9:40 AM
Unknown
Badri Khanal[1]
Abstract:
Plant
biotechnologist are facing many challenges in development of disease resistant
crop varieties. Unlike many insect resistant and herbicide tolerance cultivars,
disease resistant has only been success in few crop cultivars. Lot of efforts
have been put by conventional breeding, but genetic engineering (GE) techniques
are more effective. Some of the common techniques are: recombinant-DNA,
RNA-interference, monococol antibody, use of different proteins phytoalexins, resistance genes and RNA silencing. Each
of these techniques utilizes a different approach to deliver DNA into the
vicinity of chromosomes into which the DNA may then integrate. These techniques has resulted in
successful control of many economically important plant diseases, however these
are still to be replicated for commercial application in plant disease control.
Introduction:
Diseases of plants are one of the important threats
to world agriculture. Agricultural and horticultural crop species are attack by
many pathogens “(see Glossary)”, which cause loss of yield
significantly. Fungi alone causes more than 70 % of all major crop diseases [1]. Chemicals are mostly used for control of crop diseases. For some
diseases, chemical control is very effective for some of the diseases; whereas it
is often non-specific in its many associated effects, which kills beneficial
organisms along with pathogens. Health, safety and environmental risks are
undesirably affected by chemical control. Many cultivars resistant to various
diseases are developed using traditional breeding methods. Nevertheless, this
process is time-consuming and this means has left little room for improvements
due to limited availability of genetic resources. There are numbers of reasons
for the availability of limited genetic resources for breeding. Two most
important ones are: i) gene pool loss during the domestication and breeding of plants
and ii) many of the gene traits which may be beneficial in one plant tissue like
seeds and fruits, may be deleterious in other vegetative tissues [1].
Genetic engineering or
biotechnology has been identified as one key approach for increasing
agricultural production along with reducing losses due to both abiotic and
biotic stresses in the field as well as storage [2]. Since past
few decades, breeding possibilities have broadened by genetic engineering and
gene transfer technologies including mapping of gene and identification of the
sequence of genome of model crops and plants. Transcriptomics, proteomics
and metabolomics and similar modern technologies are now proved to be important
in knowing plant metabolic pathways and the role of important genes associated
with their regulation. This facilitates new ideas into the complex metabolic
neighborhoods which give rise to a given phenotype and may modify a given
pathway by discovery of new target genes. These new gene can then be subject to
different metabolic engineering efforts and applications [3].
Genetic
engineering is proved as potential tools to develop a cornucopia of beneficial
plant traits, particularly an enhanced capability to resist or withstand attack
by different plant pathogens. New biotechnological approaches to plant disease
control are very important particularly for pathogens that are difficult to
manage or control by existing methods [4]
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Glossary
Candidate genes: The candidate gene approach to
conducting genetic association studies focuses on associations
between genetic variation within the pre-specified genes of
interest and phenotypes or disease states.
Genetic Engineering: GE is the modification of genetic
composition of an organism by artificial means, often involving the
transfer of specific genes or traits, from one organism into a plant (or
animal) of an entirely different species.
Genetically Modified Organisms (GMOs): It can be defined as organisms (i.e.
plants, animals or microorganisms) in which the genetic material (DNA)
has been modified in such a way that does not occur naturally by any mating
and/or natural recombination.
In-vitro selection: It is
method used to screen large number of plants and/ or cells for a certain
characteristics, for example, salt tolerance (before growing in the field).
Metabolomics: It is the scientific study of the set of different metabolites
present within an organism, cell, or tissue.
Pathogens:
A pathogen or infectious agent is a biological agent that causes
illness or disease to its host. The term pathogen is most often used for
agents which disrupt the normal physiology of a multicellular animal or
plant.
Proteomics:
This is the large-scale study of the proteins, particularly their
structures and functions. Proteins are vital parts of living organisms, as they
play role as the main components of the physiological metabolic pathways of
cells.
Transcriptomics: It is the study of the transcriptome—the complete set of RNA
transcripts that are produced by the genome, under specific conditions or
in a specific cell—using high-throughput methods, for example microarray analysis.
