TRENDS IN BIOTECNOLOGY

1. 1
WELCOME
TO
SEMINAR SERIES 2018
(Odd series)
COLLEGE OFAGRICULTURE
JUNAGADH AGRICULTURAL UNIVERSITY
JUNAGADH

2. MINOR GUIDE
Dr. H. J. Kapadiya
Associate Professor
Department of Plant Pathology
College of Agriculture
Junagadh Agricultural University
Junagadh
MAJOR GUIDE
Dr. D. M. Jethva
Associate Research Scientist
Department of Entomology
College of Agriculture
Junagadh Agricultural University
Junagadh
ROLE OF GENE SILECING IN
PLANT DISEASE MANAGEMENT
Speaker
Mr. REPALLE NAGANNA
Ph. D (Agricultural Entomology)
II nd Year Ist Semester
Department of Entomology
College of Agriculture
Junagadh Agricultural University
Junagadh

3. CONTENT
1. Introduction
2. Status of Pesticides Consumption
3. Gene Silencing
✓ Application of gene silencing
✓ Cellular components of gene
silencing
✓ Types of gene silencing
✓ Mechanisms of gene silencing
4. Case Studies
5. Conclusion
6. Future Thrust

4. ➢ Agriculture is the backbone of Indian economy. In India around 70% of the
population earns their livelihood from agriculture.
➢ In view of the world’s limited croplands and growing population, it is
necessary to take all measures to increase crop production in order to ensure
food safety
➢ Crop yield losses in India due to pests, which include all biotic stresses such as
weeds, insect-pests, diseases, nematodes and rodents, range from 15 to 30%
➢ Disease are currently managed by excessive application of chemical
fungicides, which have drastic effects and their degraded products would flow
into the atmosphere, soils and rivers, resulting in the accumulation of toxic
substances and thus threatening human health and the environment.
➢ For the control of the diseases, continuous growing of the of transgenic plant
which contain resistant genes, leads to development of the resistant in the plant
pathogens
➢ In this contest, it time to know the new approaches to management of the
plant diseases, among the various approaches gene silecing have potential to
combat with diseases by suppression of the disease causing genes
4
INTRODUCTION

5. 5

7. TOP WORLD PESTICIDES CONSUMING COUNTRIES
Source: FAOSTAT 7

8. Table-1: CONSUMPTION OF CHEMICAL PESTICIDES IN INDIA
8
S. No. States 2013-14 2014-15 2015-16 2016-17
1 Andhra Pradesh 4253 4050 2713 1884
2 Bihar 765 787 831 840
3 Chhattisgarh 1016 1589 1625 1495
4 Goa 9 12 48 22
5 Gujarat 2330 1730 1980 1713
6 Haryana 4080 4070 4093 4050
7 Himachal Pradesh 344 379 450 341
8 Jammu & Kashmir 1723 1921 2251 2188
9 Jharkhand 430 650 493 541
10 Karnataka 1735 1793 1434 1279
11 Kerala 1276 910 1123 1070
12 Madhya Pradesh 987 696 732 694
13 Maharashtra 10969 8663 11665 13496
14 Orissa 1219 1075 723 770
15 Punjab 5723 5689 5743 5843
16 Rajasthan 2736 2694 2475 1252
17 Tamil Nadu 2142 2096 2096 2000
18 Telangana 3812 2862 2950 3840
19 Uttar Pradesh 10164 9736 10457 10142
20 Uttarakhand 174 172 217 131
21 West Bengal 3190 3060 3712 2624
Source: PPQS

