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1. Details of Module and its Structure
Module Detail
Subject Name Botany
Paper Name Plant Genetic Engineering
Module Name/Title Strategies for resistance to fungal pathogens Module Id
Pre-requisites Basic knowledge about plant genetic engineering and Fungal Pathology
Objectives To create awareness to students on different strategies that can be used for the control of plant fungalPathogens and the diseases caused by them.
Keywords Plant fungal pathogens, Pathogenesis-related proteins,Genetic engineering, Hypersensitive response, Systemic acquired resistance andRNAi
Structure of Module/Syllabus of a module (Define Topic / Sub-topic of module) Strategies for
resistance to fungal pathogens
<Sub-topic Name1>
2. 2. Development Team
Role Name Affiliation
Subject Coordinator Dr. Sujata Bhargava Savitribai Phule Pune University Paper Coordinator Dr.Rohini Sreevathsa ICAR-National Research Center on
Plant Biotechnology, IARI, Pusa Campus, New Delhi
Content Writers/Authors (CW)
Dr.Basavaprabhu L. Patil Mr. Chandrashekar N Mr. SiddannaSavadi
ICAR-National Research Center on Plant Biotechnology, IARI, Pusa Campus, New Delhi Content Reviewer (CR) Dr. Rohini Sreevathsa
Language Editor (LE) Dr. A.N. Latey Prof., Zoology Dept (retd) University of Pune
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TABLE OF CONTENTS 1. Introduction
2. Mechanisms of Host plant resistance:
R-Avr
Avr-proteins/elicitors
Systemic responses
3. Methods for control of fungal diseases:
Chemical methods
Cultural methods
Biocontrol methods 4. Concluding remarks 5. Suggested Reading
Introduction
Fungi are the most important pathogens causing major yield losses in cereals, pulses, oil seed crops, vegetable and fruit cropsresulting in severe yield losses both in quantity and quality. In the past, fungal diseases have caused devastating crop losses leading to most infamous Irish and Bengal famines causing death of millions of people. The losses due to fungal pathogens are the highest among all the plant pathogens in the world’s agricultural sector. There are about 1, 00,000 species of fungi, and nearly 8000 species of them cause disease/s in crop plants.The majority of the phytopathogenic fungi belong to the Ascomycetes and the Basidiomycetes and few are oomycetes, which are not true fungi but are fungus-like organisms, but have developed very similar infection strategies.These fungal pathogens can be grouped into biotrophic and necrotrphic, the biotrophic fungal pathogens colonize living plant tissue and obtain nutrients from living host cells, while the necrotrophic fungal pathogens infect and kill host tissue and extract nutrients from the dead host cells.The fungi reproduce both sexually and asexually by production of spores and other structures and thesespores are dispersed mainly by air or water, or they may be soil borne. Many soil inhabiting fungi are capable of living saprotrophically, carrying out the part of their life cycle in the soil and these are known as facultative saprotrophs.
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The major calories for the human beings and livestock are obtained from rice, wheat, maize, potato and soybean. Some of the important fungal pathogens like Magnaportheoryzae, causing rice blast, Phakopsorapachyrhizicausing soybean rust, Pucciniagraminiscausing wheat rust, Phytopthorainfestanscausing late blight in potato and Ustilagomaydiscausing smut in maize have significantly affected socio-economic status of the people across the globe.Recently, the journal Molecular Plant Pathology(Dean et al., 2012) identified top 10 plant fungal pathogens through a survey and this includes, in rank order, (1) Magnaportheoryzae; (2) Botrytis cinerea; (3) Pucciniaspp.; (4)Fusarium graminearum; (5) Fusarium oxysporum; (6) Blumeriagraminis;
(7)Mycosphaerellagraminicola; (8) Colletotrichumspp.;(9) Ustilagomaydis; (10) Melampsoralini, with honourable mentionsfor fungi just missing out on the Top 10, including Phakopsorapachyrhiziand Rhizoctoniasolani.Magnaportheoryzae, a filamentous ascomycete fungus, causing rice blast disease is the most destructive disease of rice worldwide and its importance is underscored by the fact that approximately half of the global population relies on rice as the primary source of calories.Recent data shows that, at least 125 million tones of crop harvest from these five important crops vanished every year. There is an account of nearly 60 billion US dollar loss from fungal damages on rice, wheat and maize itself every year. As of 2013, the world population has reached 7.18 billion and is expected to reach between 8.3 and 10.9 billion by 2050.
