Anuradha Prakashan, New Delhi

BIOTECHNOLOGICAL ADVANCES FOR PRODUCTION

OF

FUNGAL PATHOGEN RESISTENT PLANTS

Associate ProfessorDepartment of BotanySri Aurobindo College

University of Delhi

Dr. Payal Mago

All rights reserved.

Author

Dr. Payal

ISBN No.: 978-93-82339-73-1

First Impression, July, 2014

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BIOTECHNOLOGICAL ADVANCES FOR PRODUCTION

OF

FUNGAL PATHOGEN RESISTENT PLANTS

DEDICATEDTO

MY FATHER

SRI V. M. MAGO

THANK YOU PAPAFOR ALL THE LAUGHTERS AND SMILES,

FOR THE HAPPINESS AND GOOD TIMES,

FOR LISTENING AND CARING,

AND LOVING AND SHARING…

FOR YOUR STRONG SHOULDERS AND KIND HEART

LOVE YOU DAD

PREFACE

Pathogens and pests constantly threaten plants and cause crop

losses of significant economic importance for agricultural

production worldwide. Remember the Irish Potato feminine from

1845 to 1847 which claimed the life of about a quarter of million

people who died of starvation. This is a fitting reminder of the

devastation plant diseases can cause and a constant reminder to

improve our agricultural system and protect our crops from

massive devastation. Crops are attacked by various fungal, viral

and bacterial pathogens.

One way to reduce the damage caused by pathogens and pests is the

development of new, resistant cultivars. However, conventional

resistance breeding often suffers from limited access to suitable

resistance sources. Enhancing the disease resistance of economic

important crops to the fungal pathogens via the development of

transgenic plants is a viable approach to achieving this goal.

The development of gene technology has drastically increased the

availability of genes conferring resistance, which can be derived

from non-related plant species as well as non-plant sources.

Considerable progress has been made in identification and cloning

of genes involved in plant defense responses. With the aid of plant

molecular biology and biotechnology several genes and gene

products that are involved in signaling pathway have been

predicted. Exploitation of strategies like enhancement of plant

structural defense, neutralization of fungal toxins, and extraction of

antifungal genes from nonrelated plants and non plant sources have

been used to produce fungal pathogen resistant transgenic plants.

The main objective of this book is to highlight the potential of the

transgenic technology as a supplementary tool to resistance against

fungal pathogens and to discuss its prospects and the challenges

that lie ahead.

Dr. Payal Mago

TABLE OF CONTENTS

Preface

1.1 Modern Biotechnology: As an Integral Supplement toConventional Plant Breeding1.2 Modern Biotechnology: A Means of GeneticEnrichment of Crop Plants1.3 Plant Biotechnology and Transgenic Plants

1.3.1 Plant Biotechnology and Transgenic Plants offers1.3.2 Plant biotechnology –an emerging field, involves

1.4 Improvement of agricultural crops by GM technology1.5 Genetic Transformation: A Rapid Tool for CropImprovement

1.5.1 Resistance to Insect pests1.5.2 Resistance to Diseases1.5.3 TransgenicApproaches to Control Fungal andBacterial Pathogens1.5.4 Pathogen-Derived Resistance to Viral Diseases1.5.5 Tolerance toAbiotic Stresses

1.6 GM crops of different traits1.6.1 Herbicide tolerance1.6.2 Engineering pathogen resistance1.6.3 Stress resistance1.6.4 Fruit Quality1.6.5 Pest resistance1.6.6 Male sterility and Fertility restoration1.6.7 ImprovedYield1.6.8 Use of Marginalized Land1.6.9 Nutritional Benefits1.6.10 Reduced Environmental Impact

1.7 Other Benefits of Transgenic Plants

2.1 Plant resistance to microbial pathogens (bacteria andfungi)2.2 Biotechnology strategies to improve resistance topathogens

Chapter 1 Introduction 9

Chapter 2 Various methodologies employed indeveloping resistant plants for various kinds ofpests and fungal pathogens 32

2.2.1 Constitutive expression of PR proteins2.2.2 Constitutive expression of two or more PRproteins2.2.3 Expression of antimicrobial proteins andpeptides2.2.4 Increased production of hydrogen peroxide2.2.5 Expression of resistance genes2.2.6 Expression of a "death gene"

