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Interactions between plants, pathogens and insects: Possibilities for engineering resistance
Interactions between plants, pathogens and insects: possibilities for engineering resistance Carl J. Braun, Joseph M. Jilka, Cynthia L. Hemenway and Nilgun E. Turner M o n s a n t o C o m p a n y , St Louis, Missouri, USA Genetically engineered agricultural crops that resist viral and insect pathogens have been produced and tested under both environmentally controlled and field test conditions. Recent advances in creating plants that resist viruses, and plants that contain a bacterial protein, which confers selected insect resistance are reviewed. Recent studies on plant defense mechanisms are also discussed. In the future, plant defense mechanisms will be genetically modified to produce plants that resist insect and fungal pathogens. Current Opinion in Biotechnology 1991, 2:193-198
Introduction Rapid improvements in plant transformation techniques coupled with progress in the isolation and characterization of plant genes, provides great potential for dramatically improving agricultural crops. Indeed, genetically modified plants that resist insect damage and viral infection are likely to become commercially available within the next 5 years. Agricultural losses caused by pathogenic attack are enormous. For example, it has been estimated that up to 15% of crops worldwide are lost to insect damage alone [1]. Similar losses are attributable to fungal and viral diseases. In many cases, farmers rely on chemical applications to decrease crop losses but, because of environmental concerns, society is becoming increasingly apprehensive about the use of chemicals in agriculture. Bioteclmology has the potential to profoundly affect modem agriculture through the production of crops with increased resistance to pathogens and insects, thereby reducing the need for chemicals. In this review, we will consider recent advances in the field of viral disease resistance, and also describe the use of a bacterial protein to produce insect-resistant plants. In addition, we will briefly review plant defense mechanisms and show how they are being used to develop fungusand insect-resistant plants.
Viral protection Coat protein-mediated protection In 1986, Powell Abel et al. [2] showed that transgenic plants expressing the coat protein (CP) gene from to-
bacco mosaic virus (TMV) displayed an increased resistance towards TMV. This type of protection is called CPmediated protection. The remarkable broad scope of CPmediated protection is illustrated by recent reviews, in which more than 20 applications have been described [3",4"], and this number is certain to continue to increase. Many new applications of CP-mediated protection were reported recently at the VIII International Congress of Virology (Berlin, 1990). Viruses that infect agronomically important crops, such as potato, are being studied intensively. Potatoes are susceptible to many viruses that reduce yield and decrease tuber quality. Potato virus (PV)X, a potexvirus, can reduce yields by up to 10%, and yield losses of up to 80% can be attributed to PVY. PVY is the type member of the potyvirus group, which is not only the largest plant virus group, but also the most important group agronomically. Additional damage is incurred by multiple viral infections. For instance, a devastating synergistic effect is observed in potatoes infected by both PVX and PVY. Simultaneous protection against these two viruses has been achieved by expressing both CP genes in Russet Burbank which is the most important commercial cultivar of potato in North America [5"']. One transgenic line is completely protected when challenged mechanically with PVX and PVY. In addition, protection is observed when plants from this line are challenged with viruliferous aphids carlying PVY. The transgenic line expressing PVX and PVY CP genes also performed well under field test conditions [6°]. Together, these reports highlight several important points. First, they demonstrate commercial efficacy for protection against agronomically destructive pathogens under both environmentally controlled and field test con-
Abbreviations BNYVV---beet necrotic yellow vein virus; Bt--Bacillus thuringiensis; CAT-~chloramphenicol acetyltransferase;CP--coat protein; GUS---I~-glucuronidase;PLRV--potato leaf roll virus; PV--potato virus; SMV--soybean mosaic potyvirus; TMV--tobacco mosaic virus.
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Plantbiotechnolo~/ ditions. Second, protection against two unrelated viruses is demonstrated simultaneously and this protection is effective against the synergistic reaction between PVX and P'v-Y. Finally, resistance against a potyvims, which is the largest and most important plant virus group, is achieved. Protection against potyviruses is also demonstrated by Stark and Beachy [7°]. In this report, transgenic plants expressing the soybean mosaic potyvirus (SMV) exhibit a delay in symptoms when challenged with PVY and tobacco etch virus. Potato leafroll virus (PLRV) is another agronomically important pathogen that is transmitted to potato by aphids. PLRV infection causes yield loss and, more importantly, can severely damage tuber quality in Russet Burbank potato. Rhone-Poulenc's voluntary withdrawal of the carbamate insecticide Temik for use on potatoes has already increased the early season aphid pressure, resulting in increased PLRV infection. As a result, PLRV is a primary target for genetically engineered viral resistance. Expression of PLRV CP in tobacco and in the Desiree cultivar of potato provides these plants with significant protection against a high inoculum of viruliferous aphids [8]. Our group at Monsanto has recently demonstrated that PLRV CP expression confers resistance to PLRV under field conditions (unpublished data). Infection of sugarbeet, another agronomically important crop, by beet necrotic yellow vein virus (BNYVV) resuits in severe stunting of growth and a reduced sugar content. When protoplasts expressing BNYVV CP are infected with the virus, virus levels in these transgenic protoplasts are greatly reduced, compared with non-transformed protoplasts [9]. This is an encouraging finding and it will be interesting to see whether virus levels can be reduced at the whole plant level.
