Gigantic advancements, in understanding the extremely complex molecular events occurring in plant-pathogen interactions, have been made. This information has provided a number of opportunities and strategies which can be employed to develop transgenic plants resistant to pathogens. The utilization of agrochemicals creates many perils that include detrimental effects on the ecosystem and a raise in the input cost of the farmers.
Over and over again sexual crosses are intricate to build genetic exchange in the hybrids and are skimpy due to low incidence of pairing between chromosomes of crop species and wild species. Tribulations can also happen due to linkage drag (gene/s for resistance are linked to some deleterious genes which lower the yield of the crop variety). Genetic variability is a basically a product of genetic recombination, mutation and natural selection, so the disparity in genetic background is arbitrated by these factors (Ijaz et al., 2012).
A lot of work has been done to identify the genes which can introduce into the plants so that they may build or enhance their resistance to fungal pathogens. Similarly many genes have been identified and cloned from plants those play role in identifying and tackling the pathogens (Feys and Parker, 2000; Takken and Joosten, 2000). Various signaling pathways tracking the pathogen infectivity have been scrutinized and numerous antifungal compounds have been recognized and produced by plants to fight fungal infections. Interactions of plant pathogenic fungi with their hosts are a complex phenomenon which involve many mechanisms like the synthesis of fungal toxins and enzymes that degrade the plant cell wall (Rodriguez and Redman, 1997). Major polymers of plant cell wall are degraded by the action of various enzymes; those are secreted from fungal pathogens and these principal enzymes are cutinases, pectate-lyases and endopolygalacturonases.
Plant apply countless strategies inhibit the ingress of pathogen into cell wall. One of such strategies is reinforcement of cell wall which involve the phenolic compounds production such as lignin is accumulated in the cell wall and extra cellular matrix is strengthened by glycoproteins rich in hydroxyproline and others similarly the cell wall breakage result in the release of small oligomers which act as elicitors for initiating the plant defense responses (Wegener et al., 1996). The enzymes secreted by fungal pathogens to degrade the plant cell wall are also inhibited by plant proteins. For example polygalacturonase-inhibiting protein (PGIP) in plants specifically inhibits endopolygalacturonases and throws it to the extra-cellular matrix. This results in the production of large oligomeric degradation products. These products then act as elicitors of defense responses of plants (Cervone et al., 1989).
1 Plant Disease Resistance Mechanisms
Gene for gene interaction (Plant defense system) between resistance (R) and avirulence (avr) genes from plant and pathogen respectively is activated to control resistance (Dangl and Jones, 2001; Jones and Dangl, 2006). Plant R gene coded protein recognizes pathogen avr gene product and stimulate hypersensitive response (HR). Hypersensitive response is basically a type of programmed cell death that happens at or near the point of pathogen attack (Morel and Dangl, 1999; Heath, 2000) which leads to the death of affected plant cell and restrict the spread of pathogen to the other parts of the plant. Receptor like proteins are encoded by several of R genes which have been recognized (Bent, 1996). Most of the higher plants have innate defense mechanisms.
Activation of inducible defense response leads to the restriction of pathogen spread in incompatible interaction and resulting in system acquired resistance (SAR). These proteins Possess antifungal activity and hit the cellular components of the pathogen cell wall e.g. chitinases, β-1,3-glucanases and fungal membrane permeability is also affected by thaumatin like proteins (TLP) (Linthorst, 1991). Three chitinases isoforms were found to be induced simultaneously in maize after inoculation of maize seed by the fungus Fusarium moniliforme (Cordero et al., 1994). In wheat 7 constitutively expressed and 3 pathogen induced chitinases isoforms have been reported by Botha et al., (1998). A Chitinase (33 kD) was isolated in wheat and expressed in E.coli to demonstrate its antifungal role in vitro (Singh et al., 2007).
2 Secondary Metabolites and Stress Resistance
In response to infection or abiotic stress some secondary metabolites collectively termed as phytoalexins are also produced. They are more than 350 in number reported from plant families. They are not the ultimate solution of fungal pathogens although generated in all parts of plants including vegetative and reproductive. They control the pathogens to some extents. Most of the fungal pathogens have detoxifying mechanism for phytoalexins (KuÄ‡, 1995; Etten et al., 1995). Phenylpropanoid pathway depends basically on phenylalanine ammonia-lyase (PAL) for the production of phytoalexins and salicylic acid which are the key pathways in plant defense response. In order to check the potential of it, PAL gene was transferred into tobacco. The resulting transgenics were resistant to different Phytophthora but the transgenic themselves were showing stunted growth (Way et al., 2002).
