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TABLE OF CONTENT TABLE OF CONTENT
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TITLE PAGE -
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CHAPTER ONE INTRODUCTION-
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CHAPTER TWO MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED TRANSFORMATION -10 AGROBACTERIUM MEDIATED PLANT TRANSFORMATION PROCESS
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INTEGRATION OF T-DNA INTO PLANT GENOME
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REGULATION OF TraR GENE EXPRESSION
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CHAPTER THREE APPLICATIONS OF AGROBACTERIUM TO AGRICULTURAL BIOTECHNOLOGY-29
CHAPTER FOUR TRANSIENT GENE EXPRESSION MEDIATED BY AGROBACTERIUM
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EVALUATION OF TRANSIENT EXPRESSION USING DIFFERENT POTATO GENOTYPES SUB-CELLULAR LOCALIZATION OF VARIOUS POTATO PROTEINS
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RNai- BASED TRANSIENT GENE SILENCING MEDIATED BY AGROBACTERIUM
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RAPID LATE BLIGHT RESISTANCE ASSAY USING RNai-BASED TRANSIENT SILENCING
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CHAPTER FIVE AGROBACTERIUM MEDIATED TRANSFORMATION OF COTTON -
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GENOMICS OF PLANT GENES IMPORTANT FOR AGROBACTERIUM-MEDIATED GENETIC TRANSFORMATION
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FORWARD GENETIC SCREENS FOR PLANT MUTANTS WITH ALTERED TRANSFORMATION CHARACHTERISTICS
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MANIPULATION OFAGROBACTERIUM FOR GENETIC ENGINEERING PURPOSES-50
CHAPTER SIX TRANSFORMATION OF LEGUMINOUES PLANTS
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REASONS
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FORAGE AND PASTURE LEGUMES
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GRAINSAND PULSES
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CHAPTER SEVEN MAIZE TRANSFORMATION TO OBTAIN PLANT TOLERANT TO VIRUSES BY RAai TECHNOLOGY
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CONSTRUCTION OF RNaiTARGET GENES
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CHAPTER EIGHT GENOMIC APPROACHES TO IDENTIFY PLANT GENES THAT RESPOND TO AGROBACTERIUM INFECTION -
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CONCLUSION
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REFERENCE -
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AGROBACTERIUM TRANSFORMATION: A BOOST TO AGRICULTURAL BIOTECHNOLOGY
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CHAPTER ONE INTRODUCTION Agricultural biotechnology is any technique in which living organisms or parts of organisms are altered to make or modify agricultural merchandise, to boost crops, or develop microbes for specific uses in agricultural processes. Merely put, when the tools of biotechnology are applied to agriculture, it's termed as "agricultural biotechnology". Genetic engineering is additionally a half of agricultural biotechnology in today's world. It's currently potential to hold out genetic manipulation and transformation on virtually all plant species, including all the planet's major crops. Plant transformation is one in every of the tools involved in agricultural biotechnology, in which genes are inserted into the genetic structure or genome of plants. The two commonest methods of plant transformation are Agrobacterium Transformation - ways that use the naturally occurring bacterium; and Biolistic Transformation - involving the employment of mechanical means. Using any of these ways the popular gene is inserted into a plant genome and traditional breeding methodology followed to transfer the new trait into different kinds of crops. Genetic transformation has been a powerful tool for enhancing the productivity of novel secondary metabolites; especially by Agrobacterium rhizogenes induced hairy roots. Genetic transformation is one of the biotechnological tools used to harness the production of secondary metabolites. The recent achievement and advancement of genetic transformation is the in vitro regeneration of medicinal plants from various explants to enhance the production of secondary metabolites which has lead to production of high-quality plant based medicine.
ϰ
The recent advances and developments in plant genetics and recombinant DNA technology have helped to improve and boost research into secondary metabolite biosynthesis. Emphases have been paid on identifying enzymes of a metabolic pathway and then manipulating these enzymes to provide better control of that pathway. Transformation is currently used for genetic manipulation of more than 120 species of at least 35 families, including the major economic crops, vegetables, ornamental, medicinal, fruit, tree and pasture plants, using Agrobacterium mediated or direct transformation methods. However, Agrobacterium-mediated transformation offers several advantages over direct gene transfer methodologies (particle bombardment, electroporation, etc), such as the possibility to transfer only one or few copies of DNA fragments carrying the genes of interest at higher efficiencies with lower cost and the transfer of very large DNA fragments with minimal rearrangement. The fundamental requirement in all this is a good yield of the compound, and reduced cost compared to the natural synthesis by the plants. More recently, several fungi have been transformed with Agrobacterium tumefaciens. For insertional mutagenesis, this technique offers huge potential as an alternative tool to restriction enzyme mediated-integration. One of the principal advantages
of
Agrobacterium-mediated
transformation
over
conventional
transformation techniques is the versatility it provides in choosing which starting material to transform. Applicable to several fungi, Agrobacterium tumefaciens can transform protoplasts, hyphae, spores, and blocks of mushroom mycelial tissue. Agrobacterium tumefaciens is a widespread naturally occurring soil bacterium that causes crown gall, and has the ability to introduce new genetic material into the plant cell. The genetic material that is introduced is called TDNA (transferred DNA) which is located on a Ti plasmid. Plasmid is a circular piece of DNA found in almost all bacteria.
ϱ
This natural ability to alter the plant’s genetic makeup was the foundation of plant transformation using Agrobacterium. Currently, Agrobacterium-mediated transformation is the most commonly used method for plant genetic engineering because of relatively high efficiency. Initially it was believed that this Agrobacterium only infects dicotyledonous plants, but it was later established that it can also be used for transformation of monocotyledonous plants such as rice. The closely related species, A. rhizogenes, induces root tumors, and carries the distinct Ri (root-inducing) plasmid. Although the taxonomy of Agrobacterium is currently under revision it can be generalised that 3 biovars exist within the genus; A. tumefaciens, A. rhizogenes, and A. vitis. Strains within A. tumefaciens and A. rhizogenes are known to be able to harbour either a Ti or Ri-plasmid, whilst strains of A. vitis, generally restricted to grapevines, can harbour a Ti-plasmid. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this "natural genetic engineering" for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Research is on going for the application of plant transformation and genetic modification using A. rhizogenes, in order to boost production of those secondary metabolites, which are naturally synthesized in the roots of the mother plant. Transformed hairy roots mimic the biochemical machinery present and active in the normal roots, and in many instances transformed hairy roots display higher product yields. ϲ
Genetic transformation has been reported for various medicinal plants. Report of the successful regeneration of transgenic neem plants (Azadirachta indica) using Agrobacterium tumefaciens containing a recombinant derivative of the plasmid, the genetic transformation of Atropabelladona has been reported using Agrobacterium tumefaciens, with an improved alkaloid composition. Agrobacterium mediated transformation of Echinacea purpurea has been demonstrated using leaf explants. Genetic transformation has been a powerful tool for enhancing the productivity of novel secondary metabolites of limited yield. Hairy roots, transformed with Agrobacterium rhizogenes, have been found to be suitable for the production of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. A number of plant species including many medicinal plants have been successfully transformed with Agrobacterium rhizogenes. The hairy root culture system of the medical plant Artemisia annua L. was established by infection with Agrobacterium rhizogenes and the optimum concentration of artimisin was 4.8 mg/L. Giri et al (1997) induced the development of hairy roots in Aconitum heterophyllum using Agrobacterium rhizogenes. Pradel et al (1997) developed a system for producing transformed plants from root explants of Digitalis lanata. They evaluated different wild strains of Agrobacterium rhizogenes for the productions of secondary products obtained from hairy roots and transgenic plants. They reported higher amounts of anthraquinones and flavonoids in the transformed hairy roots than in untransformed roots. An efficient protocol for the development of transgenic opium poppy (Papaver somniferum L.) and California poppy (Eschscholzia californica Cham.) root cultures using Agrobacterium rhizogenes is also reported. Bonhomme et al (2000) has reported the tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacterium ϳ
rhizogenes. Argolo et al (2000) reported the regulation of solasodine production by Agrobacterium rhizogenes-transformed roots of Solanum aviculare. Souret et al (2002) have demonstrated that the transformed roots of A. annua are superior to whole plants in terms of yield of the sesquiterpene artemisinin. Shi and Kintzios(2003) have reported the genetic transformation of Pueraria phaseoloides with Agrobacterium rhizogenes and puerarin production in hairy roots. The content of puerarin in hairy roots reached a level of 1.2 mg/g dry weight and was 1.067 times the content in the roots of untransformed plants. Thus, these transformed hairy roots have great potential as a commercially viable source of secondary metabolites. The genus Agrobacterium has been divided into a number of species. However, this division has reflected, for the most part, disease symptomology and host range. Thus, A. radiobacter is an “avirulent” species, A. tumefaciens causes crown gall disease, A. rhizogenes causes hairy root disease, and A. rubi causes cane gall disease. More recently, a new species has been proposed, A. vitis, which causes galls on grape and a few other plant species. Today, many agronomically and horticulturally important species are routinely transformed using this bacterium, and the list of species that is susceptible to Agrobacterium-mediated transformation seems to grow daily. In some developed countries, a high percentage of the acreage of such economically important crops as corn, soybeans, cotton, canola, potatoes, and tomatoes is transgenic; an increasing number of these transgenic varieties are or will soon be generated by Agrobacterium-mediated, as opposed to particle bombardmentmediated transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. ϴ
The overall advantages of using Agrobacterium-mediated transformation over other transformation methods are: reduction in transgene copy number, and intact and stable integration of the transgene (newly introduced gene) into the plant genome. This book reviews the impact of Agrobacterium in the transformation of plants to improve the yield and productivity of plants in order to meet up with the food requirement of the world population explosion. Agrobacterium species have been used as a tool to transfer desirable genes to different economic plants hence, these plants were transformed to resist drought, viral infection etc. Today molecular biology has advanced to such an extent that if scientists of any country are not adequately imaginative they would most certainly be left behind only to put their countries to an economic disadvantage in an open market economy.
ϵ
CHAPTER TWO MOLECULAR BASIS OF AGROBACTERIUM-MEDIATED TRANSFORMATION The molecular basis of genetic transformation of plant cells by Agrobacterium is transfer from the bacterium and integration into the plant nuclear genome of a region of a large tumor-inducing (Ti) or rhizogenic (Ri) plasmid resident in Agrobacterium. Ti plasmids are on the order of 200 to 800 kbp in size. The transferred DNA (T-DNA) is referred to as the T-region when located on the Ti or Ri plasmid. T-regions on native Ti and Ri plasmids are approximately 10 to 30 kbp in size. Thus, T-regions generally represent less than 10% of the Ti plasmid. Some Ti plasmids contain one T-region, whereas others contain multiple T-regions. The processing of the T-DNA from the Ti plasmid and its subsequent export from the bacterium to the plant cell result in large part from the activity of virulence (vir) genes carried by the Ti plasmid. T-regions are defined by T-DNA border sequences. These borders are 25 bp in length and highly homologous in sequence. They flank the T-region in a directly repeated orientation. In general, the T-DNA borders delimit the T-DNA, because these sequences are the target of the VirD1/VirD2 border-specific endonuclease that processes the T-DNA from the Ti plasmid. There appears to be a polarity established among T-DNA borders: right borders initially appeared to be more important than left borders. We now know that this polarity may be caused by several factors. First, the border sequences not only serve as the target for the VirD1/VirD2 endonuclease but also serve as the covalent attachment site for VirD2 protein. Within the Ti or Ri plasmid (or T-DNA binary vectors), T-DNA borders are made up of double-stranded DNA. Cleavage of these double stranded border sequences requires VirD1 and VirD2 proteins, both in vivo, and in vitro. In vitro, however, VirD2 protein alone can cleave a single-stranded T-DNA border ϭϬ
sequence. Cleavage of the 25-bp T-DNA border results predominantly from the nicking of the T-DNA “lower strand,” as conventionally presented, between nucleotides 3 and 4 of the border sequence. However, double-strand cleavage of the T-DNA border has also been noted. Nicking of the border is associated with the tight (probably covalent) linkage of the VirD2 protein, through tyrosine 29, to the 5 end of the resulting single stranded T-DNA molecule termed the T-strand. It is this T-strand, and not a double-stranded T-DNA molecule, that is transferred to the plant cell. Thus, it is the VirD2 protein attached to the right border and not the border sequence per se, that establishes polarity and the importance of right borders relative to left borders. It should be noted, however, that because leftborder nicking is also associated with VirD2 attachment to the remaining molecule (the “non-T-DNA” portion of the Ti plasmid or “backbone” region of the T-DNA binary vector), it may be possible to process T-strands from these regions of Ti and Ri plasmids and from T-DNA binary vectors. Second, the presence of T-DNA “overdrive” sequences near many T-DNA right borders, but not left borders, may also help establish the functional polarity of right and left borders. Overdrive sequences enhance the transmission of T-strands to plants, although the molecular mechanism of how this occurs remains unknown. Early reports suggested that the VirC1 protein binds to the overdrive sequence and may enhance T-DNA border cleavage by the VirD1/VirD2 endonuclease. VirC1 and virC2 functions are important for virulence; mutation of these genes results in loss of virulence on many plant species. However, several laboratories have noted that T-strand production in virC mutant Agrobacterium strains occurs at wild-type levels. Thus, any effect of VirC must occur after T-DNA processing.
