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Role of Antisense and RNAi-based Gene Silencing in Crop Improvement

This document presents information on RNA interference (RNAi) and its application in crop improvement. It discusses the discovery of antisense RNA and RNAi technology. The mechanisms of antisense technology and RNAi are described. Advantages of RNAi include its ability to study essential genes and its high specificity. Applications include crop protection and gene therapy. Case studies demonstrate improved insect resistance in transgenic tobacco plants and the role of miRNAs in syncytium formation induced by cyst nematodes.

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Presented By: Mariya Zaman Ph.D (Biotech.) B.A.C.A., AAU Submitted To: Dr. Akarsh Parihar Sir Associate Professor B.A.C.A., AAU Role of Antisense and RNAi-based Gene Silencing in Crop ImprovementWel Come
Outlines Introduction of Antisense & RNAi Technology Discovery of RNAi Types of RNAs Mechanism of Antisense & RNAi Technology Importance of RNAi Technology Pros & Cons of RNAi Technology Application of RNAi Technology CRISPR Technology Case studies Conclusions Future Thrust 2
Antisense RNA is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. They are introduced in a cell to inhibit the translation machinery by base pairing with the sense RNA and activating the RNase H, to develop a particular novel transgenic. The first natural antisense RNAs were discovered in 1981 independently in Tomizawas and in Nordstro¨ms laboratories Small plasmid-encoded RNA regulators control the copy numbers of the Escherichia coli plasmids ColE1 and R1, respectively Antisense RNAs are small, diffusible, highly structured RNAs that act via sequence complementarity on target RNAs called sense RNAs. Antisense RNAs are encoded in cis, i.e. They are transcribed from a promoter located on the opposite strand of the same DNA molecule, and are, therefore, fully complementary to their target RNAs. Entails post transcriptional inhibition of target RNA function. Antisense RNA
Mechanism of Antisense Technology 4
Characteristics of Antisense RNA Naturally occurring antisense RNAs are between 35 and 150 nt long and comprise between 1-4 stem-loops. •Efficient antisense RNAs have 5–8 nt GC-rich loops. •Stems that are important for metabolic stability are often (if > 10 bp) interrupted by bulges to prevent dsRNase degradation and to facilitate melting upon antisense/sense RNA interaction. •Some antisense RNAs (those involved in plasmid copy number control and post segregation killing) are unstable, others (most chromosomally encoded and a few phage and transposon antisense RNAs) are stable. •Almost all naturally occurring antisense-RNA regulated systems have been found mostly in prokaryotes, and only a few systems are known from eukaryotes and one from archaea.
Antisense-RNA regulated systems in prokaryotes Inhibition of primer formation Inhibition of synthesis of a leader peptide MicF RNA, ompF-RNA DicF RNA RNAIII
Principal target sites • Transcription 1. DNA triple helix formation 2. Hybridization to DNA loops 3. Hybridization to nascent RNA • RNA processing 4. RNA splicing 5. RNA transport • Translation 6. Inhibition of initiation factor binding 7. Inhibition of ribosome assembly 8. Inhibition of movement
RNA Coding RNA mRNA Non Coding RNAs Constituent/ Transcription RNAs rRNA tRNA Regulatory/ Small RNAs siRNA miRNA (stRNA) snoRNA snRNA RNA Family RNA interference 8
RNA interference • RNA interference (RNAi) is an evolutionary highly conserved defense mechanism of post- transcriptional gene silencing (PTGS) occurring naturally against double stranded RNA (dsRNA) that causes sequence-specific gene regulation resulting in inhibition of translation or transcriptional repression of mRNA sequences. 9
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Mechanism of RNAi Technology 12
13 Structure of siRNA
miRNA Mechanism TRBP Ran GTP 14
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Summary of Players • Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) • Drosha and Dicer are RNase III enzymes • Pasha is a dsRNA binding protein • Exportein 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran & GTP • Argonautes are RNase H enzymes 16
• Dicer is a ribonuclease (RNase III family) with 4 distinct domains: 1. Amino-terminal helicase domain 2. Dual RNase III motifs in the carboxy terminal segment 3. dsRNA binding domain 4. PAZ domain (110-130 amino-acid domain present in protein like PIWI, Argonaute, and Zwille proteins) for protein- protein interaction. 17
siRNA miRNA Exogenous Endogenous 21-25 nt RNAs ~23 nt RNAs dsRNA Initially ssRNA but converted into hairpin secondary structure Most commonly responses to foreign RNA(Viral, transposable elements, repetitive elements or artificial transgene insert) Regulates post-transcriptional gene expression 100% complementary to target RNA Not often 100% complementary to target RNA Produced in large population Only single small RNA Effective but not tissue specific Silencing Precise and tissue specific Silencing 18
Advantages of RNAi Technology Dominant Phenotypes can be observed in the T1 generation Partial knockdown Facilitates the study of essential genes whose inactivation would lead to lethality or extremely severe pleiotropic phenotypes Tissue-specific (miRNA) Off-target effects can be reduced High-throughput/ Ease of transfection High-throughput vectors are designed to make an hpRNAi construct Flexible Individual or multiple genes can be silenced with single hpRNAi construct. Better than antisense The highest silencing was obtained with hpRNAi Cost effective High specificity Middle region 9-14 are most sensitive Can be labeled Stable hpRNAi has been shown to be stably inherited over several generations 19
Pitfalls of RNAi • Off-target effects • Inefficacy and instability • Validation of RNAi knockdown 20
Importance of RNAi • Powerful for analyzing unknown genes in sequenced genomes • Efforts are being undertaken to target every human gene via siRNAs • Faster identification of gene function • Gene therapy: down-regulation of certain genes/ mutated alleles • Cancer treatments  Knock-out of genes required for cell proliferation  Knock-out of genes encoding key structural proteins • Agriculture 21
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CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea • In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. • CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. 23Marraffini and Sontheimer, 2011USA
Mechanism for CRISPR interference 24 At the molecular level, CRISPR function can be divided into three phases: 1) Incorporation of new spacers into CRISPR arrays 2) Expression and processing of CRISPR RNAs (crRNAs) 3) CRISPR interference Features of CRISPR loci • CRISPRs - white boxes • Leader sequence - black box that is AT-rich but not conserved • Non-repetitive spacers -coloured boxes that share sequence identity with fragments of plasmids & bacteriophage genomes and specify the targets of CRISPR interference. • CRISPR-associated (cas) genes are conserved, different families and subtypes, & encode the protein machinery for CRISPR activity
25 1) Incorporation of new spacers into CRISPR arrays • During the adaptation phase of (CRISPR) immunity, new spacers derived from the invading DNA are incorporated into CRISPR loci. • Cas1 to generate short fragments of invading DNA 2) CRISPR interference
26 A) In the defence phase of (CRISPR) immunity, repeats and spacers are transcribed into a long precursor processed by a complex called CRISPR- associated complex for antiviral defence (Cascade) in Escherichia coli or CRISPR-associated 6 (Cas6) in Pyrococcus furiosus, which generates small CRISPR RNAs (crRNAs). • Processing occurs near the 3′ end of the repeat sequence, leaving a short (~8 nts) repeat sequence 5′ of the crRNA spacer & more heterogeneous 3′ terminus. B) RNAs serve as guides for an effector complex (Cas proteins) that recognizes invading DNA and blocks infection 3) Self versus non-self discrimination during CRISPR immunity Target DNA CRISPR DNA Target Target of non-self DNA Protection of self DNA CrRNA CrRNA RepeatRepeat Spacer Non-Complementary Complementary
27 RNA interference (RNAi) CRISPR interference siRNAs, miRNAs & piRNAs as guides for gene regulation and genome defence crRNA used Double-stranded precursors single-stranded precursors Post-transcriptionally amplified Not post-transcriptionally amplified Recognize other RNAs Recognize other DNAs No such integration required Invasive nucleic acids integrated into host genome before defense mechanism
28 Case Study
Enhanced Whitefly Resistance in Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene 1 Thakur et al., 2014India 29 • Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v- ATPaseA mRNA in whitefly. • Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. • The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. • Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. • The transgenic plants did not show drooping effect.
