Molecular Biotechnology: Principles and Applications - Glick, B., Pasternak, J. 2002

Molecular Biotechnology of Microbiological Systems
Plant Genetic Engineering: Applications
Development of Insect-, Virus-, and Herbicide-Resistant Plants

The primary goal of plant biotechnology experiments is the creation of new crop varieties. Most early research focused on obtaining high-yielding plant varieties without altering their nutritional value. Genes conferring resistance to insect pests, Viruses, herbicides, and adverse environmental conditions, as well as genes delaying senescence, were introduced into plants. Some of these efforts will be discussed below. In addition, experiments have been conducted to alter flower color and the quality of plant products, as well as to use plants as "bioreactors."

Insect-Resistant Plants

If cereal crops could be genetically engineered to produce functional insecticides, we would obtain insect-resistant varieties that do not require spraying with expensive and hazardous chemical pesticides (such spraying often has to be performed six to eight times during the growing season). It is estimated that approximately $4 billion was spent globally on chemical insecticides in 1995. Consequently, the production cost of grain from insect-resistant crops would be lower than that of non-resistant ones. Furthermore, biological insecticides typically act only on a strictly limited number of insect species and are safe for humans and other higher animals.

To create insect-resistant plants using Introduction/32.html">Genetic Engineering Methods, various strategies have been developed. One approach utilized the insecticidal protoxin Gene produced by a subspecies of Bacillus thuringiensis (Chapter 15). Another approach involved plant protein genes such as amylase or proteinase inhibitors, which are effective against a wide range of insects. An insect that ingests one of these inhibitors becomes unable to digest plant food because the inhibitors interfere with the Hydrolysis of Starch or plant Proteins.

The protoxin from B. thuringiensis is a safe means of plant protection: upon release into the environment, it loses its activity. Unfortunately, many cereal pests feed on the internal Tissues of the plant, rendering B. thuringiensis preparations sprayed onto the plant surface largely ineffective. This problem can be solved by ensuring the expression of toxin genes within the plants themselves. In this case, spraying insecticides is unnecessary, the toxins are not released into the environment, and problems related to limited persistence resulting from degradation do not arise. The task of biotechnologists is to create a transgenic plant that synthesizes an active form of the bacterial insecticide in quantities sufficient to protect the plant from pests. The cryIA(a), cryIA(b), and cryIA(c) genes responsible for the Synthesis of the insecticidal proteins of B. thuringiensis ssp. kurstaki are practically unexpressed in plants (Table 18.1), whereas The Development of commercially viable, insect-resistant plants requires these proteins to be synthesized in large amounts.

Class="center">Table 18.1. Expression of certain genes encoding Bacillus thuringiensis insecticides in Transgenic Plants^1),2)

¹⁾ Based on Ely, pp. 105-124, in Entwistle et al. (ed.), Bacillus thuringiensis, an Environmental Biopesticide: Theory and Practice, 1 < 393.

²⁾ Abbreviations: full-length — full-size protoxin gene; truncated — truncated version of the protoxin gene; WT — wild-type codons; PM — partially modified codons; FM — fully modified codons.

Attempting to solve this problem, researchers reduced the size of the inserted gene so that only the N-terminal portion of the toxin molecule was synthesized, and equipped it with a strong plant promoter to increase the expression level. As a result, The amount of synthesized toxin increased significantly, and transgenic plants acquired a degree of protection against insect pests.

The next objective was to find the minimum length of The nucleotide sequence required for toxin activity.

To determine whether different toxins share the same domain, the Amino acid sequences of protoxins produced by various B. thuringiensis strains were compared. It turned out that the N-terminal region of the protoxin molecules from different B. thuringiensis ssp. kurstaki strains is highly conserved (98% Homology), whereas the C-terminal region is more variable (45% homology). Further studies showed that all the insecticidal activity of the toxin is provided by the first 646 N-terminal amino acid residues of the protoxin molecule, which has a total length of 1156 Amino Acids. The region of the protoxin gene encoding this highly conserved Amino Acid Sequence was cloned and expressed in Bacteria; it was found that under laboratory conditions, this truncated protein is just as effective in protecting plants against Lepidoptera insects as its native form.