ϭϭ
AGROBACTERIUM MEDIATED PLANT TRANSFORMATION PROCESS A general interest in the soil bacterium Agrobacterium tumefaciens began with the observation, published in a series of papers almost 30 years ago, that wildtype strains of this organism have the unique ability to directly transform plant cells by transfer of discrete DNA fragments. This transformation causes the infected plant cells to overproduce phytohormones, causing cell proliferation, which results in the growth of tumours called crown galls. The transforming DNA (T-DNA) also encodes genes for the production of novel compounds called opines, which are sources of nutrients for the colonizing bacteria. By inducing plant cell growth and directing these cells to produce nutrients that only the colonizing bacteria can use, A. tumefaciens makes a novel niche for itself in its environment, giving itself a clear advantage over other plant-colonizing bacteria. Plant transformation technology offers an array of opportunities for basic scientific research and for modification of food and fiber crops. Transgenic plants are typically produced by complex methods that require careful preparation of plant cells or tissues, introduction of DNA using Agrobacterium tumefaciens or particle bombardment, selection of transformed cell lines, and regeneration of plants. These transformation methods require time, skilled labor and relatively expensive laboratory facilities. In contrast, the ‘Agrobacterium vacuum infiltration’ method is a relatively new and simple procedure for transformation of Arabidopsis thaliana. In its original form, the method involved the growth of Arabidopsis to flowering stage, uprooting of plants, application of Agrobacterium to whole plants via vacuum infiltration in a sucrose/hormone growth medium, re-planting, collection of seed a few weeks later, and identification of transformed progeny by selection on media containing antibiotic or herbicide. The technique, which can be viewed ϭϮ
as an extension of earlier in planta transformation methods, offered a substitute for widely utilized Arabidopsis transformation methods that involved root tissue culture and plant regeneration. With vacuum infiltration and other in planta transformation methods, most transformed progeny are genetically uniform (nonchimeric) and the somaclonal variation associated with tissue culture and regeneration is minimized. Transformed progeny are typically hemizygous for the transgene at a given locus, suggesting that transformation occurs after the divergence of anther and ovary cell lineages. Likely targets of heritable transformation
are
therefore
the
gametophyte-progenitor
tissues,
mature
gametophytes, or recently fertilized embryos. The primary reasons for the popularity of the Agrobacterium vacuum infiltration method have been its simplicity and reliability. The elimination of tissue culture and regeneration greatly reduces hands-on time, and success can be achieved by non-experts. Transformed plants can be obtained at sufficiently high rates so that the procedure can be used not only to introduce specific gene constructs into plants, but also as a random mutagenesis method for gene-tagging. The primary drawback of the method is that successful application has only been reported for one plant species, Arabidopsis thaliana. To improve this very widely used Arabidopsis transformation method and to gain insights that may facilitate transformation of other plant species, there is the need to test a number of parameters involved in the transformation method. Some of these parameters had also been suggested and explored in unpublished studies and other Arabidopsis researchers. The present report offers a substantially simplified protocol that is the product of controlled and replicated studies. Components of the infiltration medium have been eliminated, as has the laborious vacuum infiltration process and the uprooting and re-planting of plants during application of ϭϯ
Agrobacterium. Plant growth stages have been identified at which a maximal number of transformants can be obtained. Expertise with recombinant DNA methods is still required to generate the desired gene constructs, but stable introduction of a DNA construct into the Arabidopsis genome is possible with minimal labor, equipment or specialized reagents. These modifications may be most important for use in larger scale transformation projects such as enhancertrapping and other forms of gene-tagging mutagenesis, ‘focused shotgun complementation’ during the late stages of a positional cloning project, or attempts at site-specific gene replacement.
Fig. 1Structure of Ti plasmid (source: Ashish and Vineet, 2011) The Agrobacterium-mediated transformation process involves a number of steps: a. Isolation of the genes of interest from the source organism; b. Development of a functional transgenic construct including the gene of interest; promoters to drive expression; codon modification, if needed to ϭϰ
increase successful protein production; and marker genes to facilitate tracking of the introduced genes in the host plant; c. Insertion of the transgene into the Ti-plasmid; d. Introduction of the T-DNA-containing-plasmid into Agrobacterium; e. Mixture of the transformed Agrobacterium with plant cells to allow transfer of T-DNA into plant chromosome; f. Regeneration of the transformed cells into genetically modified (GM) plants; and g. Testing for trait performance or transgene expression at lab, greenhouse and field level. During transformation, several components of the Ti plasmid enable effective transfer of the genes of interest into the plant cells. These include: •
T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred into the plant genome
•
vir genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not themselves transferred, and
•
Modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest.
Bacterial colonization is an essential and the earliest step in tumor induction and it takes place when A. tumefaciens is attached to the plant cell surface. Mutagenesis studies show that non-attaching mutants loss the tumor-inducing capacity. The polysaccharides of the A. tumefaciens cell surface are proposed to play an important role in the colonizing process. The bacterial attachment could be prevented when lipopolysaccharides (LPS) solution from virulent strains is applied to the plant tissue before interaction with virulent bacteria. The LPS are an integral part of the outer membrane and include the lipid A membrane anchor and the Oϭϱ
antigen polysaccharide in their composition. A. tumefaciens, like other plantassociative Rhizobiaceae bacteria, produces also capsular polysaccharides (Kantigens) lacking of lipid anchor and having strong anionic nature and tight association with the cell. There are some evidences indicating that capsular polysaccharides may play a specific role during the interaction with the host plant. In the particular case of A. tumefaciens it was observed that the attachment of wildtype bacterium to plant cells was directly correlated with the production of an acidic polysaccharide. The chromosomal 20kb att locus contains the genes required for successful bacterium attachment to the plant cell. This locus has been extensively studied using transposon insertion mutants. Insertions in the left 10 kb side of this region produced a virulent mutant that could restore its attachment capacity if the culture medium was previously conditioned by the incubation of wild-type virulent bacterium with plant cells. This previous interaction resulted in the production and accumulation of a complement, absent in the mutant strain, conditioning the medium and allowing the attachment to the plant cell. In contrast, mutational insertion in the 10 kb right side of the att locus resulted in the irreversible loss of attachment capacity, which could not be restored by conditioned medium. These results suggest that genes, placed at att left side, are involved in molecular signaling events, while the right side genes are likely to be responsible for the synthesis of fundamental components. The att left side suggests an operon composed by nine open reading frames (ORF). Four of these ORF show homology to the genes involved in the so-called periplasmic binding protein dependent (or ABC) transports system. The mutant analysis evidenced a failure in the production and accumulation of specific compounds essential for bacterial attachment. The ABC transporter encoding genes may be involved in the secretion of these ϭϲ
substances or in the introduction into bacteria of some plant-originated activators of the synthesis of that compound specific for attachment. The T-DNA carries genes for the biosynthetic enzymes for the production of unusual amino acids, typically octopine or nopaline. It also carries genes for the biosynthesis of the plant hormones, auxin and cytokinins. These plant hormones produce opines, providing a carbon and nitrogen source for the bacteria that most other micro-organisms can't use, giving Agrobacterium a selective advantage. By altering the hormone balance in the plant cell, the division of those cells cannot be controlled by the plant, and tumors form. The ratio of auxin to cytokinin produced by the tumor genes determines the morphology of the tumor (root-like, disorganized or shoot-like).
&ŝŐ͘Ϯ. The path that the T-DNA travels from the stage of T-DNA processing from the Ti plasmid (pTi) to stable integration into the plant genome (Rossi et al., 1998).
ϭϳ
Fig. 3. Large gall formed at the base of the stem of a rose bush. B. A series of galls (arrowheads) along a branch of a grapevine. C shows the bases of two young tomato plants where a drop of A. tumefaciens bacterial suspension was placed on the stem and a pin prick was then made into the stem at this point. The photograph was taken 5 weeks later. D shows another laboratory assay, where bacterial suspension was added to the surface of freshly cut carrot disks. After 2 weeks the young galls (green-colored) developed from the meristematic tissues around the central vascular system.
Fig. 4. E, the excised T-DNA is released by the bacterium and enters the plant cells, where it integrates into the plant chromosomes and dictates the functioning of those cells. The actual mechanism of transfer is still unclear, but it seems to require a conditioning process, perhaps mediated by the production of cytokinins (plant hormones) by the bacterium. The tzs (transzeatin) gene on the Ti plasmid codes for the hormon. E. Overview of infection of a plant wound site by Agrobacterium tumefaciens. The Ti plasmid codes for a nutrient-uptake protein (opine permease) that inserts in the bacterial cell membrane. The plasmid also copies and excises part of its DNA, which enters the plant cells and causes them to produce
opines. ϭϴ
F,
Structure
of
acetosyringone.
G. Diagram of some major regions of the Ti plasmid of A. tumefaciens strain C58. T-DNA = transferred DNA; Noc = nopaline catabolising genes; Ori = origin of replication of the plasmid; Con = region governing conjugative transfer of the plasmid to other Agrobacterium strains; Acc = agrocinopine catabolising genes; tzs = transzeatin synthesis; Vir = virulence genes.
Fig. 5. H, Agar plate stab-inoculated in the centre with A. radiobacter strain K84 and incubated for 24 hours before the bacterium was killed with chloroform vapour. Then a top layer of cooled agar containing A. tumefaciens was poured over the plate (the overlay plate technique). Growth of the pathogen is inhibited in a wide zone (arrowheads) around the spot where strain K84 had grown. [Note: both bacteria produce a creamy-white growth; the plate appears yellow because the image was photo-processed to accentuate the inhibition zone].
ϭϵ
Fig. 6. J, Specificity of biocontrol by A. radiobacter strain K84. The image shows the bases of 8 tomato plants grown in pots of soil. Top row: plants stab-inoculated (arrowheads) with four different pathogenic strains of A. tumefaciens and incubated for 3 weeks. Bottom row: plants treated identically but stab-inoculated with a 1:1 mixture of cells of the pathogenic strain and A. radiobacter strain K84 The two pathogenic strains 529 and T57 have a Ti plasmid coding for nopaline production; they are controlled by strain K84. The strains 8150 and 502 have Ti plasmids coding for other opines and are not controlled by strain K84.
ϮϬ
Fig. 7. K, Mode of action of agrocin 84. Pathogenic strains of A. tumefaciens with a Ti plasmid that codes for nopaline production also cause the plant to produce agrocinopines. The plasmid codes for an agrocinopine permease, which is inserted in the bacterial membrane. The inhibitor, agrocin 84, is recognised by this permease, enters the pathogen cells and there it blocks DNA synthesis.
