

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ISB
NEWS REPORT
FEBRUARY 2010
Recruiting Genetic Engineering and Gene Silencing

Technology to Control Parasitic Weeds

Radi Aly
The parasitic weed broomrape
Parasitic weeds (over 4000 species) represent one of the most destructive and intractable problems to agricultural production,
causing heavy damage to numerous crops, reducing both crop yield and quality. The
Orobanche
species (broomrapes)
1

are obligate root parasites of important dicotyledonous crops in semiarid regions of the world. Unlike other weeds, these
parasitic weeds are directly connected through haustoria to the vascular system of crop plants that serve as their hosts, and
the parasite becomes a major sink for crop photosynthate, debilitating crop growth and yield up to a total loss. Each mature
Orobanche
plant produces tens of thousands of seeds, which can remain viable in soil for many years. Seeds germinate only
after receiving a chemical stimulus from a neighboring host root.
Broomrape control
The nature of parasitic weeds makes control extremely difficult, costly, and hazardous to the environment. Parasitic weeds
are difficult to control by conventional means due to their lifestyle: parasites are intimately involved with the host and have
so much metabolic overlap with the host that differential treatments are very difficult to develop. In some cases the parasites
are closely associated to the host root, concealed underground and undiagnosed until they irreversibly damage the crop.
Although, the simplest and most effective approach to parasitic weeds control—host resistance—remains an unrealized
goal for agriculture, a wide variety of parasitic weed control methods (chemical, biological, cultural, and resistant crops)
has been tried. Unfortunately, most are only partially effective and have significant limitations (labor intensive and time
consuming).
The lack of new sources for resistance limits the ability to cope with newly developing, more virulent, broomrape races.
Furthermore, the transfer of available resistances from a resistant crop (like sunflower) to other crops is currently impossible.
In fact, for a few years the best control method has been fumigation with methyl bromide, which affects the soil seed bank.
This method, however, is expensive, laborious, and it is now being phased out, because it is extremely hazardous to the
environment.
A novel strategy
2
has been designed to enhance host resistance to
Orobanche
based on parasite-induced expression of
a selective
sarcotoxin IA
polypeptide. Our group has generated genetically engineered tobacco plants expressing a cecropin
peptide (
sarcotoxin IA
) under the inducible control of the
HMG2
promoter. While having no obvious effect on the host
plant growth and development, transgenic plants show enhanced resistance to the parasitic weed
Orobanche
, with reduced
Orobanche
biomass and increased host biomass, compared to non-transgenic controls.
Gene silencing in plants

RNA silencing is one of the most important biological discoveries of the last decade. Double-stranded RNA (dsRNA) is a
powerful tool for suppressing gene expression
3
through a process known as RNA interference (RNAi) in animals, and post-
transcriptional gene silencing (PTGS) in plants.
4
dsRNA can be produced
in vivo
by host or virus-encoded RNA-dependent
RNA polymerases, and by transcription, either through promoters or through inverted repeats and RNA synthesis of sense
or antisense orientation. In all these cases, the main element in the silencing process is a small RNA molecule, called a short
interfering RNA (siRNA). siRNAs are short double-stranded RNA molecules that facilitate potent and sequence-specific
gene suppression by degradation of mRNA sequences to which they are homologous, thereby silencing the target gene.
Gene silencing provides plants with defenses against various pathogens, such as nematodes
5
and viruses, and is a tool
of immense importance for research on plant development. Silencing spreads mainly in the direction from carbon source to
sink, that is, from tissues that export the sugar products of photosynthesis, to tissues that import these products. Intercellular
transmittance of signals, including RNA, is usually possible through plasmodesmata, while the pathway for long-distance
translocation is the vascular system, specifically the phloem, which provides symplastic continuity between plant organs.
Another silencing tool recently recruited for characterizing the function of plant genes is virus-induced gene silencing
(VIGS). It involves cloning a short sequence of a targeted plant gene into a viral vector, which is then used for infecting plant
tissue for degradation of specific plant mRNA. Although VIGS occurs rapidly and avoids the use of plant transformation
for gene silencing by sense or antisense approaches, it does have disadvantages: (a) VIGS rarely produces a complete

ISB
NEWS REPORT
FEBRUARY 2010
suppression of expression of a target gene, so that its decreased transcript level could still be sufficient to produce enough
functional protein; and (b) the inoculation of a plant with a virus can alter plant development.
The convenience of siRNA production and its ease of use have made siRNA an important tool for the study of gene function.
siRNAs can be synthesized
in vitro
and then introduced into cells to directly suppress target genes. However, one drawback of
using synthetic siRNAs is their inability to produce stable gene knockdowns. To address this issue, siRNA sequences can be
encoded in DNA expression vectors, which induce expression in the form of short, fold-back, hairpin loop structures, known
as short hairpin RNAs. An increase in the frequency of post-transcriptional gene silencing is obtained by transformation with
constructs that include the inverted repeat of the gene of interest with an intron located between the inverted repeat elements.
Development of new effective strategies for resistance to parasitic weeds requires either the identification of 1) genes
whose products selectively inhibit parasite growth or 2) a target key-gene of the parasitic weed for silencing. Since the
silencing-signal is capable of movement in plants, even across grafts, and since the connection between
Orobanche
and its
hosts includes direct continuity of the conductive tissues with sieve pores between adjacent sieve elements of host and parasite,
we hypothesize that a silencing-signal would also move from host to parasite. siRNA expressed in lettuce roots was recently
reported to silence a homologous GUS gene in the parasite
Triphysaria
.
6

