Genetic engineering for plant improvement: Development of Pest resistance, resistance against viruses

Mitesh Shrestha

Problems with current agriculture •Increment in food production not keeping pace with population growth

•Agriculture based on monoculture

•Plants prone to various biotic and abiotic stresses which account for 40% of crop loss

•Indiscriminate use of pesticides and chemical fertilizers have led to severe environmental problems

•Land area and resources needed for increasing yield much more stretched than ever

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Important Traits for Crop Improvement

• High crop yield

• High nutritional quality

• Abiotic stress tolerance

• Pest resistance

• Adaptation to inter-cropping

• Nitrogen Fixation

• Insensitivity to photo-period

• Elimination of toxic compounds

TRANSGENIC PLANTS

NUTRITIONAL

QUALITY BIOTIC STRESS

TOLERANCE

ABIOTIC STRESS

TOLERANCE

PHARMACEUTICALS

& EDIBLE VACCINE

HYBRID DEVELOPMENT

FOR HIGHER YIELD

ENHANCED

SHELF LIFE

INDUSTRIAL

PRODUCTS

Dealing with problems: conventional vs GM approach

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Source: Tzotzos et al. (2009)

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Source:Tzotzos et al. 2009

Genetic engineering for crop improvement, achievements

•Several plants successfully engineered for various traits, but four species are commercially available: soybean, maize, cotton and oilseed rape (canola)

•Of all the transgenic crops in cultivation, three crops namely soybean (58%), maize (24%) and cotton (13%) make upto 95% (as of 2006)

•Mainly engineered with genes for herbicide resistance (75%), Insect resistance (15%), combination of both in same crop (8%); engineered plants for other traits at different stages of development prior to commercial release

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Trends in developing GM crops

Source:- Stein, A. and Rodriguez-Cerezo, E. (2010) Nature Biotechnology 28(1):23-24

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Possible targets for crop plant improvement (Nicholl, 2008)

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Plant pathogen interaction

Plants defense against pathogens

• Non Host resistance

• Host resistance (Partial, polygenic, quantitative or horizontal) or race specific

Plant defense to pathogens

• Lack of recognition between host and pathogen (Elicitors from pathogen may not be recognised by the receptors in the host)

• Lack of essential substances for pathogen

• Existence of structural and chemical defence mechanisms

• Defence mobilisation and signal transduction pathway leading to induction of structural or biochemical defence

Pre-existing structural defense

• Waxes- as water repellent and prevent germination or multiplication of pathogens

• Thick cuticle – as barrier to direct penetration by pathogen

• Thick, rough epidermal layer

• Periodicity of stomata movement

• Presence of lignified tissues

Pre-existing chemical defence • Inhibitors or toxins released by plants in the environment

• Chemicals present in plants prior to infection – mostly secondary metabolites like phenolics, saponins, etc. e.g. catechin in strawberry inhibit infection by Alternaria alternata,

• Proteins that inhibit proteinases or hydrolytic enzymes of pathogens, e.g., phytocystatins inhibit cystein proteinases of nematodes, insects; lectins cause cell lysis and growth inhibition of many fungi

Induced Structural defence • Cytoplasmic defence reactions : enlargement of cytoplasm and nucleus of the host cell leading to disintegartion of fungi as is seen in some weak virulent strains of Armillaria. • Cell wall defence: Swelling of cells and trapping of bacteria to prevent them from multiplying, thickening of cell wall due to cellulose deposition, Formation of callose papillae on inner side of the walls • Histological defence- cork formation, abscission layer formation, necrotic structural defence (hypersensitive response) • Deposition of gums, formation of tyloses, etc

Induced Structural defence

Induced Biochemical defence • Induced non-host biochemical resistance (Hypersensitive defence caused by quick mobilisation of a cascade of defence responses by the affected and the surrounding cells and the release of toxic substances that kill those cells.

• The last line of defense in plants against the pathogens is the induction of intercellular signals leading to systemic acquired response (SAR). During the initiation phase the infected cells produce signal molecules that are transported to target cells via phloem to induce the expression of SAR genes . During the maintenance phase the plants develop resistance against the virulent pathogen.