Alternative protection strategies TMV contains a coat protein gene, a movement protein gene, and one large gene coding for two proteins that presumably function as replicases. The putative replicase proteins are 183 and 126kD in size, and the larger protein is produced as a readthrough product. Golemboski and colleagues [10..] have provided evidence that the readthrough portion of the 183 kD protein may be expressed separately as a 54 kD protein. Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the 54 kD protein are unexpectedly resistant to TMV. The authors speculate that the 54 kD protein is part of the viral replication complex and that the observed protection is a result of competition between 54 and 183 kD proteins. Interestingly, transgenic tobacco plants are not protected from infection when they express the 126 kD protein [10..]. Similarly, transgenic tobacco plants expressing non-structural genes from tobacco rattle virus and alfalfa mosaic virus are not protected from viral infection [11,12]. Other strategies that interfere with viral replication have been successful. When a portion of DNA from the African cassava mosaic virus bipartite genome is transformed into
tobacco, the transformed DNA is excised in response to viral infection. Importantly, the excised DNA is disproportionally replicated at the expense of the infecting viral DNA and, consequently, viral symptoms are depressed [13]. Several other laboratories are also trying to achieve viral protection by interfering with the viral replication system. For example, it is believed that overexpression of important cis-acting DNA sequences may interfere with viral infection by disrupting viral replication. Other potential strategies include the use of viral vectors to overexpress CP genes and antisense RNAs, the expression of RNA that may interfere with viral satellites, and the use of ribozyme constructs that can cleave and inactivate target viral RNA [14]. Together, these approaches can be expected to complement and improve the levels of virus resistance already achieved with CP-mediated technology. Thus, the outlook for producing virus-resistant crops in the near future is very promising.
Bacillus thuringiensis insect control protein Varieties of Bacillus tburingiensis (Bt) produce proteins that are toxic towards several orders of insects [15,16]. In the bacterium, Btproteins are sequestered into crystalline inclusions. After ingestion by insects, they are proteolytically cleaved in the alkaline environment of the midgut, releasing active 8-endotoxins. Specificity of Bt proteins is correlated with the presence of high affinity Bt binding sites that occur in the brush border membranes of the insect midgut [17]. Bt proteins are divergent towards the carboxy terminus of the processed protein, and their specificity is thought to be a function of this diversity [18]. The toxins permeabilize the epithelial membrane within the insect midgut, disrupting the osmotic balance [16]. Perturbation of ion balance causes cells to swell and lyse with the result that insect larvae stop feeding and eventually die. Although commercial preparations of Bt proteins have been available for approximately 30 years, Indian mealmoths (Plodia interpunctella) are the only insect that have developed resistance to such a product [19]. Interestingly, this acquired resistance is limited to one type of Bt protein; other Bt proteins continue to be effective against the resistant insect. In 1987, Vaeck et al. [20] demonstrated the technical efficacy of using Bt products in transgenic plants. They showed {hat transgenic tobacco is protected from damage caused by the tobacco homworm (Manduca sexta). Progress in this area has been rapid with several crops expressing Btproteins currently being evaluated in field tests. For example, tomato plants expressing a protein from B. thuringiensis, vat kurstaki have performed well under field test conditions against a variety of Lepidopteran insects [21]. Expression of Bt at 0.0001% of soluble protein gave complete control against tabacco homworm, which is an insect that is sensitive to Bt protein. Significant control is also demonstrated against two agronomically important pests - - tomato fruitworm (Heliothis zea) and tomato pinworm (Keiferia lycopersicella).
Interactions between plants, pathogens and insects Braun, Jilka, Hemenway, Tumer Improved expression of Bt proteins in plants has been achieved by the construction of synthetic Bt genes [22-.,P1-.], which contain codons preferred by plants. In addition, polyadenylationsignals found within the coding sequence and sequences known to destabilize mRNA have been changed without altering codon specificity (F Perlak et aL, personal communication). Under field test conditions with high insect pressure, synthetic Bt protein expression in cotton provides significant protection against cabbage looper (Trichoplusia hi), beet armyworm ( Spodoptera exigua) and cotton bollworm (Heli~ this zea) [22..]. This demonstration of commercial efficacy is an important advancement because cotton is a susceptible host that normally requires between 5-8 insecticide treatments per season in order to control these insect pests. Commercial use of Bt gene-expressing cotton plants will reduce pesticide use tremendously, and will curtail the estimated $645 million dollars presently spent on insecticides per year [22..].
Plant defense mechanisms Plants respond to pathogenic attack, mechanical damage and environmental stress by activating a diverse army of defense-related genes [23,24]. Defense responses include the production of phytoalexins (small antimicrobial compounds, hydrolytic enzymes such as 13-1, 3glucanases and chitinases, and proteinase inhibitors). In addition, plant defense genes activate the extracellular modifications to primary and secondary cell walls, which strengthen the plant structural system. Most defenserelated proteins become compartmentalized, either intracellularly within the vacuole, or extraceUularly.The extracellular space that connects cells and provides the network for nutrient passage is called the apoplast, and is thought to be the primary site for defense response. Defense elicitors are known to be produced within the apoplast, and defense-related proteins accumulate to high levels within this extracellular space. Several plant defense responses affect the structure of the apoplast. Examples are lignification of the secondary cell walls that provides extensive structural support, and the expression of hydroxyproline-rich glycoproteins and glycinerich proteins which act to expand the crosslinking network within the extracellular matrix. Much attention has been paid to several of the key enzymes involved in the defense response. Phenylalanine ammonia lyase catalyzes the first step in phenylpropanoid metabolism, and is induced by ultraviolet light and by wounding [25]. This critical reaction is required for many biochemical pathways, including those for lignin and flavonoid biosynthesis. Another important enzyme that has received much attention is chalcone synthase. This enzyme is also induced by pathogenic attack, and represents the branch point for the production of isoflavonoid phytoalexins. Several of the pathogenesisrelated proteins that are expressed in response to viral infection have been identified as hydrolyases, but the
function of others remains unclear. Interestingly, many plant genes that respond to pathogenic attack are also under developmental control, and the protein products are found to accumulate in some reproductive and storage tissues.
