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1.1.RNAi studies
in Gossypium species

of allopolyploid cotton (Gossypium sp.)
is very complex, so poorly studied compared to other species. The germplasm
based cotton breeding gene pool is narrow due to following reasons: 1)
polyploidization from a common ancestor about 6–11 million years ago (Wendel, 1989); 2) a
“genetic bottleneck” occurred during domestication of cotton from a common
ancestor (Iqbal et al., 2001);
and 3) our dependence of crosses on closely-related elite domesticated
genotypes or due to the presence of limited choice for the selection of
cultivars from the existing list for high yield and superior fiber quality (Van Esbroeck et al., 1999).
Moreover, gains related to applications of marker-assisted selection (MAS) due
to existence of low level of molecular polymorphisms (Abdurakhmonov et al., 2012a). Several
genome duplications events before and after allopolyploidization was occurred (about
1.5-2 MYA) from A and D genome diploids, that caused to the gene multiplication
in the complexity of cultivated allotetraploid cotton genomes (Adams et al., 2003). This resulted with five distinct genomes (Adams et al., 2003; Chen et al.,
2007; Zhang et al., 2008), including two allotetraploid species of
cultivated cotton Gossypium hirsutum
and G. barbadense (Abdurakhmonov et al., 2012a).

Upland cotton is grown in more than 90% of world
cotton plantations (Campbell et al., 2010), with its high
yield, early maturity and other agronomic traits. In contrast, Pima cotton or
ELS cotton is grown in less than 5% of cotton growing area of the world (Liu et
al., 2015a) for its superior fiber properties. Pima cotton is
characterized with relatively lower yield and less desirable agronomic traits
such as requirement of more water and longer growing time as well as
susceptibility to various diseases. Breeders tried to transfer Pima’s fiber
quality into Upland cotton while keeping their early maturity, resistance, and
productivity of Upland genotypes (Abdurakhmonov et al., 2012b, 2014). Besides, simultaneous
improvement of all important agronomical traits in Upland cotton (G. hirsutum) is a very difficult task
using traditional crossing methods due to existence of negative correlations
between major fiber quality and yield or maturity traits (Abdurakhmonov et al., 2014).
Traditional breeding has limited successes over to solve this problem and also distorted
segregation that occurred in interspecific hybrid progenies from Upland and
Pima sexual crosses (Saha et al., 2012). Under
abovementioned complexities of cotton genome, this challenge might be more
seriuos, because higher rates of transgressive, differential and novel gene
expression patterns, and homoeologous gene silencing make it difficult to
improvement of the breeding (Yoo
et al., 2013).

World demand for
cotton products need to develop Upland cotton cultivars with high yield, early
maturity, and disease and pest resistance while producing longer and stronger
fibers in order to be competitive with synthetic fiber in the market. Scientists
must to discover novel genes which are resistance to salt, drought, and heat
stresses as well as pathogens and pests such as Verticillium/Fusarium fungi,
nematodes, and viruses (Hake,
2012; Abdurakhmonov, 2013). It requested to develop an ‘innovative new
generation crop technology’ from the cotton research community to solve this
fundamentally longstanding challenge in worldwide cotton improvement programs. Today
many innovative approaches are available for this reason. One of the sufficient
technologies is RNA intereference (RNAi) which helps to identify functions of
agricultural importance genes, and thus, solve the problems of the cotton
economy by creating “cotton varieties from biotechnology”
(“biotechnology cotton”) with the suppression of undesirable genes,
but improves the expression of desired features (s) of interest.

RNAi can be applied
for the functional studies of many agronomical, biological and physiologically important cotton genes
related to the cotton fiber development; early maturity and flowering;
increased yield potential; fertility and embryogenesis; viral, fungal, and
insect pest resistance; tolerance to various abiotic stresses; and cottonseed
and oil quality improvements.

