Nanotechnology and genetic engineering for plant pathology - By- AMOL VIJAY SHITOLE

1. We c me

4.  Whenever a new technology has emerged, it has opened many vistas to be explored.
The new Nanotechnology with materials having unique properties than their
macroscopic or bulk counter parts and has promised applications in various fields.
 Nanotechnology has the potential to increase agricultural productivity
through genetic improvement of plants, delivery of genes and drug molecules
to specific sites at cellular levels and nano-array based gene-technologies for
gene expressions in plants and animals under stress conditions.
 The essence of Nanotechnology is the ability to work at the molecular level, atom by
atom, to create large structures with fundamentally new molecular organization.
 The aim is to exploit these properties by gaining control of structures and devices at
atomic and molecular levels and to learn to efficiently manufacture and use these
devices.

5.  “Nanotechnology”, is a brain child of late Nobel laureate Richard Feynman.
 In 1959, he visualized the potential benefits of fabricating matter at nano-level.
 The heart of nanotechnology lies in the ability to compress the tools and devices to
the nanometer range and to accumulate atoms and molecules in to bulkier structures,
while the size remains very small.

6. One Japanese researcher Norio Taniguchi finally engineered materials at
nanometer scale in 1974 and coined the term ‘nanotechnology’.

7.  Nanotechnology is the art and science of manipulating matter at the
nanoscale.
 Nanotechnology is the study of making small microscopic things advance to
the future.
 Nanotechnology is the study of manipulating matter on an atomic scale.
 Nanotechnology refers to the constructing and engineering of the functional
systems at very micro level or we can say at atomic level.
Buckyball

8.  Nanos:
 Produce and manipulate matter of the size of 1 – 100 nm.
 Manufacturing at the molecular level- building things from Nano-scale
components.
 In the future, Molecular nanotechnology” will allow very complete control
over the placement of individual atoms.
 “A manufacturing technology able to inexpensively fabricate most
structures consistent with natural laws and to do so with molecular precision.”
 “The precision, placement, measurement, manipulation and modeling of
Nanometer scale matter”.

13. Fullerenes (buckyballs)
Nanotubes
Quantum dots Nanopowder
(Mesoporous silica powders)

14.  Size is reduced to less than 100 nm.
 The electronic structure of a nanocrystal critically depends on its size. For
small particles, the electronic energy levels are not continuous as in bulk
materials, but discrete.
 Quantum confinement :
 Thus, the properties of traditional materials change at nano level due to the
quantum effect and the behaviour of surfaces start to dominate the behaviour of
bulk materials.
 The optical, electrical, mechanical, magnetic and chemical properties can
be systematically manipulated by adjusting the size, composition and shape
of the nanoscale materials.
 The wide range of applications shown by nano materials is mainly due
to :
i) large surface area and (ii) small size.
Therefore, analytical tools and synthetic methods allow one to control composition
and design on this nanometer range and will undoubtedly yield
important advances in almost all fields of science.

15.  Delivery of pesticides or medicines is either provided as “preventative”
treatment or is provided once the disease organism has multiplied and
symptoms are evident in the plant or animal.
 Nanoscale devices are envisioned that would have the capability to detect
and treat an infection, nutrient deficiency or other health problem, long
before symptoms were evident at the macro-scale. This type of treatment
could be targeted to the area affected.
 Smart Delivery Systems” for agriculture can possess any combination of
the following characteristics: time-controlled, spatially targeted, self regulated,
remotely regulated, pre-programmed or multifunctional characteristics to avoid
biological barriers to successful targeting.
 It also can have the capacity to monitor the effects of the delivery of
pharmaceuticals, nutraceuticals, nutrients, food supplements, bioactive
compounds, probiotics, chemicals, insecticides, fungicides, vaccinations or
water to people, animals, plants, insects, soils and the environment.