To comprehensively study the ability of the truncated protoxin gene to protect plants from various insect pests, transgenic tomato plants were generated. The truncated gene, equipped with a strong constitutive cauliflower mosaic virus 35S promoter and the nopaline synthase gene METABOLISM/31.html">Transcription termination/polyadenylation site, was cloned into the T-DNA of a cointegrative Ti plasmid vector (Fig. 18.1). The vector also contained: 1) a spectinomycin resistance gene (Spc^r) for Selection in either E. coli or A. tumefaciens; 2) an E. coli origin of Replication; and 3) a neomycin phosphotransferase gene under the control of the nopaline synthase promoter and transcription termination/polyadenylation site, allowing the selection of transformed plant Cells in the presence of kanamycin. In addition, the cointegrative vector contained the right flanking sequence of T-DNA from a nopaline Ti plasmid and a segment of an octopine Ti plasmid that ensures cointegrate formation with a "disarmed" Ti plasmid via Homologous Recombination. E. coli was transformed with the constructed plasmid and then transferred by conjugation into an A. tumefaciens strain containing the "disarmed" Ti plasmid. Following recombination in A. tumefaciens, the truncated protoxin gene was integrated into the tomato chromosomal DNA.

Fig. 18.1. Cointegrative cloning vector carrying the insecticidal toxin gene of B. thuringiensis (B.t). The gene is under the control of the cauliflower mosaic virus strong constitutive 35S promoter (p35S) and the nopaline synthase gene transcription termination/polyadenylation site (tNOS). The vector also contains: an E. coli origin of replication (ori) and a spectinomycin resistance gene (Spc^r), ensuring its Amplification in E. coli and allowing the selection of corresponding cells; a T-DNA right border sequence; a plant selectable marker gene; and a sequence homologous to the non-oncogenic Ti plasmid that ensures the Integration of the two Plasmids. The neomycin phosphotransferase (NPT) gene is under the control of the nopaline synthase gene transcriptional regulatory elements (pNOS and tNOS) and is used for the selection of kanamycin-resistant transformed plant cells.

Both in the greenhouse and in field trials, transgenic tomato plants synthesizing the truncated form of the protoxin exhibited a degree of protection against insects such as the tobacco hornworm (Manduca sexta), the tomato fruitworm (Heliothis zea), and the tomato pinworm (Keiferia lycopersicella) (Table 18.2). The effect varied among different insects and was not absolute, being most pronounced in the first two cases. Occasionally, good results were achieved by treating protoxin-synthesizing plants with low doses of a chemical insecticide. However, further research is needed to determine how to minimize Damage caused by these and other insect pests even more effectively.

To dramatically increase the expression level, two other approaches were employed (Table 18.1). In the first approach, Site-Directed Mutagenesis was used to alter Regions of the isolated toxin gene that might be responsible for decreased transcription or Translation efficiency in the host plant (both tobacco and tomatoes were used in these experiments). The nucleotide sequence of the modified gene was 96.5% identical to that of the wild-type gene. Transgenic plants expressing this "weakly" modified gene synthesized 10 times more toxin than plants transformed with the wild-type gene.

In the second approach, a "fully" modified form of the toxin gene was chemically designed and synthesized. This gene contained codons more frequently used by plants compared to those "preferred" by Gram-positive bacteria. Changes were also introduced to prevent the Formation of secondary structures in the mRNA or to eliminate plant-like polyadenylation sites that could reduce the expression level. The GC content of the "fully" modified gene was 49% (compared to 37% for the wild-type gene), and its nucleotide sequence was only 78.9% homologous to that of the wild-type gene.

Table 18.2. Susceptibility of transgenic and wild-type tomato plants to insect pests¹⁾

1) Based on Delannay et al., Bio/Technology 7: 1265—1269, 1989.

Transgenic plants transformed with the heavily modified protoxin gene synthesized 100 times more toxin than plants transformed with the wild-type gene, showing a direct correlation with increased insecticidal activity. These findings offer hope that expression levels of many other foreign genes in plants can be successfully increased in a similar manner.