Up to date in PubMed
( database have more than 100 refereed
journal publications explained RNA interference of Gossypium species. We used some of the research efforts, methods
and results in this study to give more information about RNAi technology.

2.1.1. RNAi methods in cotton

RNA interference, known as “co-suppression,”
“quelling,” and/or “post-transcriptional gene silencing (PTGS),” is an
evolutionarily conserved, double-stranded RNA-dependent, universal eukaryotic
process to silence the expression of genes in a sequence-specific manner (Napoli et al., 1990; Romano and
Macino, 1992; Hannon, 2002; Roberts et al., 2015). RNAi is induced by
exo- or endogenous (i.e., micro RNAs) double stranded RNA (dsRNA) molecules.
RNAi is part of the normal cellular function as well as an immune response
against foreign nucleic acid signatures from viral infections (Roberts et al., 2015). In
the cells, the dsRNAs are recognized by the Dicer family of enzymes and cleaved
into short double stranded fragments of 21–25 bp long siRNAs (Hannon, 2002; Roberts et al.,
2015). Further, siRNAs are separated into two single-stranded
‘passenger’ and ‘guide’ RNAs. The guide strand is incorporated into the
RNA-induced silencing complex (RISC), which triggers the recognition and
digestion of complementary RNA sequence signatures, whereas the passenger
strand gets degraded. RISC has enzymatic digestion activity, which consists of
the key components of argonaute (AGO) and P-element induced wimpy testis (PIWI)
proteins (Hannon, 2002;
Jakymiw et al., 2005). AGO/PIWI proteins are considered to be the
critical sites for RNAi and these proteins localize within the specific P-body
regions in the cytoplasm (Jakymiw
et al., 2005; Sen and Blau, 2005). RISC and downstream RNAi machinery
are common for exogenous and endogenous dsRNA recognition and target sequences
digestion; however, there are clear differences in distinctly processing and
handling of exogenous and endogenous dsRNAs (Hannon, 2002). The regulation of gene activity of
cells at the PTGS level is the main biological function of RNAi, which occurs
either by the inhibition of translation of mRNA or by direct degradation of the
mRNA (Hannon, 2002).
Moreover, RNAi pathway components (i.e., Dicer, siRNA, and RISC) may contribute
to maintenance of genome organization and structure through RNA-induced histone
modification, which may silence gene activity at the pre-transcriptional level
(Holmquist and Ashley,
2006). At the same time, small dsRNAs may also possibly up-regulate
expression of genes through binding into a promoter region and histone
demethylation (Li et al.,
2006). Usually, RNAi can be induced by the expression of antisense RNA,
dsRNA and by virus induced gene silencing (VIGS) constructs. A stable RNAi in
cotton is achieved by employing hairpin (HP) RNAi binary Agrobacterium vector
constructs, which produce dsRNAs that induce RNAi machinery. The majority of
cotton RNAi studies highlighted here utilized HP-mediated gene silencing. For
constructing HP vectors, usually 200–500 bp long fragments of gene of interest
are used. One limitation of these HP constructs is that they sometimes could
generate “off-target” gene silencing due to the generation of multiple variants
of short interfering RNA (siRNA) from inserted target gene fragments. This is
particularly challenging when there are several paralogous, orthologous, and
highly similar gene family members in an allotetraploid genome like cultivated
cotton. To address this issue, researchers have developed short synthetic