Methodology/Principal Findings 30 (A) Construction of v-ATPaseA dsRNA expression cassette in pBI101 (B) Transgenic and control tobacco plants (C) Selection of T1 seeds on kanamycin medium, showing non-transgenic seedlings turning white (D) PCR analysis of transgenic lines • upper panel - amplification of nptII gene • lower panel- amplification of v-ATPaseA+RTM1 intron
In-vivo bioassay of transgenic and control plants with whitefly Early Stage of Whitefly infection Loss of Sugar content due to heavy infestation C D 31
Expression analysis of v-ATPase A specific RNA in different transgenic lines of tobacco Dot-blot assay of total RNA from 12 T1 transgenic lines with probe of (A) v-ATPaseA gene & (B) U6 gene (C) Dot-blot assay of RNA from control plants (expressing dsRNA of asal gene); RNA was spotted and hybridised with same probes to show specificity of hybridisation (D) Northern blot analysis of four selected transgenic lines with v-ATPaseA specific probe 32
The Arabidopsis MicroRNA396-GRF1/GRF3 Regulatory Module Acts as a Developmental Regulator in the Reprogramming of Root Cells during Cyst Nematode Infection Hewezi et al., 2012Iowa (U.S.) 2 33 • The syncytium is a unique plant root organ whose differentiation is induced by plant-parasitic cyst nematodes to create a source of nourishment. • Syncytium formation involves the redifferentiation and fusion of hundreds of root cells. soybean Cyst nematodes Root nodes infection Female cyst containing eggs
34 • A strong downregulation of Arabidopsis (Arabidopsis thaliana) microRNA396 (miR396) in cells giving rise to the syncytium coincides with the initiation of the syncytial induction/formation phase and that specific miR396 up- regulation in the developed syncytium marks the beginning of the maintenance phase, when no new cells are incorporated into the syncytium. • miR396 has a role in the transition from one phase to the other. • Expression modulations of miR396 and its Growth-Regulating Factor (GRF) target genes resulted in reduced syncytium size and arrested nematode development. • Genome-wide expression profiling revealed that the miR396-GRF regulatory system can alter the expression of 44% of the more than 7,000 genes reported to change expression in the Arabidopsis syncytium. • Thus, miR396 represents a key regulator for the reprogramming of root cells and a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways.
35 Methodology/Principal Findings miR396 regulates the expression of 7 Arabidopsis growth-regulating transcription factor genes (GRF1–4 & GRF7–9) that share the miR396-binding site •miR396 have two genes 1) miR396a (AT2G10606) 2) miR396b (AT5G35407) N =nematode S = syncytium Histochemical localization of GUS activity directed by miR396 promoters Transgenic plants expressing constructs containing the regions upstream of miR396 precursor sequences fused to the GUS reporter gene (PmiR396a:GUS and PmiR396b:GUS). Promoter activity of miR396a and miR396b during H. schachtii infection.
36 Thus, miR396 expression changes delineate the syncytium induction/formation phase, whereby a down-regulation marks the beginning of syncytium induction/formation and a subsequent strong upregulation coincides with the transition to the maintenance phase. The GRF1 and GRF3 promoters showed very strong GUS activity in syncytia at all stages, with only the GRF3 promoter becoming inactive at the J4 stage Posttranscriptional regulation of GRF1 and GRF3 by miR396 in response to H. schachtii infection. A) Mapping of the cleavage sites of GRF1 and GRF3 using 59 RLM-RACE.
37 B) miR396 mediates the down-regulation of GRF1 and GRF3 expression in response to H. schachtii infection. The expression level of pri-miR396a, pri-miR396b, mature miR396, GRF1, and GRF3 was measured by qPCR in wild-type (Col-0) root tissues. All transgenic lines overexpressing miR396a (Fig. 4A) or miR396b (Fig. 4B) were dramatically less susceptible to nematodes than the wild-type control.
38 Transgenic plants overexpressing rGRF1 (E) or rGRF3 (F) revealed reduced susceptibility to H. schachtii Overexpression of miR396 and the target genes GRF1 and GRF3 negatively impacts root development. A) & B) Transgenic plants overexpressing miR396a (line 22-5; A) or miR396b (line 15-1; B) develop shorter roots than the wild type (Col-0). C) Transgenic plants overexpressing rGRF1 (line 18-2) or rGRF3 (line 11-15) develop shorter roots than the wild type (Col-0).
Knockdown of Midgut Genes by dsRNA Transgenic Plant- Mediated RNA Interference in the Hemipteran Insect (Nilaparvata lugens) Leaves turn yellow initially and later brownish due to drying up of the plants. Under severe cases field gives a burnt appearance in concentric circles, known as "Hopper burn". RNAi is a powerful technique for functional genomics research against Hemipteran Insect (N. lugens) Transgenic plants producing dsRNA directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests. They damage rice directly through feeding and also by transmitting two viruses, rice ragge stunt virus and rice grassy stunt virus. Up to 60% yield loss is common in susceptible rice cultivars attacked by BPH (Brown Plant Hopper). Zha et al., 2011China 3 39
Methodology/Principal Findings The Hemipteran insect brown planthopper (Nilaparvata lugens) is a typical phloem sap feeder specific to rice (Oryza sativa L.) Analysis and identification of genes (Nlsid-1 and Nlaub) encoding proteins that involved in the RNAi pathway in N. lugens insect. Both genes are expressed ubiquitously in nymphs and adult insects. Nymphs40
Three genes isolated were highly expressed in the N. lugens midgut and used to develop dsRNA constructs for transforming rice. 1) NlHT1- hexose transporter gene 2) Nlcar- carboxypeptidase gene 3) Nltry- trypsin-like serine protease gene Primary expression in midgut tissues with limited transcription in salivary glands, fat body and head 41 A A) The genes were cloned at the BamHI site of the hygromycin gene expression cassette in the pCU vector of Agrobacterium B) Southern blot analysis showed that the PCR-positive plants had 1–3 copies of the target coding sequences
When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced. Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines (A) Two-week-old seedlings (B) Four leaf stage Plants (C) Mature plants RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines.
Genome-wide identification of Brassica napus microRNAs and their targets in response to cadmium • MicroRNAs (miRNAs) also regulate response to environmental stresses. • The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus). • miRNA-regulated gene silencing may be involved in plant tolerance to heavy metals. • In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. • Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Zhou et al. 2012China 43 4
44 • Some of the miRNAs were validated by RNA gel blotting. • Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure.