Fig. Hypothetical steps in hypersensitive response defence of plants

Induced Biochemical defence

Engineering Pathogen resistance in plants

• Use of plant derived genes like R genes (HM1 gene of maize, that codes for an enzyme that inactivates HC toxin produced by leaf spot fungus Cochliobolus carbonum ; The Pto gene of tomato encoding for a protein kinase involved in signal transduction and resistance to P. syringae pv. Tomato containing the avirulence gene avrPto) • Plant genes encoding enzymes or other proteins (PR proteins) found widely in plants are reported to confer resistance to various fungal pathogens. E.g. transgenic tobacco containing bean chitinase became resistant to Rhizoctonia solani (that contains chitins) but not to Pythium aphanidermatum (that does not contain chitin) • Engineering plants with constitutive genes involved in signalling networks of plant immunity like Edr (Enhanced Disease Resistance) MAPK (May lead to low yield due to metabolic overload)

Engineering Pathogen resistance in plants/2

Pathogen derived resistance (PDR) strategy

•Engineering plants with coat protein gene of viruses, and other appropriate genes of fungi and bacteria. E.g. Transgenic potatoes expressing the H2O2 generating glucose oxidase gene from Aspergillus niger, or T4 lysozyme increase resistance to various fungal and bacterial diseases in many plants.

•Expression of chitinase gene from Trichoderma harzianum, a fungus used for biological control of many pathogenic fungi, confers resistance to various fungal diseases in potato and apple

•Genes of the pathogenesis signaling and response pathways like Edr1 (enhanced disease resistance) and mpk4 (mitogen-activated protein kinase) mutants are likely candidates to be used for engineering pathogen resistance

• Post transcriptional gene silencing (PTGS) using RNA technology

Engineering Pathogen resistance in plants

How to avoid metabolic overload?- use inducible promoters

Source: Salomon and Sessa, 2012, p337

Pathogen resistant transgenic plants

Already in the market

• SunUp and Rainbow papaya resistant to Papaya Ringspot potyvirus (PRSV)

• New Leaf potato lines from Monsanto are Bt-resistant and resistant to different lines of potato viruses. But have long since been withdrawn from the market

In the pipeline

• Bananas resistant to fungal disease called black Sigatoka (By DNA plant technology corporation)

• Sclerotina resistant canola (by DuPont)

• Fungal disease resistance in Strawberry (By DNA plant technology corporation)

R genes • There has been extensive research using Resistance-genes (R-genes) for

engineering resistance in plants due to the high levels of qualitative resistance from traditional breeding programs.

• Plant R-genes and the corresponding pathogen-produced avirulence (avr) genes are part of the evolutionary arms race during establishment of disease.

• Typical basal plant defense responses are triggered by the recognition of Pathogen Associated Molecular Patterns (PAMPs) that can potentially be any part of the pathogen including cell-wall proteins or flagella, toxins, etc. PAMPs are recognized by the plant using extracellular receptor-like kinases (RLK) that can perceive the PAMP and rapidly trigger a signaling cascade through MAP kinases resulting in basal immunity.

R genes

• Pathogens that develop avr genes can override the basal immunity of the plant by either blocking perception of a particular PAMP or through the inhibition of the MAP kinase signaling cascade. This susceptibility through pathogen avr genes is termed effector-triggered susceptibility. Plants then develop and obtain immunity to the pathogens through expression of distinct R-genes targeted against individual avr genes.

• R-genes primarily code for transmembrane nucleotide-binding peptides with leucine-rich repeats (NB-LRR). These NB-LRRs can directly recognize the Avr gene product and trigger the activation of various downstream responses, including; PR-gene induction, accumulation of inhibitory metabolites and production of reac-tive oxygen species through the oxidative burst response that can lead to the hypersensitive response (HR), which is a form of pro-grammed cell death.