oligonucleotide cassette binary vectors, consisting of 19–24 bp highly specific
target-gene or sRNA/miRNA sequence with intronic loop size of 7–9 bp. These
synthetic oligonucleotide-based vectors could efficiently and selectively
silence target genes in plants (Higuchi et al., 2009) including cotton (Abdukarimov et al., 2011). However, both HP
and synthetic oligonucleotide vectors require genetic transformation where many
genotypes of cotton are recalcitrant to genetic transformation. Therefore,
there is a need for rapid in vitro and in vivo testing of RNAi effects for
targeted genes. Tang et al. (2004) experimentally tested the silencing power of
siRNA designed for targeting the green fluorescent protein (GFP) gene. The
silencing effects of two GFP derived siRNAs were tested in vitro cultured GFP
transgenic cells of rice, cotton, Fraser fir, and Virginia pine. These efforts
resulted in efficient silencing of GFP marker gene expression, which is useful
for large-scale screening of gene function and drug target validation (Tang et al., 2004). Due
to the limitation of siRNA delivery in vitro experiments, that affect the
efficiency of RNAi, researchers later developed the first efficient delivery
system of siRNA to plant cells including cotton by a nanosecond pulsed
laser-induced stress wave (LISW). This LISW-mediated siRNA delivery system was
found to be a reliable and effective method for inducing PTGS efforts in vitro
cultured cells (Tang et
al., 2006). Further, to perform a high throughput, rapid functional
validation of cotton genes and phenotypic effects researchers developed potent
RNAi assays using VIGS, facilitating transient PTGS. For example, because of
the important role in viral disease symptom modulation in cotton leaf curl
virus (CLCV) disease, VIGS-mediated RNAi was utilized to affect betasatellite
DNA of CLCV of Multan (CLCuMV). When inoculated, such VIGS system showed
efficient silencing of the target genes in tobacco, Arabidopsis and Upland
cotton plants (Kumar et al., 2014). A variety of Agrobacterium-mediated VIGS
vectors bearing various marker genes to monitor RNAi efficiency were developed
rapidly to test the gene function of interest from cotton genome. Examples
include tobacco rattle virus (TRV) vector with GrCLA1, GaPDS, and GaANR or
fused GaPDS/GaANR marker genes (Gao et al., 2011b; Gao and Shan, 2013; Pang et al., 2013)
resulting in albino or brownish plant phenotypes. In addition to the TRV-VIGS
vector, a cotton leaf crumple virus (CLCrV)-based vector was developed recently
and was shown as an effective RNAi method for gene discovery in cotton (Gu et al., 2014). As
described above and extensively referenced herein, VIGS has been widely used
for the discovery and identification of many useful genes in cotton. For
instance, RNAi of cotton phytoene synthase (GhPSY) using TRV-induced VIGS
caused a uniform bleaching of the red color in newly emerged leaves, suggesting
its role in controlling red plant phenotype (Cai et al., 2014). Similarly, the VIGS induced
RNAi was instrumental to discover the plant phenotypes of anthocyanidin
reductase (GhANR) pathway genes of cotton involved in the biosynthesis of
proanthocyanidins (PAs). In this study, the gene silencing effort has resulted
in a significant increase in anthocyanins and a decrease in the PAs,
(-)-epicatechin, and (-)-catechin in the stems and leaves of VIGS-infected
plants. Results demonstrated the role of ANR pathway in the biosynthesis of
flavan-3-ols and PAs in cotton (Zhu et al., 2015).