The T-DNA transfer two models for the translocation of T-DNA-complex The transferring vehicle to the plant nucleus is a ssT-DNA-protein complex. Is must be translocated to the plant nucleus passing through three membranes, the plant cell wall and cellular spaces. According to the most accepted model, the ssTDNA-VirD2 complex is coated by the 69 kDa VirE2 protein, a single strand DNA binding protein. This cooperative association prevents the attack of nucleases and, Ϯϭ
in addition, extends the ssT-DNA strand reducing the complex diameter to approximately 2 nm, making the translocation through membrane channels easier. However, that association does not stabilizes T-DNA complex inside Agrobacterium. VirE2 contains two plant nuclear location signals (NLS) and VirD2 one. This fact indicates that both proteins presumably play important roles once the complex is in the plant cell mediating the complex uptake to the nucleus. The deletion of NLS in one of these proteins reduces, but does not totally inhibit, the ssT-DNA transfer and its integration into plant genome, evidencing the other partner can at least partially assume the function of the absent protein. It is known that VirE1 is essential for the export of VirE2 to the plant cell, although other specific functions are still uncharacterized. Bacterial strains mutated in virE1, cannot export VirE2 which is accumulated inside the bacterium. Such mutants can be complemented if coinfected with a strain that can export VirE2, indicating that this protein can be exported independently and that the transfer of VirE2 as part of the ss-T-DNA complex is not necessary for the transmission event being possible to transfer naked T-DNA to the plant cell. From these experimental evidences, an alternative model was brought to light for ssT-DNA complex transfer. This model proposes that the transfer complex is a single-strand DNA covalently bound at its 5'-end with VirD2, but uncoated by VirE2. The independent export of VirE2 to plant cell is presented as a natural process, and once the naked ssT-DNA-VirD2 complex is inside the plant cell, it is coated by VirE2. It is also possible that the process can be performed by one of the proposed alternatives ways according to the infection conditions. Previous researches described the role of 9.5 kb virB operon in the generation of a suitable cell surface structure for the ssT-DNA complex transfer from bacterium to ϮϮ
plant. The VirD4 protein is also required for the ss-T-DNA transport. The function of VirD4 is the ATP-dependent linkage of protein complex necessary for T-DNA translocation. VirB are proteins that present hydrophathy characteristics similar to other membrane-associated proteins. VirD4 is a transmembrane protein but predominantly located at the cytoplasmatic side of the cytoplasmic membrane. Comparative studies showed a high degree of homology between the virB operon and transfer regions of broad host range (BHR) plasmids in genetic organization, nucleotide sequence and protein function. Both systems deliver non-self transmissible DNA-protein complex to recipient host cell. In addition, they have the capacity to DNA interkingdom delivery suggesting that the T-DNA transfer apparatus and conjugation systems are related and probably evolved from a common ancestral. The majority of VirB proteins are assembled as a membrane-spanning protein channel involving both membranes. Except for VirB11, they have multiple periplasmic domains. VirB1 is the only member of VirB proteins found in the extracellular milieu although it is possible that some of the other VirB proteins may be redistributed during the process of biogenesis and functioning of the transcellular conjugal channel. That could be the case of the VirB2, a protein with deduced extracellular functions. Vir B2 is translated as a 12 kDa proprotein, which is later processed by proteolysis to its mature 7 kDa functional form. VirB4 and VirB11 are hydrophilic ATPases necessary for active DNA transfer. Vir B11 lacks continuous sequence of hydrophobic residues, formiing periplasmic domains. Despite these structural characteristics, less than one third of VirB11 constitutes its soluble fraction, while the rest of the protein remains associated with the cytoplasmic membrane. These characteristics are atypical for this type of protein and evidence the possible dynamic co-existence of different conformational Ϯϯ
forms in vivo . VirB4 tightly associates with the cytoplasmic membrane. It contains two putative extracellular domains conferring transmembrane topology to this protein, which presumably allows the ATP-dependent conformational change in the conjugation channel. Probably, the functional forms of VirB4 and VirB11 are homo- and heterodimers. The VirB4 synthesis is well correlated with the accumulation and distribution of VirB3. Other protein, VirB7, seems to be crucial for the conformation of the transfer apparatus. VirB7 interacts with VirB9 forming heterodimers and probably higher-order multimeric complexes. The synthesis of VirB9 and its stable accumulation depends of heterodimer conformation, indicating that VirB9 alone may be unstable and requires the association with VirB. In this intermolecular conformation the monomeric subunits are joined by disulfide bridges. The VirB7-VirB9 heterodimer is assumed to stabilize other Vir proteins during assembly of functional transmembrane channels (Fernandez et al., 1996. Some of the initial steps of biogenesis of ssT-DNA complex apparatus have been recently identified. Firstly, VirB7 and VirB9 monomers are exported to the membrane and processed. They interact each other to form covalently cross-linked homo- and heterodimers. Although the role of both types of dimers in the biogenesis of the transfer apparatus is widely accepted, it is likely that only heterodimers are essential. Subsequently the VirB7-VirB9 heterodimer is sorted to the outer membrane. The sorting mechanism has not been elucidated the next step implies the interaction with the other Vir proteins for assembling the transfer channel with the contribution of the transglycosidase VirB1. It is known that VirB2 through VirB11 are essential for DNA transfer, suggesting that these proteins are fundamental component of the transfer apparatus while VirB1 has a lesser contribution to this process. Ϯϰ
Two accessory vir operons, present in the octopine Ti-plasmid, are virF and virH. The virF operon encodes for a 23 kDa protein that functions once the T-DNA complex is inside the plant cells via the conjugal channel or independently, as it was assumed for VirE2 export. The role of VirF seems to be related with the nuclear targeting of the ssT-DNA complex but its contribution is less important than in the case of VirF. The virH operon consists of two genes that code for VirH1 and VirH2 proteins. These Vir proteins are not essential but could enhance the transfer efficiency, detoxifying certain plant compounds that can affect the bacterial growth. If that is the function of VirH proteins, they play a role in the host range specificity of bacterial strain for different plant species. Integration of T-DNA into plant genome Inside the plant cell, the ssT-DNA complex is targeted to the nucleus crossing the nuclear membrane. Two Vir proteins have been found to be important in this step: VirD2 and VirE2, which are the most important, and probably VirF, which has a minor contribution to this process. The nuclear location signals (NLS) of VirD2 and VirE2 play an important role in nuclear targeting of the delivered ss-T-DNA complex, as early described. VirD2 has one functional NLS. The ssT-DNA complex is a large (up to 20 kb) nucleoprotein complex containing only one 5'end covalently attached VirD2 protein per complex. But the complex is coated by a large number of VirE2 molecules (approximately 600 per a 20 kb T-DNA), and each of them has two NLS. The two NLS of VirE2 have been considered important for the continuos nuclear import of ss-T-DNA complex, probably by keeping both sides of nuclear pores simultaneously open. The nuclear import is probably mediated also by specific NLS-binding proteins, which are present in plant cytoplasm.
Ϯϱ
The final step of T-DNA transfer is its integration into the plant genome. The mechanism involved in the T-DNA integration has not been characterized. It is considered that the integration occurs by illegitimate recombination. According to this model, paring of a few bases, known as micro-homologies, are required for a pre-annealing step between T-DNA strand coupled with VirD2 and plant DNA. These homologies are very low and provide jus a minimum specificity for the recombination process by positioning VirD2 for the ligation. The 3´-end or adjacent sequences of T-DNA find some low homologies with plant DNA resulting in the first contact (synapses) between the T-strand and plant DNA and forming a gap in 3'-5' strand of plant DNA. Displaced plant DNA is subsequently cut at the 3'-end position of the gap by endonucleases, and the first nucleotide of the 5' attaches to VirD2 pairs with a nucleotide in the top (5'-3') plant DNA strand. The 3' overhanging part of T-DNA together with displaced plant DNA are digested away, either by endonucleases or by 3'-5' exonucleases. Then, the 5' attached to VirD2 end and other 3'-end of T-strand (paired with plant DNA during since the first step of integration process) joins the nicks in the bottom plant DNA strand. Once the introduction of T-strand in the 3'-5' strand of the plant DNA is completed, a torsion followed by a nick into the opposite plant DNA strand is produced. This situation activates the repair mechanism of the plant cell and the complementary strand is synthesized using the early inserted T-DNA strand as a template. VirD2 has an active role in the precise integration on T-strand in the plant chromosome. The release of VirD2 protein may provide the energy containing in its phosphodiester bond, at the Tyr29 residue, with the first nucleotide of T-strand, providing the 5'-end of the T-strand for ligation to the plant DNA. This phosphodiester bond can serve as electrophilic substrate for nucleophilic 3'-OH from nicked plant DNA. When the mutant VirD2 protein is transferred attached to Ϯϲ
the T-strand, the integration process take place with the loss of nucleotides at the 5'-end of the T-strand. REGULATION OF traR GENE EXPRESSION Two groups independently discovered that the traR gene is regulated by the ‘conjugal opines’ on both the nopaline- and the octopine-type Ti plasmids. On nopaline-type Ti plasmids, traR is the fourth gene in the five-gene arc operon, which is divergently transcribed from the acc operon. The transcriptional regulator AccR is encoded by the first gene of the acc operon, the other members of which are required for catabolism of the opines agrocinopine A and B. In the presence of agrocinopine A or B, repression of both the arc and the acc operons by AccR is relieved, resulting in the transcription of all of the genes of these operons, including traR. In octopine-type Ti plasmids, the conjugal opine is octopine. Octopine binds to its intracellular target, the transcriptional regulator OccR, resulting in activation of the occ operon. The traR gene is at the distal end of this operon, while many of the genes upstream of traR are required for uptake and catabolism of octopine. When traR is expressed from a constitutive promoter, conjugation no longer requires octopine. Therefore, regulation of traR expression by OccR completely explains the requirement of octopine for Ti plasmid conjugation. Control of traR expression by opines therefore has evolved independently in these two types of Ti plasmids. The genes of the arc operon and occ operon are not similar, except for traR. Furthermore, traR is the only gene in each of these operons that is required for conjugation. The regulators of the occ and the arc operons are also dissimilar. OccR is a LysR-type transcriptional activator, which binds to promoter DNA both in the presence and in the absence of the inducing signal. Ligands binding to LysR-type proteins often results in a shift from a high-angle bend in the DNA to a low-angle bend, allowing RNA Ϯϳ
polymerase to bind to the promoter. A direct binding of OccR to occ promoter DNA and a change in OccR-DNA conformation in response to octopine have been demonstrated in vitro. In contrast, AccR is similar to the Lac repressor. Binding of the inducing signal to these proteins results in a conformational change that disrupts DNA binding, resulting in derepression. Control of traR expression by opines is a feature of all Ti plasmids that have been studied to date. Regulation of traR expression on pTiChry5 is also thought to be through an AccR homologue, although in this case derepression occurs in response to agrocinopines C and D, and traR is in a two-gene operon (also called arc;. Agrocinopines C and D are also known to induce conjugal transfer of pTiBo542, although in this case the mechanism of regulation is not known. The nonpathogenic strain A. radiobacter K84 carries a plasmid called pATK84b, which does not carry vir genes or T-DNA, but does contain genes for opine uptake and utilization. This allows A. radiobacter to compete with pathogenic strains for nutrients in or near crown galls. Conjugal transfer of pATK84b is regulated by two different traR genes. One copy, traRnoc, is in the nox operon, which is induced by nopaline, while traRacc expression is induced by agrocinopines A and B.
