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TOPIC TO BE PRESENTED: 1. GENETICS OF RESISTANCE, 2. ‘R’ GENES, 3. MECHANISM OF GENETIC VARIATION IN PLANT PATHOGEN, 4. MOLECULAR BASIS FOR RESISTANCE, 5. MARKER-ASSISTED SELECION, 6. GENETIC ENGINEERING FOR DISEASES RESISTANCE.
GENES AND DISEASES
pathogen is often specific for a particular host plant. Thus, the fungus Fusarium oxysporum f. sp. lycopersici, which causes tomato wilt, attacks only tomato and has absolutely no effect on apple, wheat, or any other plant. The gene combinations make a plant susceptible, i.e., a host to a particular pathogen, are present only in that one kind of plant and possibly a few related kinds of plants. The development of disease in a host is the presence in the pathogen of one or more genes for pathogenicity, for specificity, and for virulence against the particular genes of host. Plants may resistant to certain pathogens as because they belong to taxonomic groups outside the host range of these pathogens (non host resistance) or because they possess genes for resistance (R genes) directed against the avirulence genes of the pathogen.
TYPES OF REGISTANCE
TRUE RESISTANCE: Disease resistance that is controlled genetically by the presence of one, a few, many genes for resistance in the plant is known as true resistance. In true resistance, the host and the pathogen are more or less incompatible with one another, either because of lack of chemical recognition between the host and the pathogen or because the host plant can defend itself against the pathogen. There are two kinds of true resistance: a) Horizontal Resistance: All plants have a certain, but not always the same, level of possibly unspecific resistance that is effective against each of their pathogens. Such resistance is sometimes called partial, race nonspecific, general, quantitative, polygenic, adult-plant, field, or durable resistance, but in the past it was referred to most commonly as horizontal resistance. It is also known as Partial, Quantitative, Polygenic resistance. b) Vertical Resistance: Many plant varieties are quite resistant to some races of a pathogen while they are susceptible to other races of the same pathogen known as monogenic, qualitative, major gene, vertical or R gene resistance.
APPERENT RESISTANCE: The apparent resistance to disease of plants known to be susceptible is generally a result of disease escape or tolerance to disease. Disease Escape: Disease escape occurs whenever genetically susceptible plants do not become infected because the three factors necessary for disease (susceptible host, virulent pathogen, and favourable environment) do not coincide and interact at the proper time or for sufficient duration. Tolerance to Disease: Tolerance to disease is the ability of plants to produce a good crop even when they are infected with a pathogen. Tolerance results from specific, heritable characteristics of the host plant that allow the pathogen to develop and multiply in the host while the host, either by lacking receptor sites for or by inactivating or compensating for the irritant excretions of the pathogen, still manages to produce a good crop.
GENETICS OF VIRULENCE IN PATHOGENS AND OF RESISTANCE IN HOST PLANTS Infectious plant diseases are the result of the interaction of at least two organisms, the host plant and the pathogen. The properties of these two organisms are governed by their genetic material, the DNA. The host reaction, i.e., the degree of susceptibility or resistance to various pathogens, is an inherited characteristic. When a variety is inoculated with two appropriately chosen races of a pathogen, the variety is susceptible to one race but resistant to the other. This clearly indicates that, one race possesses a genetic characteristic that enables it to attack the plant, while the other race does not. Studies of the inheritance of resistance versus susceptibility in plants prove that genes control resistance and their absence allows susceptibility and in pathogen also genes control avirulence and their absence allows virulence. Under favourable environmental conditions, the outcome — infection (susceptibility) or noninfection (resistance) — in each host–pathogen combination is predetermined by the genetic material of the host and of the pathogen.
THE GENE-FOR-GENE CONCEPT H Flor (1942-56) had conducted a series of experiments on inheritance of resistance in the host and virulence in the parasite in relation to each other. On working with 18 different varieties of flax (linseed) in relation to rust( Melampsora lini) disease, he concluded his results in the gene for gene hypothesis.
hypothesis states that during their evolution the host and the pathogen have developed complementary genetic system so that for each gene conditioning rust reaction in the host there is complimentary and specific gene in the pathogen that determines its virulence and avirulence.
M. lini- Flax system genes conditioning resistance occur as multiple alleles at 7 loci designated as K, L, M, N, P, D and Q.
