RNAi for crop

1. AMERICAN JOURNAL OF BOTANY 102(9): 1399–1400, 2015; http://www.amjbot.org/ © 2015 Botanical Society of America • 1399
A M E R I C A N J O U R N A L O F B O TA N Y
NEWS & VIEWS
It is widely understood that our current methods of food produc-
tion will soon be insufficient to meet the demands of a growing
global population. While we increase crop yield, we must also achieve
stability in food production and ensure agriculture makes minimal
impact on fragile ecosystems. Genetic engineering is a powerful
tool to generate crops with greater yield, lower impacts, and toler-
ance to biotic and abiotic stresses, yet there is resistance to genetically
modified organisms among consumers in the developed world. In
this essay, we outline creative approaches that use mobile RNA si-
lencing for crop improvement and bypass the creation of geneti-
cally modified foodstuffs.
RNA silencing is a conserved eukaryotic mechanism for gene
regulation and viral defense that uses small RNAs to suppress
complementary messenger or viral RNAs. RNA silencing can also
repress gene transcription by inducing epigenetic modifications
such as DNA methylation or histone modification at DNA se-
quences complementary to small RNAs. Because these epigenetic
modifications are maintained through mitosis and meiosis in the
absence of the initiating small RNA signal, RNA silencing can
trigger heritable changes in gene expression. Another intriguing
aspect of RNA silencing is that locally produced small RNAs in-
duce gene silencing in distant tissues. Agrobacterium tumefaciens
can be engineered to cause production of small RNAs from genes
on its T-DNA. These small RNAs degrade homologous host mRNAs
in both infected and noninfected leaves of Nicotiana benthamiana
(Voinnet and Baulcombe, 1997). Similarly, small RNAs can travel
long distance through a graft between silenced and nonsilenced
plants (Palauqui et al., 1997; Dunoyer et al., 2010; Molnar et al.,
2010). Mobile small RNAs also induce epigenetic modification
and transcriptional silencing (Melnyk et al., 2011). The mobile
nature of small RNAs and the potential for heritable gene repres-
sion make RNA silencing an attractive tool for crop improvement
that could bypass the need to insert transgenes that may be ex-
pressed in edible tissues.
Grafting, a technique whereby scions and rootstocks with differ-
ent genomes are joined, has been used in horticulture for thousands
of years, and is commonly employed for crops like tomatoes, cu-
curbits, and fruit trees (Melnyk and Meyerowitz, 2015). One com-
pelling strategy would be to use transgenic rootstocks expressing
small RNAs to trigger RNA silencing in nontransgenic scions. Re-
cently, this approach was used to transmit virus resistance in N.
benthamiana (Md Ali et al., 2013). This strategy would most easily
be applied to improve plants that are widely cultivated using graft-
ing, such as fruit trees. Although transmission of RNA silencing
was not observed in nontransgenic scions in apple (Flachowsky
et al., 2012), a more recent study in cherry trees demonstrates that
transgene-derived small RNAs can indeed be transported into non-
transgenic scions (Zhao and Song, 2014). Some of this variation in
transmission might result from lower efficiency of silencing de-
pending on the nature of the targeted gene and the small RNAs
produced by the transgene. For example, silencing was only in-
duced in a subset of leaves when a metabolic enzyme was targeted
for graft-transmissible silencing in N. benthamiana, suggesting
weak transmission or inefficient silencing (Kasai et al., 2011). More
research is needed to identify the characteristics of gene targets that
allow for efficient silencing through grafts. Another option to improve
the efficiency of mobile silencing might be to use phloem-specific
promoters to produce small RNA in the rootstock because these
yield more potent silencing than general overexpression (Kasai
et al., 2011). Finally, because transmission of small RNAs is more
efficient from photosynthetic sources to sinks, graft-transmissible
silencing may be best applied to manipulate traits in crops such as
potato, in which the underground stem forms the edible part of the
plant.
A second approach to exploit mobile silencing to improve crops
is to transiently produce small RNAs in leaves. Infiltration of
N. benthamiana leaves with A. tumefaciens carrying a small RNA-
generating construct efficiently induces silencing in root tissue
even though the bacteria are restricted to the infiltrated leaves
(Bai et al., 2011). Similarly, infecting a leaf with viral genomes
1
Manuscript received 15 April 2015; revision accepted 18 June 2015.