Ϯϴ
CHAPTER THREE
APPLICATIONS OF AGROBACTERIUM TO AGRICULTURAL BIOTECHNOLOGY
Plant transformation mediated by Agrobacterium tumefaciens, a soil plant pathogenic bacterium, has become the most used method for the introduction of foreign genes into plant cells and the subsequent regeneration of transgenic plants. A. tumefaciens naturally infects the wound sites in dicotyledonous plant causing the formation of the crown gall tumors. The first evidences indicating this bacterium as the causative agent of the crown gall goes back to more than ninety years. Since that moment, for different reasons a large number of researches have focused on the study of this neoplastic disease and its causative pathogen. During the first and extensive period, scientific effort was devoted to disclose the mechanisms of crown gall tumor induction hoping to understand the mechanisms of oncogenesis in general, and to eventually apply this knowledge to develop drug treatments for cancer disease in animals and humans. When this hypothesis was discarded, the interest on crown gall disease largely decreased until it was evident that this tumor formation may be a result of the gene transfer from A. tumefaciens to infected plant. Agrobacterium tumefaciens infects plants by making a crown gall in region where the stem meets the roots. Researchers have discovered that the bacteria transfer part of their DNA to the plant nucleus hence integrated into the plant genetic material. The transfer DNA or T-DNA is part of a large tumour inducing plasmid. The T-DNA carries an oncogenic region that code for the production of plant growth hormones which causes the proliferation of plant cells forming gall or tumour. It also code for unusual derivatives of arginine which is a growth Ϯϵ
substance. This bacterium – plant interaction is known as genetic colonization. Scientist also discovered that the introduction of a foreign gene into the T-DNA would enable its transfer to the plant cell nucleus leading to the development of plant transformation using a disarmed, oncogenic, version of the Ti-plasmid that could transfer DNA into plants without causing the production of tumour. Having known that all that is required for gene to be introduced into a plant are the 25bp repeated sequences at the borders of oncogenic region (the left and right borders), the virulence gene of the Ti-plasmid. It was possible to separate these in a system of binary vectors. The earliest species to be transformed was tobacco, Nicotiana tabacum which rapidly became the model dicotyledonous plant. However, more recently, the workhorse has changed to Arabidopsis thaliana which has a very small genome of 120 Megabases and is easier to transform. In other to transform tobacco and most other dicotyledonous plants, leaf disk are cut and placed in a petri-dish containing liquid medium. The A. tumefaciens strain is placed on the surface of the disks and co-cultivation carried out for 2-3 days. The cutting of the leaf disks results in the plant producing wound-response compound, such as acetosyringone which induce the virulence genes. The leaf disks are then transfer to selection media, containing the herbicide or antibiotics of choice. This is often kanamycin as many binary vectors carry the Neomycin phosphotransferase gene which code for kanamycin resistance. Transformation occur along the cut edges of the disks, resulting in the formation of callus tissue which carries the DNA between the left and right border integrated at random into the plant genome. The callus tissue is then transfer into the regeneration medium also containing kanamycin, which only allow transgenic plants, expressing kanamycin resistance, to develop, the whole process takes about 3-4 months.Therefore transformation with Agrobacterium can be achieved in two ϯϬ
ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in an Agrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die. The initial results of the studies on T-DNA transfer process to plant cells demonstrate three important facts for the practical use of this process in plants transformation. Firstly, the tumor formation is a transformation process of plant cells resulted from transfer and integration of T-DNA and the subsequent expression of T-DNA genes. Secondly, the T-DNA genes are transcribed only in plant cells and do not play any role during the transfer process. Thirdly, any foreign DNA placed between the T-DNA borders can be transferred to plant cells, no matter where it comes from. These well-established facts, allowed the construction of the first vector and bacterial strain systems for plant transformation. Agrobacterium-mediated transformation has currently become a very welldeveloped science. The use of artificial pesticides that will be harmful to man, and pollute groundwater and the atmosphere, has been considerably lessened with the introduction of genetically engineered insect-resistant cotton. Herbicide-tolerant soybeans and corn have conjointly enabled the use of reduced-risk herbicides that break down more quickly in soil. These are nontoxic to plants or animals, and herbicide-tolerant crops facilitate the preservation of topsoil from erosion since they thrive better in reduced tillage agriculture systems. Papayas proof against the ring spot virus were conjointly developed through genetic engineering, which ϯϭ
saved
the
U.S.
papaya
industry.
Agrobacterium- mediated transformation might additionally be useful in improving and enhancing the nutritious quality of some crops. As an example, enhancing the levels of beta-carotene in canola, soybean, and corn improves oil compositions, and reduces vitamin A deficiencies in rice. There also are researches going on in the field of biotechnology to produce crops that cannot be laid low with harsh climates or environments which can require less water, fertilizer, labor etc. This is able to greatly reduce the stress and pressures on land and wildlife (Article click, 2011).Agrobacterium is listed as being the original source of genetic material that was transferred to foods such as soybean, cotton, corn,sugar beet, Alfalfa, Wheat,Rapeseed Oil(Canola), Creeping bentgrass(for animal feed), and Rice(Golden rice).
ϯϮ
Fig. 8. Agrobacterium vector method. The Ti plasmid of the plant bacterium Agrobacterium tumefaciens is used in plant genetic engineering. (Reprinted with permission from P. H. Raven and G. B. Johnson, Biology, 6th ed.,McGraw-Hill, New York, 2002)
ϯϯ
CHAPTER FOUR TRANSFORMATION OF POTATOES Potato (Solanum tuberosum) is the third most important food crop in the world, next only to rice and wheat. However, genetic and genomic research of potato has lagged behind most major crops. Functional discovery of genes in potato is still a lengthy process and is often hampered by the complex characteristics associated with the potato genome, including autotetraploidy, self-incompatibility, and high heterozygosity. Although several gene discovery tools have been used in potato research, including transposon based insertional mutagenesis, gene activationtagging, and map-based cloning, applications of these techniques were timeconsuming, resource-intensive, and technically challenging. RNA interference (RNAi)-based potato gene silencing has recently been reported by several laboratories. However, the RNAi technique relies on the traditional transformation procedure and is a low throughput methodology. It takes on average six months to develop a transgenic potato line using RNAi constructs. Therefore, this technique can only be used to target a limited number of potato genes. Transient gene assays are convenient alternatives to stable transformation because such techniques allow a rapid analysis of gene function. Early successful transient gene assay in potato was demonstrated using a microprojectile bombardment-based appeoach. Virus-induced gene silencing (VIGS) has been successfully used in several plant species, including potato. However, VIGS has not yet been proven to be a straightforward technique that can be readily adapted in different potato laboratories. As a similar approach to VIGS, transient gene expression was also be accomplished by infection of an Agrobacterium tumefaciens strain carrying a potato virus X (PVX)- based binary vector. This approach was successfully used in high-throughput screening for specific ϯϰ
recognition of INF elicitins of Phytophthora infestans in different Solanum species. Leaf infiltration of Agrobacterium is another popular method for transient gene functional assay. The agro infiltration has been best used in Nicotiana benthamiana, although it has also been successfully applied to several other plant species, including Arabidopsis thaliana, tobacco, tomato, lettuce, and grapevine. To our knowledge, Agrobacterium-mediated infiltration for rapid functional gene assays without involving a viral based system has not been reported in potato. An international Potato Genome Sequencing Consortium (PGSC) has been established (http://potatogenome.net) and is expected to fully sequence the 850 Mb potato genome by the end of 2010. This soon available genome sequence will dramatically change the genetic and genomic research of potato. One of the most urgently needed tools is a reliable, efficient, and high throughput technique for discovery and characterization of individual potato genes. The development of an Agrobacterium mediated infiltration procedure in potato was successful. Gene expression or RNAi-based gene silencing constructs can be delivered into potato leaf cells using agro-infiltration. The demonstration of agro-infiltration technique can be used as a rapid gene assay tool to localize protein expression in sub-cellular compartments and to determine the role of candidate genes in R-gene mediated potato late blight resistance.
Transient gene expression mediated by agro-infiltration
The efficiency and versatility of the agro-infiltration technique in N. benthamiana prompted the test of the possibility to adapt a similar approach in potato. The initial experiments using previously established protocols in N. benthamiana, only resulted in limited success with a low transformation efficiency. To optimize the procedure in potato, Katahdin, a cultivar highly amenable to whole ϯϱ
plant transformation, was used to infiltrate the leaves at various stages of growth. A potato RAR1 (GFP - Green Fluorescent Protein, Required for Mla12 Resistance) construct, which also contained the DsRED1 (Red Fluorescent Protein) reporter, was used to optimize all the infiltration conditions. The presence and spread of transgenic cells around the infiltration zones were identified based on red fluorescence under an epifluorescence microscope. In contrast, no DsRED1 fluorescence was observed from un-infiltrated potato leaves. The potato leaves were tested at various growth ages and found that the infiltration was consistently most efficient when using terminal leaflets from 5–6 week-old potato plants. It was also noticed that the leaflets from middle or lower positioned leaves with less pubescence were easier to infiltrate. The efficiency of infiltration became significantly lower when leaves from 3–4 week old plants were used in the experiments. Then the investigation whether the concentration of the Agrobacterium cultures had any effect on the outcome of infiltration was done. Cultures resuspended to OD600= 0.2–0.5 resulted in the best transient gene expression activity. In order to suppress the silencing of the transgene co-infiltrated of the silencing suppressor P19 together with the transgene was done. However, the introduction of P19 appeared to have no effect on the expression of transgenes. Two different Agrobacterium strains, GV3101 and LBA4404, were used for infiltration. GV3101 showed a higher efficiency than LBA4404 in the experiments, which confirms the high efficiency of GV3101 reported in N. benthamiana.
Evaluation of transient expression using different potato genotypes Leaves from potato cultivarsKatahdin, Atlantic, Megachip, USW1, and a wild diploidspecies Solanum bulbocastanum were infiltrated in order to investigate the influence of potato genotypes on the efficiency of transient expression. It was observed that a considerable variation for the intensity of the DsRED1fluorescence ϯϲ
among the genotypes. Katahdin showed the highest transformation efficiency. Large numbers of transgenic cells away from the infiltration zone in Katahdin leaves were consistently observed. Katahdin was followed by Atlantic and Megachip where the transgenic cells were mostly observed in close proximity to the infiltration zone. Transformation events were not observed in leaves from USW1 and S. bulbocastanum. Several previous studies reported that potato genotypes, both different potato cultivars and different Solanum species, can affect the success of the VIGS technique and agro-infection assays. Similarly, studies in grapevine also showed an effect of genotype for success of infiltration assays. The high infiltration efficiency with Katahdin leaves in our study is in accordance with the high efficiency of this cultivar in whole plant transformation. Katahdin and Agrobacterium strain GV3101were used for all further studies. The transient functional assay using agroinfection of PVX-based constructs will depend on the susceptibility of various potato cultivars to PVX. Thus, the agroinfiltration method offers an alternative strategy to the viral based systems.
ϯϳ
Fig. 9. Red fluorescence derived from DsRED1 six days after agroinfiltration into potato leaves. (A) Red fluorescence from a single infiltration site on Katahdin. (B) The same infiltration site as (A) under bright field. (C) Red fluorescence from a single infiltration site on Atlantic. (D)The same infiltration site as (C) under bright field. (E) Red fluorescence from a single infiltration site on USW1. No transgenic cells were detected on this image. The strong red fluorescence signals in this infiltration site were derived from autofluorescence associated with the necrotic tissue. (F) The same infiltration site as (E) under bright field. All bars are 10 mm. Sub-cellular localization of various potato proteins One of the main applications of the transient gene assay tools is to monitor the localization of proteins to sub-cellular compartments in living cells. To test such a function of agroinfiltration technique, several GFP-tagged potato gene constructs
were
developed.