• The study also identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. • In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd. 45
The Chloroplast Triggers Developmental Reprogramming When MUTS HOMOLOG1 Is Suppressed in Plants • To explore the control of phenotypy in higher plants, they examined the effect of a single plant nuclear gene on the expression and transmission of phenotypic variability in Arabidopsis thaliana. • MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene product that functions in both mitochondria and plastids to maintain genome stability. • Genetic hemicomplementation experiments show that this phenotypic plasticity derives from changes in chloroplast state. • The result of MSH1 suppression through RNAi, triggers a plastidial response process that involves non-genetic inheritance. Xu et al., 2012China 46 5
Loss of MSH1 by RNAi results in programmed phenotypic changes, including A) Altered phytohormone effects for dwarfed growth & reduced internode elongation B) altered leaf morphology C) Effects are partially reverted with the application of GA D) enhanced branching E) reduced stomatal density F) extended juvenility, with conversion to perennial growth pattern in short days G) delayed flowering 47
Fig. 2. Phenotypic plasticity in the Arabidopsis msh1 mutant. 48
Fig. 5. Evidence of transcriptional and metabolic changes in Arabidopsis msh1 mutant and hemicomplementation lines. A) & B) quantitative RT-PCR assay C) RNA gel blot assay D) Heat map assay of metabolites Fig. 6. Hemicomplementation analysis of the Arabidopsis msh1 altered growth phenotype. Col-0, dual-targeted, and plastid-targeted MSH1 transgenic lines flowered uniformly, whereas the msh1 mutant and the mitochondria-targeted MSH1 transgenic line showed marked variation for growth, flowering time, and maturity. The Observed Developmental Reprogramming Is the Consequence of Chloroplast Changes 49
Development of male sterility by silencing Bcp1 gene of Arabidopsis through RNA interference • Use of RNA interference (RNAi) technology to silence a male specific gene, Bcp1 in the model host Arabidopsis thaliana. • Bcp1 is active in both diploid tapetum and haploid microspores. • Three batches of explants (A. thaliana) were selected on herbicide Glufosinate ammonium and putative transgenes were confirmed through PCR and Southern hybridization. • The present study resulted in developing male sterile A. thaliana (Eco. Columbia) line through genetic engineering. Tehseen et al., 2010Pakistan 50 6
• Bcp1 gene can be divided in two parts – 163bp non conserved region – 372bp conserved region Methodology/Principal Findings • Silencing of Bcp1 gene ,587bp in size, responsible for fertility They targeted 0.77kb regulatory region of Bcp1 gene via antisense • Expression of both sense and antisense fragment separated by an intron, yields more efficient silencing than only antisense Targeting coding sequence of Bcp1 using RNAi leads to male sterility • Bgp1,female fertility gene -- 87 % homology with conserved region -- To avoid silencing of female part, only non conserved region is targeted 51
• 163bp region of Bcp1 gene cloned in both sense and antisense orientation in pFGC5941 -Cloning of gene in sense and antisense orientation in same vector produce dsRNA inverted repeat molecule which induce PTGS in plant cells •Primers design w.r.t dsRNA binary vector pFGC5941 -It has two mcs, bar gene and 35S promoter within left and right borders -Two mcs flanked by intron of 1.364kb • Construct was transformed in A.tumefaciens strain LBA4404 by electroporation Agrobacterium culture was confirmed with PCR amplification The construct was transformed in Arabidopsis using leaf disc method 52
• Formation of sharp bands confirm the presence of transgenes – Transcribed mRNA of RNAi construct will result in a dsRNA with a hairpin loop – Resultant dsRNA triggered on the RNAi machinery • 3 batches of explants were selected on herbicide glufosinate ammonium Putative transgenic plants confirmed by PCR using bar gene specific primers and southern hybridisation It gives amplification along with positive and negative controls 53
The Introgression of RNAi Silencing of γ-Gliadins into Commercial Lines of Bread Wheat Changes the Mixing and Technological Properties of the Dough •The effects on dough quality by the RNAi-mediated silencing of γ - gliadins in commercial bread wheat lines (namely ‘Gazul’, ‘Podenco’ and ‘Arpain’) along with the transgenic line A1152 (cv. Bobwhite) were compared with their respective wild types. •The protein fractions were quantified by RP-HPLC, whereas the technological and mixing properties were assessed by SDSS test and by the Mixograph instrument. •The down-regulation of γ-gliadins resulted in stronger dough and a better tolerance to over-mixing in some transgenic lines. Gil-Humanes et al., 2012Spain 54 7
Methodology/Principal Findings Wheat gliadin genes occur as tightly-linked clusters located on 1 and 6 chromosomes ω and γ-gliadins on Chromosome 1 having clusters of genes • Gli-A1 • Gli-B1 • Gli-D1 LMW-GS genes on the Glu3 loci α-gliadins on Chromosome 6 having clusters of genes • Gli-A2 • Gli-B2 • Gli-D2 Down-regulation of γ –gliadins through RNAi approach in three commercial lines and F1 hybrid obtained •After three generations of self-pollination, the gliadin and glutenin profiles analyzed by A-PAGE and SDS-PAGE. 55
Glutenin content increased and the ratio Gli/Glu reduced in the transgenic lines, provoking an increase of the dough strength and a decrease of the extensibility. Analysis of grain composition and quality parameters •The analysis of the variance (ANOVA) and LSD analysis of homogeneous groups showed significant differences in all the fractions of gliadins and glutenins. •SDSS volume showed an increase in all the transgenic lines w.r.t. the wild types, resulting in significantly higher volumes for transgenic lines G613, G626, G845 and G664. •In all parameters of Mixograph, no significantly different observed in the average of transgenic lines(except G622 & G626) to wild types, indicating that the mixing properties were not affected by the silencing of the γ-gliadins in the transgenic lines with different HMW-GS profile. 56
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Engineering of the Rose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers Accumulating Delphinidin Katsumoto et. al (2007)Japan o Rosa hybrida lacks violet to blue colour due to the absence of flavonoid 3’,5’-hydoxylase (F3’5’H) enzyme which produces delphinidin-based anthocyanins. o Other factors such as co-pigments and vacuolar pH also affect flower colour. o Expression of the viola F3’5’H gene  accumulates (~95% high) delphinidin  a novel bluish flower colour. 58 8
• For more exclusive and dominant accumulation of delphinidin irrespective of the hosts, the endogenous dihydroflavonol 4-reductase (DFR) gene was down- regulated and overexpressed the Iris3hollandica DFR gene in addition to the viola F3’5’H gene in a rose cultivar. • The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. • Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations. 59
Bi o sy n t h et ic p at h w a y O f fl a v o n oi d 60
WKS77 WKS82 WKS100 WKS116 WKS124 WKS140 Rose Varieties transformed with pSPB130 and their flower colour changed are shown Schematic representation of T-DNA region of binary vectors constructed for colour modification  for the constitutive over expression of the viola F3’5’H BP40 gene and the torenia 5AT gene in rose. 61
Host flower violet-coloured transgenic flower 98% delphinidin 98% delphinidin Madam Violet Seiryu  The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes. 62
• Northern blot analysis of LA/919-4-10. • The expected sizes of the transcripts of viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7 kb) genes & smaller size was detected for rose DFR mRNA (A). • A rose DFR probe detected about 23 bp small sized RNA, which was supposed to be a degraded endogenous rose DFR transcript with RNAi (B). • Delphinidin contents was confirmed in all transgenic (KmR) progeny of LA/919-4-10. • The flowers of F1 and F2 progeny contained exclusively delphinidin. 63 (si RNA)
Production of picotee-type flowers in Japanese gentian by CRES-T A B Wild type flower‐Solid color suppression of pigment production generates picotee type flower Nakatsuka et al., 2011Japan CRES-TChimeric repressor gene-silencing technology 9 64
• (CRES-T) is an efficient gene suppression system which worked successfully in Japanese gentian. • A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue- flowered gentian. • Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal. • HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type. • The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F35H, DFR and ANS, were strongly suppressed in petals of these transgenic plants. 65
• Expression of flavonoid biosynthetic genes in transgenic gentian plants. • The expression levels of GtMYB3-SRDX and endogenous flavonoid biosynthetic genes were determined by semi- quantitative RT-PCR analysis in wild-type and GtMYB3- SRDX expressed transgenic gentian clone nos. 7 & 11 • Schematic representation of pSMABR-AtACT2pro- GtMYB3-SRDX. • Bar  herbicide bialaphos resistance gene as a selectable marker. • NOSp  promoter of nopaline synthase (NOS) gene from A. tumefaciens. • rbcSt  terminator of RuBisCO small subunit 2B gene from Arabidopsis. • NOSt  terminator of NOS gene. • LB  left border; RB  right border. semi-quantitative RT-PCR analysis 66
Flavonoid analysis in the flowers of transgenic gentian plants by HPLC A & D- wild type B & E- transgenic gentian clones no. 7 C & F- transgenic gentian clones no. 11 Anthocyanins Flavones 67
Conclusions 68 Genetic engineering overcomes almost all the limitations of traditional breeding approaches. Recent advances in plant molecular biology provide opportunities to use techniques of genetic engineering for improvement of crop plants for disease resistance, toxic resistance, plant architecture, male sterility, dough quality parameter, flower colour, etc. Case-1 Host plant derived pest resistance was achieved at field level against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Case-2 miR396- a key regulator, provides the nematode with a single molecular target to wield power over a substantial proportion of syncytium developmental events.
