Target Cells for Gene Transformation

8/8/2019 Target Cells for Gene Transformation

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Target Cells for Gene Transformation - The first step in gene

transfer technology is to select cells that are capable of giving rise to

whole transformed plants. Transformation without regeneration and

regeneration without transformation are of limited value. In many

species, identification of these cell types is difficult.

This is unlike the situation in animals, because the plant cells are

totipotent and can be stimulated to regenerate into whole plants in

vitro via organogenesis or embryogenesis. However, in vitro plant

regeneration imposes a degree of 'genome stress', especially if plants

are regenerated via a callus phase. This may lead to chromosomal or

genetic abnormalities in regenerated plants a phenomenon referred to

as soma clonal variation.

Structure and Functions of Ti and Ri Plasmids - The most

commonly used vectors for gene transfer in higher plants are based on

tumour inducing mechanism of the soil bacterium Agrobacterium

tumefaciens, which is the causal organism for crown gall disease, A

closely related species A. rhizogenes causes hairy root disease. An

understanding of the molecular basis of these diseases led to the

utilization of these bacteria for developing gene transfer systems. Ithas been shown that the disease is caused due to the transfer of a

DNA segment from the bacterium to the plant nuclear genome.

The DNA segment, which is transferred is called T - DNA and is part of

a large Ti (tumour inducing) plasmid found in virulent strains of

Agrobacterium tumefaciens. Similarly Ri (root inducing) megaplasmids

are found in the virulent strains of A. rhizogenes. The Ti and Ri

plasmids, inducing crown gall disease and hairy root disease

respectively have been studied in great detail during the last decade.

However, we will discuss only those aspects of these plasmids which

are relevant to the design of vectors for gene transfer in higher plants.

Most Ti plasmids have four regions in common,


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(i) Region A, comprising T-DNA is responsible for tumour induction, so

that mutations in this region lead to the production of tumours with

altered morphology (shooty or rooty mutant galls). Sequences

homologous to this region are always transferred to plant nuclear

genome, so that the region is described as T-DNA (transferred DNA).

(ii) Region B is responsible for replication.

(iii) Region C is responsible for conjugation.

iv) Region D is responsible for virulence, so that mutation in this

region abolishes virulence. This region is therefore called virulene

(v) region and plays a crucial role in the transfer of T-DNA into the

plant nuclear genome. The components of this Ti plasmid have been

used for developing efficient plant transformation vectors.

The T-DNA consists of the following regions:

(i) An one region consisting of three genes (two genes tms and tms2

representing 'shooty locus' and one gene tmr representing 'rooty

locus') responsible for the biosynthesis of two phytohormones, namelyindole acetic acid or lAA (an auxin) and isopentyladenosine 5'-

monophosphate (a cytokinin). These genes encode the enzymes

responsible for the synthesis of these phytohormones, so that the

incorporation of these genes in plant nuclear genome leads to the

synthesis of these phytohormones in the host plant. The

phytohormones in their turn alter the developmental programme,

leading to the formation of crown gall

(ii) An os region responsible for the synthesis of unusual amino acid or

sugar derivatives, which are collectively given the name opines.

Opines are derived from a variety of compounds (e.g. arginine +

pyruvate), that are found in plant cells. Two most common opines are

octopine and nopaline. For the synthesis of octopine and nopaline, the


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corresponding enzymes octopine synthase and nopaline synthase are

coded by T- DNA.

Depending upon whether the Ti plasmid encodes octopine or nopaline,

it is described as octopine-type Ti plasmid or nopalinetype Ti plasmid.Many organisms including higher plants are incapable of utilizing

opines, which can be effectively utilized by Agrobacterium. Outside the

T-DNA region, Ti plasmid carries genes that, catabolize the opines,

which are utilized as a source of carbon and nitrogen.

The T-DNA regions on all Ti and Ri plasmids are flanked by almost

perfect 25bp direct repeat sequences, which are essential for T-DNA

transfer, acting only in cis orientation. It has also been shown that any

DNA sequence, flanked by these 25bp repeat sequences in the correct

orientation, can be transferred to plant cells, an attribute that has

been successfully utilized for Agrobacterium mediated gene transfer in

higher plants leading to the production of transgenic plants.

Besides 25bp flanking border sequences (with T DNA), vir region is

also essential for T-DNA transfer. While border sequences function in

cis orientation with respect to T -DNA, vir region is capable of

functioning even in trans orientation. Consequently physical separationof T-DNA and vir region onto two different plasmids does not affect T-

DNA transfer, provided both the plasmids are present in the same

Agrobacterium cell. This property played an important role in designing

the vectors for gene transfer in higher plants, as will be discussed

later.

The vir region (approx 35 kbp) is organized into six operons, namely

vir A, vir B, vir C, vir D, vir E, and vir G, of which four operons (except

vir A and vir G) are polycistronic. Genes vir A, B, D, and G areabsolutely required for virulence; the remaining two genes vir C and E

are required for tumour formation. The vir A locus is expressed

constitutively under all conditions.


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The vir G locus is expressed at low levels in

vegetative cells, but is rapidly induced to higher

expression levels by exudates from wounded

plant tissue. The vir A and vir G gene products

regulate the expression of other vir loci. The vir A

product (Vir A) is located on the inner membrane

of Agrobacterium cells and is probably a

chemoreceptor, which senses the presence of

phenolic compounds (found in exudates of

wounded plant tissue), such as acetosyringone

and -hydroxyaceto syringone.