16. Nanosized metals as diagnostic probes
Fluorescent silica nanoprobes - rapid diagnosis of plant diseases.
Fluorescent silica nanoprobes conjugated with the secondary antibody of goat anti-
rabbit IgG (Yao et al., 2009)
For detection of a bacterial plant pathogen Xanthomonas axonopodis pv. Vesicatoria
Nanoscale biosensor/ nanosensors
Small and portable - rapid response and real-time processing with accurate, quantitative,
reliable, reproducible, specific and stable results.
Detection of infection in non-symptomatic plant followed by targeted delivery of
treatment would be an essential component for precision farming.
Use of micromechanical cantilever arrays for detection of fungal spore (Aspergillus niger
and Saccharomyces cerevisiae) was demonstrated by Nugaeva et al. (2005).

17.  Quantum dots
 Few nm in diameter, roughly spherical (some rod like structures), fluorescent,
crystalline particles of semiconductors whose excitons are confined in all the
three spatial dimensions”.
 Important tool for detection of a specific biological marker in medical field with
extreme accuracy.
They have been used in cell labelling, cell tracking and DNA detection (Sharon et
al., 2010).

18.  Carbon nano material as a sensor
 Developed to act as electrode for electrochemical analysis (Sharon and Sharon,
2008).
 They have the potential to be developed as electro chemical sensor to detect
pesticide residue in plants.
 Nanofabrication
 Used in creating artificial plant parts such as stomata and xylem vessel.
 Useful for studding infection process and behavior of pathogens inside host
plant.
Eg. Uromyces appendiculatus and Colletotrichum graminicola.
 In other words, it would help formation of proper breeding strategy to screen for
or to develop disease resistant crop plants.

19.  Smart’ delivery
 An interesting and fascinating area of nanoparticles is ‘smart’ or targeted drug
delivery in the biological system.
 Gonza´lez-Melendi et al. (2008) was the first to report the penetration and
transport of nanoparticles inside whole plant. These results indicate the
possibility and potential of nanoparticles in delivery of substances inhibitory to
various plant pathogens.

20. 1) Nanosized silver:
Silver (Ag) is known to have antimicrobial activity both in ionic or
nanoparticle forms. The powerful antimicrobial effect of silver especially in unicellular
microorganisms is believed to be brought about by enzyme inactivation (Kim et al.,
1998).
E.g. Powdery mildew pathogen of rose.
2) Nanosized silica-silver:
Silica is well known to enhance stress resistance to plants including plant
diseases through promotion of plant physiological activity and growth (Kanto et al.,
2004) but it has no direct antimicrobial effect. It was found that smaller size of silver
nanoparticles was more effective against fungi.
Eg. Powdery mildew diseases of cucurbits.

21. 3) Mesoporous silica nanoparticles:
These are silica (SiO2) nanoparticles with regularly arranged pores which increase
the surface area of the nanoparticles.
Nano-copper was reported to be highly effective in controlling bacterial diseases viz.,
bacterial blight of and
leaf spot of mung.
4) Nano-iron:
Movement and behaviour of nanoparticles and their curative effect is being studied
more extensively involving humans.
Similar study to deliver the nanoparticles in the targeted site of a diseased plant has
been done by Corredor et al., (2009).
They applied iron nanoparticles coated with carbon to pumpkin plants for treating
specific plant part that is infected.

22. 5) Carbon nanotubes:
Carbon nanotubes have shown growth enhancing effect on tomato when grown in
soil containing carbon nanotubes (Khodakovsky et al., 2000).
It is believed that carbon nanotubes entered the germinating tomato seeds thus
facilitating water uptake and plant growth.

23. Manufacturers are developing nanoformulations.
Syngenta
Eg. Banner MAXX Fungicide (active ingredient propiconazole), Apron MAXX (active
ingredient fludioxonil) for seed treatments.
Similarly, cyclopropyl derivative of cyclohexenone (Primo MAXX) - plant growth
regulator but it helps the plant in withstanding abiotic as well as biotic stresses
including plant pathogens (Gogoi et al., 2009)
‘Nano-5’ is a marketed product and is projected as natural mucilage organic solution
to control several plant pathogens and pests besides improving crop yield.
A product of nanotechnology research in agriculture with the name of ‘Nano-Gro’
has been launched (Agro Nanotechnology Corp., Florida)

24. Plants treated with ‘Nano-Gro’
average yield increase of 20% with maximum of 50% in case of grain yield of
sunflower; increase in protein and sugar content by about 10% and plants can
fight various diseases.
‘Nano Green’
eliminate blast disease (Magnaporthe grisea) from infected rice plant. The test
was conducted in University of Georgia and the product was found to
outperform any other pesticide or fungicides currently in use in agriculture
(Gogoi et al., 2009).
Nano Fungicides, offered by
the company, use against all
kinds of fungal diseases.