Thionins play their role by creating hypersensitive death of the cell and do it by forming cations ion channels which bind to the Phosphatidylserine group of lipid bilayer resulting in permeability and oxidative burst of cell membrane. in vitro studies showed an inhibited growth of 20 different fungi including Botrytis cinerea, Fusarium, Phytophthora infestans and Rhizoctonia solani when they were exposed to thionins (Cammue et al., 1992; Molina et al., 1993; Bussing et al., 1998; Hughes et al., 2000; Hilpert et al., 2001; Coulon et al., 2002). Plant defensins are another class of small cystein-rich proteins and they are structural and functional homologues of insect and mammalian proteins that have well established roles in host defense. Definsins act upon fungal cell wall and permealize their cell membrane by interacting with sphingolipids but not with phosphoglycerolipids. Definsins are constitutively produced in flower and seeds and are induced upon fungal infection in other plant parts. In the known plant defensins many are related to insect’s defensins containing eight disulphide linked cysteins (Thevissen et al., 2000).
3 Chitinases and Chitosanases in plant defense response
Plants adopt a variety of ways and mechanisms to defend against pathogens and many pathways are involved in the development of disease resistance. The defense response/PR genes function in a variety of ways to hinder fungal infection and the expression of these genes in transgenic plants has been shown to augment fungal resistance (Muehlbauer and Bushnell, 2003). Chitosanase is an enzyme, capable of breaking the β-1,4 linkages between N-acetyl-D-glucosamine and D-glucosamine residues in a partially acetylated fungal cell wall polymer.
Many plants take advantage of this hydrolytic action as a component of a larger post-attack defense response when attacked by pathogenic fungi (Agrios, 1997), but these enzymes may also function in pathogenesis-related (PR) signal transduction. After hydrolysis with a chitosanase or chitinase, fungal cell wall fragments are released, which act as elicitors of plant defense responses such as stomatal closure (Lee et al., 1999), lignification (Vander et al., 1998; Moerschbacher et al., 1988), and PR gene induction (Jabs et al., 1997). The responses elicited by these molecules depend on the length and degree of acetylation of the released fragments (Vander et al., 1998).
Many plant species have been transformed with chitinases (Punja, 2001) and these studies showed great variability in the antifungal effectiveness among chitinases from different sources. The in vitro antifungal potential of chitinases has been documented (Collinge et al., 1993; Ji and Kuc, 1996) and the introduction of chitinase genes into plants under the control of a constitutive promoter has been found to augment plant resistance to fungal pathogens in greenhouse studies (Broglie et al., 1991; Lin et al., 1995; Tabei et al., 1998) and field trials (Grison et al., 1996). Transgenic rice plants with constitutive expression of a rice chitinase gene, chi11, under control of the cauliflower mosaic virus (CaMV) 35S promoter have been developed (Lin et al., 1995). The transgenic rice plants and their progeny revealed a high expression of chitinase and had improved resistance to sheath blight (Lin et al., 1995).
Chitinase genes have been cloned and the gene products of chitinase genes have been used in bioassays against many fungi of economic interest. A Trichoderma chitinase was cloned in E. coli and the transformed bacteria were used in irrigation to see the impact on S. rolfsii and a noteworthy reduction was observed in pathogen population in the irrigated field (Chet et al., 1993). In comparison with the enzymes of plant origin, chitinases and (1,3) β-glucanases of Trichoderma origin are 100 times stronger and powerful than their counterparts of plant origin and are also non toxic to plant tissues even at very high concentrations (Lorito et al., 1994; Lorito et al., 1996) and their antifungal activity is boost up synergistically when used in combination with PR proteins, biocontrol bacteria and fungicide or toxin (Lorito et al., 1998; Steyaert et al., 2004). Chitinases from Trichoderma harzianum in addition to antifungal activity have also been revealed to augment tolerance against salinity and heavy metals in transgenic tobacco (Dana et al., 2006).
Chitosan is a component of fungal cell wall and degraded by an enzyme chitosanase, and this enzyme is a proficient candidate for slowing down the infection process (Hendrix and Stewart, 2002). Oligomers produced after the hydrolysis are shorter than oligomers formed after the hydrolysis of chitin by an enzyme, chitinase and used in the elicitation of defense response in the plant system such as stomatal closure and cell wall lignification. Small oligomers are stronger elicitors of defense than larger ones (Vander et al., 1998 and Lee et al., 1999). In most fungi cell wall, chitin, chitosan, and β-(1,3) glucan are the major structural polymers. Some part of chitin is always de-acetylated to convert it into chitosan. Chitinases are imperative and significant component of plant defense system (Jones et al., 1986; Collinge et al., 1993). This group of enzyme hydrolyses β-(1,4) linkages endolytically in chitin molecules (Cabib, 1987). These enzymes are present naturally in fungi as well as in plants but in fungi they play a vital role in cell division and differentiation and help saprophytic and mycoparasitic fungi to get their food (Cabib, 1987; Kuranda and Robin, 1991). Literature supports the Co-expression of chitinase and chitosanase to enhance plant defense against fungal pathogens and proved their synergistic work (Ayoo et al., 2011; Ahmad, 2009).