Host reaction to Gene-for Gene Combination in M. lini – Flex system
Host resistance genes at Locus L Pathogen virulence genes conditioned by host genes at locus L AL AL or AL aL aL aL AL Al or AL al aL aL Type of rust reaction
LL LL ll ll
Resistance Susceptible Susceptible Susceptible
This indicates that a plant is resistant only when it is homozygous for
resistance (LL) and is attacked by a pathogen that is homozygous (ALAL) or heterozygous (AL aL) for avirulence.
Resistance (R) Genes of Plants
varieties are resistant to certain pathogen races because they possess specific resistance (R) genes that enable the plant to remain resistant to pathogens carrying the corresponding avirulence (avr) genes.
the first R gene, the maize Hm1 gene, was located, isolated, and sequenced, and its function was described at the molecular level.
The Hm1 R gene makes corn plants
of certain varieties resistant to race 1 of the fungus Cochliobolus carbonum, which causes a leaf spot disease on susceptible corn varieties. Race 1 of C. carbonum, the asexual stage of which is Bipolaris (Helminthosporium) carbonum, produces a host specific toxin, the HC toxin.
EXAMPLES OF ‘R’ GENE
Hm1 gene Pto gene
Source or host plant Target pathogen
Maize Tomato Race 1 of Cochliobolus carbonum Pseudomonas Syringae pv. Tomato that carry the avirulence gene avrPto. Cladosporium fulvum races 2, 4, 5, and 9 that carry the avirulence genes avr2, avr4, avr5, and avr9. TMV Xanthomonas oryzae Broad range of powdery mildew pathogens
Cf2, Cf4, Cf5, and Cf9 genes
N1 gene Xa21 gene RPW8
Tobacco Rice Arabidopsis
Melampsora lini race 6 carrying the avr6 gene
VARIABILITY IN PATHOGEN
One of the most dynamic and significant aspects of biology is that
characteristics of individuals within a species are not ―fixed,‖ i.e., they are not identical but vary from one individual to another. Variability in pathogens results in the evolution of the followings a) Variant: The progeny of a pathogen showing variation in characters from the parents. b) Biotype: Progeny developed by a variant having similar heredity. c) Race or strain: A group of biotypes with identical characters. d) Variety or forma specialis: When a group of races of identical morphology attack and infect only a specific host genus or group of species.
MECHANISMS OF VARIABILITY A. General Mechanisms of Variability: a. Mutation:
A mutation is a more or less abrupt change in the genetic material of an organism, which is then transmitted in a hereditary fashion to the progeny. Mutations represent changes in the sequence of bases in the DNA either through substitution of one base for another or through addition or deletion of one or many base pairs. Mutations occur spontaneously in nature in all living organisms. Since the average fungus genome consists of about 10,000 genes, one cell in a hundred could be a mutant. b. Recombination: Recombination occurs primarily during the sexual reproduction of plants, fungi, and nematodes whenever two haploid (1N) nuclei, containing genetic material that may differ in many loci, unite to form a diploid (2N) nucleus, called a zygote. The zygote, sooner or later, divides meiotically and produces new haploid cells (gametes, spores, mycelium). Recombination of (different alleles of the same genes) occurs during the meiotic division of the zygote as a result of genetic crossovers in which parts of chromatids of one chromosome of a pair are exchanged with parts of chromatids of the other chromosome of the pair.
B. Specialized Mechanisms of Variability in Pathogens
FUNGI: a. Heterokaryosis: Heterokaryosis is the condition in which, as a result of fertilization or anastomosis, cells of fungal hyphae or parts of hyphae contain two or more nuclei that are genetically different. For example, in Basidiomycetes, dikaryotic state may differ drastically from the haploid mycelium and spores of the fungus. In P. graminis tritici, the fungus causing stem rust of wheat, the haploid basidiospores can infect barberry but not wheat, and the haploid mycelium can grow only in barberry; however, the dikaryotic aeciospores and uredospores can infect wheat but not barberry, and the dikaryotic mycelium can grow in both barberry and wheat. b. Parasexualism: Genetic recombination without sexual cycle, in which there is no definite coordination between recombination, segregation and reduction as there is no meiosis, has been termed as parasexual. The essential steps of the parasexual cycle are 1. Heterokaryosis, 2. Fusion of unlike nuclei to form heterozygous diploids in the vegetative cells and 3. Segregation and recombination at mitosis. c. Heteroploidy: Heteroploidy is the existence of cells, tissues, or whole organisms with numbers of chromosomes per nucleus that are different from the normal 1N or 2N complement for the particular organism. It has been shown, for example, that some heteroploids, such as diploids of the normally haploid fungus Verticillium albo-atrum, the cause of wilt in cotton, lose the ability to infect cotton plants even when derived from highly virulent haploids.