Insect resistance Several plant genes expressed in response to tissue maceration encode serine proteinase inhibitors. Expression of these proteins provides protection against insect damage by making plant tissue more difficult to digest, and by decreasing its nutritional value [26]. Solanaceous plants contain two wound-inducible serine proteinase inhibitors, termed inhibitors I and n [27,']. When these genes are expressed in transgenic tobacco, the resulting proteinase inhibitory activity correlates with the inhibitory gene present. Johnson et al. [28°,] carried out feeding studies using tobacco homworms, which showed that inhibitor II (but not inhibitor I) inhibits larval weight gain. Because inhibitor II is active against both chymotrypsin and trypsin, whereas inhibitor I blocks chymotrypsin action only, the authors concluded that the trypsin inhibitory activity is responsible for reduction in larval growth. Hilder et aL [29] were the first to demonstrate that proteinase inhibitors are effective when expressed in transgenic tobacco. They produced transgenic tobacco plants that expressed the cowpea trypsin inhibitor gene, and these plants showed improved resistance to insect damage. Insecticidal assays show that transgenic plants expressing this gene exhibit enhanced resistance to damage caused by insects in the orders Lepidoptera, Coleoptera and Orthoptera [1]. This wide spectrum of effectiveness is encouraging because it suggests that engineering plants with more resistance to a broad range of insects might be possible. In two instances, the expression of different proteins active against insects provides additive protection [30",31"]. Expression of either pea lectin or cowpea trypsin inhibitor in tobacco yields plants that display an increased resistance to tobacco budworm (Heliothis virescens). By crossing transgenic lines, progeny plants expressing both pea lectin and cowpea trypsin inhibitor show an additive protective response [31.]. A similar response is observed in an insect growth bioassay in which serine proteinase inhibitors are mixed with Bt protein [30"] from B. thuringiensis var. Kurstaki. The Bt protein is fed to various target insects in varying amounts to produce an insect mortality rate of between 10-20%. When the same levels of Bt protein are supplemented with low concentrations (4 pM) of various serine proteinase inhibitors, insecticidal activity is increased by 2-20-fold. Together, these reports demonstrate that insect resistance can be potentiated by the application of multiple insecticidal genes. Perhaps more importantly, the use of a combination of insecticidal proteins with different modes of action greatly reduces the risk of insects developing resistance. Studies involving plant recognition and response to pathogenic attack are of great interest. Ryan and cowork-
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Plant biotechnology ers [27°.,32] have recently discovered that both sucrose and methyl jasmonate can each elicit expression of proteinase inhibitor genes. The potato inhibitor II gene is regulated in leaves by physical damage; in the tuber, expression is tumed on by developmental signals, and the protein accumulates to high levels. Leaf tissue from transgenic tobacco plants expressing a chimeric potato inhibitor II-chloramphenicol acetyltransferase (CAT) reporter gene responds with a 50-fold increase in transcription of this gene when floated on solutions containing 1% sucrose [32]. Deletional analysis of the chimeric gene demonstrates that wound-inducibility is localized to a small region within the promoter [33]. Exposure to sucrose does not affect this deletional mutant phenotype, implying that sucrose modulates, but cannot activate transcription [32]. Methyl jasmonate is a lipid-derived compound that acts as an inducer of proteinase inhibitor genes in plants [27.°]. Apart from ethylene, methyl jasmonate is the only known chemical signal that can become airborne and activate plant defense genes. Farmer and Ryan [27"] postulate that, after plants are wounded, linolenic acid, which is a common plant fatty acid, can be released by specific lipases. Methyl jasmonate, or a de-esterified derivative, jasmonic acid, is then produced by cyclo-oxygenases. Interestingly, methyl jasmonate induces approximately 2.5fold more inhibitor I protein than does wounding [27.']. Studies involving fusions between promoters and reporter genes reveal that tissue-specific expression occurs in response to wounding. When transgenic tobacco plants containing a proteinase inhibitor II promoter fused to CAT are wounded mechanically, CAT is expressed quickly and reaches highest levels in the apical section of the leaves [34"]. When aphids feed on the transgenic leaves (28 aphids/cm2), CAT activity is not induced. Conversely, tobacco hornworm feeding does induce CAT production. These larvae preferably attack the lamina portion of the leaf, and localization studies show that CAT expression occurs within the damaged region. Two important conclusions can be drawn from these studies. Firstly, the proteinase II promoter is expressed at the highest levels in tissues that are preferred by insect predators. Secondly, there must be sufficient tissue damage to elicit a wound response (the damage caused by aphid feeding is insufficient in this case). Thomburg et al. [34 °] speculate that wound induction requires the release of polygalacturonases, which are then proteolytically processed to produce proteinase inhibitor-inducing factors. Promoter deletional analysis, using the wound-inducible w u n l gene from potato fused to the reporter gene [3-glucuronidase (GUS), shows that several c~s-acting sequences affect expression. The fusion of an upstream segment of the w u n l gene (residues - 571 to - 1022) to the GUS gene enhances expression by 13-370-fold. This gene segment is also responsible for wound induction. Ix)calization studies show that GUS activity is highest in the epidermal cells of leaves and stems. Interestingly, expression of GUS is constitutive in the develop-
ing microspore, and is not affected by wounding [35]. As well as providing an understanding of how plant defense genes are induced, these studies have provided the experimental foundations for future work on the manipulation of plant defense genes in the production of pathogen-resistant crops.