More than 1.24 billion people are known to live on very low earnings of less than 1.25 dollar a day and this situation inevitably makes them to rely up on low cost food obtained from the above five crops in the developing countries. It is estimated that such losses in food production due to fungal diseases in crop plants is enough to feed at least 600 million people across the world. The Food and Agriculture Organization (FAO) has estimated that each year, 25% of the world’s crops are affected by mycotoxins with an annual loss of about 1 billion metric tons of food and food products (The American Phytopathological Society, 2014). So it is an utmost important aspect of farming community of the world to mitigate fungal diseases in order to reduce yield losses in field and also during post-harvest.
Several different control or management strategies are followed for controlling the fungal diseases of crop plants, which include cultural, chemical and biological methods.These fungal diseases can be controlled by the use of fungicides and other agriculture practices, however new races of fungi often evolve that are resistant to various fungicides. Breeders have developed crop varieties/hybrids resistant to different fungal diseases in various crop plants. Molecular marker technology or Marker
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Assisted Selection (MAS) has significantly helped the plant breeders to hasten the transfer of disease resistance genes/traits in different crop plants. Recently, with the advancement in molecular biology and genomics, transgenic technology has gained the impetus for improvement of disease resistance in crop plants.
Mechanisms of Host plant resistance
For a disease to occur there must be an interaction among virulent pathogen, susceptible host and the congenial environment. This interaction among these three determinants of disease development is called as Disease Triangle. Plants do not have a circulatory system or an immune system like in animals for defense against the invading pathogens. However, plants have developed morphological, physiological, biochemical and molecular barriers to counteract the pathogen attack.
The morphological and structural barriers like cuticle, wax, lignin, pectins, suberin and bark etc form a physical barrier for the entry of pathogen into the plant system and this mechanism provides the plant with first level of defense. When pathogen is able to overcome this barrier, the next level of defense is provided by pre-existing proteins like: defensins and defensin like proteins or phytoanticipins. When the second line of defense is also overcome by pathogen then specific defense mechanisms are evoked. The specific defense mechanisms embrace inducible systems like, R-Avr (Resistance genes-avirulence genes), PR (Pathogenesis-related) proteins and systemic responses such as SAR (Systemic Aquired Resistance) and ISR (Induced Systemic Resistance).
R-Avr: Harold Henry Flor proposed the gene-for-gene hypothesis based on genetic analysis of interaction between flax rust pathogen (Melampsoralini) and flax (Linumusitatissimum). The hypothesis says “resistance to the disease requires two complementary gene products, one from the pathogen called Avr protein other from the plant called R-protein, and whenever there is an absence of either of the gene product it can lead to disease development”. The R-proteins bind to ligands from the pathogen which include PAMPS (Pathogen-associated molecular patterns) and TTSS (Type three secretion system) proteins and are involved in sensing pathogen derived elicitors. The R-proteins are classified into different classes based on the conservation of structural domains such as LZ–NBS–LRR, LZ–NBS–LRR, NBS–LRR, TIR–NBS–LRR, LRR–TM–PK, PK, and LRR–TM.
Avr-proteins/elicitors: These proteins or elicitors are grouped into two types, based on their source of origin/production, namely exogenous and endogenous elicitors. The exogenous elicitor molecules originate from the pathogens infecting the plant, whereas the endogenous elicitor molecules are those present in the host plant itself, which are activated when there is an infection.
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Avr-proteins induce a general response or race-specific response in a host ultimately leading to the hypersensitive response (HR) when it interacts with counter R-protein in the plant. The elicitors include toxins, cell wall degrading enzymes (pectinases, endoxylanaseetc), chitins, β-glucans and Avr-proteins per se.
Systemic responses: Spread of disease from the site of infection to the distal parts of the plants is mediated by SAR and ISR mechanisms. Inter-cellular signals generated at the site of infection will systemically move to the other parts of the plant to produce a defense response. The signal generated through SAR, salicylic acid, is transported to targeted cells in distal parts of plant through phloem. The ISR mechanism is independent of salicylic acid and is mediated by jasmonic acid or ethylene pathway to induce defense response. Past research suggest that the necrotrophic fungal resistance is mediated through jasmonic acid and ethylene pathways, where as the biotrophic fungal resistance is through salicylic acid pathway.
Methods for control of fungal diseases:
Chemical methods: At present chemical fungicides are most widely used for controlling most of the fungal crop diseases in the fields. Though fungicides control the diseases effectively, the excessive use of fungicides has resulted in environmental and health hazards. Apart from it, the repeated application of same chemical fungicides results in development of resistance in pathogen and also may lead to evolution of more virulent strains of pathogens. The chemical residues might enter food chain causing health hazards in humans and animals. However, they have detrimental effects on environment by accumulating in the soil, spreading into air and entering into water bodies.