Signal transduction proceeds via activation

(possibly phosphorylation) of Vir G (product of

gene vir G), which in its turn induces expression

of other vir genes.

Transformation Techniques Using

Agrobacterium -Agrobacterium infection

(utilizing its plasmids as vectors) has been

extensively utilized for transfer of foreign DNA

into a number of dicotyledonous species. The onlyimportant species that have not responded well,

are major seed legumes, even though transgenic

soybean (Glycine mar) plants have been

obtained.

The success in this approach for gene transfer

has resulted from improvement in tissue culture

technology. However, monocotyledons could not

be successfully utilized for Agrobacterium

mediated gene transfer except a solitary example

of

The reasons for this are not fully understood,

because T -DNA transfer does occur at the


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cellular level. It is possible that the failure in

monocots lies in the lack of wound response of

monocotyledonous cells

Requirements of Transgenic Plants -Theimportant requirements for Agrobacterium

mediated gene transfer in higher plants include

the following:

(i) The plant explants must produce

acetosyringone or other active compounds in

order to induce vir genes for virulence;

alternatively Agrobacterium may be preinduced

with synthetic acetosyringone.

(ii) The induced agrobacteria should have access

to cells that are competent for transformation; for

gene transfer to occur, cells must be replicating

DNA or undergoing mitosis (wounded and

dedifferentiated cells, fresh explants or

protoplasts have these properties).

(iii) Often transformed tissues or explants do not

regenerate and it is difficult to combine

transformation competence with totipotency

(regeneration ability); therefore, the

transformation competent cells should be able to

regenerate in whole plants, a combination that

can be easily achieved only in some species, such

In some cases, undifferentiated cells of embryos

may undergo transformation, so that the embryos

may develop into chimeric plants


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Selectable Marker Genes in Vectors Used for Gene Transfer -

Selectable marker gene Substrate used for selection

I. Antibiotics

1. Neomycin phosphotransferase (npt II) G418, kanamycin, neomycin

2. Hygromycin phosphotransferase (hpt) Hygromycin B

3. Dihydrofoate reductase (dhfr) Methotrexate trimethoprim

4. Gene ble (enzyme not known) Bleomycin

5. Gentamycin acetyltransferase Gentamycin

6. Streptomycin phosphotransferase Streptomycin

II. Herbicides

7. Mutant form of acetolactate synthase (als) Chlorosulfuron imidazolinones

8. Bromoxynil nitrilase Bromoxynil

9. Phosphinothricin acetyl transferase (bar)L - phosphinothricin (PPT; also known

as bialaphos)

10. S. enolpyruvylshikimate -3 phosphate (EPSP)synthase (aro A)

Glyphosate (Roundup

Reporter Genes Used as Scorable Markers and Their Enzyme

Assays -

Reporter gene for Substrate * and assay Identification

1. Chloramphenicol acetyl-transferase (CAT)

(14C) chloramphenicol +acetyl CoA; TLC separation

Detection of acetyl

chloremphenicol byautoradiography

2. Neomycin phosphotrans-ferase (NPT II) Kanamycin + e2p) A TP (insitu assay) Radioactivity detection (also dotblots)

3. -glucuronidase (GUS)Glucuronides (PNPG, x-

GLUC, NAG, REG)

Fluorescence detection:colourimetric, fluorometric and

histochemical

4. -galactosidase (lac Z) -galactoside (X-gal)

Colour of the colony


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5. Luciferase(a) Decanal and FMNHz

(b) ATP +02 +Iuciferin

Bioluminiscence (exposure of x-

rays film)

6. Octopine synthase (OCS)Ariginine + pyruvate +NADH

Electrophoresis

7. Nopaline synthase (NOS)Arginine + ketoglutaric acid

+ NADHElectrophoresis

Explants Used for Transformation - The explants used for

inocultion or cocultivation with Agrobacterium carrying the vector,

include protoplasts, suspension cultured cells, callus cell clumps

(undifferentiated and proembryogenic), thin cell layers (epider mis),

tissue slices, whole organ sections (e. g. leaf discs, sections of roots,

stems or floral. tissues), etc. Wounding and inoculation of whole plants

may also be used.

Neomycin Phosphotransferase Gene - npt - This gene is used

both as a selectable and a scoreable marker in experiments involving

transfer of genes leading to the production of transgenic plants. It

imparts kanamycin resistance, so that the transformed tissue can be

selected on kanamycin.

An assay for NPT II enzyme is also used to detect its presence intransformed tissue or transgenic plants. The gene for NPT II enzyme is

often used with nos promoter, which drives its synthesis.

In some cases, npt II gene had adverse effect on the expression of the

desirable gene introduced (e.g. bt2 gene for insect resistance; see

next chapter), so that alternative approaches for improving its

expression had to be used.

For an enzyme assay of NPT II (Reiss et al., 1984; Gene 30; 217-230),the enzyme is first fractionated using non denaturing polyacrylamide

gel electrophoresis (PAGE). Since the enzyme detoxifies kanamycin by

phosphorylation, radioactively labelled A TP (p32) is used with

kanamycin in an agar layer, which is used to cover the gel containing

the enzyme.


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The whole set is incubated at 35C and the phosphorylation leading -to

incorporation of 32p in kanamycin can be detected by autoradiography

The filter with dot blots is incubated with the substrates and is then

subjected to autoradiography to detect the presence NPT II enzyme.