Fungal resistance Plant hydrolase enzymes, specifically [3-1,3-glucanases and chitinases, have received much attention because of their antifungal activity. These enzymes degrade the [3-1,3-glucan and chitin components of the fungal cell wall, respectively. Because they are involved in the defense against fungal attack, it is important to understand both where they are located, and how they are expressed in response to pathogenic attack. An immunocytochemical localization study has revealed that [3-1,3-glucanase is found in low levels in the vacuoles of susceptible and resistant host cells [36]. Upon fungal infection, resistant plants display a greater increase in ]3-1,3-glucanase accumulation than susceptible plants. Even though the rate of accumulation is dramatically different, the pattern of 13-1,3-glucanase localization is similar in resistant and susceptible hosts. In the resistant host, accumulation mainly occurs in the host cell walls and secondary thickenings of the xylem at the point of infection, as well as in surrounding cells. In the susceptible host, the accumulation pattern is similar, but is limited to colonized tissues [36]. Roby et al. [37"] fused the chitinase 5B gene promoter from bean to the GUS reporter gene and transformed tobacco with this construct. This promoter is induced by ethylene and the c/s-acting sequences required for ethylene induction and for maximal expression have been defined [38]. When transgenic tobacco is exposed to fungal attack, GUS activity is greatest at the site of infection, and quickly diminishes away from this area; over a distance of 3mm, GUS activity decreases by 12-fold [37"]. Although no GUS activity is observed on uninoculated leaves, the signal that induces expression must be able to migrate across the mid-rib because GUS activity is measured on the uninoculated half of the leaf. Importantly, levels of endogenenous chitinase activity correlate with levels of GUS activity. Thus, the chitinase promoter-GUS fusion gene will be a valuable tool for studying the means by which the host defense system activates chitinase expression. Obviously, the next hurdle to overcome is the development of transgenic plants that exhibit an increased resistance to fungal attack. A preliminary account of the protection of tobacco against a soil fungal pathogen has recently appeared in abstract form (R Broglie, K Broglie, M Holliday and I Chet, 1990). A US Patent [P2.] based on chitinase expression in plants and bacteria has also been granted. Part of this patent describes a fungal infection assay in which tissue from chitinase-producing, transformed and non-transformed tobacco are exposed to the fungal pathogen. Transformed tobacco exhibit decreased lesion sizes and a decreased infection rate compared with non-transformed controls.
Interactions between plants, pathogens and insects Braun, ]ilka, Hemenway, Tumer
Conclusions Despite tremendous progress in the field, several important hurdles still remain. The most important group of crop plants, the cereals, cannot yet be routinely transformed. Although recent progress has been encouraging, cereal transformation remains the most important technical obstacle for plant biotechnology. It is also clear that we must continue to focus on basic research. For example, although CP-mediated protection has been tremendously effective and has been applied in many crops, it is stiU unclear h o w expression of viral CPs in plants provides protection. Similarly, before plant defense mechanisms can be exploited to produce fungus- or insectresistant plants, a clearer understanding of plant-pathogen interactions is necessary. The last hurdle is non-scientific; government approval and, ultimately, society's willingness to accept genetically engineered food will decide the future of plant biotechnology.
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Annotated patents • •0
of interest of outstanding interest
P1. MONSANTOCOMPANY:Synthetic Plant G e n e s and Methods for 0• Preparation. 20-2-90 EPO 0385962. This patent describes an algorithm that can be used to create synthetic genes for increased expression in transgenic plants. A synthetic Bt gene was shown to be expressed at much higher levels in several transgenic crops. P2. DNA PLANT TECHNOLOGY: Chitinase-producing Bacteria and • Plants. 7-18-86 US Patent 4940840. Application of a plant defense gene to provide protection against fungal pathogens.
CJ Braun, JM Jilka, CL Hemenway and NE Turner, Monsanto Agricultural Products Company, 700 Chesterfield Village Parkway, St Louis, Missouri 63198, USA.
Introduction Rapid improvements in plant transformation techniques coupled with progress in the isolation and characterization of plant genes, provides great potential for dramatically improving agricultural crops. Indeed, genetically modified plants that resist insect damage and viral infection are likely to become commercially available within the next 5 years. Agricultural losses caused by pathogenic attack are enormous. For example, it has been estimated that up to 15% of crops worldwide are lost to insect damage alone [1]. Similar losses are attributable to fungal and viral diseases. In many cases, farmers rely on chemical applications to decrease crop losses but, because of environmental concerns, society is becoming increasingly apprehensive about the use of chemicals in agriculture. Bioteclmology has the potential to profoundly affect modem agriculture through the production of crops with increased resistance to pathogens and insects, thereby reducing the need for chemicals. In this review, we will consider recent advances in the field of viral disease resistance, and also describe the use of a bacterial protein to produce insect-resistant plants. In addition, we will briefly review plant defense mechanisms and show how they are being used to develop fungusand insect-resistant plants.