Katahdin
leaves
showed
only
minimum
autofluorescence under a GFP filter five and seven days after agroinfiltration with the pK7FWG2 empty vector. In contrast, a vector expressing GFP under the 35S
ϯϴ
promoter showed strong GFP fluorescence signals throughout the cellular compartments. Few potato genes for which the protein locations within specific sub-cellular compartments have been either known or can be predicted. Full-length coding sequences (CDS) of the target genes were amplified and cloned into the binary vector pK7FWG2. The C-terminal of the CDS was fused with the GFP gene. The constructs were then agro-infiltrated into potato leaves. The sub-cellular localization of the StRAR1::GFP fusion proteins was first determined. The GFPtagged potato RAR1 proteins were ubiquitously localized in the cytoplasm as well as in the nucleus. The GFP signals present in the nucleus were much stronger than those in the cytoplasm. These results were in agreement with the cellular localization of rice OsRAR1::GFP. StGS2::GFP (Potato Glutathione Synthetase-2, GS2), an enzyme involved in the synthesis of glutathione in plants, was found to be predominantly localized within plastids with no unambiguous signals in other cellular compartments. The GS2 protein was previously showed to be exclusively targeted to plastids in A. thaliana, Brassica juncea and Medicago truncatula. These results were in confirmation with previous studies. The cellular localization of the potato vacuolar invertase (StV-INV) protein was then tested, which was not studied previously. The StV-INV:: GFP protein was localized in cytoplasm, including endoplasmic reticulum (ER) and vacuoles. Conclusive GFP signals were not detected in the nucleus. Interestingly, the protein appeared also to be localized around the nucleus and in the cytoplasm. However, this signal pattern may be due to the strong GFP signals from ER surrounding the nuclear envelope. These results agreed with the prediction of protein localization site analysis (PSORT, www.psort.org) that the 1,920 bp CDS of potato V-Inv (639 aa of V-INV protein) to be primarily localized in ER, Golgi bodies, endosomes (peroxisomes), and membrane system within the cells. The M. truncatula DMI3 protein, which is ϯϵ
required for the initiation of legume nodulation, has previously been shown to be localized in the nucleus. We expressed the MtDMI3::GFP fusion protein into the potato cells. The GFP signals were predominantly localized in the nucleus in most of the transgenic cells. These results show that the agro-infiltration-based technique can be used to study both native and heterologous proteins in potato. RNAi-based transient gene silencing mediated by agro-infiltration Initially the established optimal agro-infiltration procedure described above was used for RNAi-based transient gene silencing experiments. However, it was observed that young and fully expanded leaves from 3–5 week-old plants were the best plant materials for transient gene silencing. Maximum silencing levels were obtained when Agrobacterium inoculums was diluted to an OD600 value of 0.3– 0.7. A low concentration (OD600, 0.1) of bacterial suspension resulted in poor transformation thus leading to lower silencing levels, while high concentration (OD600.1.0) occasionally produced necrotic spots around the infiltration zones. Previously developed Rar1-RNAi construct in the initial transient gene silencing experiments was used. This construct was agro-infiltrated into Katahdin leaves. Semi-quantitative RT-PCR was used to confirm the suppression of the potato Rar1 gene in the leaf tissues around the infiltration zone. The Rar1 transcript was significantly reduced compared with the controls. Reduction of the Rar1 transcript was generally observed 5 and 6 days post infiltration (dpi) and persisted until 8 dpi. However, partial reduction of the Rar1 transcripts started as early as 3–4 dpi in 20– 30% of the leaves analyzed. The reduction of the Rar1 transcript compared to the control leaves was as much as 90–99% in different experiments. These results are in accordance with the previous finding in N. benthamiana that the production of
ϰϬ
siRNAs for the target gene in the infiltrated zones started as early as 2 days post infiltration and reached peak abundance by day 5. Rapid late blight resistance assay using RNAi-based transient silencing Agroinfiltration assay was successfully used for screening candidate signaling components required for the activation of R-gene mediated disease resistance in N.benthamiana and tomato. This is intended to develop a similar technique to screen the candidate genes required for the late blight resistance mediated by the RB gene. Gene RB confers a broad-spectrum resistance against the late blight pathogen Phytophthora infestans and recognizes the P. infestans effector, IpiO1. In the procedure an RNAi construct developed against a candidate gene was first introduced into a RB-containing potato plant by agro-infiltration, which will silence the target gene. Four days after agro-infiltration, the second construct containing the IpiO1 gene was agro-infiltrated at the same site. This double infiltration would result in a hypersensitive response (HR) phenotype if the candidate gene is not involved in the RB mediated resistance, because the silencing of this candidate gene will not affect the resistance. However, no HR would be observed if the candidate gene is involved in RB-mediated resistance. A transgenic Katahdin line SP925, which contains the RB gene, was used in the double agroinfiltration experiments. The Agrobacterium strain GV3101 produced few or no necrotic spots on RB-Katahdin leaves 7–12 dpi. In contrast, infiltration with effector IpiO1 in the same plant resulted in a confluent necrosis response starting at 3–5 dpi. Infiltrations with silencing construct alone or mock infiltration did not produce any background effect on potato leaves observed until 10 dpi, although few necrotic spots emerged 10 dpi even in the control experiments, which may be caused by the natural senescence of the tissues around the site of infiltration. The ϰϭ
Rar1 and Sgt1 genes have been extensively studied for their roles in the regulation of disease resistance genes. It was previously demonstrated that SGT1, but not RAR1, is essential for the RB-mediated late blight resistance in potato. Double agro-infiltration was performed using Rar1-RNAi and Sgt1-RNAi constructs together with the IpiO1 gene construct. Eight days after the first infiltration, a clear HR response was observed around the infiltrated sites on potato leaves double infiltrated with Rar1-RNAi constructs and IpiO1. In contrast, no HR was observed around the infiltrated sites of Sgt1-RNAi and IpiO1. These results showed that RB activation by IpiO1 depends on SGT1 but not RAR1, which is consistent with our whole plant stable transformation results. Late blight is the most devastating potato disease worldwide and is also the most extensively studied potato disease. Several late blight resistance genes, including both race-specific and race-nonspecific genes, have been cloned in recent years. However, very limited effort so far has been devoted to understand the resistance pathways mediated by any of these genes. This is at least partially due to the lack of tools in potato for rapid analysis of candidate genes associated with resistance or signaling pathway. The double agro-infiltration technique developed in this study will provide a powerful tool to fill this need in the future.
ϰϮ
Fig, 10. Laser-scanning confocal micrographs showing GFP fluorescence from agroinfiltrated leaf cells. Katahdin leaves were agroinfiltrated with (A) pK7FWG2 empty vector; (B) 35S::GFP; (C) StRAR1::GFP; (D) StGS2::GFP; (E) StV-INV::GFP; and (F) MtDMI3::GFP. The background fluorescence derived from plastids is in blue color. All the scale bars represent 10 mm. Arrows point to the nucleus in the cells.
ϰϯ
Fig.11. A double agroinfiltration procedure to test candidate genes associated with potato late blight resistance mediated by the RB gene. All the pictures were taken at 10 days post infiltration and bars represent 2 cm. (A) Infiltration with Agrobacterium carrying pGR106 IpiO1 and HR response was observed around the infiltrated site. (B) Infiltration with Agrobacterium containing pHellsgate-8 silencing construct. (C) Double agro-infiltration with Agrobacterium carrying Sgt1-RNAi construct followed with pGR106-IpiO1. No HR was observed around the infiltrated site. (D) Double agro-infiltration with Agrobacterium carrying Rar1RNAi construct followed with pGR106-IpiO1. HR response was observed around the infiltrated site. ϰϰ
CHAPTER FIVE AGROBACTERIUM-MEDIATED TRANSFORMATION OF COTTON Cotton crop occupies 32-33 million hectares of world area with a production of 18-19 million tonnes. In India its area currently spans over 9.1 million hectares with an average yield of306 kg/ha of lint and 918 kg/ha of seed cotton. To meet the challenges of 2000 AD with an anticipated population of more than one billion, a total of at least 20 million bales would have to be produced as against the 16-17 million bales of today. This can be achieved by the use of improved crop production practices, generation of novel transgenics coupled with appropriate pest management tactics. Cotton transgenic plants were generated almost a decade ago and are currently under commercial cultivation in the United States of America and Australia since 1996 and China, Argentina, Mexico and South Africa since 1997. Field trials are now underway in Zimbabwe, Columbia, Bolivia, Brazil, EL Salvador, Greece, India, Israel, Paraguay and Thailand. Transgenic crops within built resistance to insect pests in India could result in at least 25-30% reduction in insecticide use on cotton, resulting in a benefit of about of about Rsl300 crores, apart from the favorable impact on the environment. In vitro Regeneration of cotton has been a major bottleneck in genetic engineering of cotton. So far generation of transgenic plants, from callus regeneration or somatic embryogenesis has been restricted to a few selected Coker 312 genotypes. However, simultaneously regeneration in other genotypes was also reported from China, Australia, Russia and Egypt. To circumvent the existing problem of genotypic limited regeneration of callus or leaf tissue, other methods such as transformation and regeneration from meristematic tissues have been found useful. Recently, in India, genotypes within variety MCU-5 were regenerated profusely ϰϱ
through somatic embryogenesis. Though several different methods of plant transformation have been used in various crops, two methods most widely in cotton, involve 1. Agrobacterium mediated transfer of DNA and 2. Bombardment of cells with DNA coated particles through particle acceleration gene delivery systems. Agrobacterium tumefacians was successfully used as a vector for cotton transformation. But this method was limited to only specific cultivars that could be regenerated in tissue culture. Later, particle delivery gene transfer systems were used to transform embryogenic cell suspensions and meristems of elite cotton lines which facilitate the transformation of commercial genotypes thereby eliminating the need for backcrossing with a regenerable genotype. Recently, a method to recover genetically transformed cotton plants via Agrobacterium -mediated transformation of shoot apex ex-plants, was reported. This development has resulted in the control of three cotton bollworms which are internal feeders of cotton and are difficult to control and thrive on fruiting parts of the crop, hence result in heavy economic losses. It is estimated that together the three bollworms can cause yield losses of up to 80% in cotton. Resistance to almost all groups of insecticides has led to persistent insect control problems especially with H. armigera thus necessitating the need for viable alternative methods. Insect resistant transgenic cotton is perceived as one of the best tools available to strengthen integrated pest management programmes, and is certainly considered as one of the most environment friendly option GENOMICS OF PLANT GENES IMPORTANT FOR AGROBACTERIUMMEDIATED GENETIC TRANSFORMATION Scientists have used a variety of genomic techniques to investigate plant genes important for Agrobacterium-mediated transformation. These include forward genetic screens to identify mutant plants with altered transformation susceptibility, ϰϲ
yeast two-hybrid studies to detect plant proteins that interact with Virulence effectors proteins, and transcriptional profiling to discover plant genes whose expression is altered following Agrobacterium infection. In addition, reverse genetic analyses have been used to probe the importance of candidate genes in the transformation process.
Forward Genetic Screens for Plant Mutants with Altered Transformation Characteristics Plant species, and even different cultivars/genotypes of the same species, are notoriously
varied
in
their
transformation
susceptibility.