25. Genetic manupulation for
development of resistance
Antisense RNA
Technology
Coat protein
mediated
resistance

26.  Messenger RNA (mRNA) is a single stranded molecule that is used as the
template for protein translation.
 Messenger RNA (mRNA) is single-stranded. Its sequence of nucleotides is
called "sense" because it results in a gene product (protein).
 It is possible for RNA to form duplexes, similar to DNA, with a second
sequence of RNA complementary to the first strand. This second sequence is
called antisense RNA
 The formation of double stranded RNA can inhibit gene expression in many
different organisms including plants, flies, worms and fungi.

27.  When antisense RNA (aRNA) is introduced into a cell, it binds to the already
present sense RNA to inhibit gene expression.
 The sense and antisense strands bind to each other, forming a helix. This double
helix is the actual suppressor of its corresponding gene.
 The suppression of a gene by its corresponding double stranded RNA is called
RNA interference (RNAi), or post-transcriptional gene silencing (PTGS).
 The gene suppression by aRNA is likely also due to the formation of an RNA
double helix, in this case formed by the sense RNA of the cell and the introduced
antisense RNA.

29.  Since the only RNA found in a cell should be single stranded, the presence of
double stranded RNA signals is an abnormality.
 The cell has a specific enzyme ( it is called as Dicer) that recognizes the
double stranded RNA and chops it up into small fragments between 21-25
base pairs in length.
 These short RNA fragments (called small interfering RNA, or siRNA) bind to
the RNA-induced silencing complex (RISC).
 The RISC is activated when the siRNA unwinds and the activated complex
binds to the corresponding mRNA using the antisense RNA. The RISC
contains an enzyme to cleave the bound mRNA (called Slicer ) and therefore
cause gene suppression.
 Once the mRNA has been cleaved, it can no longer be translated into
functional protein.

32.  Some viruses of both plants and animals have a genome of dsRNA. And many
other viruses of both plants and animals have an RNA genome that in the host
cell is briefly converted into dsRNA.
 So RNAi may be a weapon to counter infections by these viruses by destroying
their mRNAs and thus blocking the synthesis of essential viral proteins.
 Another instance where RNAi may be fruitfully applied is in the production of
banana varieties resistant to the Banana Bract Mosaic Virus (BBrMV), currently
devastating the banana population in Southeast Asia and India.

34.  “Coat protein-mediated resistance" is refer to the resistance caused by the
expression of a virus coat protein (CP) gene in transgenic plants.’’
 Accumulation of the CP confers resistance to infection and/or disease
development by the virus from which the CP gene was derived and by
related viruses.
 A number of plant virus nucleic acid sequences, including those encoding
virus coat proteins, have been found to be especially useful in the
development of virus-resistant plants by using CPMR.

35. 1. Isolation of cloned cDNA that encodes coat protein gene.
2. Selection of an appropriate transcriptional promoter.
3. Construction of coat protein gene.
4. Agrobacterium mediated Transformation.
5. Regeneration on selective medium.
6. Identify transformed plants.
7. Grow on general medium.
8. Coat protein expressed plant and not expressed plant then
infected with virus.
9. Compare the symptoms of disease development and time of
expression of symptoms.

36.  By using Nanotechnology and Genetic engineering techniques on hard
core basis we can arrest the loses caused due to plant diseases.
 The development of efficient and durable resistances able to withstand
the extreme genetic plasticity of plant diseases therefore represents a
major challenge for the coming years.
 Transfer of desirable genes for disease resistance can achieved through
nanobiotechnology.
 Antisense RNA Technology and coat protein mediated resistance are
helpful for developing disease resistance in crop plants which is
essential in management of plant viruses.

37.  Nanotechnology has wider uses in biotechnology, genetics,
plant breeding, disease control and allied fields etc.
 All these measures can help in improving genetic makeup of crop
plants.