Nanotechnology and its role in plant disease management

1. “Nanotechnology: A Novel Approach for Genetic Material
Directed Plant Disease Management”
ICAR-Indian Agricultural Research Institute
Division of Plant Pathology
Speaker - Pankhuri Singhal, 20854
M.Sc. 2nd year
Seminar leader: Dr. T.K.Bag
Chairman: Dr. V.K.Baranwal
Credit seminar: Pl. Path. 691
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2. CONTENT
 Introduction
 Nanoscale
 Representative Types of Nanoparticles
 Nanoparticles Applications in Plant Pathology
 Nanoparticle Based Delivery of Genetic Material
 Comparative Study of Different Delivery Materials
 Representation of Nanoparticle Mediated Gene Transfer
 Case Study I
 Case Study II
 Conclusion
 Future Issues
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3. Nano scale
• The term nanotechnology is based on the prefix ‘nano’-greek word
meaning ‘dwarf’.
• Word nano means 10-9 or 1 billionth part of a metre.
• 1 nanometre = 1 billionth(10-9 ) part of a metre
• Size range between 1 to 100 nm
• They exhibit novel properties due to their large surface area to volume ratio
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4. 4

5. M.A.Alghuthaymi et al.,2015
Nanoparticles applications in plant pathology
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6.  Biopolymer Nanoparticles eg: Chitosan
 Metallic nanoparticles eg: Silver nanoparticles
Silica nanoparticles
Zinc nanoparticles
Copper nanoparticles
 Nanocomposites eg: Chitosan Silver NP
Silica-Silver NP
Nanoparticles in crop protection
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10. Silver nanoparticles
• Silver has been used as an antimicrobial agent since ancient
civilizations; it has been used extensively due to broad
spectrum and multiple modes of antimicrobial activity(Wei et
al.,2009)
• Silver exhibit higher toxicity to microorganism and lower toxicity
to mammalian cells.
• The application of silver nanoparticles as antimicrobial agents is
beacause of its economical production and multiple modes of
inhibitory action to microorganisms(Clement and Jarrett,1994).
• NP Ag has anti-fungal activity on Bipolaris sorokiana and
Magnaporthe grisea to reduce fungal diseases on perennial
ryegrass ( Jo et al.,2009).
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15. • ZnO NPs inhibited the growth of B.cinerea by
affecting cellular functions, which caused
deformation in fungal hyphae .
• ZnO NPs inhibited the growth of conidiophores
and conidia of Penicillium expansum, which finally
led to the death of fungal mats .
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16. Antifungal activities of ZnO NPs
against Botrytis
cinerea on PDA in the presence
of different concentrations
of ZnO NPs
Antifungal activities of ZnO NPs
against Penicillium
expansum on PDA in the presence
of different
concentrations of ZnO NPs
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17. Nanoparticle based delivery of genetic
material in plants
The biggest challenge for gene delivery in agricultural
crops is the plant cell wall.
Advantages:
 Nano particle approaches are applicable to both
monocot and dicot plants irrespective of tissue and organ
type.
 They can be used to overcome transgenic silencing via
regulating the DNA copies combined with nano particles.
 Nano particles can be easily functionalized for further
enhancement of transformation efficiency
 Nano particle-mediated multigene transformation is
possible without involving traditional methods that
require complex carriers.
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18. Method Advantages Disadvantages
Electroporation Safe easy and rather efficient Needs large amount of DNA and
optimization for every cell type
Microinjection Easy and highly efficient ,
Overcomes cytoplasmic degradation of DNA
Only one cell at a time can be
transfected
Not suitable for whole plant
Gene gun Can deliver thousands of DNA copies, high
transgene expression
Shallow penetration of the DNA into
the tissues,
Short term,low level of gene
expression
Chemical method Efiicient High toxicity
Agrobacterium
mediated gene
transfer
precise integration of genes,
transfer of desired DNA along with the
marker gene,
high frequency of stable and intact gene
transfer,
limitation to carry size base pair
(<500 kb),
chances of transgene silencing,
Effective only against dicot plants.
Nanoparticles Highly efficient, effective in both dicots and
monocots,can incorporate several functional
genes
Comparative study of different delivery systems
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A. 2012 . Current Nanoscience .8 : 170-179
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19. • Through the cell wall pore
diameter ranging from 5 to 20
nm(Moore 2006; Navarro et al.
2008).
• By endocytosis process, wherein a
cavity-like structure is formed
around the nanoparticles by a
phospholipid bilayer.
• NPs may also cross the membrane
using membrane-embedded
transporter proteins or through ion
channels. When NPs are applied
on the leaf surface, they can enter
through
• Through stomatal openings or
through the bases of trichomes
(Eichert et al. 2008; Fernandez and
Eichert 2009; Uzu et al. 2010)
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Mode of entry and transport