In
addition,
Agrobacterium can transform Streptomyces, yeast, and other fungal species, and sea urchin embryos. Thus, Agrobacterium is incredibly promiscuous in its ability to mediate horizontal gene flow among numerous species of different phylogenetic kingdoms. A genetic basis for susceptibility to Agrobacterium exists in many crop species. Researchers also described a genetic basis for various degrees of susceptibility among approximately 40 Arabidopsis ecotypes. Large-scale forward genetic screening of approximately 20,000 T-DNA mutagenized Arabidopsis lines resulted in the first identification of plant genes involved in Agrobacterium-mediated transformation. These forward genetic analyses revealed >120 genes encoding proteins involved in transformation and, because the screen was not saturating (e.g. no gene was discovered more than once), the authors suggested that >200 Arabidopsis genes likely influence plant transformation susceptibility. The authors termed mutants with greatly decreased susceptibility to transformation rat (for resistant to Agrobacterium transformation) mutants and the corresponding mutant genes, rat genes. The identified genes represent most of the proposed transformation events that occur in the plant ϰϳ
(bacterial attachment/biofilms formation, T-DNA and Virulence protein transfer to the plant, cytoplasmic trafficking and targeting of the proposed T-complex to the nucleus, virulence protein removal from the T-strand, T-DNA integration into the plant genome, and transgene expression). Examples of plant proteins identified in these initial genetic screens and mediating transformation include those involved in cell wall structure and biosynthesis (Rat1 and Rat4, and arabinogalactan and cellulose synthase-like [CslA9] proteins, respectively, cytoskeleton proteins potentially involved in cytoplasmic trafficking of T-complex components (actins and a kinesin), importin Į and ȕ proteins that may mediate nuclear targeting of T-complex components, chromatin proteins such as various histones, histone acetyltransferases, histone deacetylases, and histone chaperones that may facilitate T-DNA integration into the plant genome, and histone proteins that can increase transgene expression (G. Tenea and S.B. Gelvin, unpublished data). The nature of these rat genes has stimulated reverse genetic experiments to determine the potential roles of candidate genes in the transformation process. Recently, the Gelvin laboratory further identified several Arabidopsis mutants that are hypersusceptible to Agrobacterium transformation (hat mutants and, therefore, hat genes. Arabidopsis lines containing T-DNA activation tags provide a resource for over expressed genes that may influence transformation susceptibility. When roots of these mutagenized plants were assayed at low bacterial inoculum conditions (102- to 103-fold lower than that usually used to screen for rat mutants), seven independent lines that displayed increased levels of transformation relative to that of wild-type control plants were identified. T-DNA/plant DNA junction sequences from five hat mutants identified several new genes involved in transformation susceptibility, including a cellulose synthase-like protein (CslB5), a ϰϴ
potassium transporter family protein (two independent T-DNA insertion lines), a UDP-glucosyltransferase (UGT), and a myb transcription factor (MTF). Conclusively, the soon available potato genome sequence will dramatically accelerate our pace to identify agronomically important potato genes. The power of comparative genomics will also allow us to discover potato genes based on the information from other extensively studied model plant species. Thus, a rapid and simple functional gene assay tool is urgently needed for potato genetics and molecular biology research. It has been demonstrate that Agrobacterium-mediated infiltration, which has been an effective gene delivering technique in several plant species, can be adapted in potato. Katahdin, a potato cultivar that is highly amenable for Agrobacterium-mediated whole plant transformation, showed the highest efficiency for agroinfiltration. However, it will be possible to identify potato cultivars (genotypes) that have greater efficiency for agroinfiltration than Katahdin. Agroinfiltration of GFP-based gene expression constructs into potato leaf cells is a simple and highly efficient approach to examine protein expression in sub-cellular
compartments.
Research
has
also
demonstrate
that
double
agroinfiltration of RNAi-based silencing construct and a late blight pathogen effector can be used for screening candidate genes involved in late blight resistance pathway mediated by the corresponding resistance gene. This double agroinfiltration approach is simple and fast compared to the traditional approach consisting of stable transformation followed by disease resistance evaluation. It can be readily adapted to dissect the resistance pathways mediated by a wide range of potato R genes in the future.
ϰϵ
MANIPULATION OF AGROBACTERIUM FOR GENETIC ENGINEERING PURPOSES Introduction of Genes into Plants by Using Agrobacterium Years
before
scientists
elucidated
the
molecular
mechanism
of
Agrobacterium-mediated transformation of plants, Armin Braun proposed the concept of a “tumor-inducing principle” that was stably transferred to and propagated in the plant genome. Research in the 1970s resulted in the identification of large plasmids in virulent Agrobacterium strains, although we now know that many strains contain plasmids unrelated to virulence. Genetic experiments indicated that a particular class of plasmids, the Ti (and later Ri) plasmids, were responsible for tumorigenesis and that a portion of these plasmids, the T-DNA, was transferred to plant cells and incorporated into the plant genome. It was thus obvious to propose that Ti plasmids be used as a vector to introduce foreign genes into plant cells. However, Ti plasmids are very large and T-DNA regions do not generally contain unique restriction endonuclease sites not found elsewhere on the Ti plasmid. Therefore, one cannot simply clone a gene of interest into the T-region. Scientists therefore developed a number of strategies to introduce foreign genes into the T-DNA. These strategies involved two different approaches: cloning the gene, by indirect means, into the Ti plasmid such that the new gene was in cis with the virulence genes on the same plasmid, or cloning the gene into a T-region that was on a separate replicon from the vir genes (T-DNA binary vectors). Two methods were used for cloning foreign DNA into the Ti plasmid. The first method was based on a strategy developed by Ruvkin and Ausubel. A region of DNA (either the T-region or any region of DNA targeted for disruption) ϱϬ
containing unique restriction endonuclease sites is cloned into a broad-host-range plasmid, such as an IncPĮ-based vector. These plasmids can replicate both in Escherichia coli, in which the initial cloning is performed, and in Agrobacterium. The exogenous gene of interest, along with an antibiotic resistance marker, is next cloned into a unique restriction endonuclease site within the target region of DNA. Alternatively, an antibiotic resistance gene can be introduced into the DNA fragment of interest by transposition. The resulting plasmid is introduced into Agrobacterium by conjugation or transformation. The presence of this plasmid in Agrobacterium is confirmed by selection for resistance to antibiotics encoded by both the plasmid vector backbone and the resistance marker near the gene of interest. Next, another plasmid of the same incompatibility group as the first plasmid, but harboring yet another antibiotic resistance marker, is introduced into the Agrobacterium strain containing the first plasmid. The resulting bacteria are plated on medium containing antibiotics to select for the second (eviction) plasmid and the resistance marker next to the gene of interest. Because plasmids of the same incompatibility group cannot usually core side within the same bacterial cell, the bacteria can become resistant to both these antibiotics only if either (i) the first plasmid cointegrates into the Ti plasmid and uses the oriV of the Ti plasmid to replicate or (ii) an exchange of DNA on the first plasmid and the Ti plasmid occurs by double homologous recombination (homogenotization) using homologous sequences on the Ti plasmid flanking both sides of the gene of interest plus the resistance marker. In the first case (cointegration of the entire plasmid with the Ti plasmid), the resistance marker of the plasmid backbone would be expressed; these bacteria are screened for and discarded. In the second instance (homogenotization), the resistance marker encoded by the plasmid backbone is lost. Double homologous recombination can be confirmed by DNA blot analysis of total DNA from the resulting strain. A variant of this procedure utilizes a sacB gene on the ϱϭ
plasmid backbone of the first plasmid. Only elimination of the plasmid backbone after homogenotization renders the bacterium resistant to growth on sucrose.
Fig. 12. Schematic representation of the steps involved in gene replacement by double homologous recombination (homogenotization). The green lines represent regions targeted for disruption. (A) An antibiotic resistance gene (in this case, encoding a ȕ-lactamase that confers resistance to carbenicillin) has been inserted into the targeted gene that has been cloned into an IncPĮ plasmid (containing a kanamycin resistance gene [kan] in its backbone) and introduced into Agrobacterium. Double homologous recombination is allowed to take place. (B) Following double homologous recombination, the disrupted gene is exchanged onto the Ti plasmid (pTi). (C) A plasmid of the same incompatibility group as the first plasmid is introduced into Agrobacterium. An example is the IncPĮ plasmid pPH1JI, containing a gentamicin resistance gene (gent). (D) Because plasmids of the same incompatibility group (in this case IncPĮ) cannot replicate independently in the cell at the same time, selection for gentamicin resistance results in eviction of the other IncPĮ plasmid, onto which has been exchanged the wild-type gene.
ϱϮ
Another method to introduce foreign DNA into the T-region of the Ti plasmid involves first introducing a ColE1 replicon, such as pBR322, into the Tregion of a Ti plasmid. DNA to be integrated into this T-region is cloned into a separate pBR322-derived molecule containing a second antibiotic resistance marker. This plasmid is introduced into the altered Agrobacterium strain, and the resulting strain is selected for resistance to the second antibiotic. Because ColE1 replicons cannot function in Agrobacterium, the pBR322-based plasmid would have to cointegrate into the pBR322 segment of the altered T-region for the stable expression of the plasmid-encoded resistance gene. A modification of this procedure was used to develop the “split-end vector” system. Using this system, a gene of interest is cloned into a pBR322-based vector that contains a T-DNA right border, a nos-nptII chimaeric gene for selection of transgenic plants, a spectinomycin-streptomycin resistance marker to select for the presence of the plasmid in Agrobacterium, and a region of homology with a nononcogenic portion of an octopine-type T-region. Cointegration of this plasmid with a Ti-plasmid lacking a right border but containing the T-DNA homologous region restores border activity and localizes the gene of interest and the plant selectable marker within the reconstituted T-region. Each of these cis-integration methods has advantages and disadvantages. The first strategy can target the foreign gene to any part of the T-region (or other region in the Ti plasmid). However, it is cumbersome to perform and involves somewhat sophisticated microbial genetic procedures that many laboratories shunned. The second method is technically easier but allows cointegration of the foreign gene only into Ti-plasmid locations where pBR322 had previously been placed. However, a modification of this procedure allows cointegration of a pBR322-based plasmid into any region of the Ti plasmid. An advantage of both of these systems is that they maintain the foreign gene at the same low copy number as that of the Ti plasmid in Agrobacterium. ϱϯ
Because of the complexity of introducing foreign genes directly into the T-region of a Ti plasmid, several laboratories developed an alternative strategy to use Agrobacterium to deliver foreign genes to plants. This strategy was based on seminal findings of de Frammond et al. (1983). These authors determined that the T-region and the vir genes could be separated into two different replicons. When these replicons were within the same Agrobacterium cell, products of the vir genes could act in trans on the T-region to effect T-DNA processing and transfer to a plant cell. Hoekema et al. called this a binary-vector system; the replicon harboring the T-region constituted the binary vector, whereas the replicon containing the vir genes became known as the vir helper. The vir helper plasmid generally contained a complete or partial deletion of the T-region, rendering strains containing this plasmid unable to incite tumors. A number of Agrobacterium strains containing nononcogenic vir helper plasmids have been developed, including LBA4404, GV3101 MP90, AGL0, EHA101 and its derivative strain EHA105, and NT1 (pKPSF2). T-DNA binary vectors revolutionized the use of Agrobacterium to introduce genes into plants. Scientists without specialized training in microbial genetics could now easily manipulate Agrobacterium to create transgenic plants. These plasmids are small and easy to manipulate in both E. coli and Agrobacterium and generally contain multiple unique restriction endonuclease sites within the Tregion into which genes of interest could be cloned. Many vectors were designed for specialized purposes, containing different plant selectable markers, promoters, and poly(A) addition signals between which genes of interest could be inserted, translational enhancers to boost the expression of transgenes, and protein-targeting signals to direct the transgene-encoded protein to particular locations within the plant cell (some representative T-DNA binary vector systems are described in ϱϰ
many references
at http://www.cambia.org). Hellens et al, (2000) provide a
summary of many A. tumefaciens strains and vectors commonly used for plant genetic engineering. Although the term “binary vector system” is usually used to describe two constituents (a T-DNA component and a vir helper component), each located on a separate plasmid, and the original definition placed the two modules only on different replicons. These replicons do not necessarily have to be plasmids. Several groups have shown that T-DNA, when located in the Agrobacterium chromosome, can be mobilized to plant cells by a vir helper plasmid. Some commercial releases of transgenic plants (from Birch, 1997) Crop and release Name
Company
Novel properties
Calgene
Vine-ripened
date Tomato (1994)
Flavr Savr
flavour, shelf life Tomato (1995)
Zeneca
Consistency
of
tomato paste Cotton
Bollgard
Potato
NewLeaf
thuringiensis toxin
Maize (1996-97)
YieldGuard
for
Monsanto
Bacillus
resistance Soybean
Roundup Ready
Monsanto
Glyphosate
Canola(rape)
herbicide
Cotton (1995-96)
resistance
ϱϱ
insect
The transgenic tomatoes do not express the gene for polygalacturonase, an enzyme that degrades pectin, leading to softening of the fruit tissues. As a result, the tomatoes can be left on the plant for longer to accumulate flavour components and they also give a better consistency of tomato pastes.