Attempts were made to increase the amount of protoxin synthesized in plants by expressing the "fully" modified protoxin gene under the control of the ribulose bisphosphate carboxylase small subunit gene promoter, placed downstream of the enzyme's chloroplast transit peptide, so that the overproduced protoxin would be localized in the Chloroplasts. This strategy resulted in a radical increase in the protoxin Gene Expression level, with protoxin accounting for up to 1% of total leaf protein. In another experiment, the protoxin gene was introduced directly into the chloroplast DNA of the host plant. This offers several advantages. First, the introduced gene does not need to be modified because the transcription and translation machinery of chloroplasts is of the prokaryotic type. Second, since There are many chloroplasts per Cell and many copies of chloroplast DNA per chloroplast, the protoxin gene is present in a high copy number, thereby increasing expression efficiency. Third, chloroplasts are inherited exclusively via the egg cell rather than pollen, so plants inherit chloroplast DNA maternally, eliminating any risk of unwanted protoxin gene transfer via pollen to other plants.

One form of the protoxin gene has already been introduced and expressed in such plants as tomatoes, tobacco, potatoes, rice, corn, apple, eggplant, canola, alfalfa, walnut, poplar, spruce, cranberry, and cotton. The Prospects for applying this method of plant protection look extremely promising. For instance, efficient expression of a synthetic gene based on the insecticidal toxin gene of B. thuringiensis ssp. tenebrionis, adapted to the plant codon usage, has been achieved in transgenic potato plants. The resulting plants proved to be highly resistant to the Colorado potato beetle, the primary pest of potatoes. Successful multi-year field trials of this crop have already been conducted, and approval for its commercial use has been granted in the USA. However, It is important to remember The Need for continuous monitoring of insect pest populations to detect resistant organisms in a timely manner. In the future, it may be necessary to use a more potent B. thuringiensis protoxin to protect transgenic potatoes or, more likely, to identify and clone other insecticidal genes in plants In addition to B. thuringiensis protoxin genes.

Currently, methods are being developed to reduce the selective pressure exerted by transgenic plants expressing the B. thuringiensis protoxin gene on resistant insect pests. In one approach, the expression of the B. thuringiensis gene in the transgenic plant was temporally restricted. To achieve this, it was placed under the control of the tobacco PR-1a (Pathogenesis-related) gene promoter, whose expression is part of the plant's natural defense mechanism against pathogens. The PR-1a gene is induced by any pathogen or chemical agent such as salicylic or polyacrylic acid. When transgenic plants carrying the B. thuringiensis protoxin gene under the control of the PR-1a promoter were treated with a chemical inducer, they were found to synthesize significant amounts of the insecticide within 24 hours post-Treatment, which is sufficient for subsequent protection against insect pests. Thus, protoxin synthesis can be induced by treating the transgenic plant with an inexpensive and safe chemical at a specific time during the growing season. Such periodic protoxin synthesis reduces the selective pressure on resistant insects. Similar systems may prove useful for regulating the synthesis of A wide variety of foreign proteins in transgenic plants. No single type of B. thuringiensis protoxin is effective against all insect species. Over the course of evolution, plants have developed general defense mechanisms against insects to ensure their survival, but the degree of this protection is not always sufficient. Some plants synthesize proteinases inhibitors that, upon entering the insect gut, block the hydrolysis of plant proteins. It was logical to assume that if a plant proteinase inhibitor gene were isolated and equipped with a strong promoter, transgenic crops capable of synthesizing sufficient amounts of the proteinase inhibitor to protect against insect pests could be created. In one such experiment, a clone encoding cowpea Trypsin inhibitor was isolated from a complementary DNA (cDNA) library using a chemically synthesized DNA probe (the design of the DNA probe was guided by The amino acid sequence of this protein). The full-length cDNA was subcloned into a Ti plasmid-based binary vector (Fig. 18.2) and introduced into an A. tumefaciens strain harboring a non-oncogenic Ti plasmid with active vir genes. Following infection of tobacco leaf disks with this A. tumefaciens vector, cells containing the complementary DNA were selected based on their ability to grow in the presence of kanamycin, and transgenic plants were regenerated from them. The damage caused by Heliothis virescens (budworm) larvae to transgenic plants synthesizing more than 2 µg of trypsin inhibitor per 1 mg of plant protein was significantly lower than that observed in conventional plants.