9. Table-1: STATEWISE CONSUMPTION OF BIO PESTICIDES IN INDIA
S. No. States 2013-14 2014-15 2015-16
2016-17
(Provisional)
1 Andhra Pradesh 45 53 25 27
2 Bihar 243 252 286 314
3 Chhattisgarh 224 284 370 340
4 Goa 6 12 14 3
5 Gujarat 605 279 273 278
6 Haryana 356 260 292 380
7 Himachal Pradesh 35 15 16 2
8 Jammu & Kashmir 0.04 0.05 0.50 1
9 Jharkhand 1 3 7 11
10 Karnataka 450 530 505 473
11 Kerala 626 631 606 622
12 Madhya Pradesh 160 309 395 369
13 Maharashtra 1433 486 1173 1454
14 Orissa 223 267 271 271
15 Punjab 89 136 138 174
16 Rajasthan 10 157 9 8
17 Tamil Nadu 257 286 286 301
18 Telangana 77 82 94 85
19 Uttar Pradesh 42 43 46 46
20 Uttarakhand 26 22 30 15
21 West Bengal 655 680 950 838
Source: PPQS 9

10. ➢ Gene silencing is a general term describing epigenetic processes
of gene regulation. The term gene silencing is generally used to
describe the "switching off" of a gene by a mechanism other
than genetic modification. That is, a gene which would be
expressed (turned on) under normal circumstances is switched off
by machinery in the cell.
➢ Genes are regulated at either the transcriptional or post-
transcriptional level.
➢ Both transcriptional and post- transcriptional gene silencing are
used to regulate endogenous genes.
➢ Gene silencing is often considered the same as gene knockdown.
When genes are silenced, their expression is reduced.
➢ In contrast, when genes are knocked out, they are completely erased
from the organism's genome and, thus, have no expression.
➢ Gene silencing is considered a gene knockdown mechanism since
the methods used to silence genes, such as RNAi, CRISPR, or siRNA,
generally reduce the expression of a gene
10
GENE SILENCING

11. Application of gene silencing to plant disease
management
➢ Tissue-specific or inducible gene silencing
➢ Silence several genes simultaneously and protect crops
against destructive pathogens
➢ Multiple viruses can be simultaneously targeted,
➢ Gene silencing also functions as a natural antiviral defense
mechanism
➢ To counteract the defense mechanism, many viruses encode
a protein called viral suppressor of RNA (VSR) silencing
➢ Broader resistance with high efficacy
➢ Inherently suppress the gene in the of the plants population
11

12. CELLULAR COMPONENTS OF GENE SILENCING
1. MicroRNAs (miRNAs): are gnomically encoded non-coding
RNAs that help regulate gene expression, particularly during
development.
2. Small interference RNAs (siRNAs): siRNAs derived from long
dsRNA precursors, siRNAs typically base-pair perfectly and induce
mRNA cleavage only in a single, specific target
3. Dicer: Dicer is the key enzyme initiating the RNA-silencing process.
It is a dsRNA specific Ribonuclease III-like endonuclease which
cleaves the target dsRNAs into fragments
4. RNA-induced silencing complex (RISC): are endonucleases called
argonaute proteins, which cleave the target mRNA strand
complementary to their bound siRNA. As the fragments produced
by dicer are double-stranded, they could each in theory produce a
functional siRNA. However, only one of the two strands, which is
known as the guide strand, binds the argonaute protein and directs
gene silencing
12

13. 5. Argonaute proteins: Argonaute proteins are the direct binding
partners of siRNAs and form the core of RISC
6. Histones: are highly alkaline proteins found in eukaryotic cell nuclei
that package and order the DNA into structural units called
nucleosomes. They are the chief protein components of chromatin,
acting as spools around which DNA winds, and playing a role in
gene regulation.
7. Chromatin and Heterochromatin: Chromatin is a complex
of macromolecules found in cells, consisting of DNA, protein,
and RNA. Heterochromatin is a tightly packed form
of DNA or condensed DNA, which comes in multiple varieties.
8. Transposons: A transposable element (TE or transposon) is a DNA
sequence that can change its position within a genome, sometimes
creating or reversing mutations and altering the cell's genetic
identity and genome size. Transposition often results in duplication
of the same genetic material.
13

14. 14

15. TYPES OF GENE SILENCING
1. TRANSCRIPTIONAL
1. RNA-directed DNA methylation
2. Transposon silencing (Histone Modifications).
3. Transgene silencing
2. POST-TRANSCRIPTIONAL
1. RNA interference
3. MEIOTIC
1. Transvection
2. Meiotic silencing of unpaired DNA
15

16. 16
TRANSCRIPTION
Transcription is the first step of gene expression, in which a particular
segment of DNA is copied into RNA (especially mRNA) by the enzyme RNA
polymerase.