Viral protection Coat protein-mediated protection In 1986, Powell Abel et al. [2] showed that transgenic plants expressing the coat protein (CP) gene from to-
bacco mosaic virus (TMV) displayed an increased resistance towards TMV. This type of protection is called CPmediated protection. The remarkable broad scope of CPmediated protection is illustrated by recent reviews, in which more than 20 applications have been described [3",4"], and this number is certain to continue to increase. Many new applications of CP-mediated protection were reported recently at the VIII International Congress of Virology (Berlin, 1990). Viruses that infect agronomically important crops, such as potato, are being studied intensively. Potatoes are susceptible to many viruses that reduce yield and decrease tuber quality. Potato virus (PV)X, a potexvirus, can reduce yields by up to 10%, and yield losses of up to 80% can be attributed to PVY. PVY is the type member of the potyvirus group, which is not only the largest plant virus group, but also the most important group agronomically. Additional damage is incurred by multiple viral infections. For instance, a devastating synergistic effect is observed in potatoes infected by both PVX and PVY. Simultaneous protection against these two viruses has been achieved by expressing both CP genes in Russet Burbank which is the most important commercial cultivar of potato in North America [5"']. One transgenic line is completely protected when challenged mechanically with PVX and PVY. In addition, protection is observed when plants from this line are challenged with viruliferous aphids carlying PVY. The transgenic line expressing PVX and PVY CP genes also performed well under field test conditions [6°]. Together, these reports highlight several important points. First, they demonstrate commercial efficacy for protection against agronomically destructive pathogens under both environmentally controlled and field test con-
Abbreviations BNYVV---beet necrotic yellow vein virus; Bt--Bacillus thuringiensis; CAT-~chloramphenicol acetyltransferase;CP--coat protein; GUS---I~-glucuronidase;PLRV--potato leaf roll virus; PV--potato virus; SMV--soybean mosaic potyvirus; TMV--tobacco mosaic virus.
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Plantbiotechnolo~/ ditions. Second, protection against two unrelated viruses is demonstrated simultaneously and this protection is effective against the synergistic reaction between PVX and P'v-Y. Finally, resistance against a potyvims, which is the largest and most important plant virus group, is achieved. Protection against potyviruses is also demonstrated by Stark and Beachy [7°]. In this report, transgenic plants expressing the soybean mosaic potyvirus (SMV) exhibit a delay in symptoms when challenged with PVY and tobacco etch virus. Potato leafroll virus (PLRV) is another agronomically important pathogen that is transmitted to potato by aphids. PLRV infection causes yield loss and, more importantly, can severely damage tuber quality in Russet Burbank potato. Rhone-Poulenc's voluntary withdrawal of the carbamate insecticide Temik for use on potatoes has already increased the early season aphid pressure, resulting in increased PLRV infection. As a result, PLRV is a primary target for genetically engineered viral resistance. Expression of PLRV CP in tobacco and in the Desiree cultivar of potato provides these plants with significant protection against a high inoculum of viruliferous aphids [8]. Our group at Monsanto has recently demonstrated that PLRV CP expression confers resistance to PLRV under field conditions (unpublished data). Infection of sugarbeet, another agronomically important crop, by beet necrotic yellow vein virus (BNYVV) resuits in severe stunting of growth and a reduced sugar content. When protoplasts expressing BNYVV CP are infected with the virus, virus levels in these transgenic protoplasts are greatly reduced, compared with non-transformed protoplasts [9]. This is an encouraging finding and it will be interesting to see whether virus levels can be reduced at the whole plant level.
Alternative protection strategies TMV contains a coat protein gene, a movement protein gene, and one large gene coding for two proteins that presumably function as replicases. The putative replicase proteins are 183 and 126kD in size, and the larger protein is produced as a readthrough product. Golemboski and colleagues [10..] have provided evidence that the readthrough portion of the 183 kD protein may be expressed separately as a 54 kD protein. Transgenic tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the 54 kD protein are unexpectedly resistant to TMV. The authors speculate that the 54 kD protein is part of the viral replication complex and that the observed protection is a result of competition between 54 and 183 kD proteins. Interestingly, transgenic tobacco plants are not protected from infection when they express the 126 kD protein [10..]. Similarly, transgenic tobacco plants expressing non-structural genes from tobacco rattle virus and alfalfa mosaic virus are not protected from viral infection [11,12]. Other strategies that interfere with viral replication have been successful. When a portion of DNA from the African cassava mosaic virus bipartite genome is transformed into
tobacco, the transformed DNA is excised in response to viral infection. Importantly, the excised DNA is disproportionally replicated at the expense of the infecting viral DNA and, consequently, viral symptoms are depressed [13]. Several other laboratories are also trying to achieve viral protection by interfering with the viral replication system. For example, it is believed that overexpression of important cis-acting DNA sequences may interfere with viral infection by disrupting viral replication. Other potential strategies include the use of viral vectors to overexpress CP genes and antisense RNAs, the expression of RNA that may interfere with viral satellites, and the use of ribozyme constructs that can cleave and inactivate target viral RNA [14]. Together, these approaches can be expected to complement and improve the levels of virus resistance already achieved with CP-mediated technology. Thus, the outlook for producing virus-resistant crops in the near future is very promising.