Fig. 18.2. Binary cloning vector carrying the cowpea trypsin inhibitor gene. The vector contains a broad-host-range DNA replication origin (on) and a kanamycin resistance gene (Kan^r) that Functions in both E. coli and A. tumefaciens. Between the right (R) and left (L) T-DNA border sequences are: 1) the neomycin phosphotransferase (NPT) gene under the control of nopaline synthase gene transcription regulatory elements (pNOS and tNOS), which allows for the selection of kanamycin-resistant transformed plant cells; 2) the cowpea trypsin inhibitor gene under the control of the cauliflower mosaic virus 35S promoter (p35S) and the nopaline synthase gene transcription termination/polyadenylation signal (tNOS).

Cowpea seeds containing the aforementioned amount of inhibitor are non-toxic to animals and humans. However, should such a risk exist, inhibitor gene expression could be restricted to those plant tissues preferred as food by major insect pests but not consumed by humans and animals. Thus, the cloned proteinase inhibitor gene could function in the leaves and roots of the plant, but not in its fruit.

Introduction of the potato proteinase inhibitor II gene into rice plants protects them against the pink stem borer (Sesamia inferens), a major insect pest of this crop; infestation leads to hollow stems and dead, seedless panicles. A plasmid was constructed containing the potato proteinase inhibitor II gene under the control of its own promoter and transcription termination signal. The first intron of the rice Actin gene was inserted between the promoter and the coding region of the inhibitor gene. This construct was introduced into suspended rice cells by microprojectile bombardment (Fig. 18.3), and transgenic plants were regenerated from them. When pink stem borer larvae were placed on the plants thus obtained, only 15 to 20% of the latter were damaged, compared to 70 to 100% for wild-type plants. Since plant proteinase inhibitors are normal Components of the Human and Animal diet and are rapidly inactivated during cooking, their introduction into new cereal crops can be considered safe.

Fig. 18.3. Plasmid vector carrying the potato proteinase inhibitor II gene. Abbreviations: Pin2 — potato proteinase inhibitor II gene; 5'-end — DNA segment preceding the gene; 3'-end — DNA segment following the gene; Act1 intron — first intron of the rice actin 1 gene; 35S 5'-end — cauliflower mosaic virus 35S promoter; bar — bacterial phosphinothricin acetyltransferase gene; nos 3'-end — DNA segment following the nopaline synthase gene. The bar gene serves as a selectable marker for transgenic plants, conferring resistance to the herbicide Basta (glufosinate-ammonium).

Another approach to increasing the efficiency of plant protection using B. thuringiensis toxin is based on the combined use of this toxin and a Serine proteinase inhibitor. It has been shown that a mixture of purified B. thuringiensis toxin at a concentration ensuring minimal insect mortality and a low concentration of proteinase inhibitor exhibits 20 times greater insecticidal activity than B. thuringiensis protoxin alone. To test whether this system would function in transgenic plants, a DNA fragment encoding a proteinase inhibitor/truncated toxin fusion protein was constructed. Transgenic tobacco plants that synthesized small amounts of this recombinant protein were largely protected from insect pests.

Yet another method of plant protection involves introducing a gene encoding an $\alpha$-amylase inhibitor. Cereals suffer heavy damage from seed-feeding insects such as the cowpea weevil (Callosobruchus maculatus) and the azuki bean weevil (C. chinensis). If common beans (Phaseolus vulgaris) are included in the diet of the larvae of these insects, their growth is retarded. This is due to the presence of an $\alpha$-amylase inhibitor in common bean seeds. The $\alpha$-amylase inhibitor gene isolated from the common bean was placed under the transcriptional control of a strong seed-specific bean phytohemagglutinin gene promoter and used to transform peas (Pisum sativum), which are normally highly susceptible to the aforementioned insects. Transgenic pea plants synthesizing the $\alpha$-amylase inhibitor were resistant to both insects, with the effect being proportional to the amount of inhibitor synthesized by the plant in the case of the cowpea weevil (Fig. 18.4). An alternative approach to developing insect-resistant transgenic plants is based on The Use of a bacterial Cholesterol oxidase gene. This enzyme, synthesized by various bacteria, catalyzes The oxidation of 3-hydroxysteroids to ketosteroids and hydrogen peroxide. It is frequently used in human serum cholesterol assays, and in small quantities, it exhibits high insecticidal activity against bollweevil (Anthonomus grandis grandis) larvae (Fig. 18.5). This widespread coleopteran insect causes significant damage to cotton crops. Cholesterol oxidase is less effective against lepidopteran insect pests. The action of the enzyme appears to involve the disruption of the midgut epithelial cell membrane of the insect, leading to its death. The cholesterol oxidase gene, encoding a protein with a Molecular Weight of 55,000 Da (504 amino acid residues) and a leader peptide with a molecular weight of 5,000 Da (43 amino acid residues), was isolated from a Streptomyces strain and inserted into a vector along with the figwort mosaic virus promoter and a transcription termination signal from the 3'-region of the A. tumefaciens nopaline synthase gene. When this construct was introduced into tobacco cell protoplasts, the transformed cells actively expressed cholesterol oxidase. In the future, this gene will likely be introduced into cotton plants, where — either independently or in combination with other biological insecticide genes — it will become an effective tool for protecting plants from insect pests.