a. Conjugation: Two compatible bacteria come in contact with one another and a small portion of the chromosome or plasmid from one bacterium is transferred to the other through a conjugation bridge or sex pilus. b. Transformation: Bacterial cells are transformed genetically by absorbing and incorporating in their own cells genetic material secreted by, or released during rupture of, other bacteria. c. Transduction: A bacterial virus (phage) transfers genetic material from the bacterium in which the phage was produced to the bacterium it infects next.
When two strains of the same virus are inoculated into the same host plant, one or more new virus strains are recovered with properties (virulence, symptomatology etc) different from those of either of the original strains introduced into the host. a. Pseudo-recombination: In mixed infection of virus having fragments of the genome may aggregate and re-assemble to form a new type of progeny. b. Genome masking: This can be result from direct interaction between different viruses in mixed inoculation. The genome of one virus is encapsidated in the protein of another strain of virus.
MOLECULAR BASIS FOR HOST RESISTANCE
MARKER ASSISTED SELECTION
is a process whereby a marker (morphological, biochemical or one based on DNA/RNA variation) is used for indirect selection of a genetic determinant or determinants of a trait of interest (e.g. productivity, disease resistance, abiotic stress tolerance, and/or quality). In other word, selection of a genotype carrying desirable gene or gene combination on the basis of linked-markers is called marker assisted selection. Differences in DNA sequences can be studied to identify plant varieties that carry desirable gene or trait. Breeders sometimes practice marker-assisted selection when an important trait, that is tightly linked to Mendelian trait, which can be easily scored.
purple color coleoptiles in some varieties of paddy is closely linked to presence of gene that confers resistance to BPH (Brown Plant Hopper). Tomato Tm-2 gene for resistance to tobacco mosaic virus (TMV) is linked to an anthocyaninless seedling (Robinson et al,1970). Molecular marker for Gluten strength in wheat identified as STS for Glu-1. molecular marker for grain protein content in wheat: CAPS for Gpc-B1.
Possible to screen for single trait, multigenic trait (QTL).
of multiple desirable characters into a single variety can be
performed. Eliminates environmental effects in expression of desirable trait. Accelerated selection process of plant breeding. Allows selection of traits that are difficult to evaluate phenotypically. DISADVANTAGES: Costly equipments are to be used. Expensive method of plant selection. Skilled manpower is needed.
GENETIC ENGINEERING FOR DISEASE RESISTANCE Genetic engineering/Gene cloning/Recombinant DNA technology: Integration of specific fragment of foreign DNA into a cell through a suitable vector in such a way that the inserted DNA replicate independently and transferred to progenies as a result of cell division. Vectors used in gene cloning: A vector is a DNA molecule that has the ability to replicate in an appropriate host cell, and into which the DNA fragment to be cloned (called DNA insert) is integrated for cloning. Ex: Tumor inducing (Ti) plasmid of Agrobacterium tumefaciens, Bacteriophages, cosmid vectors (derived from λ phage). Among the various method of gene transfer, 1. Ti plasmid of Agrobacterium-mediated gene transfer and 2. Biolistic transformation (particle bombardment) have been predominantly used in transformation work.
GENERAL STEPS IN GENE CLONING:
1. Identification and isolation of the desired gene or DNA fragment to be cloned(Restriction digestion and electrophoresis) 2. Insertion of the isolated gene in a suitable vector (ligation)
3. Introduction of this vector into a suitable organism or cell called host (transformation)
4. Selection of transformed host cells (selectable markers)
5. Multiplication / integration followed by expression of the introduced gene in the host
Agrobacterium tumefaciens Ti plasmid
Ti plasmid is a large conjugative plasmid or mega plasmid of about 200 kb. Ti plasmid has a T-DNA region (15-24 kb) which is bounded by a pair of 24 bp repeats. T-DNA carries genes for auxin, cytokinins and opine synthesis which are responsible for tumor formation (tumorigenesis). Agrobacterium Ti Plasmid Transfer of T-DNA depends on 35 kb virulence (vir) region of the Ti Mediated Gene Transfer plasmid. This region has 7 operons ranging from vir A to vir H (vir A, vir B, vir C, vir D, vir E, virG and vir H). The protein products of these genes respond to phenolics to generate a copy of T-DNA and mediate its transfer into the cell. The T-DNA when transferred from the Agrobacterium to the plant cell integrates with the chromosome, and the plant cells which are affected begin to synthesize opines, auxins and cytokinins. Opines are tumor specific compounds formed by the condensation of amino acid, ketoacid and sugar. The opines (octopine, nopaline, succinamopine or leucinopine) can be metabolized only by Agrobacterium. The IAA (auxin) and Isopentenyl-AMP (cytokinins) are phytohormones which cause the proliferation of plant cells and induction of the gall. Plant wound exudates contain phenolics, which attract Agrobacterium and induce vir genes. The strong vir gene inducers are syringic acid, ferulic acid, acetosyringone and sinapinic acid. Only Agrobacterium with Ti plasmid are attracted by these compounds. The exogenous DNA is inserted into the T-DNA region of the Ti plasmid by homologous recombination using an intermediate vector system or directly using binary vectors.
