Chapter 15 Exploitation of tumefaciens ICsABA KONCZand JOZEF SCHELL 15.1 Introduction - T-DNA-encoded genes are not required for the transfer of the T-DNA. During invasion of wounded plants, soil agrobac- - virABCDE and G operons of Ti and Ri plas- teria transfer a defined segment of their Ti and Ri mids encode an inducible DNA processing sys- plasrnids into the plants. The transferred DNA, tem that mediates the recognition of direct termed T-DNA, is integrated into the plant nuclear 25 bp repeats located at the borders of T-DNA genome. Genes encoded by Ti and Ri plasmid T- Segments of Ti and Ri plasmids. DNAs are expressed in plants and confer the syn- - The function of these 25 bp repeats is analo- thesis of plant growth factors as well as sugar and gous to that of conjugational transfer origins of amino acid derivatives, called opines. Expression bacterial plasmids, thus: of T-DNA genes iaaM, iaaH, and ipt (see Hohn, - Any DNA sequence flanked by these 25 bp end- Chap. 15, this Vol.) leads to production of repeats can be transferred from Agrobacterium phytohormones, auxin and cytokinin that induce into plants and: proliferation of transformed cells to form undiffer- - Separation of the T-DNA and virulence genes entiated tumors, crown galls. In contrast, cells does not influence the transformation process. transformed by rol A, B and C genes of Ri plasrnid Certain requirements are essential for T-DNA T-DNA5 differentiate to hairy roots. While genetic vector designs. Since Escherichia coli is used as a analysis of the function and expression of these T- host for construction of vectors with transferable T- DNA genes provided a key for better understanding DNA segments, T-DNA vectors should contain se- af various aspects of hormonal regulation and cell lectable markers for both E. coli and Agrobacteri- differentiation, studies of the T-DNA transfer and urn as well as a replicative or integrative main- I integration contributed directly to the development tainance function. Vectors made in E. coli can be , of T-DNA-based transformation vectors and trans- transferred to Agrobacterium by transformation, I genic plant technology. How T-DNA gene vectors electroporation, or conjugation. In the latter case, are exploited to gain more insight to molecular bi- 1 DNA sequences recognized by a suitable plasmid ology of plants is the focus of this chapter. b mobilization system have to be added to the con- I structs. Finally, for selection of transformed plant I cells, a marker gene selectable in plants should also i 15.2 T-DNA, a Universal Tool be inserted in the T-DNA. Notwithstanding its rela- I of Plant Molecular Biology2 tively short history, a great number of different T- DNA-based vectors have already been constructed along the following two lines. Development of modern plant gene vectors derived from the T-DNA was based on the observations that: 15.2.1 Recombination-Based vectors3 j - Foreign DNAs inserted in the T-DNA are accu- rately transferred from Agrobacterium to Recombination-based vectors are Ti or Ri plasmid plants. derivatives from which some or all T-DNA on- 218 Exploitation of Agrobacterium tumefaciens cogenes have been removed. Foreign DNAs are in- serted into the T-DNA by homologous recombina- tion using a target DNA sequence that is homolo- Transconjugant gous to commonly used bacterial antibiotic resis- tance genes or to diverse E. coli cloning vectors and located within the T-DNA borders of Ti or Ri plasmids. The same target DNA is also part of a second plasmid, referred to as "intermediate vec- tor? Foreign genes are subcloned into the target transformation DNA of intermediate vectors and transferred from v E. coli to Agrobacterium by plasmid conjugation A. tumehciens or mobilization. A variety of genetic methods have been developed to select or screen for the integra- tion of foreign DNA into the T-DNA using single or double cross-over recombination and diverse replicative or nonreplicative vectors in agrobacter- ia. To identify T-DNA-transformed cells, selectable Transformation - and/or screenable marker genes are provided either Conjugation within the T-DNA or in the intermediate vector car- Electroporation r rying the cloned foreign DNA. Recombination-bas- transformation ed vectors are commonly referred to as "armed" or Fig. la,b. T-DNA plant gene vectors. a Insertion of cloned "disarmed" plasmids depending whether or not DNA into recombination-based vectors by single crossover. their T-DNAs contain oncogenes. Apart from spe- Foreign DNA is cloned into an intermediate vector and trans- cial applications, the "armed" Ti plasmids are no ferred into E. coli strain containing helper plasmids that pro- mote conjugational transfer into Agrobacterium. The interme longer used as vectors. In contrast, "armed" Ri diate vector is unable to replicate in Agrobacterium but main- plasmid are frequently employed to obtain tran- tained by recombination with the T-DNA of Ti plasmid vector. sgenic plants from hairy roots. Disarmed Ti Open boxes refer the T-DNA borders and the thick line for for- plasmid vectors, such as pGV3850 or the SEV sys- eign DNA. b Use of binary vectors. Foreign DNA is cloned into the T-DNA of binary vectors and transferred into Agrobac- tem, for example, are still in use today. These vec- teriurn that contains a T-DNA-less virulence helper Ti or Ri tors are based on single cross-over recombination plasmid within a target site located inside the T-DNA bor- ders that results in an intermediate vector-Ti plasmid cointegrate (Fig. 1a). based on a plant vector cassette (Fig. 2a) that car- ries only the oriv (replication origin) and oriT (ori- gin of conjugational plasmid transfer) regions of 15.2.2 Binary vectors4 plasmid RK2 in combination with diverse T-DNAs. oriv and oriT are active only when trans-acting Binary vector systems consist of two elements: a RK2 functions trf a (replication function) and trcr helper Ti or Ri plasmid providing virulence func- (plasmid transfer) are expressed in the same cell. 'Ib tions, and a cloning vector containing bacterial and provide helper functions for replication and conju- plant selectable marker genes and cloning sites gation of PCV vectors, defective RK2 plasmid de flanked by T-DNA end-repeats (Fig. I b). Most bi- rivatives were inserted into the chromosome of E. nary cloning vectors were derived from the well- coli and Agrobacterium hosts or into a T-DNA-less characterized wide-host-range plasmid RK2 that Ti plasmid pMP90RK. In Agrobacterium hosts can efficiently be mobilized between E. coli and carrying chromosomal insertions of trfa and tm Agrobacterium. An advanced binary system, re- RK2 genes, any Ti or Ri plasrnids can be used as ferred to as PCV (Plant Cloning Vector) system, is virulence helper. The presence of RK2 tra func- Gene Transfer and Wsgenic Plant lkhnology 219