2.3 Mechanisms of pest resistance in plants2.3.1 Preformed resistance mechanisms2.3.2 Inducible resistance mechanism2.3.3 Recognition aspects in the HR2.3.4 Induced resistance responses2.5.5 Future applications2.3.6 Ecological concerns

3.1 Genetic engineering of plants to enhance resistance tofungal pathogens3.2 Strategies for development of fungus-resistanttransgenic plants3.3 Transgenics with antifungal molecules

3.3.7

3.3.10Antimicrobial proteins, peptides, and othercompounds

3.3.12 OtherAntifungal Proteins3.3.13 Compounds toxic to fungi

3.4 Transgenics engineered for hypersensitive response

Chapter 3 Transgenic plants with increased tolerance 43against fungal pathogens

3.3.1 Antifungal proteins3.3.2 Pathogenesis-related proteins3.3.3 Plant ribosome-inactivating proteins3.3.4 Small cystein-rich proteins3.3.5 Lipid transfer proteins3.3.6 2S storage albumins

Polygalacturonase inhibitor proteins (PGIPS)3.3.8 Antiviral protein3.3.9 Non-plant antifungal proteins

3.3.11 Phytoalexins

3.4.1 Resistance genes from plants3.4.2 Resistance gene-avirulence gene two-component system

3.4.3 Barnase–barstar two component system

3.4.5 Broad spectrum disease resistance using SAR

3.13 Other approaches to induce cell death

Brassica napus

3.4.4 Race-cultivar-specific resistance traits

3.5 Hydrolytic enzymes3.6 Inhibition of pathogen virulence products3.7Alteration of structural components3.8Activation of plant defense responses3.9 Resistance genes3.10 Genes used to improve resistance to fungal pests3.11 Inhibitors of fungal enzymes3.12 Crop varieties resistant to fungal diseases

4.1 Scientific challenges4.2 Commercial development4.3 Issues to be addressed4.4 Concluding remarks4.5 Future prospects

5.1 Thaumatin gene confers resistance to fungal pathogensas well as tolerance to abiotic stresses in transgenictobacco plants5.2 Tolerance to the fungal pathogen Rhizoctonia solaniAG4 of transgenic tobacco expressing the maizeribosome-inactivating protein b-325.3 Mannitol-accumulating transgenic eggplants exhibitenhanced resistance to fungal wilts5.4 Application of genes encoding antimicrobial peptidesfor the control of fungal pathogens of canola5.5 Overexpression of the rice EREBP-like geneOsBIERF3 enhances disease resistance and salt tolerancein transgenic tobacco5.6 Field tolerance to fungal pathogens ofconstitutively expressing a chimeric chitinase gene5.7 Transgenic Tobacco Plants Overexpressing Chitinasesof Fungal Origin Show Enhanced Resistance to Biotic andAbiotic StressAgents5.8 Transgenic carrots expressing enhanced tolerance toherbicide and fungal pathogen infection.

Chapter 4 100

Chapter 5 Example of Fungal Resistant Transgenic Plants 106

5.9APeroxiredoxin Q Homolog from Gentians is Involvedin Both Resistance against Fungal Disease and OxidativeStress5.10 Response of transgenic cucumber and carrot plantsexpressing different chitinase enzymes to inoculation withfungal pathogens5.11 Enhanced resistance to fungal pathogens in foresttrees by genetic transformation of black spruce and hybridpoplar with a endochitinase gene5.12 Transgenic Plants with Enhanced Resistance to theFungal Pathogen

Biblography 117

Table 1 Contribution of fungal diseases towards yield loss insome major crops of India

Table 2 PR protein genes used for making fungus-resistanttransgenic plants

Table 3 Two genes used in combination for making fungus-resistant transgenic plants

Table 4 Different classes of R genes and their examplesTable 5 Plant species genetically engineered to enhance

resistance to fungal diseases (1991–2001)Table 6 Number of initiatives to develop GMOs with

resistance to pathogens

Figure 1 Regional GM crop research activities on different traitsFigure 2 Transgenic plants with enhanced disease resistance havebeen engineered to express gene products to counterattack fungalvirulence products (from hypha on left), enhanced expression ofplant-derived gene products (inside of cell) or gene products fromnonplant sources (outside of cell)