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Box1: A simplified model of defense
illustrating successful transgenic strategies [5]
Strategy 1 related with direct interference
with pathogenicity (or inhibition of pathogen physiology). Thus 1a involves
in constitutive expression of any type of antimicrobial factors and 1b in
pathogen-induced expression of one or more genes in the transgenic plant.
Strategy 2 is related with the regulation of the natural induced host
defenses. 2a involves altering recognition of the pathogen (e.g., R-genes)
and 2b involves downstream regulatory pathways (e.g., SAR), and also
includes transcription factors. Strategy 3 is related with pathogen
mimicry: the manipulation of the plant to prime recognition of a specific
pathogen via pathogen derived gene sequences called genetic vaccination.
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Resistance against disease is the most effective way of
controlling disease. However, there are many such pathogens for which no
effective sources of disease resistance identified. Only very few genetically modified (GM) disease resistant
cultivars have been introduced to commercial agriculture although genetic
engineering has been promoted more than two decades to develop disease
resistant plants. This is in stark contrast to the development for two other
key disciplines of plant protection, (insect pest and weed control), where Bt1 and herbicide-tolerant crops
represent over 90% of all GM crops. The answer to this primarily lies in the
complexity of the biology of the traits concerned. Economics has also played a
role as the investment for transgenic insect and herbicide resistance was
considered safe due to established key technologies concerned. The
implementation of new products furthermore delayed as a result of moratoria
from negative public opinion for GM plants and expense of commercialization [5].
Various types of plant pathogens has different biological traits
creating substantial problems in development of GM resistant plants. Firstly,
organisms causing disease are highly diverse taxonomically; major being
cellular pathogens such as bacteria, fungi and the algal Oomycetes and
molecular pathogens such as viruses. They completely differ physiologically and
therefore no single gene product can be toxic effect on all of these pathogens.
Another trait is, pathogens use two major life strategies i.e.biotrophy and
necrotrophy. Biotrophic pathogens act as a sink for the host’s anabolic assimilates, and keep
alive to host. Necrotrophic pathogens in other hand consume the tissues of host
as invaded. Hemibiotrophs combine both of these strategies [5]
This
review paper highlights recent advances in understanding of role of
biotechnology in combating diseases of commercial crops and discuss future
prospects for use of biotechnology in field of plant pathology.
Techniques of Biotechnology in Plant
Diseases control:
Enhanced disease resistance has been achieved and established
using several strategies. These are depicted in Fig 1 “(see Box1)”. Some of the common and successful techniques
are tried to comprehend as below.
Tissue
Culture Techniques:
Almost all
the tissue culture techniques are used in plant pathology. Some of the
important one are as follows.
i). Protoplast Fusion: Protoplast fusion is one of
the methods that can be used to circumvent related problems in introgression
genes to develop resistance. By this, factors that contribute to crossing
barriers between species can be avoided and viable hybrids ( called Cybrids)
have been recovered even between the distantly related species [3].
ii). Chemically induced fusion: Isolated protoplast fusion
can occur in the presence of high pH (9-10) and high CA2+ but
commonly used chemical (fusogen) is polyethyleneghycol(PEG). Addition of PEG causes
adhesion of protoplast to neighbors that can be assessed by microscope [3].
Selection
for Disease Resistance:
In-vitro Selection
has an advantage over other selection system as it allows significant saving of
time, space and money. Plants diseases that are damaged through toxins can have
disease resistant genotype by cell selection for toxin resistance in cultures
and plans regeneration from descendants of the selected cell. An example is, disease resistant potato plant
have been developed through in vitro selectin to control Phytopthera infestans [3].
Recombinant
DNA Technology:
The r-DNA technology
has produced a notable success with regard to viral diseases. For example, the
transfer and expression of coat protein genes of tobacco mosaic virus(TMV) and tobacco
alfalfa mosaic virus (AMV), resulting in protection against or disease development delay in transgenic plants
[3].