Col El bacterial marker 15.3 Gene 'kansfer and hnsgenic Ofi boz----- .gene(a) . Plant Technologys

One of the first observations during the pioneering experiments with wild-type T-DNAs showed that plant promoter although T-DNA genes of agrobacteria were active in plants, other bacterial, yeast and animal genes 1-DNA left border inserted via T-DNA into the plant genome were not transcribed. It thus became evident that T-DNA-en- coded genes must contain all signals necessary for marker gene transcription in plants. Ri achieve the expression of bacterial genes in plants, transcription promoter and terminator sequences of the nopaline synthase (nos) gene of the T-DNA were used first to con- struct chimeric genes with the coding sequence of neomycin phosphotransferase [aph(3')II] and chloramphenicol acetyltransferase (cat) genes of aph (3'111 transposons IIh5 and 'Ih9. Expression of these chi- pnos meric antibiotic resistance genes could be followed by simple enzyme assays and permitted the selec- tion of transformed plant cells. This opened the way for the development of transformation tech- niques using co-cultivation of agrobacteria with plant protoplasts, leaf-disks, stem and root ex- plants; direct DNA uptake, protoplast fusion with Fig. 2a, b. Plant vector cassette for construction of binary vec- charged liposomes, macro- and microinjection or tors. a Schematic design of a plant vector cassette that contains bombardement with microprojectile-bound DNA. doning sites, plant selectable and screenable marker genes with- in the T-DNA carried by an oriv-on+ basic RK2 replicon. Ar- From cells transformed with disarmed T-DNA vec- rows 1,2, and 3 are unique cleavage sites for introduction of ad- tors fertile transgenic plants were regenerated that ditional elements into the cassette. b Plasmid pPCV002, a pro- transmitted the introduced genes to their offsprings &type of simple binary plant gene vectors. pnm nopaline syn- in a Mendelian fashion. Since little was known thase promoter; aph (3')11 coding sequence of kanamycin resis- tance gene of transposon n35;~AOCS polyadenylation signal of about gene expression in plants, most studies fo- Oetopine synthase gene; A# ampicillin resistance gene; ori and cused initially on the regulation of the expression born of ColEl replication and conjugational transfer origins of of plant genes in foreign genetic background. Ex- piasmid ColEI;pg5 promoter of T-DNA gene 5; LB and RB left ploration of maize alcohol dehydrogenase and su- and right ends of the T-DNA crose synthase, pea Rubisco and Cab, soybean lec- tin, and leghemoglobin genes, etc. in tobacco indi- cated that regulation of the transcription in re- tions in both hosts results in a ping-pong conjuga- sponse to environmental-, hormonal-, tissue-specif- tion of binary vectors that helps to test the stability ic and developmental stimuli, is similar in diverse of T-DNA constructs before plant transformation. plant species. These studies, together with the anal- Since oriv plasmids are maintained at a low copy ysis of the expression of plant virus genomes, con- number, most PCV vectors contain also a multi- tributed basic information and useful plant pro- copy ColEl replicon to facilitate the cloning in E. moters to achieve regulated expression of foreign coli (Fig. 2 b). genes in plants. 220 Exploitation of Agrobacterium lumefaciens