17. 1. Transcriptional gene silencing (TGS)
➢ Transcriptional gene silencing is the result of histone
modifications, creating an environment of heterochromatin
around a gene that makes it inaccessible to transcriptional
machinery (RNA polymerase, transcription factors, etc.).
➢ A form of silencing identified by run-on experiments showing that
transcription initiation is blocked in the nucleus
➢ In plants TGS and DNA methylation can be induced by either
dsRNA or viral infection
2. Post-transcriptional gene silencing (PTGS)
➢ Post-transcriptional gene silencing is the result of mRNA of a
particular gene being destroyed. The destruction of the
mRNA prevents translation to form an active gene product (in
most cases, a protein). A common mechanism of post-
transcriptional gene silencing is RNAi.
3. Meiotic
➢ DNA unpaired in meiosis causes silencing of all DNA homologous
to it, including genes that are themselves paired.
17

20. INTER RELATED MECHANISMS
OF
TRANSCRIPTIONAL GENE SILENCING
20

21. DNA METHYLATION
➢ DNA methylation is a biochemical process that is important for
normal development in higher organisms.
➢ DNA methylation is a process by which methyl groups are added to
the DNA molecule. Methylation can change the activity of a DNA
segment without changing the sequence.
➢ DNA methylation typically acts to repress gene transcription.
➢ DNA methylation is essential for normal development and is
associated with a number of key processes including genomic
imprinting, repression of transposabl elements, aging etc.,
➢ Two of DNA's four bases, cytosine and adenine, can be methylated.
Cytosine methylation is widespread in both eukaryotes and
prokaryotes. Adenine methylation has been observed in bacterial, plant
and recently in mammalian DNA
21

22. MECHANISM OF DNA METHYLATION
➢ DNA methylation is addition of methyl groups by a family of
enzymes, DNA Transferases (DNMTs) at the 5′-position of
cytosine residues in CMethyl pG dinucleotides (cytosine-guanosine
dinucleotides ) by transferring methyl groups from S-adenosyl
methionine (SAM), thus 5-methylcytosine is formed.
➢ Most CpG dinucleotides are often grouped in clusters at the 5′-
regulatory regions of many genes, which are called CpG islands.
DNA methylation of promoter CpG islands generally can regulate
gene expression
➢ These islands are typically found in or near promoter regions of
genes, where transcription is initiated.
➢ In CpG islands that are un- methylated, the nucleosomes are in
a more open configuration that allows access of factors that
favor transcription
➢ When CpG islands are hypermethylated, as in heterochromatin,
the nucleosomes become more tightly compacted, inhibiting
the access of regulatory 22

23. DIAGRAMMATIC REPRESENTATION OF DNA METHYLATION
23

24. 24

25. ➢ Methylation controls gene activity, it alone is insufficient to
repress transcription. The local chromatin structure also
contributes in determining whether genes are transcribed or
repressed.
➢ The chromatin structure surrounding un-methylated,
transcriptionally active genes differs from that of methylated
silenced genes.
➢ Both nucleosome structure and histone acetylation affect
chromatin structure and thus regulate gene transcription
➢ Histones are the core protein components of nucleosomes and
their acetylation status regulates, in part, gene expression.
➢ Histone acetyltransferases and histone deacetylases (HDACs)
determine the acetylation status of histones. This acetylation affects
the regulation of gene expression, and inhibitors of HDACs have
been found to cause growth arrest
25
HISTONE DEACETYLATION