R genes

Examples

• The R-gene Rxo1 from maize was successfully introduced into rice and conferred resistance against bacterial streak disease caused by Xanthomonas oryzae pv. oryzicola;

• R-gene RCT1 from Medicago truncatula that was expressed in alfalfa and conferred resistance to Colletotrichum trifolii;

• RPI-BLB2 from wild potato Solanum bulbocastanum conferring resistance to Phytophtohora infestans in cultivated potato.

Problems

• There are several potential problems with genetic engineering of resistance through the use of R-genes. These include the potential for spontaneous activation leading to cell death via HR-like response, the development of pathogens with an alternative avr gene and reduced overall fitness.

• Additionally, R-gene resistance is only useful against biotrophic pathogens, pathogens that are obligate parasites that essentially act as a sink for the host plant’s metabolism, feeding while keeping the plant cells alive. In contrast, necrotrophic pathogens actively destroy the living plant cells and then consume the dead plant material.

• In fact, certain necrotrophic pathogens including Botrytis cinerea actively trigger the activation of plant R-genes as a virulence factor, leading to programmed cell and more rapid colonization and destruction of the plant tissues.

• Unfortunately, R genes are often quickly defeated by co-evolving pathogens

Detoxification of Virulence Factors

• An example of this would be inhibiting the ability of the pathogen to degrade polysaccharides within the plant cell wall. Polygalacturonase-inhibitory proteins (PGIPs) serve to inhibit the activity of the fungal cell wall-degrading polygalacturonases.

• Overexpression of PGIPs in transgenic plants has successfully reduced disease symptoms due to B. cinerea and Bipolaris sorokininia.

• Both B. cinerea and B. sorokininia have multiple copies of polygalacturonase genes within their genome, produce vast amounts of PGs and utilize these enzymes as one of their main pathogenesis factors.

• Overexpression of PGIPs would have relatively little effect on pathogens that express limited amounts of these enzymes.

Detoxification of Virulence Factors

• The main non-host selective mechanism for Sclerotinia sclerotiorum infection involves the secretion of oxalic acid.

• Oxalic acid functions through a variety of mechanisms, including: lowering the pH of the plant to near optimal for cell-wall-degrading enzyme (CWDE) activity,repression of the oxidative burst decreasing the activity of plant defense enzymes, weakening of plant cell walls through chelating Ca++ ions, as well as being directly toxic to the plant cells, and being a potent mediator of plant-programmed cell death.

• Oxalic acid-deficient S. sclerotiorum mutants are unable to infect many plant species, indicating its significance in pathogenicity.

• Proteins that can degrade oxalic acid include wheat oxalate oxidase and oxalate decarboxylase, converting oxalic acid to CO2 and hydrogen per-oxide or CO2 and formate, respectively.

• Overexpression of these enzymes in lettuce, sunflower, soybean, rape seed, tomato and tobacco all demonstrated at least partial resistance to S. sclerotiorum.

• Oxalic oxidase-derived resistance was also achieved against pathogenic fungal species that produce lesser amounts of oxalic acid including B. cinerea in tomato and Septoria musiva in poplar.

Detoxification of Virulence Factors

• Fusarium graminearum and Fusarium culmorum are the causal agents of Fusarium head blight (FHB), a devastating disease of wheat, barley and maize.

• Trichothecene mycotoxins, in particular deoxylnivalenol (DON), produced by these pathogens contribute to virulence in addition to being a major health concern for both human and livestock grain consumption through inhibition of protein synthesis in eukaryotes.

• F. graminearum protects itself through expressing a protein that rapidly converts non-secreted DON to a less-active acetylated form through the action of trichotecene 3-O-acetyltransferase (tri101).

• Overexpressing Fusarium Tri101 in transgenic wheat resulted in reduction in wheat spike infection during greenhouse trials. Subsequently Tri101 expression resulted in overall lower DON contamination in grains of rice and barley inoculated with F. graminearum.

Expression of Antimicrobial Peptides/Metabolites

• The most commonly used approach for engineering fungal and bacterial resistance in plants is through the expression of antimicrobial peptides, PR-proteins and proteins involved in the production of antimicrobial metabolites.