15.4 Gene Expression in Plants6 (especially that of the first intron) may regulate the level of gene expression. - Ribosomes bind to the 5' leader sequence of Expression cassettes consisting of characterized mRNAs and initiate translation at the first ATG plant promoter and polyadenylation signal se- codon. The presence of nonframe ATG codons quences separated by suitable cloning sites were in chimeric gene constructs therefore greatly re- constructed using transcriptional regulatory ele- duces the translation of foreign transcripts. ments of T-DNA genes (octopine, nopaline, man- Kozak's rule for efficient initiation of transla- nopine synthase genes, etc.), 35s and 19s RNA tion at ATG/G starts can also be applied to genes of Cauliflower Mosaic Virus (CaMV) and plant genes. A consensus sequence for ribo- plant genes, such as the light-regulated SSU or Cab. some-binding sites is not yet established for These cassettes were inserted into the T-DNA of bi- plant genes. However, it was observed that lead- nary vectors in linkage with diverse chimeric antibi- er sequences of certain viral RNAs (i.e., tobacco otic resistance genes, to clone and express various or alfalfa mosaic virus) can be used as transla- foreign genes in plants. Certain empirical rules es- tional enhancers. Alteration of the coding se- tablished during these studies indicated that: quence of foreign genes according to plant codon usage may also increase the efficiency of - The active core of plant promoters contains a translation. TATA-box followed by a transcription initiation - Ribosomes do not necessarily dissociate at the site at 40 to 100 bp 3'-downstream. stop codon during scanning the transcript. In - 5'-Upstream of the core ("minimal") promoter the case of a dicistronic transcript this may lead region are cis-regulatory elements located that to initiation of the translation of the second modulate the level of transcription in a quanti- coding region. Since the translation of the first tative (i.e., SV 40 type positive enhancers) or coding region is more efficient, chimeric con- qualitative (i.e., tissue-specific enhancers and si- structs carrying a foreign gene as first, and a se- lencers) fashion by interaction with trans-acting lectable marker gene as a second cistron can fa- regulatory proteins (transcription factors). cilitate an increased production of foreign pro- - Increasing the copy number of certain enhan- teins in plants. cers results in a proportional increase in the lev- - N- and C-terminal sequences of plant proteins el of transcription. may be recognized by diverse processing mecha- - Many promoters contain AT-rich DNA se- nisms that mediate the targeting of the proteins quences (e.g., AT-boxes in heat shock, lectin, to cellular compartments, such as chloroplast, leghemoglobin, etc. promoters) in the 5'-up- mitochondrium, peroxisome, or endoplasmic stream region that probably mediate interaction reticulum. A fusion of corresponding DNA se- with common nuclear matrix proteins (e.g., quences to foreign genes can successfully be ap- HMG class) regulating conformational changes plied for targeting foreign proteins to plant or- of active chromatin. ganelles. - From genes transcribed by RNA polymerase I1 polyadenylated transcripts are synthesized. Sig- The use of T-DNA-based expression vectors result- nals for polyadenylation (i.e., AATTAA/T) are ed in a burst of applications. New selectable mark- located 3'-downstream of the translational stop ers, such as hygromycin, bleomycin, gentamycin, codon. The distance between the stop codon streptomycin, and methotrexate resistance genes and the polyadenylation site influences the were constructed by expression of diverse bacterial steady-state level of transcripts. genes and a dihydrofolate reductase gene from - Most plant genes contain introns, therefore the mouse. Reporter enzyme systems providing sensi- derived primary transcripts undergo splicing. It tive assays for monitoring gene expression in vitro, is apparent that viroids that cause serious plant in vivo, and by histological methods were developed diseases affect splicing. The length of introns by expression of ~glucuronidase(gus), pgalac- Insertional Mutagenesis: a Li Between Classical and Molecular Plant Genetics 221