Fig. 18.4. Mortality of cowpea weevil larvae developing on transgenic pea plants as a function of the amount of $\alpha$-amylase inhibitor synthesized by the plants.

Fig. 18.5. Mortality of bollweevil larvae as a function of cholesterol oxidase concentration. (Corbin et al., Appl. Environ. Microbiol. 60: 4239—4244, 1994.)

Virus-Resistant Plants

Plant viruses often cause significant damage to crops and substantially reduce yields. To avoid treating crops with chemicals, breeders have attempted to transfer natural virus resistance genes from one plant line to another. However, resistant plants often become susceptible again, and resistance to one virus does not guarantee resistance to others. Natural Immunity to viral infections is due to various factors: blocking The entry of the virus into the plant, preventing its spread, or suppressing the symptoms of viral infection. To obtain virus-resistant plants, "immunization" has been carried out using viral genes encoding coat proteins, other viral genes, or antisense sequences of the viral genome.

If a transgenic plant expresses a gene encoding the coat protein of a virus that normally infects that plant (with this protein often being the major protein component of the virus), the ability of the virus to enter and spread within the plant is frequently greatly reduced. The Mechanism of viral proliferation inhibition in the presence of coat protein genes has not been precisely established, but it is clear that the antiviral action begins to manifest at Cytology/cytology/16.html">Early stages of viral replication, so that Viral Particles are not formed. This reduces the likelihood of spontaneous viral mutants arising that are capable of replication in the presence of the viral coat protein. Using this approach, virus-resistant transgenic plants of many different cereal crops have been obtained (Table 18.3). Although absolute resistance could not be achieved in this way, the level of resistance was very high. Furthermore, it was found that the coat protein gene of one virus can sometimes confer resistance to a broad range of unrelated viruses. The value of this approach is further enhanced by the fact that transgenic plants develop similarly in both field and laboratory conditions.

An RNA molecule complementary to the transcript of a normal gene (mRNA) is called antisense RNA, whereas the mRNA involved in translation is called sense RNA. Antisense RNA forms a duplex with mRNA, thereby blocking translation so that the synthesis of the corresponding gene product is reduced in its presence. In addition, the antisense RNA-mRNA duplex rapidly degrades, reducing the Abundance of the specific mRNA within The Cell. Given all the above, one can attempt to prevent plant virus replication and protect plants against them by introducing a gene that provides for the synthesis of antisense RNAs complementary to the viral coat protein mRNA.

Table 18.3. Some virus-resistant transgenic plants synthesizing viral coat proteins¹⁾

¹⁾ Based on Fitchen, Beachy, Annu. Rev. Microbiol. 47: 739-763, 1993.

To compare the effectiveness of approaches based on the use of a viral coat protein gene, on the one hand, and antisense RNA, on the other, the coat protein cDNA of cucumber mosaic virus (CMV) was cloned into tobacco plants in two orientations, "sense" and "antisense" (each individual plant containing one of these orientations), and the susceptibility of the transgenic plants to viral infection was subsequently determined (Fig. 18.6). The CMV genome consists of three separate single-stranded RNA molecules, each encoding a specific viral protein. In vivo, one of these molecules — RNA3 — undergoes Processing; a portion of its sequence is removed to generate RNA4, which encodes the viral coat protein. The creation of transgenic plants that synthesize either normal mRNA and the viral coat protein or the corresponding antisense RNA involves the following steps:

Fig. 18.6. Procedure for introducing cucumber mosaic virus coat protein cDNA into plant cells. RNA4, encoding the coat protein, is isolated from total viral RNA preparations and used as a template for double-stranded cDNA synthesis. Linker sequences are attached to the cDNA, and it is inserted into an E. coli plasmid-based vector. Clones containing full-length cDNA are selected, excised from the E. coli vector, and inserted into a Ti plasmid vector between the cauliflower mosaic virus 35S promoter (p35S) and the transcription termination signal of the ribulose bisphosphate carboxylase small subunit gene (rbcS). Here, the RNA4 cDNA is inserted in two orientations, such that in one case the transcript is sense RNA and the coat protein is synthesized, while in the other an RNA complementary to the coat protein mRNA — antisense RNA — is formed.