Box 1 Some comman terms used by molecular plant pathologistsBox 2 Fngal diseases of some important crops worldwide

Trichoderma harzianum

Rhizoctonia solani

List of Tables

LIST OFFIGURES

LIST OFBOXES

Chapter 1: Introduction

1.1 Modern Biotechnology: As an Integral Supplement toConventional Plant BreedingThe advent of the principles of genetics at the turn of the lastcentury catalyzed the growth of breeding, making it a science-based technology that has been instrumental in substantialimprovements in crop plants. Largely through exploitation ofhybrid vigor, grain yields of several cerealcrops were substantiallyincreased. Intervarietal and interspecific hybridizations, coupledwith appropriate cytogenetic manipulations, proved useful inmoving genes for resistance to diseases and insect pests fromsuitable alien donors into crop cultivars. Plant improvement hasbeen further accelerated by biotechnological tools of gene transfer,to engineer new traits into plants that are very difficult to introduceby traditional breeding. The successful deployment of transgenicapproaches to combat insect pests and diseases of important cropslike rice ( L.), wheat ( L.), maize( L.), barley ( L.), and cotton( L.) is a remarkable accomplishment.Biofortification of crops constitutes another exciting developmentin tackling global hunger and malnutrition. Golden Rice,genetically enriched with vitamin A and iron, has, for example, thereal potential of saving millions of lives. Yet another excitingapplication of transgenic technology is in the production of ediblevaccines against deadly diseases. How these novel approaches togene transfer can effectivelysupplement the conventional breedingprograms? The current resistance to acceptance of this noveltechnology should be assessed and overcome so that its fullpotential incrop improvement can be realized.

A paramount factor in the evolution of human civilizations was asteady supply of food. Food production is therefore the oldestprofession of humanity. The processes of crop cultivation andselection were an integral part of human activity. Although early"plant breeding" was developed essentially as an art, its scientificbasis became well established with the rediscovery of laws ofgenetics at the turn of the last century. And with the application of

Oryza sativa Triticum aestivumZea mays Hordeum vulgareGossypium hirsutum

BIOTECHNOLOGICAL ADVANCES ......FUNGAL PATHOGEN 11

the principles of genetics to crop improvement, the period from1930 to 1970 witnessed a phenomenal increase in crop yields,particularly of cereal grains (Khush, 1999). Largely throughexploitation of hybrid vigor, maize, pearl millet [

(L.) R. Br.], and sorghum [ (L.) Moench]registered a considerable increase in grain yields during 1965 to1990 (Khush, 2001; Jauhar and Hanna, 1998; Jauhar et al., 2006).Improved wheat and rice varieties with reduced height developedby incorporating dwarfing genes in the 1960s and 1970s launchedthe famous Green Revolution in Asia (Khush, 1999; see alsoJauhar, 2006). Around the same period, the advent of the tools ofcytogenetics greatly facilitated wide hybridization andchromosome-mediated gene transfers from wild species into cropplants (Jiang et al., 1994; Jauhar, 1993, 2003a; Friebe et al., 1996;Fedak, 1999). Chromosome engineering methodologies, based onthe manipulation of pairing control mechanisms and inducedtranslocations, were, for example, applied to transfer into wheatcultivars specific disease and pest resistance genes of alien origin(Ceoloni and Jauhar, 2006; Jauhar, 2006; Mujeeb-Kazi, 2006).Thus, cytogenetic tools were instrumental in the geneticimprovement of several crop plants,particularly cereals.

The development, in the last decade and a half, of novel tools ofdirect gene transfer, collectively termed , hasadded new dimensions to breeding efforts. Genetic engineering isdefined as any nonconventional tool aimed at mobilizing specificgenetic information from one member of the plant kingdom (or, forthat matter, any organism) into another. (Any nonconventional toolof today may of course become conventional in the future.) Theseasexual techniques of biotechnology help engineer intoplants newcharacters that are otherwise very difficult to introduce byconventional breeding. The molecular techniques, including therecombinant DNA methods, involve the introduction of well-characterized alien DNA into the recipient plant cells ofregenerable embryogeniccalli to permanently transform the plant'sgenetic makeup. Geneticengineering has the potential to acceleratecrop improvement and has already yielded encouraging results(e.g., Jauhar and Chibbar, 1999; Muthukrishnan et al., 2001;Repellin et al., 2001; Dahleen et al., 2001;Janakiraman et al., 2002;Patnaik and Khurana, 2003; Wesseler, 2003; Sharma et al., 2004).