(LacZ) and light-producing luciferase (luc by plant transcription factors that modulate their enzymes from fireflies and Vibrio harveyi. activity in a hormone-regulated and tissue-specific resistant to or tolerant of insects, fashion, the T-DNA itself provides an excellent tool des, the crystal toxin protein of to gain more insight into hormone signal transduc- ins, an- tion and transcriptional regulation of plants.

synthase, and phosphinotricine acetyl- 15.6 Insertional Mutagenesis: a Link Between Classical and Molecular Plant Genetics*

From a genetic point of view, T-DNA is a unique insertion element that is integrated into the plant nuclear genome after transfer from agrobacteria, and therefore may cause insertional inactivation of ental, hormonal, or plant genes. To identify T-DNA insertions in func- lopmental signals are transmitted through vari- tional plant genes, a gene fusion approach was de- veloped. A promoterless reporter gene was linked regulatory elements to the right border of a T-DNA, which also carried d transcription factors is studied using promoter a bacterial plasmid replicon and a plant selectable d enhancer test T-DNA vectors. These vectors marker gene. The ATG start codon of the reporter ivity of which can gene was either retained or deleted in order to gen- be followed in transformed protoplasts (tran- erate either transcriptional or translational gene fu- sions (Fig. 3). Following selection of transformants using the selectable marker, the frequency of T- DNA-induced gene fusions was determined. In to- bacco and Arabidopsis about 40% of all insertions resulted in transcriptional fusions, while 15 to 20% of T-DNA inserts induced translational gene fu- sions. Differences in the complexity and distribu- hich suitable clon- tion of transcribed DNA sequences between tobac- n of putative cis- co and Arabidopsis excluded the possibility that a approach is used similar frequency of gene fusions in both plant spe- analysis of individual promoter ele- cies resulted from random T-DNA insertions. The sible interactions data rather indicated that T-DNA is preferentially ranscription fac- integrated in plant chromosomal loci that are po- tentially transcribed. ically regulated The gene fusion technique has the advantage es that are tar- that the' expression of T-DNA-tagged plant genes can be followed in vitro and in vivo throughout the ture emerging life cycle of plants or under influence of various ex- ar regulatory ternal stimuli. Both transcriptional regulatory ele- nt promot- ments and coding sequences of T-DNA-tagged ction between transcriptional fac- genes can be rescued from the nuclear DNA of transgenic plants with the help of a bacterial plas- cription. mid replicon carried by the T-DNA. Plant DNA5 f all T-DNA genes are recognized are digested with a restriction endonuclease that 222 Exploitation of Agrobacterium tumt$aciens

promoterless bacterial reporter gene plasmid

plant aelectabie marker gene

II. 1.111. plant gene v AT0 b promoter codlng region end of transcript

Transcriptional gene fusions

Translational gene fusion

*1 1. CAATTAT)- ATG -1 ATT >

Fig. 3%b. Insertional mutagenesis with T-DNA gene fusion vec- ATG codon of the reporter gene, FDNA insert in I results in a tors. a Schematic structure of T-DNA gene fusion vectors. A dicistronic transcript. The first coding region of this transcript promoterless reporter gene is linked to the right border (RB)of encodes a truncated plant protein, while the second one encodes the T-DNA that also contains a bacterial plasmid replicon (A# the reporter enzyme. T-DNA insert in II leads to a and ori) and a plant-selectable marker gene joined to the left T- monocistronic transcript starting at + 1 position of the plant DNA border (LB). Underneath the T-DNA vector a hypotheti- gene and terminating at the polyadenylation site of the reporter cal plant gene is depicted with promoter region (CAATTATA), gene within the T-DNA. From both Zand Iztranscriptional gene transcription start (+I), translation start (ATG) and transcrip- fusions an intact reporter protein is synthesized. IZZ shows a T- tion termination site. Arrows I, II, and ZII indicate T-DNA inte- DNA insert with a promoterless reporter gene that does not con- gration sites. b Principle of T-DNA-induced transcriptional and tain ATG translational start codon. T-DNA insertion in the cod- translational fusions. Line I shows a T-DNA insert in the coding ing region of a plant gene may lead to in-frame fusion between region of a plant gene, while in line II a T-DNA insert is depicted plant and reporter genes. This results in the translation of a fu- after integration in the transcribed but untranslated leader re- sion protein that consists of an N-terminal plant protein domain gion of a plant gene (located between + 1 and ATG). Due to the and a C-terminal reporter enzyme domain presence of stop codons in all reading frames upstream of the has no recognition site within the T-DNA, circular- or used as probes to isolate wild-type alleles of the ized by self-ligation and transformed into E. coli, tagged genes from genomic and cDNA libraries. where the T-DNA and flanking plant DNA is re- Ambidopsis thaliana, a plant with excellent ge- covered as a plasmid. Plant DNA sequences res- netics, became a model for plant cued in linkage with the promoterless reporter gene in general and for insertional mutagenesis studies of the T-DNA are dissected and inserted into pro- in particular. A search for T-DNA-induced muta- I moter and enhancer test vectors for further studies tions in Arabidopsis showed that while insertions 4 of the regulation of the identified plant promoters, in diverse genes can be obtained at a fairly high fre- 1 Summary 223