• Chitinases (PR-3, 4, 8 and 11) and β1–3 glucanases (PR-2) have been investigated extensively since they are hydrolytic enzymes that serve to break down the main structural components of fungal cell walls, chitin and laminarin.

• Overexpression of both chitinases and glucanases from a wide range of donor organisms has been examined in a variety of plant species.

• Chitinase overexpression has been moderately successful in increasing tolerance to diseases caused by both biotrophic and necrotrophic fungal pathogens. However, this chitinase-derived resistance was rarely at a level high enough to pursue commercial development.

• There has been limited success reported from overexpression of β-1, 3 glucanases, with little to no increased disease resistance reported in nearly all cases.

• However, combined expression of a chitinase and β-1, 3 glucanase often resulted in a synergistic effect, further enhancing the resistance in several plant species.

• Additionally, chitinases originating from mycoparasitic biocontrol agents, most notably Trichoderma harzianum, that can exhibit higher anti-fungal activity than plant chitinases have been proven to be more effective source for enhancing fungal disease resistance in transgenic plants.

Expression of Antimicrobial Peptides/Metabolites

• Overexpression of genes encoding for other PR-proteins or antimicrobial peptides in a variety of plants has met with some success.

• Defensins (PR-12) and thionins (PR-13) are low molecu-lar weight (~5 kDa) cysteine-rich peptides which are thought to modify ion uptake within the microorganism cell membrane, although the exact mechanism of action is not completely under-stood.

• Defensins and thionins have been used as peptides for overexpression within transgenic plants, generally resulting in moderate levels of resistance to a wide range of pathogens.

• However, there are recent reports overexpressing a radish defensin, RS-AFP2, in tomato and a Dahlia merckli defensin DM-AMP1 in rice that resulted in up to 90% reduction in disease symptoms against agriculturally important pathogens.

Expression of Antimicrobial Peptides/Metabolites

• Plant Class III peroxidases (Prx) exist as a large multi-gene family present in all land plants. Prx catalyzes the oxidation of a broad range of organic substrates using H2O2 as a reductant.

• A large number of Prx transcripts are upregulated during pathogen attack, with 10 Prx genes increased in rice challenged with Magnaporthe grisea.

• There seems to be at least three main functions that Prxs can perform:

– Increased basal resistance through generation of lignin or phe-nolic compounds;

– Increased induced resistance through the generation of H2O2, thereby increasing resistance against biotrophs;

– Removal of H2O2 resulting in increased resistance towards necrotrophic pathogens.

• Despite the uncertainty of the mechanism of Prx action, resistance towards pathogens appears to be at very high levels and it makes it difficult for pathogens to overcome this resistance.

Expression of Antimicrobial Peptides/Metabolites

• Overexpression of Swpa4 in transgenic tobacco resulted in constitutive increases in H2O2, lignin, total phenolics and heightened PR-gene expression. These plants also exhibited enhanced resistance to the biotrophic pathogen Phytophthora parasitica var. nicotinae.

• In bell pepper (Capsicum annuum), the Prx CaPO2 was identified to be involved in increased H2O2 accumulation during Xanthomonas campestris pv. vesicatoria attack.

• Overexpression of CaPO2 in Arabidopsis resulted in increased H2O2 accumulation, HR and PR-gene expression, resulting in increased resistance to virulent Pseudomonas.

• These findings indicate that increased accumulation of H2O2 induced downstream effects, including HR, that significantly enhanced resistance towards biotrophic pathogens.

Expression of Antimicrobial Peptides/Metabolites

• The plant phenylpropanoid biosynthetic pathway has also attracted interest from the perspective of genetic engineering to enhance disease resistance, since the pathway is responsible for the biosynthesis of a large number of low molecular weight polyphenolic compounds.

• These compounds include flavanoids which have been associated with many aspects of plant growth and development including pathogen resistance.

• Phenylpropanoid metabolism is controlled primarily through the action of phenylalanine ammonia lyase (PAL), and therefore overexpression of PAL has been investigated extensively as a source for increased genetic resistance.