Luciferase Gene - lux - In the presence of a suitable substrate, the

enzyme luciferase confers on the organism carrying it, the ability to

glow in the dark. In other words the organism carrying this enzyme

exhibits luminiscence on an assay for the enzyme luciferase.

The enzyme luciferase differs in bacteria and firefly. While in bacteria,

it consists of two peptide subunits coded by genes lux A and lux E, in

firefly it consists of a single polypeptide coded by a gene lux.

The enzyme luciferase in these two systems uses different substrates

for luminiscence. Therefore, depending upon the source of luciferase

gene used as a scoreable marker gene, the assay for detection of its

presence also differs

When the source of the gene is bacteria, the substrate used is an

aldehyde (decanal), which is supplied exogenously with FMNH2.

Alternatively, when the source is firefly, luciferin (a heterocyclic

carboxylic acid) is instead used as a substrate, and is supplied with

ATP and oxygen.

Chloremphenicol Acetyl Transferase Gene - cat - This gene is

primarily used as a reporter gene or a scoreable marker and not as a

selectable marker. The gene first isolated from E. coli codes for an

enzyme (CAT), which is absent in mammals and higher plants, so that

whenever transferred in a gene construct, its presence can be detectedby enzyme assay.

Rarely screening for this enzyme may also be used for selection of

transformed regenerants, although no selection pressure can be

applied


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The enzyme uses acetyl CoA + chloramphenicol (32p) as substrates

and helps in the transfer of acetyl group to chloramphenicol. The

presence of acetyl chloramphenicol is detected through

autoradiography. The gene cat has also been used for identification of

a number of regulatory sequences.

Glucuronidase GUS Gene - The enzyme -glucuronidase, popularly

described as GUS, breaks down glucuronidase giving a coloured

reaction, so that its presence can be detected in situ, i.e. inside the

plant tissue, used either as thin section or in any other form.

Several glucuronidase, which can be used as substrates, include p-

nitro phenyl glucuronide (PNPG), 5-bromo, 4-chloro, 3-indolyl /

glucuronide (BCIG), naphthol AS-B1 glucuronide (NAG) and resorufin

glucuronide (REG).

The enzyme GUS is coded by a gene gus, first isolated from E. coli.

The major advantage of using this reporter gene (scoreable marker)

lies in its assay, which requires no DNA extraction, electrophonesis or

autoradiography.

Agroinfection and Gene Transfer - Agroinfection is a phenomenon,

in which a virus infects a host as a part of T-DNA of Ti plasmid carried

by Agrobacterium. Viral DNA can be integrated into the T-DNA and can

be delivered into plant cells with the normal Agrobacterium T-DNA

transfer process. After infection viral DNA is released to form

functional virus that replicates and spreads systemically.

Agroinfection may also lead to the integration of viral DNA so that

transgenic plant containing integrated viral DNA can be produced.In

maize, agroinfection with maize streak virus has been demonstrated.


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This suggested that Agrobacterium based vector system can be used

for genetic engineering in cereals, although ordinarily Agrobacterium

does not infect monocotyledons. Thus agroinfection can lead to the

production of transgenic plants, even though it has no better chances

of yielding transgenic cereals than does Agrobacterium infection alone.

However, agroinfection has great potential for studies in virus biology,

because it can transfer deletion mutations or even single viral genes.

T DNA Transfer Process- An early event in the T-DNA: transfer

process is the nicking of Ti plasmid at two specific sites, each between

the third and fourth base of the bottom strand of each 25bp repeat.

This initiates DNA synthesis from the nick in the right hand 25bp

repeat sequence in 5' - 3' direction, thus is displacing a single T-DNA

strand.

This T-DNA single strand forms a complex with protein Vir E and gets

transported to the plant nucleus. The vir D operon encodes an

endonuclease that produces the nicks in the border sequences.

Several gene products of the vir B operon have been identified in the

bacterial envelope, a location, which suggests that they may playa role

in directing T -DNA transfer extracellularly.

The functions of several other vir gene products are largely

unknown.Apart from the role of Ti plasmid, the genes located on

Agrobacterium chromosome also help in virulence.

These genes are involved in the synthesis and secretion of glucons,

cellulose fibrils and cell surface proteins. These loci are constitutivelyexpressed and are also found in other soil bacteria associated with

higher plants.


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Thus, these loci playa more general role in the virulence of

Agrobacterium, and thus also in the Agrobacterium mediated gene

transfer

Vectors Based on Ti and Ri Plasmids - The following properties ofTi plasmids did not allow their direct use as vectors: (i) large size,

(ii) absence of uniqe restriction enzyme sites and

(iii) tumour induction property.

Therefore, only the useful attributes of Ti plasmid have been used in

designing several plant transformation vectors. Since tumour cells can

not develop into normal shoots, disarming of Ti plasmids was anessential step in designing useful vectors. This was achieved by

replacement of tumour inducting genes in T-DNA (T-DNA is found in

both Ti and Ri plasmids) by selectable markers (like npt II), providing

resistance against antibiotics like kanamycin.

Promoters and polyadenylation signal isolated from octopine or

nopaline synthase genes were used for expression of selectable

markers. Other powerful promoters included CaMV35S and CaMV19S

isolated from cauliflower mosaic virus.