1. Isolation of RNA4.

2. In vitro enzymatic synthesis of cDNA on RNA4.

3. Attachment of linker sequences to the cDNA.

4. Insertion of the full-length cDNA into cloning vectors in both orientations, placing each under the control of the cauliflower mosaic virus 35S promoter and the transcription termination regulatory signals of the plant ribulose bisphosphate carboxylase small subunit gene.

5. Regeneration of individual transgenic plants with the cDNA integrated into their genome in one of the two possible orientations. To introduce cDNAs encoding sense (protein-coding) and antisense RNA into individual tobacco cells, a binary vector system based on Ti plasmids was used (Fig. 18.7). In transgenic plants synthesizing the CuMV coat protein, viral particles did not accumulate, and infection symptoms failed to appear, regardless of the inoculum titer. In contrast, transgenic plants synthesizing antisense RNA for the CuMV coat protein showed resistance only at low concentrations of viral particles in the inoculum.

Fig. 18.7. Binary cloning vectors based on Ti plasmids containing cucumber mosaic virus (CuMV) coat protein cDNA in either "sense" (A) or "antisense" (B) orientation. The cDNAs are under the control of the cauliflower mosaic virus 35S promoter (p35S) and the transcription termination/polyadenylation signal of the ribulose bisphosphate carboxylase small subunit gene (tRBC). The vectors also contain the neomycin phosphotransferase gene (NPT gene) under the control of nopaline synthase gene transcription regulatory elements (pNOS and tNOS), a spectinomycin resistance gene (Spc^r), the right and left T-DNA flanking sequences, and a broad-host-range DNA replication origin (ori). A → Z — "sense" cDNA orientation, Z → A — "antisense".

Similar results were obtained in other laboratories that generated transgenic plants synthesizing antisense RNA copies of viral coat protein genes to test whether these plants could withstand viral infection. In all cases, the plants showed resistance to infection only when the titer of the inoculum used was low. The general Conclusion from such experiments is that antisense RNA copies of viral coat protein genes provide much poorer protection to transgenic plants against viral infections than sense copies of viral coat protein genes. While the antisense RNA-based protection strategy should perhaps not be entirely abandoned, it requires significant improvement before this technique can be implemented.

Agricultural crops are frequently exposed to multiple viral infections; any of these can damage the plants and reduce yield. Ideally, transgenic plants should be resistant to more than one virus. To achieve this goal, binary vectors based on Ti plasmids carrying one or more coat protein genes of CuMV, zucchini yellow mosaic virus, and watermelon mosaic virus 2 were used to transform yellow crookneck squash (Cucurbita pepo) plants (Fig. 18.8). Transgenic plants expressing all three genes were resistant to all these viruses under laboratory conditions. Plants expressing the coat protein genes of zucchini yellow mosaic virus and watermelon mosaic virus 2 were tested in the field for resistance to aphids—natural insect vectors of these viruses to growing plants. Plants expressing both coat protein genes exhibited complete resistance to simultaneous infection by these viruses (Fig. 18.9), whereas plants expressing only one of the viral coat proteins showed delayed symptom development, but eventually all symptoms of viral infection appeared, and the plant lost its commercial value. Thus, it is clear that the most effective strategy for developing transgenic plants resistant to all major viruses that retard their GROWTH AND DEVELOPMENT is the introduction of multiple genes determining the synthesis of viral coat proteins.

Fig. 18.8. A. T-DNA carrying the neomycin phosphotransferase gene (NPT II) as a selectable marker, the ß-glucuronidase gene (GUS) as a reporter gene, two copies of the watermelon mosaic virus 2 (WMV2) coat protein gene, and the cucumber mosaic virus (CMV) coat protein gene. The left and right T-DNA flanking sequences are designated as L and R, respectively. B. A construct analogous to construct A, but lacking CMV and NPT II, containing a single copy of WMV2 and the zucchini yellow mosaic virus (ZYMV) coat protein gene. C. A construct analogous to construct B, but containing CMV. All three constructs contain appropriate promoters and transcription termination signals.