• Tobacco plants overexpressing endogenous or exogenous PAL cDNA had increased resistance to Cercospora nicotinae and Phytophthora parasitica pv. nicotianae, corresponding to increased accumulation of the antimicrobial phenolic compounds.

Modification of Signaling Pathways

• Overexpression of a single or combination of a small number of individual genes is generally unlikely to provide high levels of resistance against a broad range of pathogens.

• Modifications of existing innate signaling pathways, including SAR and ISR, can activate a number of transcription factors, increasing the expression of a large number of defense genes.

• Potential candidate genes for genetic engineering include transcription fac-tors like WRKY, ERF1 or Whirly factors, mitogen-activated pro-tein kinases (MAPK ) or key signaling nodes, most notably NPR1.

• WRKY transcription factors are involved in SA-mediated defense pathways. Several WRKYs have the potential for increas-ing disease resistance, with WRKY70 from Arabidopsis being the most studied.

• Arabidopsis constitutively overexpressing WRKY70 had increased resistance to powdery mildew, Pseudomonas syringe and Pectobacterium carotovora.

Modification of Signaling Pathways

• The major drawback to activating entire signaling pathways is the high fitness cost and potential yield reduction associated with constitutive expression of a large number of genes.

Engineering virus resistance in plants

Virus-resistant plants

• Overexpression of the virus coat protein (e.g. cucumber mosaic virus in cucumber and tobacco, papaya ringspot virus in papaya and tobacco, tobacco mosiac virus in tobacco and tomato, etc.)

• Expression of a dsRNase (RNaseIII)

• Expression of antiviral proteins (pokeweed)

Fig. 18.7 Procedure for putting CuMV coat protein into plants

Genetically engineered Papaya to resist the Papaya Ringspot-Virus by overexpression of the virus coat protein

Genetic engineering of plants for resistance to insect pests

•The genes targeted to be used for engineering resistance against insect pest should be specific in action and should work only for the intended organisms and should not affect the predators or parasites that depend on that particular insect group •Genes encoding the endotoxins (like bt), protein inhibitors (PIs) targeting the specific proteases in the insect gut, or other compounds like lectins, chitinases from various sources are successfully used for confering insect resistance in transgenic plants

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Genetic engineering of plants for resistance to insect pests

•The genes encoding the crystal proteins (cry genes) Bacillus thuringiensis (Bt) endotoxin are the commercially successful and most important among the insect resistant genes

•Various cry genes encode for the proteins with varying degree of specificity to different insect groups

•Most of the insect resistant transgenic lines are engineered with this gene. So far no resistance is reported in transgenic lines against bt endotoxins

•These endotoxins bind to specific receptors in the cell membrane of the insect gut and exert their effect by forming lytic pores there

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Engineering of plants for resistance to insect pests

•The genes encoding the inhibitors of specific proteases present in insect body confers insect resistance in transgenic plants by depriving the insects of the most limiting of all the nutrients, Nitrogen, due to inhibition of proteolysis of food especially in insects with chewing habit (Lepidoptera and coleoptera)

•Some examples of genes encoding the protease inhibitors (PIs) that have been successfully used to engineer resistance to various insects are cowpea serine PI (cpTI), potato serine PI (PPI-II), tomato serine PI (TI-II), rice cystein PI (OC-1), Mustard trypsin inhibitor (MTI-2)

•Partial success is also achieved by engineering the genes encoding various enzymes for manipulating the secondary pathways like chitinases, peroxidases,etc. For example transgenic tobacco lines expressing ipt have been reported to show resistance against lepidoptera and homoptera

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Benefits of engineering insect resistance

•Environmentally sustainable (significant reduction in chemical pesticides)

•No significant risk (especially that of bt toxins) posed to non target insects or herbivores or pollinators

•No reports of field resistance reported so far

•No negative effects from exposure to insecticidal transgene products

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Insect resistance

Anti-Insect Strategy - Insecticides

a) Toxic crystal protein from Bacillus thuringensis

• Toxic crystals found during sporulation

• Alkaline protein degrades gut wall of lepidopteran larvae

– Corn borer catepillars

– Cotton bollworm catepillars

– Tobacco hornworm catepillars

– Gypsy moth larvae

• Sprayed onto plants – but will wash off

The Bt toxin isolated from Bacillus thuringiensis has been used in plants.