As is obvious from the discussion in the previous section, T-DNA

(disarmed) and vir genes are two essential elements for designing

transformation vectors. While T-DNA with border sequences allows

manipulation of DNA sequences intended to be transferred, vir genes

allow infection of host plant.

Since vir genes can function even in trans configuration, it is not

essential that vir genes be present on the same plasmid, which

functions as a vector and carries DNA segment to be transferred. In

view of this, basically two types of Agrobacterium vectors are currently

in use:


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(i) Cointegrative vectors recombine, via DNA homology, with an

intermediate cloning vector, which is used for manipulation and cloning

of the gene in E. coli. Agrobacterium containing cointegrative vector

and E. coli containing intermediate cloning vector are allowed to

undergo conjugation, but the intermediate vector can not replicate in

Agrobacterium so that it has to transfer the marker genes as well as

the DNA segment to the resident Ti plasmid (cointegrative vector)

through recombination in the region of DNA homology.

One of the first cointegrative vectors, pGV3850 was developed from a

nopaline type Ti plasmid (C58), where almost all T-DNA has been

deleted and replaced by pBR322, a common small E. coli cloning

vector. The intermediate vector (e.g. pGVllO3) based on pBR322 is

conjugated into pGV3850 at the region of pBR322 homology.

After cointegration, pGV3850 : : 1103 T-DNA contains the whole of

intermediate vector including the unwanted DNA, which is also

transferred to host plant along with the gene intended to be

transferred. To overcome this difficulty the right border repeat or both

borders are introduced to flank a cloning site in the intermediate

vector, so that only a part of intermediate vector cointegrates. The

utility of this vector system is however limited.

(ii) Binary vectors are based on the principle that vir genes may be

located on a 'helper' Ti plasmid having the whole of T-DNA deleted

(e.g. pAL4404), because vir genes can function even in trans

configuration.

In this case, T-DNA is found on a separate vector (binary vector)

designed to replicate in both E. coli and Agrobacterium and capable of

conjugal transfer between these two bacterial species. Many binaryvectors have been developed, which differ in size and the source of

25bp repeat sequences, plant selection marker, bacterial selection

marker and cloning sites. pBin19 which was designed in 1984, is still

popular and is based on wide host range replicon pRK252.


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This vector contains the following elements: kanamycin resistance

gene (APH-l)- for selection in bacteria, T-DNA borders derived from

pTiT37, a plant selectable transformation marker (npt-11) isolated

from transposon, TnS (this marker is associated with promoter and

polyadenylation signal derived from nopaline synthesis gene or nos), a

multiple cloning site derived from pUC19 and housed within lac Z (-

galactosidase gene) region.

Bacterial colonies containing pBin19 are recognized by loss of blue

colour on IPTG/X-GAL plates. Unlike the cointegrative vectors, binary

vectors need not have ai1Y homology with resident Ti plasmid and are

capable of autonomous replication.

Binary vectors based on Ri plasmid of A.

rhizogenes have also been prepared. One such

binary vector (pARS 8) in a virulent strain (A4) of

A. rhizogenes was used for the production of

transgenic plants in tomato. Other examples of

vectors based on Ri plasmids are also available in

literature.

Virus Vectors - Many viruses or their isolatedgenomes are capable of infecting intact plant

tissue. This made them suitable for use as plant

transformation vectors. But since viral genomes

can not integrate with plant genome, they can be

used only for a study of transient expression of

transferred genes.

All the three kind of viruses including

caulimoviruses (double stranded DNA), geminiviruses (single stranded DNA) and tobacco mosaic

virus (RNA) are capable of delivering genes into

intact plant tissues, where they are expressed.

However, virus vectors can not be used for the

production of stably transformed plants, popularly


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described as transgenic plants.

Crystal (Cry) Proteins -

The cry gene of B. thuringiensis produces a protein,which forms crystalline inclusions in the bacterial

spores. These crystal proteins are responsible for the

insecticidal activities of this bacterium. The cry genes

(or Cry proteins) have so far been grouped into 16

distinct groups, which either code for a 130 kD a or a

70 kD a protein.

These proteins are solubilized in the alkaline

environment of insect midgut and are thenprototypically processed to yield a 60 kD a toxic, core

fragment (except in the case of cry IVD). The toxin

function is localized in the N-terminal half of tile 130

kD a proteins; the C-terminal half of these proteins is

highly conserved and is most likely involved in the

crystal formation.

The Cry I proteins are insecticidal to Lepidopteron

insects; all the proteins, even the Cry IA subfamily,have a distinctive insectidal spectrum. The CryIIA

proteins are active against both Lepidoptera and

Diptera, while Cry IIB is specific to Diptera.

The Cry III proteins are active against Coleoptera

species, while CryIV proteins are specific to Diptera.

But the CytA protein does not show any insecticidal

activity, is cytolytic for a variety of vertebrate and

invertebrate cells, and exhibits no homology withother Cry proteins.


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Toxic Action of Cry Proteins - When Cry proteins are ingested by insects,

they are dissolved in the alkaline juices present in the midgut lumen. The

gut pro teases process them hydrolytically to release the core toxic

fragments. The toxic fragments are believed to bind to specific high affinity

receptors present in the brush border of midgut epithelial cells.

As a result, the brush border membranes develop pores, most likely

nonspecific in nature, permitting influx into the epithelial cells of ions and

water, which causes their swelling and eventual lysis.