20. Representation of Nanoparticle Mediated Gene
Transfer
Source: Rai,M., Deshmukh, S. , Gade, A., Elsalam, K.A. 2012 . Current Nanoscience .8 : 170-179
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21. Nanoparticle Review
Gold nanoparticle
Iron Oxide nanoparticle
Carbon nanotubes
Calcium phosphate
nanoparticle
Silica nanoparticle
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Diversity of NPs for Nucleic acid delivery

22. Purpose: To prove MSNs can function as DNA delivery
agents
Plasmid containing a green fluorescent protein (GFP) gene under the control of constitutive
promoter is used
Optimal coating ratio for DNA/Type-II MSNs was 1/10
Type-II MSN bound DNA not digested by restriction enzyme
Transient GFP expression observed 36 hr after protoplasts were incubated with DNA coated Type-
II MSN
Conclusion:
 Type-II MSN system can serve as efficient delivery system for
protoplasts and make DNA accessible to transcription machinery
(Torney et al., 2007) 22

23. CASE STUDY I :
AIM : To investigate the potential use of sheet like clay nanoparticles,
specifically positively charge nanosheets as a dsRNA carrier for prolonged and
effective protection against plant viruses.
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24. Characteristics of LDH
nanosheets
dsRNA loading into
LDH nanosheets
SHAPE : Hexagonal
DIAMETER: 15-120nm
LATERAL DIMENSION : 20-80nm
LDH-bound dsRNA does not migrate and
can be seen as fluorescence in the well.
Complete loading was achieved at a
dsRNA–LDH mass ratio of 1:4 (lane 6)
dsRNA chain can be
observed between
LDH nanosheets
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25. Degradation of LDH and release of dsRNA
After exposure for 7 days, LDH residue
on the leaf showed a decrease in
amounts of aluminum and magnesium
by 28%and 22% respectively.
Droplets of LDH suspension were placed
on detached Nicotiana tabacum leaves
incubated at 27oC, 95% RH,5% CO2
CMV2b-dsRNA-LDH suspension was
incubated for a week under 5% CO2 or
normal atmospheric conditions
4 replicates exposed to 5% CO2 showed decrease
in amount of residual LDH-bound CMV2b-dsRNA
compared with sample incubated in normal
atmospheric CO2 conditions
This indicates that under environmental condition LDH degrades due to formation of
carbonic acid from CO2 in the water film on the leaf surface resulting in slow and
sustained release of dsRNA. 25

26. Uptake of dsRNA into plant cells and induction of RNAi
Bright field image (1st column), Cy3 florescence image (2nd
column), natural florescence of chlorophyll (3rd column)
and merged imaged of all three (4th column)
GUS activity in 12-day-old seedlings of
Arabidopsis transgenic line 6b4 treated with
MilliQ water, LDH and/or 100 μg of RNA 5 days
after germination; RNA treatments included total
RNA from E. coli HT115 carrying an empty L4440
vector (L4440), non-GUS-specific CMV2b-dsRNA,
GUS-dsRNA and GUS-dsRNA–LDH
These results demonstrate that both naked
dsRNA or dsRNA released from a dsRNA–LDH
complex was taken up by seedlings and induced
post-transcriptional downregulation of GUS
expression.
These observations suggest that dsRNA can be
taken up by the plant cells, either by passive
diffusion or by an active transport process. 26

