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Welcome

ROLE OF NANOTECHNOLOGY AND

GENETIC ENGINEERING IN PLANT

PATHOLOGY

Introduction 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.

“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.

One Japanese researcher Norio Taniguchi finally engineered materials at

nanometer scale in 1974 and coined the term ‘nanotechnology’.

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

WHAT IS NANOTECHNOLOGY?

WHAT IS NANOTECHNOLOGY?

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”.

Fullerenes (buckyballs) Nanotubes

Quantum dots Nanopowder (Mesoporous silica powders)

NANOPARTICLES

MINIATURIZATION 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.

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.

SMART TREATMENT DELIVERY SYSTEMS

DETECTION AND OTHER USES OF NANO-TECHNOLOGY IN PLANT PATHOLOGY

Nanosized metals as diagnostic probesFluorescent 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/ nanosensorsSmall 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).

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).

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.

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.

NANO-PARTICLES CONTROLLING THE PLANT DISEASES

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.

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.

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.

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)

NANOFORMULATIONS

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.

Genetic manupulation for development of resistance

Coat protein mediated resistance

Antisense RNA Technology

GENETIC ENGINEERING IN MANAGEMENT OF PLANT DISEASES

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.

1. ANTISENSE RNA TECHNOLOGY

Christine Antler (2003)

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.

aRNA and RNAi

HOW ANTISENSE RNA BLOCKS TRANSLATION PROCESS?

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.

ANTISENSE RNA MECHANISM

Figure :Formation of antisense RNA blocks translation.

Christine Antler (2003)

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.

(Rodoni et al.,

1999).

WEAPON FOR MANAGEMENT OF PLANT VIRUSES.

2. COAT PROTEIN MEDIATED RESISTANCE AGAINST VIRUSES

“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.

(Roger N. Beachy et al., 1990)

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.A. Srivastava And S. K. Raj (2008)

STRATEGIES TO CREATE COAT PROTEIN-MEDIATED RESISTANCE

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.

CONCLUSION

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.