Bacillus thuringiensis insect control protein Varieties of Bacillus tburingiensis (Bt) produce proteins that are toxic towards several orders of insects [15,16]. In the bacterium, Btproteins are sequestered into crystalline inclusions. After ingestion by insects, they are proteolytically cleaved in the alkaline environment of the midgut, releasing active 8-endotoxins. Specificity of Bt proteins is correlated with the presence of high affinity Bt binding sites that occur in the brush border membranes of the insect midgut [17]. Bt proteins are divergent towards the carboxy terminus of the processed protein, and their specificity is thought to be a function of this diversity [18]. The toxins permeabilize the epithelial membrane within the insect midgut, disrupting the osmotic balance [16]. Perturbation of ion balance causes cells to swell and lyse with the result that insect larvae stop feeding and eventually die. Although commercial preparations of Bt proteins have been available for approximately 30 years, Indian mealmoths (Plodia interpunctella) are the only insect that have developed resistance to such a product [19]. Interestingly, this acquired resistance is limited to one type of Bt protein; other Bt proteins continue to be effective against the resistant insect. In 1987, Vaeck et al. [20] demonstrated the technical efficacy of using Bt products in transgenic plants. They showed {hat transgenic tobacco is protected from damage caused by the tobacco homworm (Manduca sexta). Progress in this area has been rapid with several crops expressing Btproteins currently being evaluated in field tests. For example, tomato plants expressing a protein from B. thuringiensis, vat kurstaki have performed well under field test conditions against a variety of Lepidopteran insects [21]. Expression of Bt at 0.0001% of soluble protein gave complete control against tabacco homworm, which is an insect that is sensitive to Bt protein. Significant control is also demonstrated against two agronomically important pests - - tomato fruitworm (Heliothis zea) and tomato pinworm (Keiferia lycopersicella).
Interactions between plants, pathogens and insects Braun, Jilka, Hemenway, Tumer Improved expression of Bt proteins in plants has been achieved by the construction of synthetic Bt genes [22-.,P1-.], which contain codons preferred by plants. In addition, polyadenylationsignals found within the coding sequence and sequences known to destabilize mRNA have been changed without altering codon specificity (F Perlak et aL, personal communication). Under field test conditions with high insect pressure, synthetic Bt protein expression in cotton provides significant protection against cabbage looper (Trichoplusia hi), beet armyworm ( Spodoptera exigua) and cotton bollworm (Heli~ this zea) [22..]. This demonstration of commercial efficacy is an important advancement because cotton is a susceptible host that normally requires between 5-8 insecticide treatments per season in order to control these insect pests. Commercial use of Bt gene-expressing cotton plants will reduce pesticide use tremendously, and will curtail the estimated $645 million dollars presently spent on insecticides per year [22..].
Plant defense mechanisms Plants respond to pathogenic attack, mechanical damage and environmental stress by activating a diverse army of defense-related genes [23,24]. Defense responses include the production of phytoalexins (small antimicrobial compounds, hydrolytic enzymes such as 13-1, 3glucanases and chitinases, and proteinase inhibitors). In addition, plant defense genes activate the extracellular modifications to primary and secondary cell walls, which strengthen the plant structural system. Most defenserelated proteins become compartmentalized, either intracellularly within the vacuole, or extraceUularly.The extracellular space that connects cells and provides the network for nutrient passage is called the apoplast, and is thought to be the primary site for defense response. Defense elicitors are known to be produced within the apoplast, and defense-related proteins accumulate to high levels within this extracellular space. Several plant defense responses affect the structure of the apoplast. Examples are lignification of the secondary cell walls that provides extensive structural support, and the expression of hydroxyproline-rich glycoproteins and glycinerich proteins which act to expand the crosslinking network within the extracellular matrix. Much attention has been paid to several of the key enzymes involved in the defense response. Phenylalanine ammonia lyase catalyzes the first step in phenylpropanoid metabolism, and is induced by ultraviolet light and by wounding [25]. This critical reaction is required for many biochemical pathways, including those for lignin and flavonoid biosynthesis. Another important enzyme that has received much attention is chalcone synthase. This enzyme is also induced by pathogenic attack, and represents the branch point for the production of isoflavonoid phytoalexins. Several of the pathogenesisrelated proteins that are expressed in response to viral infection have been identified as hydrolyases, but the
function of others remains unclear. Interestingly, many plant genes that respond to pathogenic attack are also under developmental control, and the protein products are found to accumulate in some reproductive and storage tissues.
