Authors: Neelam Geat1 and Devendra Singh2
1Ph.D Scholar, Division of Plant Pathology, IARI, New Delhi, 110012
Biotechnology is defined as the manipulation, genetic modification and multiplication of living organisms through novel technologies, such as tissue culture and genetic engineering, resulting in the production of improved or new organisms and products that can be used in a variety of ways. Traditional plant breeding methods have been used to develop cultivars resistant to various diseases. But this process is time consuming and limited availability of genetic resources for most of the crops are available and has left little room to continued improvement by this means. Development of crop varieties which are resistant against many economically important diseases is a major challenge for plant biotechnologists, worldwide. Plant diseases are a threat to world agriculture and general food security. Significant yield losses due to the attack of pathogen occur in most of the agricultural and horticultural crop species.
Plant biotechnology helps plant pathology in many ways:
1. To obtain pathogen-free mother plants through rapid clonal propagation.
2. New plants to which genes have been incorporated through genetic engineering are likely to show instability towards environmental conditions and towards the pathogenic microflora of their habitats. Here, pathology plays its part.
3. The main vehicle for transferring genes from donor to recipient, in plant pathogens, particularly the bacterium Agrobacterium tumefaciens and the cauliflower mosaic virus.
4. Control of plant diseases by inserting resistance genes into plants by genetic engineering techniques.
5. The study of plants genes for resistance to disease and of pathogen genes for virulence to pathogen has already added considerably by genetic engineering techniques.
Tissue Culture Techniques:
Almost all tissue culture techniques are used in plant pathology. Some of the importance tissue culture techniques and their importance to plant pathology are briefly described here:
Disease resistance in breeding program may come either from closely related species or from more distantly related species. Problems are generally encountered if an effort is made in crossing distantly related species. Protoplast fusion is one of the methods that can be used to circumvent problems in introgression genes for resistance. By this method, factors that contribute to crossing barriers between species can be avoided and viable hybrids (Cybrids) have been recovered even between distantly related species.
Table 1: Disease Resistant Plants Produced from Protoplast Fusion Species used for fusion Diseases
|Species used for fusion||Diseases|
|Lactusa sativa||Dowmy mildew (Bremia lactucae)|
|Brassila oleracea and Raphanus sativus||Club root (Plasmodiophora brassicae )|
|Brassica napus and Brassica nigra||Black leg (Phoma lingum) club Root|
|Solanum brevidens and Solanum tuberosum||Bacterial soft rot (Erwinia spp)|
Chemically induced fusion
Isolated protoplasts are sticky, tend to aggregate in suspension and show fusion spontaneously during incubation. Chemicals tend to increase the fusion frequency. Fusion can occur in the presence of high
CA2+ and high pH (9-10) but a commonly used chemical (Fusogen) is polyethyleneghycol (PEG). Due to the addition of PEG there is adhesion of protoplast to their neighbors which can be assessed by microscope. Subsequent dilution of stabilized PEG, either stepwise or at once results in fusion and mixing of the cytoplasm. PEG causes slight dehydration of the protoplasts and crinkling of the membrane. The level of fusion is usually 1-10% as chemical fusion agents are toxic and therefore damaging to the cell. The bacterium Agrobacterium tumefaciens or its modified T-plasmid and the double-stranded DNA virus cauliflower mosaic virus have been used to introduce foreign genetic material into plant cell.
Recombinant DNA Technology:
Advances in molecular biology have opened up possibilities of identifying and isolating any gene for an organism, and mobilizing and expressing it in a different organism of one's choice.
(i) Engineering Plants for Resistance to Disease:
A notable success has been made with regard to viral diseases following use of r-DNA technology. For example, a major achievement has been the transfer and expression of coat protein genes of tobacco mosaic virus (TMV) and alfalfa mosaic virus (AMV) in tobacco, resulting in protection against or delay of disease development in the transgenic plants. The purpose of introducing coat protein genes to give resistance against the virus is that the multiplication of infecting viral RNA is somehow checked by coat protein synthesized in the plant cells. Engineered plants synthesized chitinase which breaks down the fungal cell wall and this kills the soil borne pathogen, Rhizoctonia solani.
(ii) Engineering Plants for Resistance to Pest
The best way of insect control has been the use of insecticides. These insecticides were effective but proved to be environmental hazards and forced development of resistant strains of insects. There are genes in bacteria (Bacillus thuringiensis) that encode insecticidal proteins. Bacillus thuringiensis strains toxic to dipteran, lepidopteran, and coleopteran insects have been identified and the insecticidal protein gene cloned. Using T plasmid vectors of Agrobacterium tumefaciens, the gene encoding the insecticidal protein has been transferred to tobacco, potato, tomato, rice and corn. Such transgenic plants incorporate resistance to specific insects that feed on these crops.
During the last decade, RNA-mediated functions has been greatly increased with the discovery of small non-coding RNAs which play a central part in process called RNA silencing. Ironically, the very important phenomenon of co-suppression has recently been recognized as a manifestation of RNA interference (RNAi), an endogenous pathway for negative posttranscriptional regulation. RNAi has revolutionalized the possibilities for creating custom "Knock down" of the gene activity. RNAi operates in both plants and animals, and use double stranded RNAi (dsRNA) as a trigger that targets homologous mRNAs for degradation or inhibiting its transcription translation. It has been emerged as a method of choice for gene targeting in fungi, viruses, bacteria and plants as it allows the study of the function of hundreds of thousands of genes to be tested.