26. MECHANISM OF HISTONE DEACETYLATION
➢ Acetylation is the process where an acetyl functional group is
transferred from one molecule (in this case, Acetyl-Coenzyme A)
to another.
➢ Deacetylation is simply the reverse reaction where an acetyl
group is removed from a molecule.
➢ Histone acetylation is determined by the activities of two classes
of enzyme: Histone Acetyl Transferases (HATs) and Histone
Deacetylases (HDACs).
➢ Deacetylated histones are generally associated with silencing
gene expression
➢ Acetylation of histones is generally associated with of gene
expression
➢ Several types of compound have been identified that inhibit
HDACs
26

27. ➢ Acetylation removes the positive charge on the histones,
thereby decreasing the interaction of the N termini of
histones with the negatively charged phosphate groups
of DNA.
➢ As a consequence, the condensed chromatin is
transformed into a more relaxed structure that is
associated with greater levels of gene transcription.
➢ This relaxation can be reversed by HDAC activity.
Relaxed, transcriptionally active DNA is referred to
as euchromatin. More condensed (tightly packed) DNA is
referred to as heterochromatin.
27

28. 28

29. 29

31. RNA INTERFERENCE (RNAi)
➢ RNA interference (RNAi) refers to the ability of double-stranded
RNAs to shut down the expression of a messenger RNA with which
they have sequence in common.
➢ RNA interference (RNAi) is a biological process in
which RNA molecules inhibit gene expression or translation, by
neutralizing targeted mRNA molecules.
➢ Historically, it was known by other names, including co-
suppression, post-transcriptional gene silencing (PTGS), and quelling.
➢ Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in
Physiology or Medicine for their work on RNA interference in
the nematode worm Caenorhabditis elegans, which they published in
1998. Since the discovery of RNAi and its regulatory potentials, it has
become evident that RNAi has immense potential in suppression of
desired genes.
31

32. APPLICATIONS OF RNAi IN PLANT IMPROVEMENT
✓Alterations in plant architecture related to plant
height, shoot branching, stem elongation, and leaf
and inflorescence morphology
✓Abiotic stress tolerance
✓Biotic stress resistance
✓Deletion of allergens
✓Development of male sterile plants
✓Engineering of secondary metabolites
✓Removal of toxic compounds
32

33. SALIENT FEATURES OF RNAi
✓ Double stranded RNA rather than single–stranded
antisense RNA is the interferring agent.
✓ High degree of specific gene silencing with less
effort.
✓ Highly potent and effective, only a few double stranded
RNA molecules per cell are required for effective
interference.
✓ Silencing can be introduced in different developmental
stages systemic silencing
✓ Avoids problems with abnormalities caused by a
knocked out gene in early stages which could mask
desired observations.
✓ Silencing effects passed through generations
33

34. Mechanism of RNA interference (RNAi)
➢ RNAi is an important and natural anti defense mechanism
➢ Two types of small ribonucleic acid (RNA) molecules – micro
RNA (miRNA) and small interfering RNA (siRNA) – are central to
RNA interference. RNAs are the direct products of genes, and these
small RNAs can bind to other specific messenger RNA (miRNA)
molecules and either increase or decrease their activity, Both miRNAs
and siRNAs share a common RNase-III processing enzyme, Dicer, and
closely related effector complexes and both can regulate gene expression
at the post transcriptional level.
➢ Dicer is one of the enzymes involved in RNAi mechanism that is
encoded by a variable number of genes and presents distinct specificity
among organisms
➢ The most recognized RNAi pathways are the siRNA and miRNA;
despite being triggered by different molecules, both precursors are long
double-stranded RNAs (dsRNAs).
34

35. ➢ An siRNA-containing effector complex is referred to as an “RNA
Induced Silencing Complex” (RISC), and an miRNA-containing
effector complex is referred to as an miRNP
➢ In these complexes, the regulation is at a post transcriptional level
and every RISC or miRNP contains a member of the Argonaute
(Ago) protein family . For the regulation at the transcriptional level
as guided siRNAs, a specialized nuclear Argonaute-containing
complex, known as the RNA-Induced Transcriptional Silencing
complex (RITS) mediates gene silencing
➢ In general, one strand of the short-RNA duplex (the guide strand) is
loaded onto an Argonaute protein at the core of the effectors
complexes. During loading, the non guide strand is cleaved by an
Argonaute protein and ejected. The Argonaute protein then uses the
guide RNA to associate with target RNAs that contain a perfectly
complementary sequence and then catalyzes the slicing of these
targets, either to be cleaved by RISC, to be blocked for translation in
miRNP or by inducing histone modifications in RITS.
CONT…..
35