RNA-interference
Techniques:
RNA interference defined collectively to diverse RNA
based processes that results in sequence-specific inhibition of gene expression
at any of the transcription, mRNA stability or translational level. The unifying feature are the production of small
RNAs(21-26 nucleotides(nt)) that act as specific determinants for
down-regulating gene expression and required for one or more members of
Agronaute protein. RNAi triggers the action of dsRNA intermediates, that
processed into RNA duplexes of 21-24 nucleotides by Dicer( a ribonuclease III-
like enzyme).After that, the small RNA molecules or short interfering
RNAs(siRNAs) are incorporated in to a multi-subunit complex known as RNA
induced silencing complex(RISC). This RISC
is formed by an endonuclease and siRNA among
other component [3].
Methods to
Induce RNAi in Plants
Agroinfiltration:
The
injection of Agrobacterium which carries similar DNA constructs
into the leaves intracellular spaces for triggering RNA silencing is called as
agroinoculation or agroinfiltration [1]
Micro-Bombardment:
A
linear or circular template is transferred by micro-bombardment into the
nucleus, in this method. Synthetic siRNAs are delivered into plants by
biolistic pressure to silence GFP expression. Bombarding cells with particles which
are coated with dsRNA, siRNA or DNA that encode hairpin constructs as well as
sense or antisense RNA, plays to activation of
the RNAi pathway [1].
Virus Induced Gene Silencing (VIGS):
RNA
is induced in plants using modified viruses as RNA silencing triggers. Many RNA
and DNA viruses have been altered to act as vectors for gene expression Some
viruses, for example, Tobacco
mosaic virus (TMV) and Potato
virus X (PVX) can be used for both gene silencing and protein
expression [1].
Monococol Antibodies
Technique:
In this
technique there is the fusion of cancer cells (mycloma cells) with
antibody-body producing while blood cell (B- lymphocytes). The resulting hybrid
celled is called a hybridoma. This, techniques have been used to produce large
quantities of identical antibodies. [3].
Pathogenesis-related (PR) proteins:
PR protein genes are potential source for candidate genes for resistance of fungi. Host plants contribute an
enormous number of resistance genes of diseases such as those encoding
pathogenesis-related (PR) proteins, which have been used against fungal
diseases. These proteins are induced
during hypersensitive response (HR) and also during systemic acquired
resistance (SAR). Thus, these are thought to have a role in natural defense or plants
resistance against pathogens [6].
Antifungal proteins:
Fungal resistance in plants has been enhanced by introduction of
the chitinase gene in tobacco and rice. The major constituents of the fungal
cell wall (chitin and α-1, 3 glucan) is degraded by chitinase enzyme.
Coexpression of chitinase and glucanase genes in tomato and plants confers a
higher level of resistance than that imparted by individual genes[6].
Phytoalexins:
Phytoalexins are low molecular weight compounds, that possess
antimicrobial properties and that develops plant resistance to fungal and
bacterial pathogens. Active oxygen species (AOS) and hydrogen peroxide, play an
important role in plant defense responses to infection of pathogen. Transgenic
potato plants which express an H2O2-generating fungal gene for
glucose oxidase were found to have resistance against both to fungal and
bacterial pathogens, mostly in case of verticillium wilt pathogen [6].
Antimicrobial Proteins:
Botrytis cinerea growth was subsequently
reduced when expressed in transgenic geranium with an antimicrobial protein
with homology to lipid transfer protein. Antimicrobial peptides are synthesized
in the laboratory to develop smaller (10-20 amino acids in length) molecules
that have gained potency against fungi [6].
Plant ribosome-inactivating proteins and other peptides:
Plant enzymes with 28S rRNA N-glycosidase activity are
ribosome –inactivating proteins which depending on their specificity will
inactivate conspecific or foreign ribosomes, ultimately shutting down protein
synthesis. Plant RIPs cause inactivation of foreign ribosomes of distant species
along with other eukaryotes including fungi. A purified RIP from barley crop
inhibits growth of several fungi in vitro. Resistance levels improved
when RIP was used in addition to either PR2 or PR3[6].