Mechanism of RNAi
RNA interference refers collectively to diverse RNA based processes that all result in sequence-specific inhibition of gene expression at the transcription, mRNA stability or translational level. The unifying features of this phenomena are the production of small RNAs (21-26 nucleotides (nt)) that act as specific determinants for down-regulating gene expression and the requirement for one or more members of Argonaute family of protein. RNAi operates by triggering the action of dsRNA intermediates, which are processed into RNA duplexes of 21-24 mucleotides by a ribonuclease III like enzyme called Dicer. Once produced, these small RNA molecules or short interfering RNAs (siRNAs) are incorporated in a multi-subunit complex called RNA induced silencing complex (RISC). RISC is formed by a siRNA and an endonuclease among other component. The siRNAs within RISC acts as a guide to target the degradation of complementary messenger RNAs (mRNAs). When dsRNA molecules produced during viral replication trigger gene silencing, the process is called virus-induced gene silencing (VGS). One interesting feature of RNA silencing in plants is that once it is triggered in a certain cell, a mobile signal is produced and spread through the whole plant causing the entire plant to be silenced. This silencing process is also enhanced by the enzymatic activity of the RISC complex, mediating multiple turnover reaction. Furthermore, production of the secondary siRNAs leads to enrichment of silencing via its spread from the first activated cell to neighbouring cells, and systematically through system. The cell to- cell spread can be mediated as passive spread of the small RNAs via plasmodesmata, since it does not spread into meristematic cells. The discovery of RNA binding protein (PSRPI) in the phloem and its stability to build 25 nt ssRNA species add further to the argument that siRNAs (24- 26nt) are the key components for systemic silencing signal.
Disease Management in RNAi
In this sense, RNAi technology has emerged as one of the most potential and promising strategies for enhancing the building of resistance in plants to combat various fungal, bacteria, viral and nematode diseases causing huge losses in important agricultural crops. The nature of this biological phenomenon has been evaluated in a number of host-pathogen systems and effectively used to silence the action of pathogen. Many of the examples illustrate the possibilities for commercial exploitation of this inherent biological mechanism to generate disease resistant plants in the future by taking advantage of this approach e.g. including; Cladosporium fulvum Magnaporthae oryzae, Venturia inaequalis and Neurospora crassa.
Transgenic Plant Disease Management:
Diseased resistance genes could be sourced from plant pathogens themselves, as was possible with coat protein-mediated plant viral resistance and with toxin inactivating protein-mediated bacterial resistance. Host plants also contribute an enormous number of disease resistance genes such as those encoding pathogenesis-related (PR) proteins, which have been used against fungal disease.
Candidate Genes against Viral Pathogens
One of the most successful examples, as of date of the use of transgenic resistance against plant disease is that was accomplished in the management of papaya ring spot virus (PRSV) in Hawaii. Traditional breeding in bringing about resistance against this disease was of no avail as crossability barriers were a big problem. Under these circumstances, coat- protein- mediated resistance using coat protein genes sourced from a Hawaiian strain of PRSV was attempted. One transgenic line was found to be completely resistant to PRSV.
Candidate Genes against Bacterial Pathogens
Wild Fire disease of tobacco caused Pseudomonas syringae pv. tabaci is a serious disease. A phytotoxin secreted by the pathogen drastically modifies the amino acid metabolism of the plant with the eventual accumulation of ammonia in tobacco leaves, which causes extensive blighting. Interestingly, the pathogen that synthasises the phytotoxin remains unaffected by the toxin. This formed the basis for a search of the candidate gene from the pathogen itself. A toxin-inactivating gene, which was named 'ttr' was successfully isolated from the pathogen and the same was cloned into tobacco cultivars, which showed excellent wildfire resistance.
Candidate Genes against Fungal Pathogens
PR protein genes appear to be a very profential source for candidate genes for fungal resistance. These proteins may play a direct role in defense by attacking and degrading pathogen cell wall components. Typical candidate genes are that encoding chitinases and B - 1, 3 glucanases increasing expression of individual and multiple PR proteins in various crops have demonstrated some success in enhancing disease resistance in particular pathogens (e.g. in rice against (Rhizoctonia solani, the shealth blight pathogen).
Meristem or shoot tip culture:
Meristem and shoot tip culture are used to eliminate virus from infected germplasm. It has long been observed that the rapidly growing meristems of plants are usually free of viruses, or at least have much lower concentration of viruses than nonmeristem cells. This situation has been exploited for the production of virus-free plants by meristem culture. It is commonly used in cassava, potato, sweet potato and ornamental plants. "Virus-free" the term has been loosely used in literature. Plants infected with more than one type of virus and also may carry some unknown viruses. Thus, a plant can be claimed as free of only those viruses for which specific tests have given negative results. In infected plants, the apical meristems are generally either free or carry a very low concentration of the viruses. In the older tissues, the titre of the viruses increases with increasing distance from the meristem tips.
Five main possibilities have been suggested to explain the mechanisms underlying the Resistance of meristems to viruses.
(i) Exclusion of the viruses from the meristems by lack of suitable vascular or plasmodesmatal connections.
(ii) Competition for key metabolites by the rapidly dividing meristem cells.
(iii) The production of substances in meristem cells that result in breakdown of the virus.
(iv) Deficiency in some key components of the machinery of virus replication.
(v) Presence of inhibitors of virus replication.
About Author / Additional Info:
I am pursuing Ph.D in Plant Pathology from IARI, New Delhi