Types of vector
1. The Co- integrated vector For this purpose a suitably modified E coli plasmid or vector is integrated into disarmed Ti plasmid, this gives rise to co-integrate vector. During disarming oncogenes of T-DNA are replaced by gene insert and other sequences of E coli plasmid. For this both plasmids are introduced into same Agrobacterium cell. Because of homology through recombination pBR plasmid is integrated into the TDNA region. The gene to be cloned is therefore inserted into unique restriction site on the small pBR plasmid, introduced into A tumefaciens cell carrying a Ti plasmid and natural recombination process left to integrate the new gene into T-DNA.
2. Binary (Two) Vector strategy (Agrobacterium containing two different recombinant plasmids): First is Agrobacterium E coli shuttle vector containing disarmed T DNA
with 25 bp repeats flanking the gene insert to be introduced and a selective marker (often a Neo gene which provide cells resistance to
antibiotic Kanamycin ) This Mini plasmid (pBIN19) is designed to
replicate in both E coli and Agrobacterium and is capable of conjugal transfer between two bacterial species also has a copy of lacZ gene
containing multiple cloning sites Second
is helper Ti plasmid ( p
AL4404) from which T DNA segment has been removed. It has a functional Vir region. The Vir genes of the helper Ti induce the transfer of T DNA present on first plasmid in the same bacterial cell. As a result a gene insert within T-region is also transferred into plant cell.
DEVELOPMENT OF DISEASE RESISTANT TRANSGENIC PLANTS THROUGH TI PLASMID MEDIATED GENE TRANSFER:
The appropriate gene construct is inserted within the T-DNA of a disarmed Ti plasmid; either a co-integrate or binary vector is used. The recombinant gene construct is placed in Agrobacterium, which is co-cultured with the plant cells or tissues to be transformed for about 2 days.
In case of many plant species, small (a few mm diameter) leaf discs are excised from surface sterilized leaves and used for co-cultivation. In general the transgene construct involves a selectable reporter gene (Bacterial neo gene), the presence of which confers resistance to kanamycin.
the leaf disc-Agrobacterium co-culture, acetosyringone released by plant cells induces the vir genes which bring about the transfer of recombinant T-DNA into many of the plant cells. The T-DNA would become integrated into the plant genome, and the transgene would be expressed. As a result, the transformed plant cells would become resistant against a particular pathogen. After 2 days, the leaf discs are transferred onto a regeneration medium containing appropriate concentrations of kanamycin and carbenicillin. Kanamycin allows only transformed plant cells to divide and regenerate shoots in about 3-4 weeks, while carbenicilin kills Agrobacterium cells. The shoots are separated, rooted and finally transferred into soil.
Biolistic transformation (particle bombardment)
DNA is bound to tiny metal particles like tungsten or gold particles (0.5-5 mm), and a gunpowder driven piston is fired at the target cell with a velocity of about 430 meters per second. Some of the cells that survived the bombardment incorporated the DNA into the plant genome.
EXAMPLE: GENETICALLY MODIEIEF CROP
Crop Name of Company Novel Traits variety
Tomato (1994) Lacks Flavr polygalacturonase enzyme Savr
Cotton Potato (1996) Soybean (1996)
Vine-ripened longer shelf life
Bollgard Monsanto Bt toxin New Leaf resistance
Roundup Monsanto Glyphosphate herbicide Ready resistance
THANKS ALL OF YOU
For your kind patience
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