The presence of specific receptors in the midgut epithelium is most likely the

chief reason for Cry toxin specificity. The specificity seems to be lost upon

reduction of the cysteine residues of the protoxin, but can be restored by

reoxidation of these residues.


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Gene Protein

(mol.mass)

Bacterial

strain

Target Remarks

cryIA(a) 133.2 HD-1 Lepidoptera 1. All Cryl

proteins have a

distinctinsecticidal

spectrum

cryIA(b) 131.0 Berliner

1715

Lepidoptera 2. Most cry genes

are situated on

large conjugative

plasmids

cryIA(c) 133.3 HD-73 Lepidoptera

cryIB 138.0 HD-2 Lepidoptera

cryIC 134.8 HD-110 Lepidoptera

cryID 132.5 HD-68 Lepidoptera

cryIE 132.5 HD146 Lepidoptera

cryIIA 70.9 HD-263 Lepidoptera

and Diptera

cryIIB* 70.8 HD-1 Diptera

cryIIIA 73.1 tenebrionis Coleoptera

cryIIIB 74.2 tolworthi Coleoptera

cryIIIC 129.4 gallariae Coleoptera

cryIVA 134.4 israelensis Diptera

cryIVB 127.8 israelensis Diptera

cryIVC 77.8 israelensis Diptera

cryIVD 72.4 israelensis Diptera

cytA** 27.4 israelensis --

Insect Resistance to Cry Proteins -

There are some reports on development of resistance in some insects to Cry

proteins. For example, Plodia interpunctella and Cadra cautella were selected

by continuous exposure to high levels of B. thuringiensis. There was a >250-

fold increase in resistance in P. interpunctella after 36 generations, and a


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seven fold increase in C. cautella after 21 generations.

The problem of development of insect resistance to Cry proteins may be

managed by

(i) combining or(ii) alternating two or more kinds of these proteins, and by

(iii) reducing the selection pressure on insects by limiting cry gene

expression to only the economically important plant parts.

Low Levels of Expression of Cry Genes -

The low expression level of cry genes seems to be

due to the presence in them of sequences not

commonly found in plant coding regions. For

example, cryI(A)b contains:

(1) localized A + T rich regions resembling plant

introns,

(2) 18 potential plant polyadenylation signal

sequences,

(3) 13 ATTTA sequences, which destabilize mRNA,

and

(4) codons rarely used in plants.

CryIA(b) and cryIA(C) sequences have been either partially or fully

modified to remove the above difficulties as far as possible without

affecting the amino acid sequences of the Cry proteins encoded by them.

The transgenic plants containing these modified cry genes showed greatly

enhanced levels of expression; in case of tobacco and tomato, there was a

100-fold increase over the expression level of the wild type genes.


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Other Genes for Insect Resistance -

Several important insect pests are not susceptible to the currently available

Cry proteins. For such insects, alternative insecticidal proteins will be

needed; inhibitors of digestive enzymes, e.g., cowpea trypsin inhibitor(CpTI), serine protease inhibitor (aprotinin), cystein protease inhibitor, and

proteinase inhibitor II, and lectins appear to be quite promising.

These genes have been transferred into certain crop plants where they

produce resistance to different insects, e.g., members of Lepidoptera,

Coleoptera, etc.

Virus Resistance -Several approaches have been used to engineer plants for virus resistance,

which are as follows:

(1) coat protein gene,(2) cDNA of satellite RNA,

(3) defective viral genome,

(4) antisense RNA approach, and

(5) ribozyme mediated protection.

Of these strategies, use of coat protein gene has been the most successful.

Transgenic plants having virus coat protein gene linked to a strong

promoter have been produced m many crop plants, tobacco, tomato,alfalfa, sugarbeet, potato, etc.The first transgenic plant of this type was tobacco produced in 1986; it

contained the coat protein gene of tobacco mosaic virus (TMV) strain U I.When these plants were inoculated with TMV U I, symptoms either failed to

develop or were considerably delayed.Further, there was a much less accumulation of virus than in the control

plants in both inoculated and systemically infected leaves. In addition,

these plants showed delayed expression of disease symptoms when

inoculated with the related tomato mosaic virus (ToMV) and with tobacco

mild green mosaic virus (TMGMV).

It appears to be a common feature that expression of a virus coat protein

gene not only confers resistance to the concerned virus but also gives ameasure of resistance to related viruses.

The effectiveness of coat protein (CP) gene in conferring virus resistance can

be affected by both the amount of coat protein produced in transgenic plants


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and by the concentration of virus inoculum.

Most likely the resistance generated by CP is due to the blocking of the

process of uncoating of virus particles, which is necessary for viral genome

replication as well as expression. However, other effects seem to be involved

in producing coat protein mediated virus resistance; one such mechanismappears to be the prevention or delay of systemic spread of the viruses.

But at least in some cases, the resistance mechanism does not involve the

coat protein itself since CP genes even in antisense orientation produce

resistance to the virus.

Resistance to Bacterial and Fungal Diseases - In case of bacterial and

fungal pathogens, resistance has been sought to be generated by expression

of the following transgenes:

(1) genes encoding insensitive target enzymes,

(2) genes specifying toxin inactivation,

(3) expression of antibacterial peptides,

(4) expression of bacterial lysozymes,

(5) genes specifying artificially programmed cell death (in items 1-5,

transgenes are from non plant sources),

(6) expression of heterologous phytoalexins,

7) genes encoding ribosome inactivating proteins,

(8) expression of heterologous thionins,

(9) ectopic (out of the natural place) expression of pathogenesis related

proteins, and

(10) ectopic expression of chitinases (items 6-10 use plant genes). In almost

all the approaches, transgenic plants showed increased resistance to the

concerned diseases.