Insect resistance Several plant genes expressed in response to tissue maceration encode serine proteinase inhibitors. Expression of these proteins provides protection against insect damage by making plant tissue more difficult to digest, and by decreasing its nutritional value [26]. Solanaceous plants contain two wound-inducible serine proteinase inhibitors, termed inhibitors I and n [27,']. When these genes are expressed in transgenic tobacco, the resulting proteinase inhibitory activity correlates with the inhibitory gene present. Johnson et al. [28°,] carried out feeding studies using tobacco homworms, which showed that inhibitor II (but not inhibitor I) inhibits larval weight gain. Because inhibitor II is active against both chymotrypsin and trypsin, whereas inhibitor I blocks chymotrypsin action only, the authors concluded that the trypsin inhibitory activity is responsible for reduction in larval growth. Hilder et aL [29] were the first to demonstrate that proteinase inhibitors are effective when expressed in transgenic tobacco. They produced transgenic tobacco plants that expressed the cowpea trypsin inhibitor gene, and these plants showed improved resistance to insect damage. Insecticidal assays show that transgenic plants expressing this gene exhibit enhanced resistance to damage caused by insects in the orders Lepidoptera, Coleoptera and Orthoptera [1]. This wide spectrum of effectiveness is encouraging because it suggests that engineering plants with more resistance to a broad range of insects might be possible. In two instances, the expression of different proteins active against insects provides additive protection [30",31"]. Expression of either pea lectin or cowpea trypsin inhibitor in tobacco yields plants that display an increased resistance to tobacco budworm (Heliothis virescens). By crossing transgenic lines, progeny plants expressing both pea lectin and cowpea trypsin inhibitor show an additive protective response [31.]. A similar response is observed in an insect growth bioassay in which serine proteinase inhibitors are mixed with Bt protein [30"] from B. thuringiensis var. Kurstaki. The Bt protein is fed to various target insects in varying amounts to produce an insect mortality rate of between 10-20%. When the same levels of Bt protein are supplemented with low concentrations (4 pM) of various serine proteinase inhibitors, insecticidal activity is increased by 2-20-fold. Together, these reports demonstrate that insect resistance can be potentiated by the application of multiple insecticidal genes. Perhaps more importantly, the use of a combination of insecticidal proteins with different modes of action greatly reduces the risk of insects developing resistance. Studies involving plant recognition and response to pathogenic attack are of great interest. Ryan and cowork-
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Plant biotechnology ers [27°.,32] have recently discovered that both sucrose and methyl jasmonate can each elicit expression of proteinase inhibitor genes. The potato inhibitor II gene is regulated in leaves by physical damage; in the tuber, expression is tumed on by developmental signals, and the protein accumulates to high levels. Leaf tissue from transgenic tobacco plants expressing a chimeric potato inhibitor II-chloramphenicol acetyltransferase (CAT) reporter gene responds with a 50-fold increase in transcription of this gene when floated on solutions containing 1% sucrose [32]. Deletional analysis of the chimeric gene demonstrates that wound-inducibility is localized to a small region within the promoter [33]. Exposure to sucrose does not affect this deletional mutant phenotype, implying that sucrose modulates, but cannot activate transcription [32]. Methyl jasmonate is a lipid-derived compound that acts as an inducer of proteinase inhibitor genes in plants [27.°]. Apart from ethylene, methyl jasmonate is the only known chemical signal that can become airborne and activate plant defense genes. Farmer and Ryan [27"] postulate that, after plants are wounded, linolenic acid, which is a common plant fatty acid, can be released by specific lipases. Methyl jasmonate, or a de-esterified derivative, jasmonic acid, is then produced by cyclo-oxygenases. Interestingly, methyl jasmonate induces approximately 2.5fold more inhibitor I protein than does wounding [27.']. Studies involving fusions between promoters and reporter genes reveal that tissue-specific expression occurs in response to wounding. When transgenic tobacco plants containing a proteinase inhibitor II promoter fused to CAT are wounded mechanically, CAT is expressed quickly and reaches highest levels in the apical section of the leaves [34"]. When aphids feed on the transgenic leaves (28 aphids/cm2), CAT activity is not induced. Conversely, tobacco hornworm feeding does induce CAT production. These larvae preferably attack the lamina portion of the leaf, and localization studies show that CAT expression occurs within the damaged region. Two important conclusions can be drawn from these studies. Firstly, the proteinase II promoter is expressed at the highest levels in tissues that are preferred by insect predators. Secondly, there must be sufficient tissue damage to elicit a wound response (the damage caused by aphid feeding is insufficient in this case). Thomburg et al. [34 °] speculate that wound induction requires the release of polygalacturonases, which are then proteolytically processed to produce proteinase inhibitor-inducing factors. Promoter deletional analysis, using the wound-inducible w u n l gene from potato fused to the reporter gene [3-glucuronidase (GUS), shows that several c~s-acting sequences affect expression. The fusion of an upstream segment of the w u n l gene (residues - 571 to - 1022) to the GUS gene enhances expression by 13-370-fold. This gene segment is also responsible for wound induction. Ix)calization studies show that GUS activity is highest in the epidermal cells of leaves and stems. Interestingly, expression of GUS is constitutive in the develop-
ing microspore, and is not affected by wounding [35]. As well as providing an understanding of how plant defense genes are induced, these studies have provided the experimental foundations for future work on the manipulation of plant defense genes in the production of pathogen-resistant crops.