69 Case-3 These genes (Nlsid-1 and Nlaub) were identified and knockdown in N. lugens through RNA interference. The results demonstrate the potential of this technique at field- level control of plant-hoppers. Case-4 Identification of many high-quality target transcripts will help in the regulatory mechanism for plant tolerance to Cd. Case-5 Suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves non-genetic inheritance. Conclusions Case-6 Bcp1 gene is responsible for fertile pollen development and active in both diploid tapetum and haploid microspores. Silencing Bcp1 gene lead to transgenic male sterile plants.
70 Case 7 The down-regulation of γ-gliadins resulted in stronger doughs and a better tolerance to over-mixing in some transgenic lines. Case-8 Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors and knowledge of flower coloration at the biochemical and molecular level has made it possible to develop novel color. Case-9 (CRES-T) is an efficient gene suppression system which worked successfully in Japanese gentian. Conclusions
Future Prospects and New Avenues 71 •Careful consideration of the interplay factors (such as the selection of the target gene, most effective site within the gene for knockdown, length of the targeted nucleotide sequence, and minimization of the off-target effects) is expected to deliver a competent technology in insect/pest resistance in the near future. •The regulatory unit represents a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways. •More research efforts are needed for tolerance of crop plants toward heavy metal toxicity.
72 • RNAi can be employed to obtain hybrid seeds of commercially high valued crops. •New genes should be isolated that will have utility in floriculture, and new transformation methods for flower crops should be further optimized. • More advancement required for dough quality, as the reduction of γ-gliadins seems not to have a direct effect on the mixing and bread-making properties, the compensatory effect on the synthesis of the other prolamins has resulted in stronger doughs with improved over-mixing resistance in some transgenic lines. Future Prospects and New Avenues
Earth is our unique Masterpiece, can neither be replicate, transcribe nor translate it. We are only regulatory components of Earth, can save our planet by Downregulating environmental pollutions and Upregulating natural resources. - Mariya Zaman 73

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Role of Antisense and RNAi-based Gene Silencing in Crop Improvement

  • 1.
    Presented By: Mariya Zaman Ph.D(Biotech.) B.A.C.A., AAU Submitted To: Dr. Akarsh Parihar Sir Associate Professor B.A.C.A., AAU Role of Antisense and RNAi-based Gene Silencing in Crop ImprovementWel Come
  • 2.
    Outlines Introduction of Antisense& RNAi Technology Discovery of RNAi Types of RNAs Mechanism of Antisense & RNAi Technology Importance of RNAi Technology Pros & Cons of RNAi Technology Application of RNAi Technology CRISPR Technology Case studies Conclusions Future Thrust 2
  • 3.
    Antisense RNA isa single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. They are introduced in a cell to inhibit the translation machinery by base pairing with the sense RNA and activating the RNase H, to develop a particular novel transgenic. The first natural antisense RNAs were discovered in 1981 independently in Tomizawas and in Nordstro¨ms laboratories Small plasmid-encoded RNA regulators control the copy numbers of the Escherichia coli plasmids ColE1 and R1, respectively Antisense RNAs are small, diffusible, highly structured RNAs that act via sequence complementarity on target RNAs called sense RNAs. Antisense RNAs are encoded in cis, i.e. They are transcribed from a promoter located on the opposite strand of the same DNA molecule, and are, therefore, fully complementary to their target RNAs. Entails post transcriptional inhibition of target RNA function. Antisense RNA
  • 4.
  • 5.
    Characteristics of AntisenseRNA Naturally occurring antisense RNAs are between 35 and 150 nt long and comprise between 1-4 stem-loops. •Efficient antisense RNAs have 5–8 nt GC-rich loops. •Stems that are important for metabolic stability are often (if > 10 bp) interrupted by bulges to prevent dsRNase degradation and to facilitate melting upon antisense/sense RNA interaction. •Some antisense RNAs (those involved in plasmid copy number control and post segregation killing) are unstable, others (most chromosomally encoded and a few phage and transposon antisense RNAs) are stable. •Almost all naturally occurring antisense-RNA regulated systems have been found mostly in prokaryotes, and only a few systems are known from eukaryotes and one from archaea.
  • 6.
    Antisense-RNA regulated systemsin prokaryotes Inhibition of primer formation Inhibition of synthesis of a leader peptide MicF RNA, ompF-RNA DicF RNA RNAIII
  • 7.
    Principal target sites •Transcription 1. DNA triple helix formation 2. Hybridization to DNA loops 3. Hybridization to nascent RNA • RNA processing 4. RNA splicing 5. RNA transport • Translation 6. Inhibition of initiation factor binding 7. Inhibition of ribosome assembly 8. Inhibition of movement
  • 8.
    RNA Coding RNA mRNA Non CodingRNAs Constituent/ Transcription RNAs rRNA tRNA Regulatory/ Small RNAs siRNA miRNA (stRNA) snoRNA snRNA RNA Family RNA interference 8
  • 9.
    RNA interference • RNAinterference (RNAi) is an evolutionary highly conserved defense mechanism of post- transcriptional gene silencing (PTGS) occurring naturally against double stranded RNA (dsRNA) that causes sequence-specific gene regulation resulting in inhibition of translation or transcriptional repression of mRNA sequences. 9
  • 10.
  • 11.
  • 12.
    Mechanism of RNAiTechnology 12
  • 13.
  • 14.
  • 15.
  • 16.
    Summary of Players •Drosha and Pasha are part of the “Microprocessor” protein complex (~600-650kDa) • Drosha and Dicer are RNase III enzymes • Pasha is a dsRNA binding protein • Exportein 5 is a member of the karyopherin nucleocytoplasmic transport factors that requires Ran & GTP • Argonautes are RNase H enzymes 16
  • 17.
    • Dicer isa ribonuclease (RNase III family) with 4 distinct domains: 1. Amino-terminal helicase domain 2. Dual RNase III motifs in the carboxy terminal segment 3. dsRNA binding domain 4. PAZ domain (110-130 amino-acid domain present in protein like PIWI, Argonaute, and Zwille proteins) for protein- protein interaction. 17
  • 18.
    siRNA miRNA Exogenous Endogenous 21-25nt RNAs ~23 nt RNAs dsRNA Initially ssRNA but converted into hairpin secondary structure Most commonly responses to foreign RNA(Viral, transposable elements, repetitive elements or artificial transgene insert) Regulates post-transcriptional gene expression 100% complementary to target RNA Not often 100% complementary to target RNA Produced in large population Only single small RNA Effective but not tissue specific Silencing Precise and tissue specific Silencing 18
  • 19.