Chemical sprays such as copper and sulphur based fungicides have profound effects on broad range of organisms as their residues accumulate in the soil and enter into the waterways.
Cultural methods: In the past, cultural practices like summer deep ploughing of soil, removal of weeds and infected crop residues, crop rotations and mixed cropping were followed by the farmers.
Modern agriculture has encouraged intensive and mono-cropping practices which is an outcome of mechanization in agriculture. However, monoculture practices have resulted in development of resistance in fungal pathogens and even a resistant variety turning to be susceptible. Monoculture provides conditions and time for a pathogen to evolve alternative defense mechanisms (eg: Southern
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corn leaf blight caused by Bipolarismaydis). The practice of crop rotation and mixed farming with different crop species would reduce the development of resistance in pathogens.
Biocontrol methods:The growing public awareness about health and nutrition has resulted in demand for organically grown foods. There are serious concerns about the safety of fungicides due to the adulteration of crop produce sprayed with fungicides, cost implications, limited efficacy and development of fungicide resistance. As an alternative to chemical control, an environmental friendly means for the control of fungi was devised using fungal antagonists. Fungal antagonists are the microbes which inhibit the growth or kill the fungal pathogens and several such microbial fungicides have been identified. However, these biological control agents have lower persistence, efficacy or speed of control in the field conditions hindering their commercialization and optimum utilization in controlling pathogens. They can be employed as a part of Integrated Disease Management practices to effectively control fungal pathogens and also reduce mycotoxins in field and storage food produce. Some examples of important microbial fungicides are Pseudomonas syringe, Streptomyces griseoviridis, and Trichoderma harzianum are used to control diseases of field and horticultural crops.
Breeding and MAS approaches: Most effective way of reducing crop yield losses due to fungal diseases is to have crop cultivars with inherent potential to overcome attack of disease causing fungi.
In the long term, the fungal disease control or management practices followed are not economical as they have to be repeated in each cropping season. Resistant cultivars have inherent genetic ability of resistance against diseases. Breeders screen available crop germplasm or mutant populations for the identification of resistant lines and utilize those lines in breeding programmes in order to transfer the disease resistance trait into the commercial crop varieties using breeding techniques like backcross breeding and mutation breeding. The success of breeding for disease resistance is influenced by factors, such as, the nature of the pathogen and diversity of virulence in the population; availability, diversity and type of genetic resistance; screening and selection methods.
Methods followed for breeding of disease resistance include: 1) Selection, 2) Introduction, 3) Mutation, 4) Hybridization, and 5) Polyploidisation. Some examples of improved varieties developed using conventional breeding techniques for fungal disease resistance are,Wheat:
KalyanSona, Sonalika, DBW 17, PBW 550, and Lok 1, PUSA Vishesh; Rice: Makassane, Jaya and
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Ratna, Rohini, Karishma, Madhu; Sugarcane: Saccharumbarberi, Sachharumofficinarum; Rapeseed mustard Brassica: Pusaswarnim, PusaKarishma. Although development of resistant variety is cumbersome with higher initial investment, but a resistant cultivar is highly economical in long term, and also environmental friendly in managing diseases to reduce crop losses. However breeding strategies take longer period of time (10-15 years) for the development of successful disease resistant variety and it depends on the availability of resistant germplasm lines. There is a risk of evolution of newer virulent pathotypes breaking the available resistance of the developed variety because there is a continuous battle between pathogen and host to overcome each other’s strategies to attack and defend respectively. So, faster and easier means of incorporation of disease resistance genes into susceptible cultivars are required. Recently, MAS technique is used to aid traditional breeding approaches by improving effectiveness of selection as well as pace of breeding. With the development of molecular markers and mapping techniques, genes controlling resistance have been mapped to the particular locus (chromosomal locations). Research advancements in the area of omics; genomics, proteomics, metabolomics, ionomicsetc has hastened the phase of identification and location of R-genes and QTL’s. R-genes and QTL’s from the donor parents can be transferred to the commercially valuable but susceptible to the fungus by marker assisted breeding. For instance, Pi54 and Piz-5, R-genes from Blast resistant donors developed by marker assisted breeding namely Pusa 1602 and Pusa 1603, have been incorporated into blast susceptible basmati rice variety, Pusa basmati 1121 by marker assisted breeding. In tomato, several resistance genes like Cf-2, Cf-4, Cf-5, Cf-9, Pto, Mi, I2, and Sw-5 have been cloned. These cloned R genes provide new tools for plant breeders to improve the efficiency of plant breeding strategies, via marker assisted breeding and also using genetic engineering. However both conventional and MA breeding suffer with problems like reduced yield or quality, sterility, seed abortions etc when the donor parent is genetically divergent from the commercial cultivar. Both MAS and breeding depend on availability of resistant genes in germplasm so when there are no resistance gene/s sources breeding has to depend upon mutations to create new variability. MAS suffers from linkage of undesirable trait with the resistant genes leading to the dilution of genetic background of the commercial variety. Rapid evolution of new pathogenic strains due to increased intensive monocultures, excessive use of fungicides and negligence of proper sanitary cultural practices has complicated breeding for resistance in crop plants.