36. Diagrammatic representation of RNAi Mechanism
36

37. 37

38. 1. Agroinfiltration: The injection of Agrobacterium carrying similar
DNA constructs into the intracellular spaces of leaves for triggering
RNA silencing is known as agroinoculation or agroinfiltration
2. Micro-Bombardment : A linear or circular template is transferred
into the nucleus by micro-bombardment. Synthetic siRNAs are
delivered into plants by biolistic pressure to cause silencing.
Bombarding cells with particles coated with dsRNA, siRNA or
DNA
3. Virus Induced Gene Silencing (VIGS): Modified viruses as RNA
silencing triggers are used as a mean for inducing RNA in plants.
Different RNA and DNA viruses have been modified to serve as
vectors for gene expression
4. Host Induced Gene Silencing (HIGS): Host plants have siRNA
carrying the fungal target gene can move from plant cell to fungal
cell via extrahaustrial matrix and triggers the RNAi in fungal cells.
METHODS TO DELIVER OR INDUCE RNAi IN PLANTS
38

39. AGROBACTERIUM MEDIATER AND MICROBOMBARDMENT
39

40. AGROINFILTRATION
40

41. VIRUS INDUCED GENE SILENCING (VIGS):
41

42. HOST INDUCED GENE SILENCING (HIGS)
42

43. METHODS TO DELIVER OR INDUCE RNAi IN PLANTS
43

44. 44
Pathogen Targeted region
Magnaporthae oryzae eGFP
Cladosporium falvum cgl 1 and cgl 2
Venturia inaequalis Multiple inverted repeats
Blumeria graminis Mlo
Table-3: RNAi EFFECTS ON TARGETED REGION IN PLANT
PATHOGENS.

45. Table-4: RNAi IN VARIOUS PLANT-VIRUS SYSTEMS
Virus Targeted region
Barley stripe mosaic virus pds
Cabbage leaf curl virus gfp, CH42, pds
Potato virus X pds, gfp
Tobacco mosaic virus pds, psy
Pea early browning virus pspds, uni, kor
Tomato golden mosaic virus su, luc
Tobacco rattle virus Rar1, EDS1,
Tomato yellow leaf curl China pcna, pds, su, gfp
45

46. Table-5: RNAi EFFECT ON TARGETED REGION OF NEMATODES
Nematode Targeted region RNAi affect
M. incognita
Cysteine proteinase
Delayed development, Decrease in established
nematodes
Secreted peptide
16D 10
Reduction in gall formation and established
nematode population
H. glycines
Cysteine proteinase Increased male: female ratio.
C-type lectin Reduction in established nematodes population
Secreted peptide
SYV46
Decrease in established nematode population
G. pallida
Cysteine proteinase Increase in male: female ratio.
FMR Famide-like
peptides
Motility inhibited
G.rostochiensis Chitin synthase Delay in egg hatch
Hschachtii
Suc transporter
genes
Reduction of female nematode development
46

49. Overexpression of CaAMP1 for control of Fusarium
oxysporum f. sp. matthiolae and Alternaria brassicicola.
Figure-1: Inhibitory effect of the CaAMP1 protein on conidial germination and hyphal
growth of A. brassicicola and F. oxysporum f. sp. matthiolae.
Lee et al. (2008)
Korea 49

50. Host induced gene silencing (HIGS) of vital fungal genes in
transgenic banana plants against Fusarium wilt
Figure-2: Transgenic Fusarium wilt resistant banana plants expressing the two ihpRNAs (targeted
against vital Fusarium genes, Petunia floral defensins, antimicrobial peptides genes) show efficient
resistance towards Fusarium wilt whereas the untransformed control plants show marked Fusarium
wilt symptoms after a 6 weeks long bioassay with Foc race 1 inoculate
Upendra et al. (2014)
India 50