4 Synergistic Effect of PR Genes Proteins and Antifungal Potential
Production of various cell wall degrading enzymes of various classes by biocontrol fungi Gliocladium virens and Trichoderma harzianum suppresses germination of spores of Botrytis cinerea in vitro assay (Lorito et al., 1994). In reaction mixture, by adding chitinolytic as well as glucanolytic enzymes, showed synergistic effect in the enhancement of antifungal responses of five toxins of fungi against B. cinerea. These toxins were benomyl, miconazole, gliotoxin, captan and flusilazole.
Dose response curves were made for each enzyme-toxin combination and observed the ED values of the mixtures were substantially lower than ED values of the two compounds used alone in all combination. For example, the addition of endochitinase at a concentration of 10 pg/mL cause 86 fold reduction in the ED value of toxin. The level of synergism appeared to be higher when enzymes were combined with toxins having primary sites of action associated with membrane structure, compared with pesticides having multiple or cytoplasmic sites of action. Among enzymes tested, the maximum levels of synergism with synthetic fungicides were observed for the endochitinase from T. harzianum strain PI, which, when used alone, was the most effective chitinolytic enzyme against phytopathogenic fungi of those tested. The utilization of hydrolytic enzymes to synergistically improve the antifungal potential of fungitoxic compounds may decrease the effect of pesticides on animals and plants.
The majority of pathogenic fungi have cell wall with chitin and callose (Bartnicki-Garcia, 1968), and dissolution of these structural polymers has adverse impact upon the growth and development of fungi (Poulose, 1992). Biocontrol microbes (Gliocladizmz and Trichoderma) produce enzyme for degrading cell wall especially chitinolytic, to control pathogenic fungi of plants (Broglie et al., 1991; Ordentlich et al., 1988; Di Pietro et al., 1993; Lorito et al., 1993). When these enzymes are present together, then they have synergistic effect (Lorito et al., 1993). Additionally these enzymes should not have toxic effects for plants as well as other vertebrates, since they have not any target polymer. For the control of pathogenic fungi, these cell wall degrading enzymes show synergistic effects in combination with pesticides (Collins and Pappagianis, 1974; Köller, 1992; Roberts et al., 1988; Watanabe et al., 1988). Cell wall break down with enzyme may improve the chemical uptake into the target cell (Poulose, 1992).
Strain (PI and 41) of T. harzianum has affectivity against B. cinerea and Pbtophtbora diseases (Tronsmo, 1991; Smith et al., 1990). Endochitinase (41 kD) and chitobiosidase (40 kD) are from strain PI of T. harzianum and both enzymes have strong inhibitory effect on fungi, which have chitin in their cell wall, especially, when both are used in combination (Lorito et al., 1993 and Lorito et al., 1994). These enzymes in combination have synergistic effect for increasing the affectivity of biocontrol agent of bacteria (Lorito et al., 1993). Glucosidase (78 kD), third enzyme from P1 strain Trichoderma, also showed antifungal potential against various chitinous fungi, in in vitro assay (Lorito et al., 1994). From strain 41 of G. viren, an enzyme endochitinase, inhibits elongation of germ tubes and germination of spores in B. cinerea, but at low level than chitinase enzymes of T. harzianum (Di Pietro et al., 1993).
There are several reports according to which the cell wall breaking enzymes, act synergistically and cause various morphological syndromes within hyphae, even vegetative cell is burst (Lorito et al., 1993; De La Cruz et al., 1992; Lorito et al., 1993; Mauch et al., 1988; Nevalainen et al., 1991). Additionally plant chitinases were synergistic with inhibitors of chitin synthesis in fungi, such as polyoxin B and nikkomycin (Roberts et al., 1988; Poulose, 1992). Chitinolytic enzymes from T. harzianum have been shown to act synergistically against a wide range of disease-causing fungi in the Ascomycetes, Deuteromycetes and Basidiomycetes (Lorito et al., 1993).