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Biotechnological strategies for control of fungal diseases:Genetic engineering is an alternative strategy for control of diseases in an ecologically safe way. Stress responsive gene(s) can be introduced and over-expressed to combat the crop adversities.Understanding the molecular mechanisms involved in disease resistance is necessary for designing appropriate strategies to control the pathogens and the diseases caused by them. Progress has been made in the translation of knowledge gained on the mechanisms of resistance into designing of strategies against plant pathogens. Strategies range from resistance against individual pathogens to that against several, providing durable and broad range resistance. Strategies for single pathogen resistance include exploitation of PR-proteins and antifungal compounds. Whereas general resistance includes switching on of disease resistance pathways providing durable resistance. Compounds of durable resistance include, induction of elicitor/receptor, switching on of cascade of defense genes. Apart from these the other techniques like RNAi and SCFV (single chain variable fragment) are being utilized for generating transgenics for fungal resistance. Genetic engineering includes transformation techniques, preparation of gene constructs with suitable promoter and signal sequences.
Genetic engineering for fungal resistance: Antifungal compounds include proteins derived from plants, lower organisms and metabolites like phytoalexins having inhibitory action on fungal growth, development and disease spread. PR proteins, RIP’s, cysteine rich proteins, lipid transfer proteins, polygalacturonase inhibitor proteins and non-plant antifungal proteins etc. PR proteins with ability to inhibit fungal growth include PR-1 (Jayaraj and Punja, 2007), chitinases (Swegleet al., 1992), β-1,3-glucanases (Velazhahanet al., 2003), thionins (Carmona et al., 1993), thaumatin- like proteins (Roberts and Selitrennikoff, 1990), ribosome inactivating proteins (Leah et al.,1991), defensins and lipid transfer proteins (Cammueet al., 1995).
Many first generation transgenic plants are being made all over the world using CaMV35S promoter. For imparting fungus resistance to plants, over expression of PR genes is one approach which has been exploited by using 35S promoter. But the constitutive over-expression of such transgenes is known to hamper the growth of plants and reduce their productivity. Which is understandable as transgene over-expression can compete for energy and building blocks for the synthesis of protein or RNA that are also required for plant growth under normal conditions. Thus, it is desirable to generate transgenic plants that accumulate the transgenic products only under stress
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conditions. Therefore stress inducible promoters play a key role in regulating the expression of the stress-responsive genes in plants.
PR-proteins:Pathogenesis related (PR) proteins are defined as proteins encoded by the host plant induced under pathological or related conditions. They are set of novel low molecular weight compounds associated with host defense mainly in incompatible interaction which impedes the pathogen progress.
RIP’s: Ribosome inactivating proteins (RIP) from plants are proteins having N-glycosidase activity which remove adenine residue from 28S RNA there by inhibiting protein elongation. They have an ability to disrupt translational process even in other species. RIP from barley is constitutively expressed in tobacco showed better resistance to Rhizoctoniasolani(Logemannet al., 1992). RIP expressed in combination with PR-2 or PR-3 showed better resistance levels against Rhizoctoniasolanithan either with PR-2 or PR-3 alone.
Small cysteine rich proteins: The Small cysteine rich proteins such as plant defensins, thionins, and chitin binding proteins exhibit antifungal activity, which have been tried in various transgenic studies in an effort to improve resistance against fungal diseases. They also play an important role in pathogenicity and specificity towards fungal pathogens. This helps to understand plant-pathogen interaction at molecular level.
Polygalacturonase inhibitor proteins (PGIP): PGIP’s are inhibitor proteins of polygalacturonases, enzymes which help fungi to infect host by degrading their cell walls.
Transgenic tomato expressing pear PGIP’s reduced infection of Botrytis cinerea.