gene silencing for early functional studies of cotton genes

It is noteworthy to mention that the first
pioneering attempts on silencing of cotton genes were performed by using
antisense constructs. The pioneering effort is dated to John (1996) who studied
two E6 genes isolated from Upland and Pima cottons. A 60–98% suppression of E6
activity using antisense transgenic cotton plants revealed no noticeable
phenotypic changes in fiber development. This research demonstrated that E6 was
not critical to the fiber development process (John, 1996). Antisense suppression of cotton small
GTPase Rac (RAC13) genes decreased the levels of H2O2 in fiber cells, which in
turn affected secondary wall formation of cotton fibers (Potikha et al., 1999). Ruan et al. (2003)
successfully constructed antisense vectors for a 70% suppression of cotton
sucrose synthase (SUS) gene that resulted in fiberless and shrunken seed
phenotypes. Later, the functions of several cotton genes such as cotton steroid
5-alpha-reductase (GhDET2;
Luo et al., 2007), cotton myeloblastosis (GhMYB109; Pu et al., 2008), and GhPEL gene,
encoding a pectate lyase (Wang
et al., 2010a) were studied using antisense technology, which resulted
in significant reduction of fiber elongation and shorter fibers. These results
have suggested important roles of these genes in fiber development. Antisense
technology was also utilized to regulate other aspects of cotton besides fiber
genes. Inverted-repeat-based gene constructs designed for two key cotton
seed-specific fatty acid desaturase genes, GhSAD-1 and GhFAD2-1, resulted in
increased levels of stearic and oleic acids in RNAi cotton lines, respectively.
Results also showed that the content of palmitic acid significantly decreased
in both high-stearic and high-oleic lines, providing a promising opportunity
for the development of nutritionally improved cottonseed oil (Liu et al., 2000). CLCV
significantly reduces boll formation and development in cotton. Antisense
vectors were constructed for important genes of CLCV with the aim of affecting
the virulence of this virus. Such targeted genes were 50 and 30 fragments of
DNA replication gene (AC1) as well as a transcription activator (AC2) and
replication enhancer (AC3) genes. Transformation of these antisense constructs
into tobacco plant helped to obtain virus resistant transgenic plants (Asad et al., 2003).
Gossypol is a general biocide that provides protection from insect predation,
but it restricts the usefulness of cottonseed as a feed and protein source for
human and monogastric animals. In the efforts to manipulate gossypol, Martin et
al. (2003) developed and transformed cotton with an antisense construct of
CDN1-Cl, a member of a complex gene family of delta-(C) cadinene (CDN)
synthase. These efforts resulted in a reduction of CDN synthase gene activity
and decreased up to 70% of gossypol content. Further analyses of the
first-generation transgenic cotton plants demonstrated a significant amount of
reduction of gossypol (92%), hemigossypolone (83%) and heliocides (68%) in
leaves (Martin et al.,
2003) and seeds (Benedict
et al., 2004), negatively influencing the biosynthesis of cadinene
sesquiterpenoids and heliocides in cotton plants. Later, Townsend et al. (2005)
reported that the Agrobacterium-mediated genetic transformation of constitutive
or seed-specific antisense constructs demonstrated the suppression of CDN1-C4
genes in a response to bacterial blight infection of cotyledons in the
constitutive antisense plants, suggesting a specific role of particular cotton sesquiterpene
in the bacterial blight pathogenesis in cotton (Townsend et al., 2005).

Similar antisense technology was also used
for improvement of cottonseed oil by Sunilkumar et al. (2005) where authors developed the homologous
alpha-globulin B promoter driven antisense construct for FAD2 gene that reduced
the expression of delta -12 desaturase in cottonseed. Efforts resulted in
two-fold increase of the oleic acid level with an accompanying decrease of
linoleic acids in transgenic cottonseeds. Further, with the discovery and
understanding of the mechanisms, RNAi has been applied widely in cotton
research, and it has become a major research tool for studying gene functions
and breeding of novel cotton cultivars. For the past 15-years, more than 60
RNAi related studies have been published in cotton where we see dramatic
increase of efforts after 2010. These studies have targeted functional genomics
of important cotton genes including, but not limited to the fiber development,
fertility and somatic embryogenesis (SM), abiotic and biotic stress, cottonseed
and oil quality genes of cotton. These efforts have resulted in the development
of efficient RNAi methodologies, and RNAi-derived novel biotech cotton lines
and cultivars that were subsequently utilized for the functional studies,
breeding, and farming of cotton, which we have reviewed below in detail.

and future perspectives

Thus, replacing the anti-sense technology,
RNAi has been proven to be a valuable tool for functional genomics of cotton
for the past decade. RNAi revolutionized the discovery of many key functions
and biological roles of cotton genes involved in fiber development, fertility
and SM, resistance to key biotic and abiotic stresses, oil and seed quality and
other important cotton genes as well as in the improvement of key agronomic
traits including yield and maturity. Future studies most likely will target the
identification and RNAi of more complex, multi-functional, and developmental
genes with cross-talking features among many interconnected networks and
biochemical pathways of cotton ontogenesis (Abdurakhmonov, 2013) that generate simultaneous
improvements of key agronomic traits (Abdurakhmonov et al., 2014). Some efforts
highlighted herein demonstrate early examples to show the power of RNAi for
cotton improvement that needs to be extended in the future.