Lorito et al (1994) suggested that the utilization of cell wall degrading enzymes of fungi in combination with pesticides can be fruitful for various applications of agriculture or even for veterinary and human sciences. Davies and Pope (1978) suggested that a mixture of chitinolytic enzymes and chemical antifungal agents had fruitful antimycotic action in immuno-suppressed laboratory animals. Plant-pathogen interactions cause host-specific biochemical responses, therefore the plant could tackle the attack from pathogens (Dangl and Holub, 1997). Chitin and glucan are cell-wall molecules of various pathogenic fungi and depolymerization of cell-wall by the combined action of chitinases and glucanases could kill fungi in vitro (Mauch et al., 1988).
Chitin is abundantly occurring natural polymer of β-1,4-linked N-acetylglucosamine (GlcNac). Chitin hydrolysate may be utilized as a carbon and nitrogen source in the synthesis of single-cell proteins (Revah-Moiseev and Carroad, 1981). These two enzymes act synergistically in the partial degradation of fungal cell-walls, and reported that these two enzymes in combination strongly suppress growth of various fungi like those suppressed by chitinase or β-1,3-glucanase alone (Vögeli et al., 1988). A similar enhance in the behavior of these enzymes is significant for their finest role in plant defense (Pan et al., 1991).
It is also reported that chitinase is synthesized by a number of microbes, those living in chitin-containing habitats like soil, sediments and marine environment (Gooday, 1990). In plants, chitinases are present constitutively and are induced systematically also upon treatment with biotic as well as abiotic inducers (Viswanathan and Samiyappan, 2001). PR-proteins accumulation is related with SAR in plants (Ryals et al., 1996). Chitinase and β-1,3-glucanase have not only the potential to hydrolyze cell components like chitin and β-1,3-glucan.They release elicitors from the walls of fungi, which in turn arouse various defense responses in plants (Ren and West, 1992).
The first report on developing fungus-resistant transgenics came in 1991. Broglie et al., (1991) constitutively expressed bean chitinase gene in tobacco and Brassica napus and the plants showed enhanced resistance to Rhizoctonia solani. Since then there have been a number of reports on transgenics developed by constitutively expressing PR-protein genes. Various PR proteins may be acting synergistically in vivo and reveal improve restriction of fungal growth and development when tested in combinations in vitro. Transgenic plants expressing more than one PR protein genes in a constitutive manner were developed. Desire gene introduction with host plant under constitutively high expressing promoter may cause silencing of the transgene as well as its endogenous homologue leading to a high proportion of progeny losing its enhanced resistance. Plant defensins are another class of small cystein-rich proteins and they are structural and functional homologues of insect and mammalian proteins that have well established roles in host defense.
Over-expression of genes encoded for chitinase, did not show resistance for chitin deprived fungi. Fungus can alter its cell wall by biosynthesis of chitosan and glucan in place of chitin and may be pathogenic. It can evolve mechanisms for detoxification of certain phytoalexins. Sexually reproducing fungi may build up resistance rapidly. Plants have passive defense lines such as cell walls, wax layers and chemical barriers against pathogens. When the pathogens invade this first defense line, there is also a second defense line, which is mounted by proteins encoded by specific resistance (R) genes. This line of defense is best attributed genetically by the gene for gene model. It entails a pathogen protein encoded by avr gene to be identified by a plant protein encoded by a resistance (R) gene. This stimulates an array of defense mechanisms, like the hypersensitive response. During the last decade above 30 resistance genes which confer resistance against a variety of pathogens, like bacteria, nematodes, viruses fungi and even aphids have been cloned from both monocots and dicots. These genes for resistance have homology with each other and their products are also highly similar. R proteins have leucine-rich repeat (LRR) domain. H2O2 revealed its detrimental effect on the growth and development of microbes. An enzyme, glucose oxidase (GO), is present in the microbes causes the oxidation of β-D-glucose, producing gluconic acid and H2O2. GO has not been found in animals and plants.
Synthesis of lytic enzymes, like β-1,3-glucanase and chitinase by many PGPR strains possess principal antagonistic characteristics. Chitinase and β-1, 3-glucanase can work in defense against various fungal pathogens. These lytic enzymes have hydrolytic activity and breakage the cell-wall of various pathogenic fungi. Growth suppression of fungi entails the occurrence of β-1,3-glucanase and chitinase in combination. Actions of enzymes are stimulated in various plants in response to infection with fungal pathogens and have correlation with increased resistance.
A prolonged solution to control plant diseases is only the development of resistant varieties because chemical control is not ecosystem friendly and biological control is tedious and intricate to management point of view. Various molecular approaches to confer resistance against many fungal and bacterial pathogens have been conjure up such as the use of antimicrobial proteins genes that inactivate pathogenicity natural disease resistance genes, phytoalexins, and antimicrobial peptides and many of these genes have been effectively utilized to control plant bacterial and fungal infections.
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