School of Plant Sciences, The University of Arizona, Tucson, Arizona 85721 USA
2
Author for correspondence (e-mail: rmosher@email.arizona.edu)
doi:10.3732/ajb.1500173
ON THE NATURE OF THINGS: ESSAYS
New Ideas and Directions in Botany
Exploiting mobile RNA silencing for crop improvement1
Jochen Gohlke and Rebecca A. Mosher2
KEY WORDS crop improvement; epigenetics; genetic engineering; grafting; RNA silencing

2. ON THE NATURE OF THINGS: ESSAYS AMERICAN JOURNAL OF BOTANY
1400 • SEPTEMBER 2015, VOLUME 102 • GOHLKE AND MOSHER—EXPLOITING MOBILE RNA SILENCING TO IMPROVE CROPS
engineered to contain a host gene fragment induces RNA silencing
systemically when the RNA virus is processed into mobile small
RNAs (Lindbo et al., 1993). This approach, called virus-induced
gene silencing, has been adapted to a wide range of species, includ-
ing many crops. Just like graft-transmissible silencing, the efficiency
and specificity of transiently induced silencing depend on the na-
ture of the target gene and must be individually optimized. One
hurdle to the widespread use of infiltration or viral infection is the
labor associated with manual treatment of plants. Transient viral
transformation systems have been optimized for high-yield pro-
duction of complex proteins such as antibodies for human thera-
peutics (Giritch et al., 2006; Qiu et al., 2014); however, translating
similar processes to edible plants might only be feasible for the
most high value crops. Virus infection might be more practicable,
due to the viral replication process, which results in amplification
of the inducing signal. However, this approach raises additional
concerns, such as viral transmission to other plants and the evolu-
tion of new infectious viruses through recombination or mutation.
Despite these challenges, transient small RNA production may allow
the application of mobile silencing in crops where grafting is not
routinely used and could be applied to established genetic stock,
such as orchards and vineyards.
Although potentially useful to regulate gene expression without
creating transgenic crops, graft-transmissible and transiently in-
duced RNA silencing are labor-intensive approaches. A powerful
alternative is to trigger heritable RNA silencing before propaga-
tion. When small RNAs are complementary to upstream regulatory
sequences rather than coding sequences, they trigger epigenetic
modifications that are heritable in the absence of the trigger (Jones
et al., 2001). For some crops, vegetative propagation of silenced
tissues would be feasible (Bai et al., 2011), and seed companies
could use this approach to engineer silenced seeds from induced
stock plants (Kanazawa et al., 2011). However, while transgenes
are readily targeted to create a heritable, silenced state, epigenetic
silencing of endogenes is more difficult and requires additional
optimization (Kanazawa et al., 2011). There is also the question of
how efficiently meristematic or meiotically active tissues receive
small RNA signals from distant source tissue. In some cases, small
RNAs are excluded from the shoot apical meristem (Liang et al.,
2012), while small RNA movement into flowers was detected in
other cases (Zhang et al., 2014). Movement of small RNAs into
meristems and meiotically active cells is necessary for persistence
of silencing throughout the plant and its transmission to the next
generation.
As we grapple with a changing climate and a growing popula-
tion, we must explore every opportunity to produce more and better
food with fewer resources. Although not yet ready for widespread
application, graft-transmissible silencing, transiently induced silenc-
ing, and heritable epigenetic silencing are promising approaches
to improve crops. Critical questions that remain to be answered
include how specific sequences evade silencing, what proteins influ-
ence intra- and intercellular small RNA trafficking, especially into
reproductive tissue, and how to establish robust heritability of si-
lencing. Additional research in these areas may yield new methods
for generating transgene-free plants with favorable traits, poten-
tially transforming current approaches for plant breeding and crop
improvement.
ACKNOWLEDGEMENTS
J.G. is supported by a Deutsche Forschungsgemeinschaft Research
Fellowship. R.A.M is supported by National Science Foundation
Grant MCB 1243608.
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