Fig. 18.9. Disease incidence in transgenic yellow crookneck squash and wild-type plants under field conditions. Aphids were used to transmit a mixture of zucchini yellow mosaic virus (ZYMV) and watermelon mosaic virus (WMV) to the squash plants. (Adapted from Fuchs, Gonsalves, Bio/Technology 13: 1466-1473, 1995.)

Preliminary data indicate that transgenic plants expressing viral genes other than coat protein genes (such as viral satellite RNA genes or viral replication genes) are also conferred some degree of protection against viral infections, though the efficacy and practical applicability of these approaches remain unclear.

Protecting plants from pathogenic viruses can be achieved not only by "immunizing" them with viral protein genes, but also through the action of antiviral proteins synthesized by the plants themselves. For instance, the cell walls of the pokeweed plant (Phytolacca americana) contain three different antiviral proteins: PAP, synthesized in leaves in the spring; PAPII, found in leaves in the summer; and PAP-S, present in seeds. These proteins are easily isolated from aqueous extracts of ground plant tissues. Applying a small amount of PAP to the leaves of other plants renders them resistant to several viruses as well. Therefore, the PAP gene is a promising candidate for generating transgenic plants resistant to a broad spectrum of plant viruses.

The isolated PAP cDNA was introduced into the tobacco and potato genomes using Ti plasmid-based binary vectors. Transformants synthesizing high levels of PAP (> 10 ng per 1 mg of total protein) were stunted, mottled, and sterile, whereas plants with lower PAP content (1–5 ng per 1 mg of protein) had a normal appearance and were fertile. These findings suggest that exceeding a certain threshold concentration of PAP disrupts normal cellular function. The antiviral effect of the PAP protein in transgenic plants manifests primarily as a reduction in the number of lesions; however, once a lesion did form, the plant became systematically infected. This implies that PAP inhibits viral infection at an early stage. Nevertheless, when transgenic tobacco and potato plants expressing low levels of PAP were infected with potato virus X or Y, their leaves exhibited significantly fewer lesions than those of non-transformed control plants. Because the antiviral activity of PAP is evident at relatively low concentrations, it is feasible to develop transgenic plants that synthesize this protein in small amounts while concurrently employing other plant defense strategies against viruses.

Herbicide-Resistant Plants

Although over $10 billion is spent globally each year on The production of more than 100 different chemical herbicides, approximately 10% of crop yields are still lost due to weed infestation. Furthermore, many herbicides affect weeds and crops alike; fields frequently must be treated even before weeds emerge, and certain herbicides persist in the environment. Developing herbicide-resistant crops offers a way to address at least some of these challenges.

This can be achieved by:

✵ reducing herbicide uptake by the plant

✵ ensuring the synthesis of a herbicide-susceptible protein in quantities sufficient to perform its normal functions in the presence of the herbicide

✵ decreasing the binding affinity of the herbicide-susceptible protein for the herbicide

✵ ensuring metabolic inactivation of the herbicide within the plant.

The last three of these approaches have been successfully implemented. Herbicide-resistant transgenic plants developed using these strategies are listed in Table 18.4.

Plants resistant to glyphosate have been successfully developed. Glyphosate is an environmentally safe herbicide that rapidly degrades in the soil into non-toxic components. It acts as an inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a crucial enzyme in the synthesis of aromatic amino acids in both bacteria and plants. The gene encoding EPSPS was isolated from a glyphosate-resistant strain of E. coli, placed under the control of a plant promoter and transcription termination/polyadenylation signals, and introduced into plant cells. Transgenic tobacco, petunia, tomato, potato, and cotton plants that synthesized sufficient EPSPS to replace the herbicide-inhibited plant enzyme were resistant to glyphosate and survived treatment, unlike weeds.

Another resistance strategy—herbicide inactivation—was implemented for bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), a Photosynthesis-inhibiting herbicide. Resistant plants were created by introducing a bacterial gene encoding nitrilase into their genome, which inactivates bromoxynil before it can take effect (Fig. 18.10). The nitrilase gene was isolated from the soil bacterium Klebsiella ozaenae, placed under the control of the light-inducible promoter of the ribulose bisphosphate carboxylase small subunit gene, and integrated into the tobacco genome. Transgenic plants synthesized active nitrilase and were resistant to bromoxynil.