    Advantages of RNAiTechnology Dominant Phenotypes can be observed in the T1 generation Partial knockdown Facilitates the study of essential genes whose inactivation would lead to lethality or extremely severe pleiotropic phenotypes Tissue-specific (miRNA) Off-target effects can be reduced High-throughput/ Ease of transfection High-throughput vectors are designed to make an hpRNAi construct Flexible Individual or multiple genes can be silenced with single hpRNAi construct. Better than antisense The highest silencing was obtained with hpRNAi Cost effective High specificity Middle region 9-14 are most sensitive Can be labeled Stable hpRNAi has been shown to be stably inherited over several generations 19
  • 20.
    Pitfalls of RNAi •Off-target effects • Inefficacy and instability • Validation of RNAi knockdown 20
  • 21.
    Importance of RNAi •Powerful for analyzing unknown genes in sequenced genomes • Efforts are being undertaken to target every human gene via siRNAs • Faster identification of gene function • Gene therapy: down-regulation of certain genes/ mutated alleles • Cancer treatments  Knock-out of genes required for cell proliferation  Knock-out of genes encoding key structural proteins • Agriculture 21
  • 22.
  • 23.
    CRISPR interference: RNA-directed adaptiveimmunity in bacteria and archaea • In many bacteria and most archaea, clustered, regularly interspaced short palindromic repeats (CRISPRs) are involved in a more recently discovered interference pathway that protects cells from bacteriophages and conjugative plasmids. • CRISPR sequences provide an adaptive, heritable record of past infections and express CRISPR RNAs — small RNAs that target invasive nucleic acids. 23Marraffini and Sontheimer, 2011USA
  • 24.
    Mechanism for CRISPRinterference 24 At the molecular level, CRISPR function can be divided into three phases: 1) Incorporation of new spacers into CRISPR arrays 2) Expression and processing of CRISPR RNAs (crRNAs) 3) CRISPR interference Features of CRISPR loci • CRISPRs - white boxes • Leader sequence - black box that is AT-rich but not conserved • Non-repetitive spacers -coloured boxes that share sequence identity with fragments of plasmids & bacteriophage genomes and specify the targets of CRISPR interference. • CRISPR-associated (cas) genes are conserved, different families and subtypes, & encode the protein machinery for CRISPR activity
  • 25.
    25 1) Incorporation ofnew spacers into CRISPR arrays • During the adaptation phase of (CRISPR) immunity, new spacers derived from the invading DNA are incorporated into CRISPR loci. • Cas1 to generate short fragments of invading DNA 2) CRISPR interference
  • 26.
    26 A) In thedefence phase of (CRISPR) immunity, repeats and spacers are transcribed into a long precursor processed by a complex called CRISPR- associated complex for antiviral defence (Cascade) in Escherichia coli or CRISPR-associated 6 (Cas6) in Pyrococcus furiosus, which generates small CRISPR RNAs (crRNAs). • Processing occurs near the 3′ end of the repeat sequence, leaving a short (~8 nts) repeat sequence 5′ of the crRNA spacer & more heterogeneous 3′ terminus. B) RNAs serve as guides for an effector complex (Cas proteins) that recognizes invading DNA and blocks infection 3) Self versus non-self discrimination during CRISPR immunity Target DNA CRISPR DNA Target Target of non-self DNA Protection of self DNA CrRNA CrRNA RepeatRepeat Spacer Non-Complementary Complementary
  • 27.
    27 RNA interference (RNAi)CRISPR interference siRNAs, miRNAs & piRNAs as guides for gene regulation and genome defence crRNA used Double-stranded precursors single-stranded precursors Post-transcriptionally amplified Not post-transcriptionally amplified Recognize other RNAs Recognize other DNAs No such integration required Invasive nucleic acids integrated into host genome before defense mechanism
  • 28.
  • 29.
    Enhanced Whitefly Resistancein Transgenic Tobacco Plants Expressing Double Stranded RNA of v-ATPase A Gene 1 Thakur et al., 2014India 29 • Transgenic tobacco lines were developed for the expression of long dsRNA precursor to make siRNA and knock down the v- ATPaseA mRNA in whitefly. • Molecular analysis and insecticidal properties of the transgenic plants established the formation of siRNA targeting the whitefly v-ATPaseA, in the leaves. • The transcript level of v-ATPaseA in whiteflies was reduced up to 62% after feeding on the transgenic plants. • Heavy infestation of whiteflies on the control plants caused significant loss of sugar content which led to the drooping of leaves. • The transgenic plants did not show drooping effect.
  • 30.
    Methodology/Principal Findings 30 (A) Constructionof v-ATPaseA dsRNA expression cassette in pBI101 (B) Transgenic and control tobacco plants (C) Selection of T1 seeds on kanamycin medium, showing non-transgenic seedlings turning white (D) PCR analysis of transgenic lines • upper panel - amplification of nptII gene • lower panel- amplification of v-ATPaseA+RTM1 intron
  • 31.
    In-vivo bioassay oftransgenic and control plants with whitefly Early Stage of Whitefly infection Loss of Sugar content due to heavy infestation C D 31
  • 32.
    Expression analysis ofv-ATPase A specific RNA in different transgenic lines of tobacco Dot-blot assay of total RNA from 12 T1 transgenic lines with probe of (A) v-ATPaseA gene & (B) U6 gene (C) Dot-blot assay of RNA from control plants (expressing dsRNA of asal gene); RNA was spotted and hybridised with same probes to show specificity of hybridisation (D) Northern blot analysis of four selected transgenic lines with v-ATPaseA specific probe 32
  • 33.
    The Arabidopsis MicroRNA396-GRF1/GRF3Regulatory Module Acts as a Developmental Regulator in the Reprogramming of Root Cells during Cyst Nematode Infection Hewezi et al., 2012Iowa (U.S.) 2 33 • The syncytium is a unique plant root organ whose differentiation is induced by plant-parasitic cyst nematodes to create a source of nourishment. • Syncytium formation involves the redifferentiation and fusion of hundreds of root cells. soybean Cyst nematodes Root nodes infection Female cyst containing eggs
  • 34.
    34 • A strongdownregulation of Arabidopsis (Arabidopsis thaliana) microRNA396 (miR396) in cells giving rise to the syncytium coincides with the initiation of the syncytial induction/formation phase and that specific miR396 up- regulation in the developed syncytium marks the beginning of the maintenance phase, when no new cells are incorporated into the syncytium. • miR396 has a role in the transition from one phase to the other. • Expression modulations of miR396 and its Growth-Regulating Factor (GRF) target genes resulted in reduced syncytium size and arrested nematode development. • Genome-wide expression profiling revealed that the miR396-GRF regulatory system can alter the expression of 44% of the more than 7,000 genes reported to change expression in the Arabidopsis syncytium. • Thus, miR396 represents a key regulator for the reprogramming of root cells and a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways.
  • 35.
    35 Methodology/Principal Findings miR396 regulatesthe expression of 7 Arabidopsis growth-regulating transcription factor genes (GRF1–4 & GRF7–9) that share the miR396-binding site •miR396 have two genes 1) miR396a (AT2G10606) 2) miR396b (AT5G35407) N =nematode S = syncytium Histochemical localization of GUS activity directed by miR396 promoters Transgenic plants expressing constructs containing the regions upstream of miR396 precursor sequences fused to the GUS reporter gene (PmiR396a:GUS and PmiR396b:GUS). Promoter activity of miR396a and miR396b during H. schachtii infection.
  • 36.
    36 Thus, miR396 expressionchanges delineate the syncytium induction/formation phase, whereby a down-regulation marks the beginning of syncytium induction/formation and a subsequent strong upregulation coincides with the transition to the maintenance phase. The GRF1 and GRF3 promoters showed very strong GUS activity in syncytia at all stages, with only the GRF3 promoter becoming inactive at the J4 stage Posttranscriptional regulation of GRF1 and GRF3 by miR396 in response to H. schachtii infection. A) Mapping of the cleavage sites of GRF1 and GRF3 using 59 RLM-RACE.