The strategy of artificially programmed cell death has been designed tomimick hypersensitive response. A programmed cell death is brought about

by endogenous gene action, particularly in response to some specific

stimulus, e.g., the elicitor specified by (avirulence) avr genes of the

pathogen in the case of hypersensitive response.

However, hypersensitive response depends on specific pairs of avr genes of


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pathogens and R (resistance) genes of the host. Therefore, each such pair

specifies resistance to a single race of a pathogen and is not of general

applicability.

In contrast, the artificiallyprogrammed cell death is so

designed as to cover all the races of

a pathogen and possibly, more than

one pathogen as well. There are two

schemes for artificial cell death, viz.,

(1) two-component and

(2) single-component systems.

The single-component system isbased on the expression of a toxic

polypeptide in response to pathogen

infection. The transgenes usable in

this scheme may be those that

encode toxins, ribonucleases, or

other enzymes, whose products are

toxic to plant cells.

The barnase gene from Bacillusamyloliquefaciens was placed under

the control of infection specific

promoter prp1-1and was transferred

into potato. Transgenic potatoes

showed effective control of

Phytophthora infestans. Promoter

prp1-1j ensures the expression of

barnase gene in such cells that are

infected by a fungal pathogen.

Synthesis of Barnase protein, an

RNase, in such cells leads to their

death; the pathogen would also die

along with the dying host cells.


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Obviously, the strategy of artificially

programme cell death will be

effective against obligate parasites,

but not against facultative

parasites; in fact, facultativeparasites may be pleased to use it

to their own advantage.

Suppression of Endogenous Genes -

In many crops, certain quality related traits can be improved by

reducing eliminating the expression of specific genes. The level of

endogenous gene expression can be reduced by the following 4approaches:

(1) antisense gene,

(2) ribozyme,

(3) gene disruption and,

(4) overtranscription leading to co-suppression

Antisense Gene Approach -

In any gene, the DNA strand oriented as 3-->5' in relation to its promoter istranscribed; this strand is called the antisense strand. The mRNA base

sequence, therefore, is complementary to that of the antisense strand. The

remaining DNA strand of the gene, called sense strand, is naturally

complementary to the antisense strand of the gene.

Therefore, the base sequence of sense strand of a gene is the same as that

of the mRNA produced by it. Hence the mRNA produced by a gene in normal

orientation is also known as sense RNA.

An antisense gene is produced by inverting, i.e., reversing the

orientation of the protein encoding region of a gene in relation to its

promoter. As a result, the natural sense strand of the gene

becomes oriented in the 3'--> 5' direction with reference to its

promoter, and is transcribed. (The normal antisense strand is not


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transcribed since now its orientation is 5' --> 3'.)

The RNA produced by this gene has the same sequence as the

antisense strand of the normal gene (except for T in DNA in the

place of U in RNA), and is therefore known as antisense RNA. When

an antisense gene is present in the same nucleus as the normal

endogenous gene, transcription of the two genes yields antisense

and sense RNA transcripts, respectively.

Since the sense and the antisense RNAs are complementary to each

other they would pair to produce double-stranded RNA molecules.

This event makes

l) the mRNA unavailable for translation. At the same time,

(2) the RNA double-strand is attacked and degraded by double-stranded

RNA specific RNases. Finally,

(3) these events may somehow lead to the methylation of the promoter and

coding regions of the normal gene resulting in silencing of the endogenous

gene.

The application of antisense RNA technology is explained using the example

slow ripening tomato. In tomato, enzyme polygalacturonase (PG) degradespectin which is the major component of fruit cell wall. This leads to the

softening of fruits and a deterioration in fruit quality.

Transgenic tomatoes have been produced which contain antisense construct

of the gene encoding PG. These transgenics show a drastically reduced

expression of PG and markedly slower ripening and fruit softening. This has

greatly improved the shelf-life and the general quality of tomato fruits. Such

tomatoes are being marketed in U.S.A. under the name 'FlavrSavr'.

Co Suppression of Genes -

In case of many endogenous plant genes, an overexpression of the

sense RNA or mRNA surprisingly leads to a drastic reduction in the


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level of expression of the genes concerned; this is called co-

suppression.

One way of achieving an overexpression of the mRNA is to

introduce a homologous sense construct of the gene concerned sothat it also produces sense RNA or mRNA (in addition to the

endogenously present gene).

The efficiency of co-suppression seems to vary among plant genes.

Co-suppression has never been observed for the petunia chalcone

isomerase gene, while tobacco glutamine synthetase nuclear gene

is always co-repressed; CHS gene (petunia) represents the

intermediate situation.

The mechanism of co-suppression is not understood. According to

a threshold model, when RNA transcripts of a get1e accumulate

beyond a critical, threshold level, they are selectively degraded by

RNases. An accumulation of high levels of RNA transcripts of a

gene may lead to the production of aberrant sense RNA transcripts

of the transgene.

An accumulation of aberrant RNA transcripts is proposed to

activate RNA-dependent RNA polymerase of plant origin, whichtranscribes the RNA transcripts to produce antisense RNA. The

antisense RNA transcripts would associate with the accumulated

normal and aberrant RNA transcripts of the transgene as well as

the endogenous gene.