ϱϲ
CHAPTER SIX TRANSFORMATION OF LEGUMINOUS PLANTS Reason Legumes are a large, diverse family ranging from herbaceous annuals to woody perennials that, because of their capacity to fix nitrogen, are essential components in natural and managed terrestrial ecosystems. Legumes have been domesticated for the production of food, feed, forage, fiber, industrial and medicinal compounds, flowers, and other end uses. Understanding the molecular basis of nitrogen fixation and the unique metabolic pathways that result in the myriad of end users of legumes is both a matter of scientific curiosity and of economic necessity because of their importance in the biosphere and to the sustainability of the human race. In accordance, model legumes are being rapidly developed as experimental systems to pursue a number of important biological questions unique to these plants using molecular tools including genomics. Legume species are much more difficult to transform than others. Legume transformation systems, like transformation in all organisms, require development of: (a) a source of totipotent cells or gametes that serve as recipients of delivered DNA, (b) a means of delivering DNA into the target cells, and (c) a system for selecting or identifying transformed cells. For legumes that have been regarded as recalcitrant to transformation, regeneration in vitro is highly genotype specific and only rarely is cultivated varieties amenable to regeneration. In these cases, plant regeneration remains an “art” that requires considerable training of the practitioner to develop the skills needed to generate sufficient transgenic plants for a thesis or ϱϳ
publication. In addition, regeneration is often slow and the frequency of transformation (no. of transformed plants generated from each explant) is often low. In species that are amenable to in vitro somatic embryogenesis such as alfalfa, (lucerne; Medicago sativa), relatively rapid and efficient transformation methods have been developed based on cocultivation of tissue pieces (explants) with Agrobacterium tumefaciens. Because inducing somatic embryogenesis or organogenesis in many legume species is difficult, a variety of transformation methods have been reported that use cultures of meristematic cells as sources of totipotent cells. Most commonly, transformation has been based on infection by A. tumefaciens, although Agrobacterium rhizogenes is used for transformation of some species. Regeneration of shoots from the cotyledonary node or from other meristematic explants after Agrobacterium infection is emerging as a rapid and relatively efficient method of transformation in a number of legume species including soybeans,Lotus japonicus, barrel medic, and Trifolium repens. A number of legume species also have been transformed by direct DNA transfer methods including microinjection, electroporation, and microprojectile bombardment. In some species, the difficulty in regenerating transgenic plants has been circumvented by development of rapid and efficient transformation protocols using A. rhizogenes to produce hairy roots on “composite” plants (an untransformed plantlet with hairy roots). These composite plants have been used in studies focused on root characteristics such as nodulation and root diseases. Examples have been reported in L. japonicus, soybean, and barrel medic. Composite plants do not transmit the transgenic trait to their progeny and, thus, are of little use in crop improvement efforts. There are recent advances in transformation of forage species since 1997. Chinese milk vetch is grown as a green manure, for animal fodder, as a nectar ϱϴ
source for bees, and can be used to volatilize selenium from soil. A. rhizogenes inoculation of seedlings in vitro results in formation of hairy root, which spontaneously produce shoots in culture. Similarly, a number of protocols using A. rhizogenes for production of transgenic Lotus corniculatus have been described. Transformation of L. corniculatus via cocultivation of leaf explants with A. tumefaciens followed by callus formation and shoot organogenesis was reported by Webb et al. (1996). In contrast, transformation of red clover is based on regeneration via somatic embryogenesis after cocultivation of petiole explants with A. tumefaciens using genotypes selected for high frequency of this culture response. L. japonicus was suggested as a model system for legume genomics by Handberg and Stougaard (1992). In addition to other positive attributes as a model system, transformation of hypocotyls with A. tumefaciens is relatively efficient via shoot organogenesis. This method was further optimized and the time to produce whole plants reduced by Stiller et al. (1997). Somaclonal variation and sterility were significantly reduced by use of the bar gene and selection with PPT. A highly efficient transformation method has enabled initiation of a T-DNA insertional mutagenesis program for barrel medic. Each explant of line R1081(C3), a genotype selected for superior regeneration, produces large numbers of somatic embryos, and up to 80% of the embryos regenerate into plants 3 to 4 months after culture initiation. Methods with the potential to reduce tissue culture manipulations for transformation of barrel medic have been reported. Trieu and Harrison (1996) described a method based on cocultivation of A. tumefaciens with cotyledonary node explants followed by culture to induce multiple shoots from explants. Transgenic plantlets were produced in 2.5 months. Two in planta transformation systems were described by Trieu et al. (2000); one method is based on infiltration of flowers with A. tumefaciens, similar to the Arabidopsis flower infiltration protocol, and the other on infiltration of seedlings. Both methods were ϱϵ
reported to result in high transformation frequencies. Although promising, these results have not been repeated or further extended by this group, nor have they been corroborated by other laboratories.
Progress in transformation of large-seeded legumes has been extensively achieved. .Historically, both microprojectile bombardment and Agrobacterium have been used for DNA delivery into either embryogenic or organogenic cultures of some species that have been subjects of extensive research. However, the majority of the most recent reports are focused on A. tumefaciens-mediated transformation. This trend is evident forArachis hypogaea and soybean. On the other hand, pea transformation systems historically have been based mostly on A. tumefaciens. In contrast, we could find no reports of transgenic bean plants produced via Agrobacterium. This latter observation suggests inefficient transformation due to problems with Agrobacterium infection, T-DNA delivery, or both in this species. Cowpea (Vigna unguiculata) appears to be the most recalcitrant large-seeded legume. Although there is a report of successful production of transgenic plants, further evidence of transmission of the transgene genotype to progeny has not been reported. Leguminous trees are a rich source of wood, paper pulp, and animal fodder in many locations around the world. Recently, transformation methods have been developed for Acacia mangium and Robinia pseudoacacia. For transformation of A. mangium, rejuvenated shoots were cultured from axillary buds and shoot apices ϲϬ
of mature trees and shoot pieces cocultured with A. tumefaciens. Regeneration and culture of shoots required approximately 13 months. Transgenic R. pseudoacacia plants were obtained approximately 12 weeks after inoculation of hypocotyl segments with A. rhizogenes. Shoots arose spontaneously from hairy root cultures. Regenerated plants showed phenotypic abnormalities. In contrast, phenotypically normal plants were obtained approximately 2 months after cocultivation of stem segments with A. tumefaciens.
SEED GERMINATION I 5 days f V2xB5
A, rhizogenes INFECTION 2 - 4 weeks >/2xB5
AXENIC CULTURE I 2-4 weeks f B5 + 2% sucrose
SHOOT INDUCTION 8-12 weeks, B5 + 0 3 ug mL > BA + t 0.05 ug mL' NAA + 10 mM NH,*
SHOOT ELONGATION I 4-6 weeks, B5 + 0.5 ixg mL' BA + * 0.025 ug mL-i NAA
ROOT INDUCTION i 1 week y V, x B5 + 1.0 ug mL1 NAA
ROOT ELONGATION 3 - 4 weeks V2xB5
TRANSFER TO POT The flowchart for hairy root transformation regeneration in L. japomcus.
ϲϭ
CHAPTER SEVEN MAIZE TRANSFORMATION TO OBTAIN PLANTS TOLERANT TO VIRUSES BY RNAi TECHNOLOGY Maize is one of the most cultivated cereals in the world. The main maize producer’s countries are the United States, China, and Brazil, followed by Mexico, France, Argentina and India. Among the big losses faced by agriculture are the attacks of pests and diseases. For maize, these problems have worsened since 1990 because of the increase of the cultivated areas in both the normal growing season and the off season, mainly due to intensive cultivation of maize in the irrigated areas, and lack of adoption of crop rotation in certain fields. In recent years, diseases that were not a problem, increased in importance such as the viruses. Among the strains of the virus complexes, potyviruses cause significant losses in grain and forage of maize susceptible genotypes. Plants have different mechanisms for protection against invasion by pathogens, and different genes directly related to tolerance to viruses have been described in maize. Works have been published using methods of obtaining plants resistant to viruses by antisense, co-suppression and, more recently, RNA interference (RNAi). Agrobacterium is one of the methodologies that have been used to introduce the RNAi construct in maize cells, aiming to produce transgenic maize plants tolerant to SCMV. With this development, maize regeneration in tissue culture, transformation mediated by Agrobacterium and microprojectile bombardment, isolation and cloning of the target DNA into RNAi based vectors was made possible, some results already
ϲϮ
obtained with this technology and its application to crop improvement.
ϲϯ
Fig. 13. Timeline of maize transformation protocol, It is assumed that 100 immature embryos are collected in Step 3 and that materials derived from the 100 embryos are handled in the subsequent steps by a single, skilled technician. Similarly, seeds from ten transgenic plants are examined in Steps 32–35. Transgenic maize plants were first obtained from protoplasts by an electroporation method, but fertile plants have never been produced by this method. Other direct gene transfer methods, which did not require the prior culture of protoplasts, were then tried, and microprojectile bombardment of cells in suspension cultures or immature embryos became quite popular in basic and applied studies. Efficiency of transformation by microprojectile bombardment has been higher than other direct methods, and quite a few fertile plants have been generated to date. Microprojectile bombardment is also useful for the analysis of the transient expression of foreign genes in intact, fully developed tissues. However, high copy numbers and extensive rearrangement of the foreign DNA have frequently been found in plants transformed with direct gene transfer methods. CONSTRUCTS OF RNAi TARGET GENES Transgenes or genes that are inserted via molecular biology techniques in plants such as maize are basically composed of (i) regulatory sequences that control gene expression, (ii) the selection marker gene and, (iii) the gene of interest. The main sequences controlling gene expression are promoters, enhancers, introns and terminators. Promoters are DNA sequences, normally present in the 5 'end of a coding region, used by RNA polymerase and transcription factors to initiate the process of gene transcription. Depending on the ability to control gene expression, the promoters are classified as weak or strong, according to the binding affinity of transcription factors with the promoter sequence. Strong or weak promoters can be further classified as constitutive, tissue and / or organ-specific ϲϰ
and inducible. A constitutive promoter directs expression of a gene in all tissues of a plant during the various stages of development. The viral 35S mosaic virus promoter isolated from cauliflower (CaMV35S) is one of the most used to drive high constitutive expression in plants; however its function in monocots is not as efficient as in dicotyledons. The promoter used to drive the over expression of a protein constitutively in maize is currently the promoter isolated from maize ubiquitin gene Ubi1. A tissue-specific promoter directs gene expression only in certain tissue, which may or may not be activated during all stages of development. The use of this type of promoter may be advantageous to prevent an unnecessary waste of energy and nutrients by the transgenic plant when the protein of interest is not required throughout the plant. For example, the expression of genes related to absorption of nutrients is required only at the root. An inducible promoter initiates gene expression in response to chemical, physical, or biotic and abiotic stresses. Similar to specific promoters, inducible ones avoid the unnecessary consumption of energy and nutrients, since the protein is only produced in response to right stimulus. An example of an inducible promoter is the one isolated from the AtPHT1; 4 phosphate transporter genes from Arabidopsis thaliana, which was shown to direct expression of the uidA reporter gene only in roots of maize subjected to phosphorus stress. These features of promoters allow the expression of the transgenic protein be controlled according to the project objectives. Enhancers are regions of DNA that bind transcription factors responsible for an increase in transcription of a gene, and consequently by an increase in protein expression. Enhancers can be located before or after the coding region. In the genome, sequences of plant enhancers can be located physically distant from the gene which they are controlling, however because of the packaging of DNA in the nucleus; these sequences are geometrically positioned near the promoter. This position allows for an interaction between transcription factors and RNA polymerase II. ϲϱ
Introns are non-coding sequences within a gene that are removed during transcription. Although the mechanisms underlying the phenomenon are not completely clear, the incorporation of introns in genes can increase or decrease promoter activity and the levels of transcription. Typically, the intron is inserted between the 3 'end of the promoter and the initial codon of the protein of interest. Introns such as the rice actin Actl, Ubi1 of ubiquitin from maize, SH1 sucrose synthase from maize, and Adh1 corn alcohol dehydrogenase has been used in gene construct in order to increase the expression of transgenes. The regions 3 'UTRs also known as terminator regions are used to confer greater stability to the mRNA, and to signal the end of the transcript preventing the occurrence of the production of chimeric RNA molecules and consequently the formation of new proteins, if the polymerase complex continues transcribing beyond the end of the gene. 3' UTRs sequences used in most gene constructs for transformation of maize include the nopaline synthase gene from Agrobacterium, the 3 'region of CaMV35S, and inhibitor gene proteinase pinII from potato. The selection gene is a sequence encoding a protein that when expressed in transgenic cells confer an adaptive advantage. The selection gene is used to identify and select cells that have the heterologous DNA integrated into their genome. Selection genes are fundamental to the development of technologies for plant transformation because the process of transferring a transgene to a recipient cell and its integration into the genome is very inefficient in most experiments, and the chances of recovery transgenic lines without selection are generally very low. Currently, the most used selection markers for the production of transgenic maize are those that confer tolerance to herbicides. Among these, the bar gene, isolated from Streptomyces hygroscopicus and the pat gene, isolated from Streptomyces viridochromogenes, both encoding the enzyme phosphinothricin acetyltransferase (PAT). In majority the gene of interest is a coding sequence or ORF (Open ϲϲ
Reading Frame) of a certain protein that when expressed define a characteristic or phenotype of interest. In other cases, is a gene sequence used to silence gene expression, such as the RNAi technology. An important aspect regarding the use of RNAi for plant biologists is the ability to decide the target region of the gene that should be used to efficiently produce the dsRNA. In 2002 the company Dharmacon (www.dharmacom.com) was the first to develop an algorithm as a tool for rational design of a potent silencing, based on data. Today, there are several companies that have developed algorithms for analysis of gene sequence based on a number of parameters that predispose to more effective use of this technology.