Parasitic weeds such as
Orobanche
accumulate high amounts of mannitol during their development. The regulation of
mannitol in
Orobanche
by Mannose 6-Phosphate Reductase (M6PR) is a process essential for water and nutrient uptake from
its host. In our study, we attempted to control
Orobanche
by engineering a potential host (tomato) to produce a systemic
signal to effect the silencing of this critical metabolic activity in the parasite (
Fig. 1
). To do this we expressed a gene construct
containing a specific fragment from
O. aegyptiaca

M6PR mRNA in the host.
7
We used the inverted
repeat technique for gene silencing of M6PR, a
key-gene in
Orobanche
spp., in order to provide the
host plant with resistance against the parasite.
Our results showed that the endogenous M6PR
mRNA from
O. aegyptiaca
tubercles or shoots
grown on transgenic tomato plants harboring the
M6PR silencing construct was reduced by 60 –
80%. Concomitant with M6PR mRNA suppression,
the number of
Orobanche
dead tubercles was
significantly increased in the transgenic lines (
Fig.
2
), and a significant decrease in mannitol level was
measured in the parasite tissue grown on transgenic
plants compared to the control plants.
7

Conclusions and future perspectives
Optimal parasitic weed control could be achieved
either by the use of parasite-resistance crops (from
conventional breeding), or by crops genetically
engineered for resistance. So far only a few crop
varieties with stable resistance have been developed
after decades of conventional plant breeding, and
the genetic resources for resistance genes are rare.
Because genetic resistance based on silencing
a key-gene in the parasite is now feasible,
alternative biotechnological approaches using
genetic engineering could be ideal for parasitic
weed control.
Unlike other resistances, the gene silencing
approach could be applied to various other
susceptible crops not necessarily of the same
plant family. Also, this method could be effective
against a broad spectrum of broomrape species

ISB
NEWS REPORT
FEBRUARY 2010
and have implications for the control of other plant parasites and pathogens. Accordingly, the advantages of the
silencing technology will be to reduce labor, lower expenses, increase cropping choices, and eliminate the need
for chemicals that may be harmful to the environment.
Recently great progress has been made on researching the genomic and molecular genetics of parasitic
weeds. The ongoing Parasitic Plant Genome Project (PPGP) research should facilitate the identification of key
parasite genes to target via silencing technology. In the future, multiple silencing constructs can be pyramided
in susceptible hosts. This new biotechnological approach may also be combined with genetic or transgenic
resistances, significantly reducing the risk of developing virulent broomrape populations and thus reducing
broomrape damage, which would produce a significant gain in yields to the benefit of the farmers and industries
involved.
References
Parker C and Riches CR (1993)
Parasitic Weeds of the World: Biology and Control
. CAB International, Wallingford-UK
Hamamouch N, Westwood JH, BannerI, Cramer CL, Gepstein S and Aly R (2005) A peptide from insects protects transgenic tobacco from a parasitic weed.
Transgen. Res.
14, 227-236
Mansoor S, Amin I, Hussain M, Zafar Y and Briddon RW (2006) Engineering novel traits in plants through RNA interference.
Trends Plant Sci.
11, 559-565
Voinnet O (2008) Post-transcriptional RNA silencing in plant-microbe interactions: a touch of robustness and versatility.
Curr. Opin. Plant Biol.
11, 464-470
Atkinson HJ, Urwin PE and McPherson MJ (2003) Engineering plants for nematode resistance.
Annu. Rev. Phytopathol.
41, 615-639
Tomilov AA., Tomilova NB, Wroblewski T, Michelmore R and Yoder JI. (2008) Trans-specific gene silencing between host and parasitic plants.
Plant J.
56,
389-397
Aly R, Cholakh H, Joel DM, Leibman D, Steinitz B, Zelcer A, Naglis A, Yarden O and Gal-On A. (2009). Gene silencing of mannose 6-phosphate reductase in the
parasitic weed
Orobanche aegyptiaca
through the production of homologous dsRNA sequences in the host plant.
Plant Biotechnology Journal
7, 487-498
Dr. Radi Aly
Head, Department of Weed Science
Newe Yaar Research Center, Agricultural Research Organization (ARO)
B. O. Box 1021, Ramat yeshai 30095, Israel
radi@volcani.agri.gov.il
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