51. Plant-mediated gene silencing restricts growth of the potato
late blight pathogen, Phytophthora infestans
Figure-4: Silencing of hp-PiGPB1 targeting the G
protein β-subunit (PiGPB1) important for
pathogenicity resulted in most restricted disease
progress.
Figure-5: Phenotypes of sporangia collected at
24, 48, 72, and 96 hpi from wild-type and hp-
PiGPB1 transgenic plants
Sultana et al. (2015)
Sweden 51

52. Effects of BMV-derived host-induced gene silencing (HIGS) on Magnaporthe
oryzae genes
Lin et al. (2017)
China 52
Figure-3: Rice blast disease phenotype in the control and silenced rice plants. Plants were treated with
BMV vectors harboring the MoABC1, MoMAC1, or MoPMK1 pathogenicity genes

53. Host Delivered RNAi, an efficient approach to increase rice
resistance to sheath blight pathogen, Rhizoctonia solani
Tiwari et al. (2017)
India 53
Figure-6: Silenced of Pathogenicity Map Kinase 1 (Pmk1) , RPMK1-1 and RPMK1-2
genes shows decrease in fungal infection levels compared to non-transformed controls
and the observed delay in disease symptoms

54. Host-induced silencing of essential genes in Puccinia triticina through
transgenic expression of RNAi sequences reduces severity of leaf rust
infection in wheat
Figure-7: Leaf rust disease resistance in transgenic wheat T1 lines. Pt- inoculated leaves of
representative transgenic wheat T1 lines expressing the hp-PtMAPK1RNAi (a) or hp-PtCYC1RNAi
(b) construct show suppressed disease development with low infection types (IT), compared to
nontransformed control (NTC) plants. %, represents per cent fungal biomass reduction
Panwar et al.(2018)
India 54

55. Transgenic Arabidopsis plants expressing Bc-siR37
exhibited plant defense against B. cinerea
Wang et al.(2018)
USA
Figure-8:The host target genes of Bc-siR37 are involved in plant immunity
against B. cinerea. The lesion sizes were calculated 4 dpi
55

57. RNA-interference against Rice tungro bacilliform virus in rice plants
Figure-10: Transgenic rice plants expressing DNA encoding ORF IV of RTBV.
C. Non-transgenic control plants. T. Progenies of line RTBV-O-Ds2, panicle emergence in one RTBV-
O- Ds2 plant is indicated by arrow
Tyagi et al. (2008)
India 57

58. RNAi mediated Transgenic strategies to confer resistance
against viruses in rice plants
Figure-9: The transgenic rice plants with the introduced RNAi construct targeting the RDV gene for
Pns 6, P8. and Pns12 were almost immune to RDV infection. (A) Rice stripe virus (RSV). (B) Rice
grassy stunt virus (RGSV). Rice plants in pots from left to right: mock-inoculated non-transgenic rice
plants (Nt) normal growth; virus-inoculated transgenic rice plants (T), showing healthy growth and
fertility after inoculation; virus-inoculated non-transgenic rice plant (Nt), showing typical symptoms
caused by RSV or RGSV infection.
Sasaya et al. (2014)
Japan 58

59. Resistance to viral yellow leaf curl in tomato through RNAi
targeting two Begomovirus species strains
Figure-11: RNAi-transformed R1 plants expressed fewer symptoms than untransformed control plants
after exposure to the virus. R1 plants carrying the bi-viral RNAi construct Ty-11 three WAE to
ToLCTWV (a) and TYLCTHV (b) incomparison to the untransformed control CLN1621L
Chen et al. (2016)
Taiwan 59

60. RNAi-mediated transgenic rice resistance to Rice stripe virus
Figure- 12: The RNAi expression vector construct of full RSV NCP gene
A. typical disease symptoms in infected plants (a1 & a2 wild type (WT). B. T0 of
transgenic seedlings (b1 is WT plant, the others are transgenic seedlings categorized as
class 0 or 1). C. T2 of transgenic seedlings (c1, transgenic line &c2, WT plant). D. T5
of transgenic seedlings (d1, d2, transgenic line: d3, WT seedlings).
Cheng et al. (2016)
China 60