This will produce RNA duplexes, which present targets for double-

stranded RNA specific RNases like RNase H. Degradation of the RNA

transcripts of a gene is postulated to somehow lead to a

hypermethylation of the DNA sequences homologous to thedegraded RNA sequences.

This often leads to a drastic reduction in the level of expression of

the transgene in question and also of homologous endogenous

gene(s), if any; this is called gene silencing.

Ethylene is an important phytohormone and is involved, among

other things, in fruit ripening, e.g., in banana, tomato, avocado,


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etc., leaf abscission and flower senescence. It is produced from

amino acid methionine, the terminal two reactions of ethylene

biosynthesis being as follows.

A reduced ethylene production results in delayed petal senescence

in carnation and slow ripening of tomato fruits. Drastically reducedethylene production has been achieved in one of the following ways:

(i) expression of antisense constructs of ACC synthase or ACC oxidase,

(ii) co-suppression of either of these enzymes, and

(iii) expression of enzymes that metabolize S-adenosyl methionine (SAM),

e.g., SAM hydrolase from bacteriophase T3 (in tomato), or ACC, e.g., ACC

deaminase (over-expression in tomato). A carnation variety with longer

vase life has its ACC synthase gene co-suppressed. A similar co-

suppression approach has been used to block the onset of fruit ripening intomatoes.

RNA Mediated Interference RNAi - Silencing of homologous gene

expression triggered by double stranded RNA (dsRNA) is called RNA

mediated interference or RNA interference (RNAi). Introduction of long

double stranded RNA into the cells of plants, invertebrates as well as

mammals leads to a sequence specific degradation of the homologous gene

transcripts.

The long dsRNA molecules are cleaved by an RNase III enzyme called Dicer;

this generates small 21-23 nucleotide long dsRNA molecules called small

interfering RNAs (siRNAs).

The siRNA molecules bind to a protein complex called RNA-induced silencing

complex; this complex contains a helicase activity that unwinds the two

strands of RNA molecules.

The antisense RNA strands so generated pair with the target RNA

molecules, and an endonuclease activity then hydrolyzes the target RNA at

the site where the antisense strand is bound.

The RNAi is a recent but potent technology and is rapidly gaining wide

acceptance. The main applications of RNAi are as follows:

(1) RNAi serves as an antiviral defence mechanism.


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(2) RNAi is becoming a powerful and is widely used tool for the analysis of

gene function in invertebrates, plants, and mammals.

(3) DNA vector based strategy allows the suppression of endogenous genes

and to produce transgenic lines with suitably modified traits. RNAi has beenused to produce low caffeine coffee.

Insect resistant transgenic plants

Serious losses in crop yields are caused due to insect pests. There are

about 67,000 pest species that damage crops. Of these, 9,000 species are

insects and mites, which are responsible for major yield losses in several of

our important crops, particularly the tropical crops. Therefore, transfer of

genes(s) providing resistance against insects in crop plants leading to theproduction of insect resistant transgenic crops has been major application

of biotechnology in agriculture sector.

The insect resistant transgenic crops carrying Bt toxin genes have been

shown to be very effective in controlling insect damage and are actually

better than the currently used methods of insect control through insecticidal

sprays (consult Chapter 47 for biopesticides).

This not only allows saving in cost and time, but also reduces health risks

and provides ecological benefits, since Bt toxins are highly specific against

certain insects without affecting other specific insects (insecticides instead

kill a broad spectrum of insects).

In classical plant breeding, following three systems for insect resistance

are utilized: (i) morphological barriers to insects (e.g. hairy leaves); (ii)

insect repellant or toxic substances released constitutively by the plant, or

induced by insect damage, and (iii) toxins that have either a repellant

affect (e.g. quinonin) or dreadly effect (e.g. proteolytic enzymes such astrypsin inhibitor found in peas).

Several insect control strategies have been proposed in biotechnological

approaches. These strategies are listed which Bt is the most effective of all

strategies used so far.


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A new insect resistance technology involving the use of smart proteins (computer

designed) and VIPs (discovered in 1997) will certainly be utilized in future.

Pyramiding of different genes active against the same insect will also become

common in future. It is predicted that as many as five or more foreign genes

against the same insect will be available in most insect resistant transgenic crops

by th year 2005.

Some of the novel systems include the following : (i) Control of insect growth

through toxicity of their purine metabolic pathway, (ii) use of Beauveria virulent

with an active avaricide against Lepidoptera; (iii) Use of 3-hydroxy-steroid oxidase

against Lepidoptera and boll weevil; (iv) use of a gene for carbonarin anti

insectant metabolites isolated from Aspergillus carbonarius.

However, the use of smart proteins, VIPs, engineered proteins (computer aided)

and manipulation of metabolic pathways (e.g. production of azadirachtin, a toxic

compound from neem tree) are believed to provide long term insect resistant

transgenic plants.Bt endotoxins and their gene

Bt toxins, symbolized as Cry meaning crystalline (reflecting the crystallin

appearance of -endotoxin), were initially classified into four distinct classes

based on their host range. These are CryI (active against Lepidpoptera)

Cryll (active against Lepidoptera and Diptera), CryIII (active agains

Coleoptera) and Cry IV (active against Diptera).

Many more classes have been added to these classes (e.g., CryV, CryVI an

Cry IX) and new strains have been shown to be active against nematode

and other pests.