Fungal resistance Plant hydrolase enzymes, specifically [3-1,3-glucanases and chitinases, have received much attention because of their antifungal activity. These enzymes degrade the [3-1,3-glucan and chitin components of the fungal cell wall, respectively. Because they are involved in the defense against fungal attack, it is important to understand both where they are located, and how they are expressed in response to pathogenic attack. An immunocytochemical localization study has revealed that [3-1,3-glucanase is found in low levels in the vacuoles of susceptible and resistant host cells [36]. Upon fungal infection, resistant plants display a greater increase in ]3-1,3-glucanase accumulation than susceptible plants. Even though the rate of accumulation is dramatically different, the pattern of 13-1,3-glucanase localization is similar in resistant and susceptible hosts. In the resistant host, accumulation mainly occurs in the host cell walls and secondary thickenings of the xylem at the point of infection, as well as in surrounding cells. In the susceptible host, the accumulation pattern is similar, but is limited to colonized tissues [36]. Roby et al. [37"] fused the chitinase 5B gene promoter from bean to the GUS reporter gene and transformed tobacco with this construct. This promoter is induced by ethylene and the c/s-acting sequences required for ethylene induction and for maximal expression have been defined [38]. When transgenic tobacco is exposed to fungal attack, GUS activity is greatest at the site of infection, and quickly diminishes away from this area; over a distance of 3mm, GUS activity decreases by 12-fold [37"]. Although no GUS activity is observed on uninoculated leaves, the signal that induces expression must be able to migrate across the mid-rib because GUS activity is measured on the uninoculated half of the leaf. Importantly, levels of endogenenous chitinase activity correlate with levels of GUS activity. Thus, the chitinase promoter-GUS fusion gene will be a valuable tool for studying the means by which the host defense system activates chitinase expression. Obviously, the next hurdle to overcome is the development of transgenic plants that exhibit an increased resistance to fungal attack. A preliminary account of the protection of tobacco against a soil fungal pathogen has recently appeared in abstract form (R Broglie, K Broglie, M Holliday and I Chet, 1990). A US Patent [P2.] based on chitinase expression in plants and bacteria has also been granted. Part of this patent describes a fungal infection assay in which tissue from chitinase-producing, transformed and non-transformed tobacco are exposed to the fungal pathogen. Transformed tobacco exhibit decreased lesion sizes and a decreased infection rate compared with non-transformed controls.
Interactions between plants, pathogens and insects Braun, ]ilka, Hemenway, Tumer
Conclusions Despite tremendous progress in the field, several important hurdles still remain. The most important group of crop plants, the cereals, cannot yet be routinely transformed. Although recent progress has been encouraging, cereal transformation remains the most important technical obstacle for plant biotechnology. It is also clear that we must continue to focus on basic research. For example, although CP-mediated protection has been tremendously effective and has been applied in many crops, it is stiU unclear h o w expression of viral CPs in plants provides protection. Similarly, before plant defense mechanisms can be exploited to produce fungus- or insectresistant plants, a clearer understanding of plant-pathogen interactions is necessary. The last hurdle is non-scientific; government approval and, ultimately, society's willingness to accept genetically engineered food will decide the future of plant biotechnology.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest •• of outstanding interest 1.
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2.
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DELANNAYX, LAVALLEEBJ, PROKSCH RK, FUCHS RL, SIMs SR, GREENPLATE JT, MARRONE PG, DODSON RB, AUGUSTINE JJ, LAYTON JG, FISCHHOFF DA: Field Performance o f Transgenic Tomato Plants Expressing t h e Bacillus thuringten, sis var. Kurstaki Insect Control Protein. Biotechnology 1989, 7:1265-1269.
3, •
BEACHYRN: Coat Protein Mediated Resistance in Transgenic Plants. In Viral Genes and Plant Pathogenesis [book] edited by Pirone TP, Shaw JG. New York: Springer-Vedag, 1990, pp13-22. A short but thorough review of CP-mediated protection. 4. •
5. ••
LAWSONC, KANmWSm W, HALEY L, ROZMAN Ps NEWELL C, SANDERS P, TUMER NE: Engineering Resistance to Mixed Virus Infection in a Commercial Potato Cultivar: Resistance to Potato Virus X and Potato Virus Y in Transgenic Russet Burbank. Biotechnology 1990, 8:127-134. This study reports CP-mediated protection against two unrelated viruses in an agronomically important crop. KANIEWSKIW, LAWSON C, SAMMONS B, HALEY L, HART J, DELANNAYX, TUMER NE: Field Resistance of Transgenic Russet Burbank Potato to Effects o f Infection by Potato Virus X and Potato Virus Y. Biotechnology 1990, 8:750-754. This paper shows that CP-mediated protection observed under controlled conditions can also be demonstrated under field conditions. 6. •
STARK D, BEACHY RN: Protection Against Potyvirus Infection in Transgenic Plants: Evidence for Broad Spectrum Resistance. Biotechnology 1989, 7:1257-1262. Demonstration that a CP gene from one potyvirus (SMV) can confer some level of protection against unrelated potyviruses. 7. •
22. ••
PERLAKFJ, DEATON RW, ARMSTRONG TA, FUCHS RL, SIMS Sl~ GREENPLATE JT, FISCHHOFFDA: Insect Resistant Cotton Plants. Biotechnology 1990, 8:939-943. Commercial efficacy of Bt insect-control protein is demonstrated in ttansgenic cotton plants. Under abnormally high insect pressure, significant levels of protection against target insect pathogens were shown. High levels of Bt protein were produced by using synthetic genes, optimized for expression in plants.
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38.
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Annotated patents • •0
of interest of outstanding interest
P1. MONSANTOCOMPANY:Synthetic Plant G e n e s and Methods for 0• Preparation. 20-2-90 EPO 0385962. This patent describes an algorithm that can be used to create synthetic genes for increased expression in transgenic plants. A synthetic Bt gene was shown to be expressed at much higher levels in several transgenic crops. P2. DNA PLANT TECHNOLOGY: Chitinase-producing Bacteria and • Plants. 7-18-86 US Patent 4940840. Application of a plant defense gene to provide protection against fungal pathogens.
CJ Braun, JM Jilka, CL Hemenway and NE Turner, Monsanto Agricultural Products Company, 700 Chesterfield Village Parkway, St Louis, Missouri 63198, USA.