Pennisetumglaucum Sorghum bicolor

genetic engineering

BIOTECHNOLOGICAL ADVANCES ......FUNGAL PATHOGEN 12

Value-added traits engineered into cropplants include resistance tofungal and viral diseases, and biofortification of their nutritionalstatus (Jauhar and Khush, 2002; Schubert et al., 2004; Bajaj andMohanty, 2005). However, as with any new technology, geneticengineering is encountering resistance from some sections of thepublic. There are concerns about the potential adverse impact ofgenetically modified (GM) foods or organisms on human healthand the environment. Although some of the public concerns maynot be well founded (Jauhar and Khush, 2002), they will need to beproperly addressed. To alleviate some of these fears, perceived orreal, we will need to do a betterjob of informing the public. Some ofthese issues are raisedin this

Conventional plant breeding (Duvick, 1984; Jauhar, 1988; Khush,1999, 2001), sometimes assisted by marker-assisted selection(Dubcovsky, 2004; Lapitan and Jauhar, 2006), and widehybridization coupled with manipulation of chromosome pairing(Friebe et al., 1996; Fedak, 1999;Jauhar and Chibbar, 1999; Jauhar,2003) has clearly been instrumental in producing superior cropcultivars. However, these procedures are time consuming.Conventional breeding may take 10 or moreyears to transfer a traitfrom a donor species into a crop cultivar. Wide hybridization isundoubtedly an effective means of incorporating desirable aliengenes into crop cultivars, but it has several limitations. It results intransmission of unwanted alien chromosomes and adverse geneticinteractions can lead to sterility. Other efficient means of genetransfer have therefore been explored.

covers the latesttechnologies and current research on the engineering, synthesis,utilization, and control of primary and secondary plant metabolitessuch as carbohydrates, amino acids, lipids, polymers, proteins, andphytochemicals for industrial, pharmaceutical, and food and feedapplications—detailing methodologies for plant cell culture, cropenhancement, and large-scale production of new plant materials.

1.2 Modern Biotechnology: A Means of GeneticEnrichment of Crop Plants

1.3 Plant Biotechnology and Transgenic PlantsPlant Biotechnology and Transgenic Plants

BIOTECHNOLOGICAL ADVANCES ......FUNGAL PATHOGEN 13

1.3.1 Plant Biotechnology and Transgenic Plants offers:

1.3.2 Plant biotechnology – an emerging field, involves:

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Approaches to improve the nutritional quality,

agronomical characteristics, and oxidative stress andchemical tolerance of plants

Recent methods in farming, plant propagation, and

breeding

Modern procedures to formulate more effective

biopharmaceuticals

New bioreactors for increased production of plant-derived

products

Engineered plant resistance to pests, as well as viral,

bacterial, and fungal diseases

The development and optimization of the DNA transfer

process

The use of hairy roots for investigation of secondary

metabol i te and fore ign prote in product ion ,phytoremediation, and phytomining

Engineered plants for production of immunotherapeutic

compounds

Plant-derived drugs and extracts.Industrial strategies for the discovery of bioactivecompounds from plants.Plant cell and tissue culture techniques used in plantbreeding.Plant cell cultures as producers of secondary compounds.Genetic transformation of plants and their cells.Properties and applications of hairy root cultures.Bioreactors for plant cell and tissue cultures.The potential contribution of plant biotechnology toimproving food quality.Engineering plant biochemical pathways for improvednutritional quality.Transgenic plants as producers of modified starch and othercarbohydrates.Improving the nutritional quality and functional propertiesof seed proteins by genetic engineering.Transgenic plants as sources of modified oils.

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BIOTECHNOLOGICAL ADVANCES ......FUNGAL PATHOGEN 14

Biotechnological Advances ForProduction Of Fungal Pathogen

Resistent Plant

Publisher : Anuradha Prakashan ISBN : 9789382339731 Author : Dr. Payal Mago

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