The gene has been placed in corn, cotton, and potato, and has been marketed.

Insect Resistance

Corn hybrid with a Bt gene Corn hybrid susceptible to European

corn borer

Various insect resistant crops have been produced. Most of

these make use of the Cry gene in the bacteria Bacillus

thuringiensis (Bt); this gene directs the production of a protein

that causes paralysis and death to many insects.

δ -endotoxin gene (Cry gene) of Bacillus thuriengenesis

GENE FOR Bt TOXIN WAS TRANSFERRED

TO OBTAIN BT TRANSGENIC PLANTS

PLANT SYNTHESIZES INACTIVE PROTOXIN

PROTEINASE

DIGESTION IN

INSECT GUT

MAKES THE

ACTIVE TOXIN

Toxin binds a receptor on the gut epithelial cells, forms a channel

on the membrane. This causes electrolyte leakage and insect death

INSECT FEEDS ON TRANSGENIC PLANT

Clone gene coding for BT toxin - pesticide (several companies)

Protein toxin from Bacillus thuringiensis Kills larvae of Lepidopterans (butterflies, moths) Dipterans (2 winged flies (gnats, mosquitos)) Coleopterans (beetles)

Agricultural importance - Kills corn borer, corn root worm and cotton bollworm larvae

Insect resistant plants

Corn borer

Corn root worm

Bt Corn from Phillipines

Mechanism of toxin action: Binds to receptors in insect gut Ionophore- ion channel that allows ions to flow across plasma membrane Note: organic farmers spray crops with intact Bt bacterium

Insect resistant transgenic lines

• Rogers® brand Attribute® Bt Sweet Corn (Syngenta Seeds)

•Herculex™ I Insect Protection (Dow Agro-Sciences and Pioneer Hi-Bred Intl., Inc.) Broadest

spectrum insect resistance against including first- and second-generation European corn borer, southwestern corn borer, black cutworm, western bean cutworm, fall armyworm, sugarcane borer, southern corn stalk borer and lesser corn stalk borer

• YieldGard® Corn Borer/ YieldGard® Rootworm-Protected Corn / Yield Gard Plus corn (Monsanto)

• BollGard cotton (Monsanto)

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National Academy of Science Report on GE Crops - May 2016

In 2015, almost 180 million hectares of GE crops were planted globally, which was about 12% of the world’s planted cropland that year. There were herbicide-resistant varieties of maize (corn), soybean, cotton, canola, sugar beet, and alfalfa, and insect-resistant varieties of maize, cotton, poplar and egglplant.

Figure 1. Commercially Grown Genetically Engineered Crops Worldwide.

http://nas-sites.org/ge-crops/ full report; short report; slides from news release

Crop Resistance a

Company Date of Nonregulated Status

Insect resistance

Potato Bt IR Monsanto March 1995

Corn Bt IR Ciba-Geigy May 1995

Cotton Bt IR Monsanto June 1995

Corn Bt IR Monsanto August 1995

Corn Bt IR Northrup King January 1996

Corn Bt IR Monsanto March 1996

Potato Bt IR Monsanto May 1996

Corn Bt IR Dekalb Genetics March 1997

Cotton Bt IR, HR Calgene April 1997

Corn Bt IR, HR Monsanto May 1997

Tomato Bt IR Monsanto March 1998

Corn Bt IR, HR AgrEvo May 1998

Virus and insect resistance

Potato Bt IR, VR Monsanto December 1998

Potato Bt IR, VR Monsanto February 1999

Virus resistance only

Squash VR Upjohn/Asgrow December 1994

Squash VR Asgrow June 1996

Papaya VR Cornell University September 1996

Bt IR = Bt

endotoxin-

based insect

resistance;

VR = virus

resistance;

HR =

herbicide

resistance