-quencyby gene fusions, only a portion of these mu- genetically, may also be applied as experimental tations result in morphological alterations or other tools in studies of hormone signal transduction, visible mutant phenotypes. Selectable antibiotic re- cell division, or organ differentiation. Further de- sistance genes carried by T-DNA gene-tagging vec- velopment of insertional mutagenesis techniques tors provide suitable markers for mapping of the involving the use of plant transposable elements induced mutations even in the absence of visible may facilitate simple identification of new plant mutant phenotypes. A large number of mutations genes. TDNA-induced gene fusions to suicide induced by irradiation or by chemical mutagenesis marker genes, that cause cell lethality, might help (e.g., EMS) is also available to test allelism with the the isolation of genes that are expressed only in cer- T-DNA-induced mutations. Wild-type alleles tain cell types or during certain stages of develop- kloned on T-DNA plant gene vectors (or corre- ment. Alternatively, T-DNA-mediated insertion of 8ponding cDNAs cloned in T-DNA-based expres- strong promoters into the plant genome may be sion vectors) are used for complementation of the used for dominant activation of life defense genes 1 induced mutations. Insertional mutagenesis in involved in tolerance to drought, heat, salinity, or I giants thus offers nearly as much flexibility as simi- toxic chemicals. Tkansformation with T-DNAs car- I Jwapproaches in bacteria or in yeast. Recent isola- rying segments of the plant genome may result in 1 tion and characterization of chlorata (ch-42), recombination with homologous chromosomal loci : Jrgamous (ag), apetala (ap2), and glabrous (gl-1) that can be exploited in development of site-specif- wes of Ambidopsis demonstrated that T- ic mutagenesis techniques for plant. Last but not :DNA-tagging is an efficient approach to identify least, combination of T-DNAs with telomeric ele- k ' fenes regulating basic processes, such as photosyn- ments of plant chromosomes might facilitate the ' thesis or differentiation in plants. identification of centromeric and autonomously

!# replicating (ARS) DNA sequences to achieve chro- ! +' mosome engineering in plants. 15.7 Outlook 15.8 Application of T-DNA gene vectors in basic and Summary applied plant science is virtually unlimited. As in the past, studies of the mechanism of T-DNA trans- The properties of TDNA transfer and integration fer are expected to facilitate further improvement into the plant genome make it the system of choice of T-DNA vectors and plant transformation sys- for engineering of stably transformed plants. The tems. Some rules established for Agrobacterium- versatility of TDNA vectors that exploit the natural plant interaction may lead to the discovery of new gene transfer process between agrobacteria and forms of interspecies gene transfer. Exploiting the plants is such that TDNA can be used for a variety growing knowledge on regulation of plant gene ex- of purposes other than simple gene transfer into pression may help to achieve cell type-, tissue-, de- plants. Over the years the unique ability of Agrobac- velopmental stage-specific; hormone-, light-, heat-, terium to transfer the T-DNA to the plant cell has gravity- or drought-induced; osmotically or chemi- provided us with means to study plant-bacterial in- cally regulated expression of foreign or modified teraction, gene transfer and control of gene expres- plant genes in a great variety of plants. Studies of sion, differentiation, and development of plants. transcription factors and corresponding genes will With the T-DNA vectors and marker genes currently give more insight to the regulation of gene expres- available it is predictable that the collection of vari- sion during plant development. T-DNA genes, ex- ous approaches using TDNA as a tool to investigate ploited to alter plant morphology and development plant biology is far from being exhausted. 224 Exploitation of Agrobacterium turndaciens

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