Phytoalexins: These are the products of secondary metabolism upon exposure to external stimuli (eg. pathogens, insects etc) from plants having low molecular weight and are known for their antimicrobial activity. Phytoalexins such as alkaloids, isoprenoids and Phenylpropanoids are produced in an interconnected pathway system in plants.
Even though fungal resistance approaches such as SAR, ISR, PAMPS, ETI and R gene approaches have been understood, exploitation of these strategies has been met with limited success through genetic engineering and classical breeding. Since here our emphasis is on crop yields, nutrient composition, quality parameters, taste etc but not on exclusively for fungal resistance. So there is need for an alternative strategy for development of fungal resistant crop plants. One such promising approach is to understand signaling pathways, biosynthesis, function and regulation of phytoalexins. As the area of molecular engineering has been improved a lot in recent years, there is
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enough scope to utilize this strategy for the development of fungal resistant crops through genetic engineering.
a) Simply by inserting last enzyme coming under particular phytoalexin pathway works out to produce it in the plant system giving broad spectrum resistance.
b) Genetic engineering of plant hormones and effector proteins for the production of phytoalexins.
Transgenic plants for induced hypersensitive response and SAR: Since introduction of single resistance gene would end up with little or no resistance to the pathogens. Identification of key steps in signal transduction pathways is required to achieve more durable resistance by switching on the defense pathways. The pto gene encoding for serine/threonine kinase has been expressed in tomato under 35S promoter. Earlier it was known that pto-avrpto interaction along with other plant gene prf is required for the development of HR and SAR. Now it is clear that from the examination of transgenic plants over-expressing pto activates defense signaling cascade even in the absence of pto-avrpto interaction. It is also clear that pto expressing lines showed differential expression of around 600 genes when compared with plants not harboring this protein. This constitutive expression gives broad resistance toPsuedomonassyringaepv. tomato,Xanthomonascampestris, Cladosporiumfulvumetc.
Attempts are being made all over the world to introduce stimuli responsive genes regulating range of signal transduction pathways. NPR1 is one such gene from Arabidopsis thaliana that has been utilized in crops such as tomato, rice, wheat and Brassica etc. It is known that the protein product of this gene is a connecting bridge for several signaling pathways viz; SAR, ISR, R-gene mediated resistance, salicylic acid, jasmonic acid and ethylene. It interacts with TGA family of transcription factors (bZIP) to increase gene expression to impart resistance to range of fungal pathogens.
RNAi as a potential tool for engineering the fungal resistance in crop plants: Development of disease upon attack by fungal pathogens requires the products of fungal genes during pathogenesis.
Using RNAi strategy one can target these pathogen genes involved in invasion, growth and pathogenicity in plant system by introducing sequences of these genes for their down regulation.
The strategy now popularly regarded as host plant mediated pathogen gene silencing. This approach is now being widely employed to develop plants resistant to fungal pathogens. It was possible to develop transgenic tomato plants resistant to vascular wilt disease by RNAi targeting of ODC gene
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of Fusarium oxysporumf. sp. Lycopersici. Similarly by RNAi approach it was possible to control the powdery mildew fungi Blumeriagraminis in wheat and barley. In another report, the virus induced gene silencing (VIGS) approach was used to introduce the gene fragments from the rust fungi Pucciniastriiformis f. sp. tritici or P. graminis f. sp. tritici to plant cells and some reduced the expression of the corresponding genes in the rust fungus. The invading plant fungus takes up the dsRNA/siRNA molecules transgenically expressed in the host plant by introduction of an RNAi construct, thus ultimately leading to the silencing of target gene expression and such inhibition of fungal growth results in the durable resistance to crop plants. Thus the RNAi-based host plant mediated pathogen gene silencing appears to be very potential and promising method for durable and long lasting resistance in crop plants to combat the phytopathogenic fungi and the diseases caused by them in crop plants.
Somaclonal variations for fungal resistance: Plant tissue culture is an important tool in plant biotechnology to develop disease resistant plants. The plant tissues subjected to tissue culture are known to result in genetic variability known as somaclonal variation. This somaclonal variation has been successfully utilized for the development of fungal resistant crop plants such as, potato and alfalfa resistant to verticillium fungus, rye grass for crown rust, eye spot of sugarcane and Brassica napus resistant to sclerotinai stem root.
Somatic hybridization for fungal resistance: The tissue culture approach where one can successfully overcome sexual barriers by somatic cell fusion for genome recombination. Protoplast can be positively utilized in crops regenerated asexually or plants producing sterile seeds to get fertile plants. The cytoplasmic genes can be combined from different genomic backgrounds.