A
B
Test with the Finale herbicide (ammonium glyfosinate) in maize leaves. (A) Sample sensitive and (B) insensitive to the herbicide.
ϲϳ
Transgenic and non-transgenic plants inoculated with SCMV in the greenhouse. (A) Plant with symptoms and (B) transgenic plants with no symptoms; Black arrow indicates the symptoms.
The transgenic T1 plants arise in the frequency around 1% relative to the original number of explants. The first confirmation of the transgenic is done by spraying leaves with 3 mg/L Finale herbicide (ammonium glyfosinate - AgrEvo Environmental Health, Montvale, NJ). The bar gene present in the pCAMBIA3301 plasmid confers resistance to this herbicide Transgenic plants that express this selectable marker gene survive herbicide spraying whereas the non transgenic plants die. The second confirmation of the transgenic is done by PCR using primers specific to the gene construct. To produce high-quality, stable transgenic lines it is necessary to define individuals with a single copy insertion and in homozygosity. This decision is based on the premise that expression of one copy is more stable and reliable than multi copy in the following generations. DNA purified from a single leaves (~100 mg of tissue) of T1 transformed plants is screening in a Southern blot analysis to identify events that possess single copy insertion. DNA is digested with restriction enzyme and subjected to gel electrophoresis. After the transfer of the DNA to the nylon membrane it is hybridized either with the bar ϲϴ
gene or any other fragment present on the genetic cassette. The choice of the enzyme depends on the way the cassette was prepared. If there is no site in the cassette of the restriction enzyme used for the initial digestion of the DNA, the number of bands reflects the number of copies of the fragment integrated into the genome. Even for the self pollinatedT0 plants many of the T1 generation are still heterozygous specially if there is more than one insertion. In this case, the test of herbicide and PCR in a sample of the following generation will help identify the one that are homozygous. If 100% of the T2 progeny of a single T1 plant are resistance to the herbicide (or show positive for the PCR) it indicates that the T1 parent (as well as all the T2 sibs) is homozygous for the transgene. Recent works at Embrapa Maize and Sorghum (Brazil) obtained SCMV resistant transgenic maize plants by transforming friable callus of maize HiII using a construction based on the RNAi technology (data not published). Previous study on the SCMV gene family identified the region of the coat protein as a conserved region that might be used to produce the cassette to silence the expression of the SCMV virus in maize. Once this fragment from the SCMV genome was choose and isolated, it was cloned twice, in inverted position, into the vector pKANNIBAL containing a spacer, transferred to a binary vector pCAMBIA 3301containing the ubiquitin promoter and NOS terminator and used to transform maize by particle bombardment. The phenotypic evaluation of the transgenic plants was done by inoculation of the SCMV virus complex every week for three consecutive weeks starting in a maize V5 stage. The inoculation was confirmed by PCR and microscopy. From the 20 events obtained 30% of the plants did not show any viruses symptoms and in approximately 46% the symptoms reduces along the plant life cycle. These results indicated that the technique of RNAi based on the Coat protein sequence was capable of generating transgenic maize resistant to the SCMV virus. ϲϵ
CHAPTER EIGHT GENOMICS APPROACHES TO IDENTIFY PLANT GENES THAT RESPOND TO Agrobacterium INFECTION Plants may respond to infection by Agrobacterium, and this response may involve differential plant gene expression. Genes that are induced or repressed during the early stages of Agrobacterium-mediated transformation may provide targets for manipulation of the host to improve the efficiency of transformation of recalcitrant plant species. Several laboratories have consequently begun investigations to identify these differentially expressed plant genes. Ditt et al. (83) recently investigated the response of Ageratum conyzoides suspension cell cultures to infection by a nontumorigenic supervirulent A. tumefaciens strain. Using cDNAamplification fragment length polymorphism (AFLP) to amplify 16,000 fragments, they identified 251 bands that were differentially regulated 48 h after infection. Reverse transcription-PCR analysis of some of these genes confirmed the results of the cDNA-AFLP analysis. Some of these bands were also induced or repressed 24 h after inoculation. Whereas most of the bands investigated (encoding, e.g., an RNase, a putative receptor kinase, a peroxidase, and a pathogenesis-related protein) were also differentially regulated following incubation of plant cells with E. coli, four genes, including one encoding a nodulin-like protein, responded specifically to Agrobacterium infection. The authors speculated that this nodulin gene may respond to signals from the bacterium to regulate plant cell division or differentiation. Our laboratory has conducted a similar study, using tobacco BY-2 suspension cell cultures inoculated with five different non-tumorigenic Agrobacterium strains (Veena, H. Jiang, R. W. Doerge, and S. B. Gelvin, submitted for publication). One strain could transfer T-DNA but not VirE2 protein, one could transfer virulence proteins but not T-DNA, one could transfer neither, ϳϬ
and two could transfer both. Using suppressive subtractive hybridization followed by DNA and RNA macroarray analyses of RNA samples from eight different time points following inoculation (from 0 to 36 h), more than 400 genes that were identified differentially regulated after various periods of infection. Most of these genes showed a general differential response to Agrobacterium inoculation; however, some genes responded specifically to a T-DNA and Vir protein transfercompetent strain. A few genes responded specifically to T-DNA or Vir protein transfer only. Among the genes that were induced (or whose expression was maintained at a high level) were those encoding histones and ribosomal proteins. The activity of several plant defense and stress response genes was repressed by Agrobacterium infection. Because of the importance of histones in the T-DNA integration process, it was proposed that Agrobacterium infection induces the expression of plant genes necessary for transformation while simultaneously repressing the host defense response. Further analysis of these differentially expressed genes will indicate whether they play a direct or indirect role in Agrobacterium- mediated plant. The ability to introduce desirable genes into plants has enabled the genetic manipulation of crop plants to modify them to our advantage and is also an essential component of molecular approaches to plant biology. Genetic transformation has now become a routine technique for several plant species. Introduction of foreign DNA into plants has been achieved using the Ti plasmid of Agrobacterium tumefacians or by direct gene transfer methods such as electroporation, PEG (polyethylene glycol) and microprojectile bombardment. The expression of a foreign gene in a multicellular organism could be assayed through the use of selectable markers, which play an important role in confirmation of stable transformation and quantification and visible expression of transformed genes in plant cells. Reporter genes such as chloramphenicol acetyl transferase ϳϭ
(cat), neomycin phosphotransferase (nptII), b-glucuronidase (gus), b-galactosidase (lacZ), luciferase (lux) and green fluorescent protein (gfp) have been commonly used in gene expression systems in transformed plants. Confirmation of gene integration is often done using antibiotic markers, enzyme assays of the reporter genes, Southern blotting and labelled probes and PCR of reporter gene or the target gene itself. Expression of the gene is determined by either Northern blotting or use of labelled probes, ELISA (Enzyme linked immunosorbent assay) for the expressing protein and bioassays for the desirable trait. CONCLUSION Conventional breeding methods alone cannot feed the extra hungry mouths despite the successes of the green revolution with substantial strides in food grains production, as a result of world population explosion. Agricultural biotechnology has the potential to reduce crop losses from pest and diseases, to improve the nutrient efficiency of food and animal feeds; to extend post harvest losses with increased shelf life of fruits and vegetables; and to increase the stress tolerance of crop plants allowing them to tolerate various environmental extremes such as cold and drought. Agrobacterium – mediated transformation has been used as a tool to transfer transgenic genes with desirable characteristics to varieties of crop plants which has succeeded in boosting agricultural production in other to meet up with global population explosion. In less than 20 years, the use of Agrobacterium to genetically transform plants has advanced from a dream to a reality. Modern agricultural biotechnology is heavily dependent on using Agrobacterium to create transgenic plants, and it is difficult to think of an area of plant science research that has not benefited from this technology. However, there remain many challenges. Many economically important plant species, or elite varieties of particular species, ϳϮ
remain highly recalcitrant to Agrobacterium-mediated transformation, and the day has not yet arrived when flowers will be the only things seen coming from the barrels of gene guns. However, I feel that such a day is not too far in the distant future. I also feel that Agrobacterium evolved millions of years ago to genetically transform a very wide range of organisms; it is now up to the scientist to harness the natural ability of this bacterium. In addition to extending the host range and transformation efficiency of plants by Agrobacterium, some of the remaining challenges to the scientific biotechnology community are summarized below. (i) The first is the use of Agrobacterium for homologous or site-directed recombination. Many scientists consider homologous recombination to be one of the remaining “holy grails” of plant molecular biology. The ability to perform gene replacement experiments has become a staple of bacterial, fungal, and even animal cell and molecular biology research. However, homologous recombination in plants generally occurs at 105 the frequency of illegitimate recombination. We need an Agrobacterium-mediated transformation system that delivers T-DNA to the plant nucleus efficiently, but is deficient in random T-DNA integration. (ii) The second involves stable and predictable transgene expression in plants. Too often, the level of transgene expression in plants is highly variable. Often, lines of transgenic plants that are “good expressers” lose this characteristic after several generations of growth under field conditions. We need to understand the roles of position effects, chromatin effects, and T-DNA integration patterns in transcriptional and posttranscriptional gene silencing in order to develop strategies to enhance the extent and stability of transgene expression. (iii) The third is manipulation of the Agrobacterium genome. The availability of the complete A. tumefaciens C58 genomic sequence presents us with an unparalleled opportunity to investigate Agrobacterium gene expression patterns and the ways in which they may be altered during cocultivation of the bacterium with various plant species. ϳϯ
Such information may provide clues to methods to further manipulate Agrobacterium in order to effect higher levels of transformation of recalcitrant plant species. (iv) The fourth is plastid genetic transformation by Agrobacterium. Although a few scattered references to chloroplast transformation by Agrobacterium existed in some literatures, these reports have not been confirmed by the scientific community. The existence of NLS sequences in VirD2 and VirE2 proteins may ensure T-DNA targeting to the nucleus. Even if these NLS sequences could be removed without altering other essential functions of these proteins, the recent finding that the plant actin cytoskeleton is involved in Agrobacteriummediated transformation (not published) may preclude redirection of the T-DNA from the nucleus to plastids. (v) The fifth is genetic transformation of animal and plant pathogenic fungi. Many medically or agronomically important pathogenic fungi remain highly recalcitrant to genetic trans-formation. Recent reports of Agrobacterium-mediated transformation of several filamentous fungal species suggest that Agrobacterium may be a useful “gene-jockeying tool” for more than just plant species. (vi) The final challenge involves genetic transformation of human and animal cells. The recent report of Agrobacterium mediated genetic transformation of human cells suggests the exciting possibility of using Agrobacterium, or Agrobacterium- like processes, for human and animal gene therapy.
ϳϰ
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