Resistance genes (R-gene):
The plant disease resistance genes(R genes) help for protein encoding
to detect pathogens[7].The R-gene products are cloned from many crops like tomato,
tobacco, rice and several other plant species, which shares one or more similar
motifs: a serine or threonine kinase domain, a leucine zipper, a nucleotide binding site, or a leucine-rich repeat region, that may
contribute to recognition specificity The plant’s resistance R-gene product
plays role as a signaling receptor for
the pathogen’s avirulence (Avr) gene product when there is presence of
resistance-regulating factors such as RAR1 and SGT. This leads to a form of
cell death termed hypersensitive response [6].
Degradation of Phytotoxic metabolites:
The penetration of fungal pathogens are blocked by plant cell wall
and numerous techniques have evolved to overcome this. Production of phytotoxic
metabolites of fungi for example, oxalic acid and mycotoxins, have been shown
to facilitate infection of host tissues following the cell death. The enzymes
expressed in transgenic plants when degrades these compounds provides an
opportunity to develop resistance to disease [6].
RNA silencing:
In all plant diseases caused by viral, fungal and bacterial
pathoges, RNA-mediated gene silencing is being tried as a reverse tool for
targeting gene. Homology-based gene silencing that is induced by transgenes (i.e. co-suppression),
dsRNA and/or antisense RNAhas been demonstrated in many plant pathogenic fungi,
along with Cladosporium fulvum Magnaporthae oryzae and many other
species. hairpin-vector technology have been used in Venturia inaequalis
and is able to induce high frequency silencing of a green fluorescent protein (GFP)
transgene along with an endogenous trihydroxynaphthalene reductase gene (THN)
simultaneously[6].
Conclusion
and Future Perspectives
Recent advancement in biotechnological tools has
made possible to develop and establish techniques to develop plant resistant to
many diseases. Mostly resistance against fungi, bacteria, virus and nematode
has been developed. Techniques like tissue culture are equally successful as of
recombinant DNA techniques. New techniques like RNA interference has been
successfully implemented. RNAi techniques are used either by agrobacterium
mediated, bombardment or virus mediated method of gene transformation. Other successful techniques are use of different
kinds of proteins, like antifungal proteins, antimicrobial and plant ribosome
inactivating. Gene silencing and use of resistance gene are also used
extensively in plant disease control.
Though the use of these techniques are found to be
successful technically, commercially they are found less established. Only
traits like Insect resistance (IR) and herbicides tolerance (HT) has been
commercially produced in case of crops. There may be couple of reasons like (a)
Complexity of biology of traits concerned, due to differing biology of the
various types of pathogens, no single gene product can be toxic to all types of
pathogens. (b) High Investment in IR and HT GMO and technology developed
quickly and (c) Public opinion against GMO was high in later years.
Thus successful commercialization of disease
resistance crops are needed in future. This is only possible with development
of GMO crops and creation of scientific base to make people ensure that GMOs
are safe for our health and environment.
References
[1] Wani,
S.H. et al. (2010) Biotechnology and plant disease control- role of RNA interference, American J of Plant Sc, 1, 55-68
[2] Mundembe, R. et al. (2012) Genetic engineering
of plants for resistance to viruses, genetic engineering - Basics, new applications
and responsibilities, Prof. Hugo A. Barrera-Saldaña (Ed.), ISBN:
978-953-307-790-1
[3] Fagwalawa, L.D. et al. (2013) Current issues in
plant disease control: biotechnology and plant Disease, Bayero J of pure and
App Sc, 6(2), 121-126
[4] Shamin, Md. et al. (2013) Role of biotechnology
in plant diseases management: An overview, J of Gen and Env Res Cons,
1(1),215-220
[5]
Collinge, D.B. et al. (2008) What
are the prospects for genetically engineered, disease resistant plants? Eur J Plant Pathol,121,217–231
[6] Wani, S.H. (2010) Inducing Fungus-Resistance
into Plants through Biotechnology, Notulae Scientia Biologicae, 2(2), 14-21
[7] McDowell , J.M. and Woffenden, B.J. (2003) Plant
disease resistance genes: recent insights and potential applications, Trends in
Biotechnology, Vol.21 No.4, 178-183
[1]
Author: Badri Khanal FVSU, GA, USA
Keywords: plant diseases, biotechnology, recombinant
DNA, RNA interference, gene silencing