  • 37.
    37 B) miR396 mediatesthe down-regulation of GRF1 and GRF3 expression in response to H. schachtii infection. The expression level of pri-miR396a, pri-miR396b, mature miR396, GRF1, and GRF3 was measured by qPCR in wild-type (Col-0) root tissues. All transgenic lines overexpressing miR396a (Fig. 4A) or miR396b (Fig. 4B) were dramatically less susceptible to nematodes than the wild-type control.
  • 38.
    38 Transgenic plants overexpressingrGRF1 (E) or rGRF3 (F) revealed reduced susceptibility to H. schachtii Overexpression of miR396 and the target genes GRF1 and GRF3 negatively impacts root development. A) & B) Transgenic plants overexpressing miR396a (line 22-5; A) or miR396b (line 15-1; B) develop shorter roots than the wild type (Col-0). C) Transgenic plants overexpressing rGRF1 (line 18-2) or rGRF3 (line 11-15) develop shorter roots than the wild type (Col-0).
  • 39.
    Knockdown of MidgutGenes by dsRNA Transgenic Plant- Mediated RNA Interference in the Hemipteran Insect (Nilaparvata lugens) Leaves turn yellow initially and later brownish due to drying up of the plants. Under severe cases field gives a burnt appearance in concentric circles, known as "Hopper burn". RNAi is a powerful technique for functional genomics research against Hemipteran Insect (N. lugens) Transgenic plants producing dsRNA directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests. They damage rice directly through feeding and also by transmitting two viruses, rice ragge stunt virus and rice grassy stunt virus. Up to 60% yield loss is common in susceptible rice cultivars attacked by BPH (Brown Plant Hopper). Zha et al., 2011China 3 39
  • 40.
    Methodology/Principal Findings The Hemipteraninsect brown planthopper (Nilaparvata lugens) is a typical phloem sap feeder specific to rice (Oryza sativa L.) Analysis and identification of genes (Nlsid-1 and Nlaub) encoding proteins that involved in the RNAi pathway in N. lugens insect. Both genes are expressed ubiquitously in nymphs and adult insects. Nymphs40
  • 41.
    Three genes isolatedwere highly expressed in the N. lugens midgut and used to develop dsRNA constructs for transforming rice. 1) NlHT1- hexose transporter gene 2) Nlcar- carboxypeptidase gene 3) Nltry- trypsin-like serine protease gene Primary expression in midgut tissues with limited transcription in salivary glands, fat body and head 41 A A) The genes were cloned at the BamHI site of the hygromycin gene expression cassette in the pCU vector of Agrobacterium B) Southern blot analysis showed that the PCR-positive plants had 1–3 copies of the target coding sequences
  • 42.
    When nymphs werefed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced. Growth phenotypes of wild type (WT) plants, empty transformation vector plants and transgenic lines (A) Two-week-old seedlings (B) Four leaf stage Plants (C) Mature plants RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines.
  • 43.
    Genome-wide identification ofBrassica napus microRNAs and their targets in response to cadmium • MicroRNAs (miRNAs) also regulate response to environmental stresses. • The toxic heavy metal cadmium (Cd) induces expression of several miRNAs in rapeseed (Brassica napus). • miRNA-regulated gene silencing may be involved in plant tolerance to heavy metals. • In this study, four small RNA libraries and four degradome libraries were constructed from Cd-treated and non-Cd-treated roots and shoots of B. napus seedlings. • Using high-throughput sequencing, the study identified 84 conserved and non-conserved miRNAs (belonging to 37 miRNA families) from Cd-treated and non-treated B. napus, including 19 miRNA members that were not identified before. Zhou et al. 2012China 43 4
  • 44.
    44 • Some ofthe miRNAs were validated by RNA gel blotting. • Most of the identified miRNAs were found to be differentially expressed in roots/shoots or regulated by Cd exposure.
  • 45.
    • The studyalso identified 802 targets for the 37 (24 conserved and 13 non-conserved) miRNA families, from which there are 200, 537, and 65 targets, belonging to categories I, II, and III, respectively. • In category I alone, many novel targets for miRNAs were identified and shown to be involved in plant response to Cd. 45
  • 46.
    The Chloroplast TriggersDevelopmental Reprogramming When MUTS HOMOLOG1 Is Suppressed in Plants • To explore the control of phenotypy in higher plants, they examined the effect of a single plant nuclear gene on the expression and transmission of phenotypic variability in Arabidopsis thaliana. • MutS HOMOLOG1 (MSH1) is a plant-specific nuclear gene product that functions in both mitochondria and plastids to maintain genome stability. • Genetic hemicomplementation experiments show that this phenotypic plasticity derives from changes in chloroplast state. • The result of MSH1 suppression through RNAi, triggers a plastidial response process that involves non-genetic inheritance. Xu et al., 2012China 46 5
  • 47.
    Loss of MSH1by RNAi results in programmed phenotypic changes, including A) Altered phytohormone effects for dwarfed growth & reduced internode elongation B) altered leaf morphology C) Effects are partially reverted with the application of GA D) enhanced branching E) reduced stomatal density F) extended juvenility, with conversion to perennial growth pattern in short days G) delayed flowering 47
  • 48.
    Fig. 2. Phenotypicplasticity in the Arabidopsis msh1 mutant. 48
  • 49.
    Fig. 5. Evidenceof transcriptional and metabolic changes in Arabidopsis msh1 mutant and hemicomplementation lines. A) & B) quantitative RT-PCR assay C) RNA gel blot assay D) Heat map assay of metabolites Fig. 6. Hemicomplementation analysis of the Arabidopsis msh1 altered growth phenotype. Col-0, dual-targeted, and plastid-targeted MSH1 transgenic lines flowered uniformly, whereas the msh1 mutant and the mitochondria-targeted MSH1 transgenic line showed marked variation for growth, flowering time, and maturity. The Observed Developmental Reprogramming Is the Consequence of Chloroplast Changes 49
  • 50.
    Development of malesterility by silencing Bcp1 gene of Arabidopsis through RNA interference • Use of RNA interference (RNAi) technology to silence a male specific gene, Bcp1 in the model host Arabidopsis thaliana. • Bcp1 is active in both diploid tapetum and haploid microspores. • Three batches of explants (A. thaliana) were selected on herbicide Glufosinate ammonium and putative transgenes were confirmed through PCR and Southern hybridization. • The present study resulted in developing male sterile A. thaliana (Eco. Columbia) line through genetic engineering. Tehseen et al., 2010Pakistan 50 6
  • 51.
    • Bcp1 genecan be divided in two parts – 163bp non conserved region – 372bp conserved region Methodology/Principal Findings • Silencing of Bcp1 gene ,587bp in size, responsible for fertility They targeted 0.77kb regulatory region of Bcp1 gene via antisense • Expression of both sense and antisense fragment separated by an intron, yields more efficient silencing than only antisense Targeting coding sequence of Bcp1 using RNAi leads to male sterility • Bgp1,female fertility gene -- 87 % homology with conserved region -- To avoid silencing of female part, only non conserved region is targeted 51
  • 52.
    • 163bp regionof Bcp1 gene cloned in both sense and antisense orientation in pFGC5941 -Cloning of gene in sense and antisense orientation in same vector produce dsRNA inverted repeat molecule which induce PTGS in plant cells •Primers design w.r.t dsRNA binary vector pFGC5941 -It has two mcs, bar gene and 35S promoter within left and right borders -Two mcs flanked by intron of 1.364kb • Construct was transformed in A.tumefaciens strain LBA4404 by electroporation Agrobacterium culture was confirmed with PCR amplification The construct was transformed in Arabidopsis using leaf disc method 52
  • 53.