The novel hybrid combination showing resistance to Peronosporatabacina was created by combining two sexually incompatible Nicotiana species through protoplast fusion creating useful germplasm for future breeding applications.
Recent Advances in the field of plant disease resistance
Recently, use of effectoromics and synthetic R genes has gained importance for developing durable resistance in crop plants. Effectoromics is a high throughput functional genomics approach that uses effectors to screen germplasm for specific recognition by R proteins. It has recently emerged as a powerful tool for identification of Avr and R genes. Effectoromics was used for discovery and functional profiling of Potato late blight disease resistance and Phytophthorainfestansavirulence
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genes (Vleeshouwerset al., 2008). Genome sequencing of several fungal pathogens have been completed and identifying effector proteins from newly sequenced genomes is under progress for many diseases.
The availability of cloned genes has permitted investigation of which domains contribute to function. Domains contributing to resistance against different pathogens could be utilized to design synthetic R-genes providing broad and durable resistances. The gene-shuffling approach could be utilized for generating a library of recombinants, and used for the production of libraries of potential synthetic R genes (Jones, 2001). The host R genes could be modified in vivo to modify its level and range of resistance against pathogens. For in vivo genome modifications TALEN technology could be utilized. TALEN (Transcription activator-like effector nucleases) technology facilitates targeted genome mutagenesis and editing. Recent studies have shown that custom TALEs (Transcription activator-like effectors) can be designed to recognize specific DNA sequences in a number of different cell types including plant and mammalian cells (Zhang et al., 2011). Custom TALEs can be fused to a variety of effector domains to modulate transcription of endogenous genes in the genome as well as generate site-specific double strand break to catalyze homologous recombination for genome engineering applications (Miller et al., 2010; Li et al., 2011; Mahfouz et al., 2011).
Current status of transgenic crops resistant to fungal pathogens (Source of information: http://www.fao.org/)
The current stage of successful research across the world on identification of genes conferring durable resistance to fungal pathogensof crop plants is not very encouraging, particularly in the developing countries with several limitations. In the African continent, only two initiatives for fungal resistant transgenics are reported, a field trial of transgenic strawberry with phytoalexin synthesis genes (e.g. Vst1, Vst2) and a laboratory study on transgenic maize for resistance to cob rot (Stenocarpellamaydis), both of which are from South Africa. The three research initiatives for development of fungal resistant transgenic varieties reported in Eastern Europe are all from Bosnia and Herzegovina, wherethe transgenic potato are being tested for resistance to Fusarium, Verticilium and Rhizoctonia. In Asia,a field trial is underway for transgenic cotton with resistance to Verticilium and Fusariumin China. Other transgenic research efforts are reported from India,
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Malaysia and Pakistan for sheath blight resistance inrice and resistance for rice blast and leaf rust of coffee in Indonasia.
Inthe South American continent, the countries like Argentina, Brazil and Cuba, are carrying out a number of research activities on transgenic resistance to fungi, particularly on tropical fruit trees, with some results under field trial. Most of the activities for transgenic fungal resistance are from Cuba, in particular the field trials of transgenic potato for late blight resistance, and fungal-resistant sugar cane. Other field trials for transgenic fungal resistance reported are for maize, sunflower and wheat in Argentina, and tobacco in Mexico. Other crops subject to transgenic research for fungal resistance in Cuba are banana, plantain, pineapple, tomato, papaya, citrus and rice. Other countries involved in research on transgenic fungal resistance are Argentina on alfalfa, Brazil on rice, barley and cocoa, Chile on grape and apple, Colombia on tree tomato, Peru on potato for late blight resistance and Venezuela on sugar cane.
Concluding remarks:
Fungal pathogens mainly encounter two different kinds of resistance displayed by plants, namely host resistance and non-host resistance. Host resistance means individual plant variety, cultivar or land race is able to give resistance to a particular fungus. Nevertheless the other cultivars or varieties of a species are susceptible for the fungus in question. Now it is clear that host resistance is mediated through the interaction between single gene product of plant and a single gene product of fungus that is popularly called as gene-gene interaction. As it is under the control of single gene it is easy for the scientists to work with and at the same time it is vulnerable to give up resistance by plants. The present day research could only get some success in the area of host resistance through the above discussed strategies but there are no path-breaking advancements to achieve non-host resistance. So in future, significant efforts are required to achieve non-host resistance although it is complex in nature. Here entire population of plants belonging to all the species of the genera show resistance for a particular fungus. It is more robust than host resistance to circumvent fungus spread and its outbreak.