The development of RNAi cotton lines
targeting core RNAi machinery including proteins with important DNA methylase
and demethylase activity such as DOMAINS REARRANGED METHYLASE 1/2 (DRM1/2),
CHROMOMETHYLASE 2/3 (CMT 2/3), ROS1, and DEMETER (DME) in Upland cotton will
aid in elucidating the key regulatory mechanisms in WGD, chromosomal
rearrangements, dosage compensation, and evolutionary advantage of being
polyploids. Further, screening the epigenetic modulators for specific traits
such as fiber and yield and comparing against the genetic standard TM-1 will
aid in understanding the epigenetic landscape of Upland cotton. There is
limited information available on downstream stage of RNAi cotton cultivar
development, conducted field trials, or targeting for its commercialization.
This indicates that possibly RNAi-based “biotech cotton” cultivar development
is in its very early stage that requires more attention, effort, investment and
partnership of the cotton research community and private seed companies.
However, there are some commercially viable and already ongoing, targeted
applications of RNAi technologies, which are passing the successful small- or
large-scale field trials, and safety/risk assessment studies. These RNAi-based
cotton cultivars, highlighted in this paper, present substantial benefit in
cotton production with increased seed and oil quality, fiber, and key agronomic
trait improvements. These efforts are believed to boost cotton production and
its sustainability worldwide in the era of global climate change and increased
crop biosecurity threats. Despite general safety, RNAi based cultivars are
subject for risk assessment before commercialization as per available (Heinemann et al., 2013)
and highlighted guidelines herein. The existence and use of ARM genes in
current RNAi-based cotton cultivars is subject for rising “unjustified”
warnings and requests for removal of ARM genes from RNAi cultivars, and this
perhaps continues to be a main roadblock for future commercialization of RNAi
cotton cultivars. Therefore, there is a need for designing ARM-free RNAi
transformation experiments and development of ARM-free RNAi cotton cultivars.
RNA interference studies, reviewed herein, mostly have utilized stable HP or
transient VIGS vector constructs. There is a need for application of novel
genome modification and editing tools such as artificial microRNA amiRNA; Liang
et al., 2012), short synthetic interfering siRNA oligonucleotides (Higuchi et al., 2009;
Abdukarimov et al., 2011), Transcription Activator-like Effector
Nucleases (TALENs; Zhang
et al., 2013b), and Clustered Regularly Interspaced Short Palindromic
Repeats (CRISPRs/Cas9;
Larson et al., 2013) system to generate more effective, fine-tuned,
native knockdowns/knockouts than currently used RNAi methods. There is no doubt
that the cotton research community is already targeting these objectives,
having several diploid (Paterson
et al., 2012; Wang et al., 2012; Li et al., 2014) and key allotetraploid
(Li et al., 2015a; Liu et
al., 2015a; Zhang et al., 2015) genome sequences in hand. All of these
will require more coordinated efforts, wider international collaborations,
larger investment, and understandings of regulatory and stakeholder agencies.

Wendel JF. New
World cottons contain Old World cytoplasm. Proceedings of the National Academy
of Sciences. 1989;86:4132-4136. DOI:

Van Esbroeck, GA,
Bowman DT, May OL, and Calhoun DS. Genetic similarity indices for ancestral
cotton cultivars and their impact on genetic diversity estimates of modern
cultivars. Crop Science. 1999;39:323–328.

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