So far 50 genes for 50 Cry proteins are known, of which 28 genes were

isolated from 14 different Bt subspecies that have been shown to be active

against insects. Additional 30 Cry proteins described in the literature do not

have any insecticidal properties, although they may be effective againstnematodes and mites.

These Cry genes are being variously modified to acquire new activities,

thus extending the range of their insecticidal/nematicidal properties. The

different Bt endotoxins (Cry) and their activity against specific insects are

listed.


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Insect resistant plants based on Bt

The above toxin genes (bt2) from B. thuringiensis have been isolated and

used for Agrobacterium Ti- plasmid mediated transformation of a number of

crop plants including tobacco, cotton and tomato plants. The transgenicplants were resistant to Manducta sexta, a pest of tobacco.

Experiments of feeding the leaves of these plants to larvae of M. Sexta,

showed 75%100% mortality of the larvae, while the control plants carrying

no transgenes were severely damaged. The presence of the gene bt2 as well

as that of the toxin protein synthesized under its control was also

demonstrated by appropriate experiments.

When inheritance of insect resistance was studied using crosses withnormal control plants, F1 showed resistance and F2 generation exhibited

expected segregation.

Since, a native gene often does not express the gene at high level,

truncated versions of genes are synthesized with altered codon usage and

elimination of certain sequences (codon usage means that for same amino

acids, codons that are efficient in prokaryotes but not in eukaryotes are

replaced by other degenerate codons known to be efficient in eukaryotes).

As many as 410 Btrelated patents have been issued during 1985-96, 52%

of them issued in the North America, 30% in Europe and Russian countries

and the remaining 18% mainly in Japan.

Further, about only l/3rd of these patents were related to Bt- biopesticides

and the remaining 2/3rd were related to genes or Bt-based transgenic crops.

The major Bt genes include CryIA(b), CryIA(c) and Crylll(a).

Bt-based insect resistant transgenic crops, the genes employed, and thecommercialization status of these crops are listed.

Management strategies of Bt deployment.

One of the disadvantages of the development of insect resistant crops is the

emergence of insect strains that may possess resistance against the toxin.


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This needs management strategies so that the resistance in the Bt-crops is

durable.

These strategies may be at the level of genes (pyramiding of genes or

chimeric genes), at the level of gene promoters (tissue specific or woundinducible promoters), at the level of gene expression (low dose or mixtures)

or at the level of field (mixture of genes, gene rotation, mosaic planting,

refuge spatial or temporal).

Considerable success has been achieved in pyramiding. of genes and in

monitoring the expression of genes. But in every strategy, the refuge

approach is considered necessary to avoid development of resistance in the

insects. This is achieved by ensuring that there are always plenty of

susceptible insects nearby for the few resistant ones to mate with, so thatthe population of resistant insects does not increase (susceptibility being

dominant).

Refuge areas are provided in two possible ways: (i) only part of the field is

planted with Bt-crop, the remaining part planted with conventional crop

treated with insecticide; (ii) the whole field is planted with Bt-crop and a

small area nearby is planted with conventional crop, totally untreated.

In both cases, non-transgenic fields will generate susceptible insects in large

number to mate with resistant ones developed in the transgenic field, so

that resistant ones leading to establishment of resistant strain in the

population.

Mixtures of transgenic resistant and non-transgenic lines in the same field

have also been suggested and this approach is described as mosaics. This

approach is described as mosaics. This approach has been considered

unviable by the industry.


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Genes for protease inhibitors

(a) Gene for cowpea trypsin inhibitor or CpTI

In cowpea (Vigna unguiculata), trypsin inhibitor (CpTI) level was shown

to be responsible for its resistance to attack by the major storage pest of

its seeds (i.e. bruchid beetle = Callosobrunchus maculates). CpTI was

later shown to ne toxic to be variety of insects but cowpea seeds with

high level of CpTI are not toxic to humans. It was, therefore, considered

desirable to transfer gene(s) for CpTI for production of transgenic insect

resistant plants.

A number of binary vectors were developed using Ti plasmid, where

CpTI gene was joined with plasmid, where CpTI gene was joined with

CaMV35S promoter, and one or more marker genes, The vector was

mobilised into Agrobacterium, which was used to infect tobacco leaf

discs, which led to the production of transgenic tobacco plants

expressing high level of CpTi (a shown by western blotting) imparting

resistance against a variety of insects.

The CpTI gene in transgenic plants is stably inherited and there is no

serious yield penalty. Thus like B toxin, CpTI can also be used as a

protectant against insect attack in transgenic plants. However, extensive

field trails will be necessary before these transgenic plants can be

released to the farmers for cultivation

| Gene for alpha-amylase inhibitor (AI-Pv)

The insect pest resistant transgenic plants produced using Bt gene, protect themfrom insects mainly in the field, but not during storage. The first transgenic plants,

whose seeds, during storage, were resistant to pests (bruchid beetles), were

produced in the year 1994 in pea.

This was achieved by inserting a gene for -amylase inhibitor (AI-Pv), isolated


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from common bean (Phaseolus vulgaris), and driven by a strong seed-specific

promoter (from phytohemagglutinin gene of bean; phytohemagglutinin is a lectin).

These transgenic plants produced seeds that contained 1.0-1.2% AI-Pv and were

resistant to cowpea weavils and Azuki bean weavils.

Documents

Target Cells for Gene Transformation