    • Formation ofsharp bands confirm the presence of transgenes – Transcribed mRNA of RNAi construct will result in a dsRNA with a hairpin loop – Resultant dsRNA triggered on the RNAi machinery • 3 batches of explants were selected on herbicide glufosinate ammonium Putative transgenic plants confirmed by PCR using bar gene specific primers and southern hybridisation It gives amplification along with positive and negative controls 53
  • 54.
    The Introgression ofRNAi Silencing of γ-Gliadins into Commercial Lines of Bread Wheat Changes the Mixing and Technological Properties of the Dough •The effects on dough quality by the RNAi-mediated silencing of γ - gliadins in commercial bread wheat lines (namely ‘Gazul’, ‘Podenco’ and ‘Arpain’) along with the transgenic line A1152 (cv. Bobwhite) were compared with their respective wild types. •The protein fractions were quantified by RP-HPLC, whereas the technological and mixing properties were assessed by SDSS test and by the Mixograph instrument. •The down-regulation of γ-gliadins resulted in stronger dough and a better tolerance to over-mixing in some transgenic lines. Gil-Humanes et al., 2012Spain 54 7
  • 55.
    Methodology/Principal Findings Wheat gliadingenes occur as tightly-linked clusters located on 1 and 6 chromosomes ω and γ-gliadins on Chromosome 1 having clusters of genes • Gli-A1 • Gli-B1 • Gli-D1 LMW-GS genes on the Glu3 loci α-gliadins on Chromosome 6 having clusters of genes • Gli-A2 • Gli-B2 • Gli-D2 Down-regulation of γ –gliadins through RNAi approach in three commercial lines and F1 hybrid obtained •After three generations of self-pollination, the gliadin and glutenin profiles analyzed by A-PAGE and SDS-PAGE. 55
  • 56.
    Glutenin content increasedand the ratio Gli/Glu reduced in the transgenic lines, provoking an increase of the dough strength and a decrease of the extensibility. Analysis of grain composition and quality parameters •The analysis of the variance (ANOVA) and LSD analysis of homogeneous groups showed significant differences in all the fractions of gliadins and glutenins. •SDSS volume showed an increase in all the transgenic lines w.r.t. the wild types, resulting in significantly higher volumes for transgenic lines G613, G626, G845 and G664. •In all parameters of Mixograph, no significantly different observed in the average of transgenic lines(except G622 & G626) to wild types, indicating that the mixing properties were not affected by the silencing of the γ-gliadins in the transgenic lines with different HMW-GS profile. 56
  • 57.
  • 58.
    Engineering of theRose Flavonoid Biosynthetic Pathway Successfully Generated Blue-Hued Flowers Accumulating Delphinidin Katsumoto et. al (2007)Japan o Rosa hybrida lacks violet to blue colour due to the absence of flavonoid 3’,5’-hydoxylase (F3’5’H) enzyme which produces delphinidin-based anthocyanins. o Other factors such as co-pigments and vacuolar pH also affect flower colour. o Expression of the viola F3’5’H gene  accumulates (~95% high) delphinidin  a novel bluish flower colour. 58 8
  • 59.
    • For moreexclusive and dominant accumulation of delphinidin irrespective of the hosts, the endogenous dihydroflavonol 4-reductase (DFR) gene was down- regulated and overexpressed the Iris3hollandica DFR gene in addition to the viola F3’5’H gene in a rose cultivar. • The resultant roses exclusively accumulated delphinidin in the petals, and the flowers had blue hues not achieved by hybridization breeding. • Moreover, the ability for exclusive accumulation of delphinidin was inherited by the next generations. 59
  • 60.
  • 61.
    WKS77 WKS82 WKS100 WKS116WKS124 WKS140 Rose Varieties transformed with pSPB130 and their flower colour changed are shown Schematic representation of T-DNA region of binary vectors constructed for colour modification  for the constitutive over expression of the viola F3’5’H BP40 gene and the torenia 5AT gene in rose. 61
  • 62.
    Host flower violet-coloured transgenic flower 98%delphinidin 98% delphinidin Madam Violet Seiryu  The vector pSPB919 is to down-regulate the endogenous rose DFR gene using RNA interference (RNAi) and to over express the iris DFR and the viola F3’5’H genes. 62
  • 63.
    • Northern blotanalysis of LA/919-4-10. • The expected sizes of the transcripts of viola F3’5’H BP40 (1.8 kb) and iris DFR (1.7 kb) genes & smaller size was detected for rose DFR mRNA (A). • A rose DFR probe detected about 23 bp small sized RNA, which was supposed to be a degraded endogenous rose DFR transcript with RNAi (B). • Delphinidin contents was confirmed in all transgenic (KmR) progeny of LA/919-4-10. • The flowers of F1 and F2 progeny contained exclusively delphinidin. 63 (si RNA)
  • 64.
    Production of picotee-typeflowers in Japanese gentian by CRES-T A B Wild type flower‐Solid color suppression of pigment production generates picotee type flower Nakatsuka et al., 2011Japan CRES-TChimeric repressor gene-silencing technology 9 64
  • 65.
    • (CRES-T) isan efficient gene suppression system which worked successfully in Japanese gentian. • A chimeric repressor of the anthocyanin biosynthetic regulator gene GtMYB3, under the control of the Arabidopsis actin2 promoter, was introduced into blue- flowered gentian. • Of 12 transgenic lines, 2 exhibited a picotee flower phenotype with a lack of pigmentation in the lower part of the petal. • HPLC analysis showed that the petals of these lines contained less anthocyanin and more flavone than the wild-type. • The expressions of ‘late’ flavonoid biosynthetic genes, including F3H, F35H, DFR and ANS, were strongly suppressed in petals of these transgenic plants. 65
  • 66.
    • Expression offlavonoid biosynthetic genes in transgenic gentian plants. • The expression levels of GtMYB3-SRDX and endogenous flavonoid biosynthetic genes were determined by semi- quantitative RT-PCR analysis in wild-type and GtMYB3- SRDX expressed transgenic gentian clone nos. 7 & 11 • Schematic representation of pSMABR-AtACT2pro- GtMYB3-SRDX. • Bar  herbicide bialaphos resistance gene as a selectable marker. • NOSp  promoter of nopaline synthase (NOS) gene from A. tumefaciens. • rbcSt  terminator of RuBisCO small subunit 2B gene from Arabidopsis. • NOSt  terminator of NOS gene. • LB  left border; RB  right border. semi-quantitative RT-PCR analysis 66
  • 67.
    Flavonoid analysis inthe flowers of transgenic gentian plants by HPLC A & D- wild type B & E- transgenic gentian clones no. 7 C & F- transgenic gentian clones no. 11 Anthocyanins Flavones 67
  • 68.
    Conclusions 68 Genetic engineering overcomesalmost all the limitations of traditional breeding approaches. Recent advances in plant molecular biology provide opportunities to use techniques of genetic engineering for improvement of crop plants for disease resistance, toxic resistance, plant architecture, male sterility, dough quality parameter, flower colour, etc. Case-1 Host plant derived pest resistance was achieved at field level against whiteflies by genetic transformation of tobacco which generated siRNA against the whitefly v-ATPaseA gene. Case-2 miR396- a key regulator, provides the nematode with a single molecular target to wield power over a substantial proportion of syncytium developmental events.
  • 69.