Table 18.4. Selected Examples of genetically engineered herbicide resistance

Fig. 18.10. Inactivation of the herbicide bromoxynil by *K. ozaenae* nitrilase.

Plants Resistant to Fungi and Bacteria Phytopathogenic fungi cause significant damage to agricultural crops. According to estimates, the losses incurred by farmers in Southeast Asia, Japan, and the Philippines due to rice blast—a disease affecting one of the region's main cereal crops—are roughly $5 billion per year. Currently, the primary method for combating phytopathogenic fungi involves treating plants with chemicals that accumulate in the environment and pose risks to animals, including humans. Therefore, it is crucial to develop alternative, simple, inexpensive, effective, and environmentally friendly non-chemical methods for protecting crops from fungal pathogens.

Fig. 18.11. Plasmid vector containing the rice chitinase gene cluster and the hygromycin resistance gene cluster, used for rice protoplast transformation. Transformation was carried out by treating protoplasts with polyethylene glycol in the presence of the plasmid vector. Cells resistant to hygromycin were then selected, and tested for the presence of the chitinase gene by Southern blot analysis and for chitinase expression by Western blotting. Subsequently, whole plants were regenerated from the cells.

Often, in response to pathogen invasion, plants begin synthesizing a group of specific pathogenesis-related (PR) proteins. This group includes ß-1,3-glucanases, chitinases, thaumatin-like proteins (thaumatin is a small, exceptionally sweet protein), and proteinase inhibitors; all of these interact with pathogens in one way or another. With this in mind, scientists have attempted to develop disease-resistant plants capable of constitutively expressing The genes of one or more PR proteins. For instance, transgenic plants have been produced that synthesize high levels of chitinase—an enzyme that hydrolyzes ß-1,4-linkages in the N-acetyl-D-glucosamine molecule, the primary component of the fungal Cell wall (Fig. 18.11).

These plants included rice, tobacco, and canola. The corresponding genes introduced into the plant genome were placed under the control of the cauliflower mosaic virus 35S promoter. In addition, transgenic tobacco plants were generated that constitutively synthesized not only chitinase but also ß-glucanase. Such plants were obtained by crossing a transgenic plant expressing the chitinase gene with one expressing the ß-glucanase gene. Transgenic plants synthesizing chitinase were more resistant to pathogenic fungi than control plants, even though the latter synthesized their own PR proteins in response to fungal infection. Furthermore, the ability of the beneficial fungus *Glomus mosseae* to colonize plant roots was not impaired in any way. This is likely due to differences in The cell wall composition of these fungi. Crucially, transgenic plants constitutively synthesizing chitinase were free from fungal diseases under field conditions. Apparently, this approach will prove to be a highly effective method for protecting plants against fungal pathogens. It is estimated that crop losses in potato caused by the soil-borne phytopathogenic bacterium *Erwinia carotovora* amount to approximately $100 million annually. The situation is exacerbated by the fact that plants lack any natural defense mechanisms against this infection that could be utilized for breeding resistant commercial varieties. To address this problem, a group of researchers developed transgenic potato plants actively expressing the T4 bacteriophage Lysozyme gene. In this system, lysozyme was secreted into the apoplast (the intercellular space), which is the compartment where *E. carotovora* invades and spreads. To ensure targeted secretion, a sequence encoding the barley α-amylase signal peptide was fused to the T4 phage lysozyme gene, and the gene was placed under the transcriptional control of the cauliflower mosaic virus 35S promoter, along with a transcription termination signal and a polyadenylation site. Although the lysozyme gene was driven by such a strong promoter, only very low levels of lysozyme were synthesized. However, transgenic plants carrying this genetic construct demonstrated resistance to high loads of *E. carotovora* under both laboratory and greenhouse conditions. Under natural conditions, these pathogenic bacteria are present in much smaller numbers than those used in Laboratory tests, offering hope that this genetic construct will provide reliable plant protection. Moreover, because lysozyme lyses various Gram-positive and Gram-negative bacteria, this approach could potentially be used to protect plants against a wide variety of bacterial pathogens.