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23. Van Kan, J. A. 2006. Licensed to kill: the lifestyle of a necrotrophic plant pathogen.Trends Plant Sci.11:247-253.
24. Van Loon, L. C., Rep, M., and Pieterse, C. M. J. 2006. Significance of inducible defense related proteins in infected plants. Annu. Rev. Phytopathol. 44:135.
25. Vleeshouwers, V. G., and Oliver, R. P. 2014.Effectors as Tools in Disease Resistance Breeding Against Biotrophic, Hemibiotrophic, and Necrotrophic Plant Pathogens. Mol. Pl. Microbe Interactions27(3):196-206.
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26. Zhang, Y., Lubberstedt, T., and Xu, M.2013. The Genetic and Molecular Basis of Plant Resistance to Pathogens. Journal of Genetics and Genomics40:23-35.
27. Zhibing, L., and Tesfaye, M.2013. Genetic and cellular mechanisms regulating plant responses to necrotrophic pathogens. Current Opinion in Plant Biology16:505–512.
Table 1. List of major plant fungal diseases and their causal organism in important crop plants.
Crop Disease Fungal pathogen Impact of the disease
Rice Blast Magnaportheoryzae Globally, rice blast causes annual yield losses of up to 50%
Maize Smut Ustilagomaydis More than 20% yield loss
Soybean Rust Phakopsorapachyrhizi Yield losses of 30 to 80%
Potato Late blight Phytopthorainfestans Upto 30% yield loss
Wheat Leaf rust Pucciniatriticina Losses upto 50% and in susceptible varieties upto 100% losses
Stem rust Pucciniagraminis Stripe rust Pucciniastriiformis
Mustard White rust Albugo candida 17% to 60% yield loss Block spot Alternariaspp Upto 35% yield losses Groundnut Tikka
disease
Cercosporaarachidicola 20-50% yield losses
Table 2. Pathogenesis-related proteins (Modified from Van Loon et al., 2006).
Family Type member Properties Gene symbols
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PR-1 Tobacco PR-la Unknown Ypr1
PR-2 Tobacco PR-2 β-1,3 glucanase Ypr2,
[Gns2 (‘Glb’)]
PR-3 Tobacco P,Q Chitinase type I, II, IV, V, VI, VII
Ypr3, Chia
PR-4 Tobacco ‘R’ Chitinase type I, II Ypr4, Chid
PR-5 Tobacco S Thaumatin-like Ypr5
PR-6 Tomato Inhibitor 1 Proteinase-inhibitor Ypr6, Pis (‘Pin’)
PR-7 Tomato P69 Endoproteinase Ypr7
PR-8 Cucumber chitinase Chitinase type III Ypr8, Chib PR-9 Tobacco “lignin forming
peroxidase”
Peroxidase Ypr9, Prx
PR-10 Parsley “PRI” Ribonuclease-like Ypr10
PR-11 Tobacco “class V”
chitinase
Chitinase, type 1 YPr11, Chic
PR-12 Radish Rs-AFP3 Defensin Ypr12
PR-13 Arabidopsis THI2.1 Thionin Ypr13, Thi
PR-14 Barley LTP4 Lipid-transfer protein Ypr14, Ltp PR-15 Barley OxOa (germin) Oxalate-oxidase Ypr15 PR-16 Barley OxOLP Oxalate-oxidase-like Ypr16
PR-17 Tobacco PRp27 Unknown Ypr17
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Table 3. Mechanisms of resistance to fungal pathogens in crop plants.
Mechanisms Mode of Action
Anatomical defense
The first line of defense exerted by plants against fungal invasion.
E.g.: Bark, Cuticle and Waxes etc.
Pre-existing chemical and protein protection
Wide array of antifungal compounds synthesized during plant growth and development provides plants with second line of defense if pathogen overcomes first line of defense. Eg: Defensins and defensin like proteins, phytoalexins, phytoanticipins and glycosides etc
Inducible systems Elicitor
response
Defense mechanism activated by the production of proteins in vivo in response to fungal infection in order to give third level of defense. This involves interaction of exogenous elicitors or endogenous elicitors with host specific receptor molecules (such as R-Avr), leading to general defense. It can be operated in host and non- host plants or also of race-specific interactions resulting in the hypersensitive response (HR) to restrict pathogen spread. This intern leads to SAR (Systemic Acquired Resistance).
General response
As the pathogens infects the plants there will be general damage to the cell wall.