27. Stability of dsRNA loaded into LDH
Stability and adherence of the dsRNA–LDH on the leaf surface
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28. Stability of dsRNA loaded into LDH
These results clearly show that the LDH nanosheets can protect large
dsRNA from nucleases, withstand washing and mediate sustained release
on the leaf surface over a period of at least 30 days.
The capability of LDH nanosheets to
protect dsRNA from Rnase
degradation was tested using control-
dsRNA
The stability of CMV2b-dsRNA versus
CMV2b-dsRNA–LDH sprayed onto N.
tabacum plants was assessed by
northern blot analysis
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29. BioClay spray for crop protection
These results clearly demonstrate that BioClay provides a longer
protection window than naked dsRNA
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30. BioClay spray for crop protection
BioClay provides significantly
higher and longer protection
than naked dsRNA
This systemic protection against CMV correlated
with the presence of large CMV2b-specific RNAs
in extracts from newly emerged, unsprayed leaves
of CMV2b-dsRNA–LDH-sprayed plants.
Collectively, our results show that CMV2b-BioClay application greatly
suppresses the impact of a subsequent virus infection in both the short
and long term, whereas CMV2b-dsRNA application is only effective for
short-term protection against the virus.
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31. Findings
LDH nanosheets can load large dsRNA to form dsRNA–LDH complexes referred to
as BioClay.
The dsRNA in BioClay is protected from nuclease activity, and can be detected on
the leaf surface even 30 days after topical spray.
BioClay facilitates sustained release of dsRNA on the leaf surface under ambient
conditions like CO2 and RH.
LDH nanosheets can be completely degraded over a period of time.
Topical spray of BioClay provides RNAi-based systemic protection to sprayed and
newly emerged unsprayed leaves against targeted viruses even when challenged
20 days after a single spray.
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33. CASE STUDY II :
HYPOTHESIS : The use of dsRNA as a template for growing AgNPs on GO
enhances the synergistic effect between AgNPs and GO by controlling
size, aggregation and distribution of AgNPs and by increasing the
adhesive force between Ag@GO composites and bacterial cell
membranes.
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34. Antibacterial activity of Ag@dsDNA@GO
The changes in the rod shape of X.perforans cell is due to wrapping of
Ag@dsDNA@GO around the X. perforans cell, leading to rapid cell
deformation, in agreement with the synergism between GO and AgNPs
The well-defined, intact membrane of rod
like X. perforans cells and the newly
divided normal X. perforans cell
After 20 hours incubation of X.perforans
cells with 20 ppm
Ag(18nm)@dsDNA@GO composites
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35. Mechanism of Antibacterial activity of Ag@dsDNA@GO
The GO sheet itself tends to nonspecifically attach and wrap bacteria, thereby
increasing the interaction between the bacteria and GO. Eventually the AgNPs
on GO cause direct and irreversible damage to the cell membrane by
denaturing proteins located on the cell wall and then entering the cell through
the bacterial cell wrapping process. The AgNPs and Ag+ ions released from
AgNPs then react with thiol, carboxyl, hydroxyl, amino, phosphate and
imidazole groups existing on and in the cell for cell inactivation and death.
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36. Time-dependent antibacterial effect of 20 ppm
Ag@dsDNA@GO composite on X. perforans
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37. To investigate the antibacterial properties of bare Ag NPs,
GO and Ag@GO,Ag@dsDNA@GO on X. perforans
The,Ag(5nm)@dsDNA@GO composites
displayed higher antibacterial activity
than the Ag(18nm)@dsDNA@GO
composites
The antibacterial activity towards
X. perforans of ~18 nm bare AgNPs
at different concentrations.
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38. Effectiveness of the Nanoparticles against Bacterial Spot
on Tomato
Treatment of tomato
transplants with
Ag(18nm)@dsDNA@G
O. (a) 16 ppm. (b) 50
ppm. (c) 100 ppm. (d)
Copper + Mancozeb
control, and (e)
Untreated control.
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39. Findings
The antibacterial activity of Ag@dsDNA@GO composites towards X. perforans, is
enhanced by the synergistic effect between AgNPs and GO.
Optimal antibacterial activity was observed with 20 ppm Ag(18nm)@dsDNA@GO
and 16 ppm Ag (5 nm)@dsDNA@GO composites in vitro after an incubation of
only 60 minutes.
The Ag@dsDNA@GO displayed higher antibacterial behavior compared to
Ag@GO composite synthesized by different methods, bare AgNPs solution or
bare GO solution.
Ag@dsDNA@GO at 100 ppm was applied on tomato transplants in a greenhouse
experiment, and significant reduction of disease caused by bacterial spot was
visually observed compared to the untreated control and the control treated with
copper + mancozeb.
Application of Ag@dsDNA@GO did not induce any phytotoxic effect on plant
leaves. 39

40. Conclusions
Nanoparticle mediated gene transfer methods are relatively
new and have potential to directly transfer DNA into
nucleus.
Nanoparticles have the capability to protect the nucleic
acid from nucleases.
Nanoparticle mediated gene transfer depends on defined
nanoparticle size, surface functionalization, nucleic acid
protection, and biocompatibility of nanoparticles.
Nanoparticles are biodegradable and target specific, so
they can be successfully employed in the production of
nano capsules for delivery of pesticide as well as genetic
material.
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41. Future Issues
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42. The country is in the need of ‘Second Green
Revolution ‘ (Singh 2012)
Nanoscale science and nanotechnologies are
envisioned to have the potential to revolutionize
agriculture and food systems(Norman and
Hongda,2013)
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