    69 Case-3 These genes (Nlsid-1and Nlaub) were identified and knockdown in N. lugens through RNA interference. The results demonstrate the potential of this technique at field- level control of plant-hoppers. Case-4 Identification of many high-quality target transcripts will help in the regulatory mechanism for plant tolerance to Cd. Case-5 Suppression of MSH1, which occurs under several forms of abiotic stress, triggers a plastidial response process that involves non-genetic inheritance. Conclusions Case-6 Bcp1 gene is responsible for fertile pollen development and active in both diploid tapetum and haploid microspores. Silencing Bcp1 gene lead to transgenic male sterile plants.
  • 70.
    70 Case 7 The down-regulationof γ-gliadins resulted in stronger doughs and a better tolerance to over-mixing in some transgenic lines. Case-8 Spectral difference in flower colour is mainly determined by the ratio of different classes of pigments and other factors and knowledge of flower coloration at the biochemical and molecular level has made it possible to develop novel color. Case-9 (CRES-T) is an efficient gene suppression system which worked successfully in Japanese gentian. Conclusions
  • 71.
    Future Prospects andNew Avenues 71 •Careful consideration of the interplay factors (such as the selection of the target gene, most effective site within the gene for knockdown, length of the targeted nucleotide sequence, and minimization of the off-target effects) is expected to deliver a competent technology in insect/pest resistance in the near future. •The regulatory unit represents a powerful molecular target for the parasitic animal to modulate plant cells and force them into novel developmental pathways. •More research efforts are needed for tolerance of crop plants toward heavy metal toxicity.
  • 72.
    72 • RNAi canbe employed to obtain hybrid seeds of commercially high valued crops. •New genes should be isolated that will have utility in floriculture, and new transformation methods for flower crops should be further optimized. • More advancement required for dough quality, as the reduction of γ-gliadins seems not to have a direct effect on the mixing and bread-making properties, the compensatory effect on the synthesis of the other prolamins has resulted in stronger doughs with improved over-mixing resistance in some transgenic lines. Future Prospects and New Avenues
  • 73.
    Earth is ourunique Masterpiece, can neither be replicate, transcribe nor translate it. We are only regulatory components of Earth, can save our planet by Downregulating environmental pollutions and Upregulating natural resources. - Mariya Zaman 73

Editor's Notes

  • #30 (A) Physical map of dsRNA expression cassette in pBI101. (B) transgenic and control tobacco plants showing comparable morphology. (C) selection of T1 seeds on kanamycin (300 mg/l) medium, showing non-transgenic seedlings turning white. (D) PCR analysis of transgenic lines in T1 generation by amplification of nptII gene (upper panel) and amplification of v-ATPaseA+RTM1 intron (lower panel), M; 100 bp DNA ladder; lanes 2–9, different transgenic tobacco lines, lane 10; positive control.
  • #36 Figure 1. Histochemical localization of GUS activity directed by miR396 promoters. A to F, Spatial expression patterns of miR396a and miR396b. A to C, GUS staining of miR396a:GUS plants showing expression in leaf hydathodes (A), vascular root tissues and the root cap (B), and absence of expression in the emerging lateral roots (C). D to F, GUS staining of miR396b:GUS plants showing expression in leaf hydathodes (D), vascular root tissues (E), and emerging lateral roots (F). G to N, Promoter activity of miR396a and miR396b during H. schachtii infection. Time-course experiments compared the expression of PmiR396a:GUS (G–J) and PmiR396b:GUS (K–N) transgenic plants at the second-stage (J2), early and late third-stage (J3), and fourth-stage (J4) juvenile time points. N indicates nematode, and S indicates syncytium. Bars = 2 mm in A and D and 100 mm in B, C, and E to N.
  • #37 Cyst nematodes are obligate parasitic roundworms that induce the formation of novel plant cell types that are associated in a unique feeding organ, the syncytium. The developmental stages of nematode is enabled by feeding from the syncytium. Syncytium formation encompasses the reprogramming of differentiated root cells, and these redifferentiations are accompanied and mediated by massive gene expression changes, which have been documented in diverse research approaches using soybean (Glycine max) and the soybean cyst nematode Heterodera glycines (Alkharouf et al., 2006; Ithal et al., 2007; Klink et al., 2009) and probably most extensively in Arabidopsis infected by the sugar beet cyst nematode Heterodera schachtii (Szakasits et al.,2009).
  • #39 Overexpression of miR396 and the target genes GRF1 and GRF3 negatively impacts root development. A and B, Transgenic plants overexpressing miR396a (line 22-5; A) or miR396b (line 15-1; B) develop shorter roots than the wild type (Col-0). C, Transgenic plants overexpressing rGRF1 (line 18-2) or rGRF3 (line 11-15) develop shorter roots than the wild type (Col-0). Homozygous T3 plants were planted on modified Knop’s medium along with the wild type, and 10- d-old seedlings were photographed.
  • #40 BPH infest the rice crop at all stages of plant growth. As a result of feeding by both nymphs and adults at the base of the tillers, plants turn yellow and dry up rapidly. At early infestation, round, yellow patches appear, which soon turn brownish due to the drying up of the plants. This condition is called 'hopper burn'. Temperature is a critical factor in the life activities of the insect. The hatchability and survival rate are the highest around 25 °C. Eggs are very sensitive to desiccation and soon shrivel when the host plant starts wilting. The population growth of brown plant hopper is maximum at a temperature range of 28 to 30 °C.
  • #49 Figure 2. Phenotypic plasticity in the Arabidopsis msh1 mutant. A, The msh1 mutant, grown under a 12-h daylength, is dwarfed and the frequency of the dwarf delayed-flowering phenotype increases with selection. B and C, Under a 10-h daylength, vegetative growth in the msh1 mutant is markedly extended, with plants displaying aerial rosettes (B) and extensive branching (C). These plants are 4 to 5 months old. D and E, The msh1 plants also display thickened stems (D) and secondary growth (E). Autofluorescence in stem cross-sections from the msh1 mutant and the Col-0 wild type shows plastids (red) in the cortex and lignin (blue-green) in the xylem. Light microscopy of toluidine blue-stained sections shows extensive secondary xylem in the mutant relative to the wild type (2003). F, Three-week-old Col-0 and msh1 plants grown in a 12-h daylength and showing evidence of a prolonged juvenile phase in the msh1 mutant. G, Dwarf msh1 plants show flowering time response to treatment with 100 mM GA twice per week. Arabidopsis flowering time was recorded as the date of first visible flower bud appearance (DAG, days after germination). At this time, total rosette leaf number was also recorded. Data shown are means 6 SE from 10 plants.
  • #50 Figure 5. Evidence of transcriptional and metabolic changes in Arabidopsis msh1 mutant and hemicomplementation lines. A, Results from quantitative RT-PCR analysis of the msh1 mutant showing transcript-level changes in several genes controlling growth (cyclin P4:1 and Expansin), GA3 (Gibberellin2 Oxidase6 and GA-STIMULATED ARABIDOPSIS6), and auxin levels (PIN1/PIN7 AUXIN EFFLUX CARRIERS, IAA7 AUXIN-RESPONSIVE PROTEIN, and CYTOCHROME P450 79B3) in the plant. B, Quantitative RT-PCR assay of transcript levels from the four flowering-related genes MIR156, FLC, SOC1), and SHORT VEGETATIVE PHASE (SVP) in Col-0 and msh1 plants. Data are shown as fold change relative to the wild type (Col-0) with means 6 SE from three biological replicates. C, RNA gel blot assay of rosette leaf and flower tissues for the flowering-related microRNAs miR156 and miR172. U6 was used as a loading control. D, A heat map with a subset of metabolites assayed in the study, comparing relative accumulation patterns in msh1, the mitochondrial hemicomplementation line (AOX), and the plastid hemicomplementation line (RUBP).