The fungus is now able to release proteolytic enzymes which causes cell wall fragments to be released as pectic-oligomers. They act as general signals (endogenous elicitors) and bind to specific receptors and induces for the expression of specific defense genes encoding for the synthesis of cell wall thickening structural components to repair the damage. It also induces for synthesis of enzymes of secondary metabolism, lectins and pathogenesis related proteins (PR-proteins).
PR-proteins: Pathogenesis related proteins are defined as proteins encoded by the host plant induced under pathological or related conditions. They are set of novel low molecular weight compounds associated with host defense mainly in incompatible interaction which impedes the pathogen progress.
E.g.: Chitinases, glucanases, ribosomal inactivating proteins, plant defensins and protease inhibitors etc (Table 1)
Race-specific response
According to Harold Floor, for the development of disease there should be the interaction of Avr protein (present in pathogen) and R-protein synthesized in specific host. Upon their interaction there will be induction of HR (hypersensitive response) in the form of necrosis at the point of infection.
Systemic responses
Induced local defense pathways triggers the intercellular signals for the production of systemic response called as SAR (Systemic acquired resistance). It is mediated by the salicylic acid (SA) through phloem transport in the plant system enabling induction of PR genes and synthesis of PR proteins to provide broad spectrum resistance against fungal pathogens.
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E.g.: PR-1 and Glucanases. On the other hand ISR (Induced Systemic Resistance) is independent of SA mediated and is mediated by JA (Jasmonic acid) and ethylene. It induces the expression of other specific PR-protein encoding genes.
E.g.: Chitinases and Plant defensins
Table 4.Most commonly used genes for transgenic fungal disease-resistant crops.
(Source: Collingeet al., Annu. Rev. Phytopathol. 2010. 48:269–91)
Gene/Trait Mode of Action Donor/s Transgenic Crop
Polygalacturonase inhibitor protein (PGIP)
Inhibitor of polygalacturonase
Bean, pear Grape, raspberry,
tomato
Protein kinase Resistance gene Soybean Soybean
R-gene Resistance gene Barley (Rpg1), rice (Pi9), Solanum
bulbocastanum(RB2), soybean
(Rps1-k)
Barley, festuca, potato, soybean
Cell death regulator Cell death regulator
Baculovirus, chicken, nematode
Wheat Toxin detoxifier Fusarium toxin
detoxifier
Fusarium sporotrichioides (Deoxynivalenol
acetyltransferase, 3-hydroxyl trichoecene acetyltransferase)
Barley, wheat
PR proteins Pathogenesis-
related proteins
Alfalfa (PR-2), Arabidopsis (PR-2),
grape (PR-5), pea (PR-2), rice (PR-5), tobacco (PR-1)
Cotton, barley, grape, peanut, potato,
rice, sweet potato, sorghum, tobacco, wheat
Chitinase Chitin degradation Alfalfa, barley, bean, petunia, rice,
tobacco
Alfalfa, apple, carrot, cotton, melon,
onion, papaya, peanut, rice, squash,
tobacco, tomato, wheat
Oxalate oxidase Reactive oxygen production
Barley, wheat Cowpea, bean,
lettuce, peanut, potato,
soybean, sunflower, tobacco
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Thionin Plant defensin Barley, tobacco Barley, potato,
rice Antimicrobial
peptide
Antimicrobial proteins
African clawed frog (Xenopuslaevis)
(magainin), cow (lactoferrin), Gastrodiaelata(mannose- binding
lectin, gastrodianin), Ustilagomaydis (KP4), wheat (PGL)
Cotton, grape, plum, poplar, tobacco, wheat
Cecropin Antimicrobial
proteins
Giant silk moths
(Hyalophoracecropia)
Cotton, maize, papaya
Stilbene synthase Polyphenol Grape Potato, tobacco
Antimicrobial metabolite
Antimicrobial metabolite
Pea (lignan biosynthesis protein),
tomato (coenzymeA reductase,
divinyl ether synthase)
Grape, potato, strawberry,
tobacco
Figure 1.A view of important fungal diseases in crop plants and their symptoms.
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Source: Compiled from the pictures procured from various online sources.
Figure-2: Plant-pathogen interaction and signal transduction mechanisms and molecular mechanisms of plant resistance to pathogens: Defense response to virulent pathogens (X) who’s PAMPS or effectors does not have counterparts in plants resulting in disease development. Defense response to pathogen (Y) produced/induced P/DAMPs or Effectors and resistance conferred by R- Proteins, PR-Proteins, phytoalexin, and JA/ET signaling leading to resistance.
SAR and ISR Systemic responses produced by SA and Ethylene/JA dependent signaling respectively leading to blocking of infection at distance tissues from site of infection.