62. RNAi-mediated gene silencing confers resistance to crown gall
disease in tomato
Figure-13: A. tumefaciens tumorigenesis on tomato Transgenic (a) and wild-type (b) tomatoes were
infected with A. tumefaciens by piercing the stem with a syringe and extruding a small amount of
bacterial suspension into the wound.
Matthew et al. (2001)
USA 62

63. Involvement of the Pepper Antimicrobial Protein CaAMP1 Gene in
resistant against Pseudomonas syringae pv tomato.
Figure-14: (L) Inhibition of fungal pathogen infection in CaAMP1-OX transgenic Arabidopsis
lines Leaves from wild-type (Ws-0) and CaAMP1 transgenic plants at 5 d after inoculation. (R)
Lesion diameters were measured at 5 d after inoculation with A. brassicicola.(#1, #2, and #3
significant transgenic lines)
Lee et al. (2008)
Korea 63

65. RNAi silencing root-knot nematode parasitism gene
Figure-15: (L). RNAi silencing of 16D10 in pre parasitic M. incognita J2. Fluorescence
microscopy showing ingestion of FITC in the treated J2. (R). Wild-type Arabidopsis roots
inoculated with control J2 (Upper) or full-length 16D10 dsRNA treated J2 (Lower) showing
numerous larger galls (Upper) or fewer small galls (Lower) 7 weeks after inoculation, respectively.
Huang et al. (2006)
USA 65

66. Host Delivered RNAi of Two FMRF Amide Like
Peptides, flp-14 and flp-18, for the Management of Root
Knot Nematode, Meloidogyne incognita
Figure-16: Stained nematodes in the infected tomato roots (i) Roots inoculated with J2s
soaked in water (control) (ii) Roots inoculated with J2s soaked in dsRNA of GFP
(Unrelated control) (iii) Roots inoculated with J2s soaked in dsRNA of flp-14 (iv) Roots
inoculated with J2s soaked in dsRNA of flp-1
Pradeep et al. (2013)
India 66

67. Effect of in vitro RNAi on nematode attraction and penetration.
Figure-17: (B) Penetration into tomato roots at 24 h (C) Percentage reduction in
nematode penetration in tomato roots due to RNAi compared to control
Pradeep et al. (2013)
India 67

68. CONCLUSION
➢ Minimize the chemical fungicides for the disease management
in food crops is essential component of the sustainable
agriculture and environment protection.
➢ By adoption of the gene silecing approaches for the
management of the plant diseases, we can achieve UN
sustainable development goals of reduce the poverty, hunger
and food insecurity of the growing world population
➢ The field of gene silencing is moving at an impressive pace
and generating exciting results associated with RNAi,
transgene silencing and transposons mobilization.
➢ This technology can be considered an eco-friendly, biosafe
and ever green technology as it eliminates even certain risks
associated with development of transgenic plants carrying
first generation constructs
➢ Finally, it cab be conclude that the gene silencing is emerging tool
for the for the plant disease management and protection of the
environment
68

69. ➢ Gene silencing technology has been successfully applied in
a number of studies to develop plants with improved
resistance to biotic stresses.
➢ However, there are substantial gaps in our knowledge
about the RNAi mechanism in agriculturally important
pathogenic fungi and their host fungal interactions.
➢ Further, research is needed to expand the usefulness of this
valuable genomics research tool for important fungal
pathogens.
➢ However, the major obstacles hindering its immediate
applications include selection of targeting sequences and in
the delivery methods
➢ Logical questions will require to be answered about off-
target effects, non-target effects, and the impact of genetic
mutations
➢ In future opportunities, gene silencing may even hold
guarantee for development of gene-specific therapeutics or
a complete understanding of genomics.
FUTURE THRUST
69

70. THANK YOU