WO2007000579A1

WO2007000579A1 - Transgenic plants over-expressing extensin with increased plant pathogen resistance

Info

Publication number: WO2007000579A1
Application number: PCT/GB2006/002332
Authority: WO
Inventors: Anil Shirsat; Guo Wei; Karen Brown
Original assignee: University Of Wales, Bangor
Priority date: 2005-06-28
Filing date: 2006-06-23
Publication date: 2007-01-04
Also published as: GB0513079D0

Abstract

The invention relates to a transgenic plant cell. The genome of said cell is modified to over-express production of extensin when compared to a non-transgenic reference cell of the same species. The invention also extends to a transgenic plant, a seed and also a DNA construct which encodes an extension protein. Processes for producing a transgenic plant are also described.

Description

IMPROVEMENTS IN AND RELATING TO PLANT PATHOGEN RESISTANCE

Field of the Invention

This invention relates to transgenic plant cells, transgenic plants, and methods of increasing plant resistance to pathogen attack. The invention further relates to methods of over-expression of extensin in plants to provide pathogen resistance.

Background to the Invention

In general, plants challenged by mechanical wounding or pathogen attack induce rapid expression of certain genes, for example, proteinase inhibitor and pathogenesis-related genes that are expressed locally, as well as systemically in unaffected parts of the plant (Yang et al 1997) . It has also been shown that increased levels of extensin transcripts are effected as a result of mechanical wounding in many plants. . Many of the inducible defence responses are not exclusive to mechanical wounding but are also initiated by pathogen attack.

Extensins have been proposed to be structural cell wall proteins important in plant development, but have also been directly implicated in plant defence against mechanical and pathogen-induced wounding (Shirsat et al 1996, Sauer et al 1990) . However, at this present time, there has been no positive evidence that high levels of extensin serve to induce pathogen resistance in plants nor acts to mitigate pathogen attack itself; instead, it has previously been thought that high levels of extensin present in plants undergoing pathogen attack is as a result of other plant defence mechanisms which have a side effect of increased transcription of extensin.

Applicant has surprisingly found that construction of transgenic plant cells and plants in which the over- expression of extensin is induced in the plant, confers excellent resistance to pathogen attack in the transgenic plants .

It would be advantageous to provide pathogen resistance to plants by utilising over-expression of extensin within a plant, in order to confer both mechanical and pathogen resistance.

It is therefore an aim of preferred embodiment of the present invention to overcome or mitigate at least one problem in the prior art, whether expressly disclosed herein or not .

Summary of the Invention

According to a first aspect of the invention there is provided a transgenic plant cell, wherein the genome of said cell is modified to over-express production of extensin when compared to a non-transgenic reference cell of the same species .

Thus, the transgenic plant cell of the invention is transformed with and expresses a gene encoding the extensin protein at levels above expression of extensin in a non-transgenic reference plant cell of the same species .

Preferably the transgenic plant cell is a plant cell of a dicot species or a monocot species . According to a second aspect of the invention there is provided a transgenic plant characterised in that the genome of said plant is genetically modified to provide at least one extensin gene which is over-expressed when compared to a non-genetically modified plant.

Preferably the cell or plant is transformed with a nucleic acid molecule comprising an expression cassette, which cassette comprises a nucleic acid sequence operably linked to a promoter, said sequence comprising a sense or anti- sense sequence having at least 85% homology with the sequence shown in SEQ ID 1.

Preferably the nucleic acid sequence comprises a sequence having at least 90% sequence identity with the sequence shown in SEQ ID 1, more preferably at least 95% sequence identity, and yet more preferably at least 98% sequence identity, and especially at least 99% sequence identity.

Preferably the promoter is a constitutive or inducible promoter. Preferably the promoter is the CaMV35S promoter, but may be any suitable promoter known to persons skilled in the art. Alternative promoters may include the or any tissue specific promoter, such as ubiquitin promoter and zein promoter.

Preferably the cassette vector includes additional regulatory units, which may include a transcription termination signal. The transcription termination signal may comprise the NOS terminator from Agrobacteriυm tumefaciens.

According to a further aspect of the invention there is provided a DNA construct comprising a vector comprising a promoter, and a nucleic acid sequence which encodes an extensin protein. Preferably the vector is a plasmid. Suitably the plasmid comprises the pBIN plasmid.

Preferably the nucleic acid sequence encoding the extensin protein comprises the extensin gene atExtl from A. thaliana comprising the sequence shown in SEQ ID 1, or a gene having at least 85% homology with the atExtl gene, more preferably at least 90% homology, yet more preferably at least 95% homol.ogy, still more preferably at least 98% homology, and most preferably at least 99% homology.

According to a further aspect of the present invention, there is provided a process for producing a transgenic plant, said transgenic plant effecting over-expression of extensin in comparison with a wild-type non-transgenic plant. The process comprising the steps of:

(a) obtaining a nucleotide sequence coding for extensin;

(b) inserting the coding nucleotide sequence in a DNA construct in sense or anti-sense orientation next to a promoter as a regulatory unit;

(c) transforming a plant cell of a plant, with the DNA construct; and

(d) cultivating the plant cell and regenerating a plant, wherein the plant over-expresses extensin.

According to a further aspect of the present invention, there is provided a process for producing a transgenic plant, said plant over-expressing extensin in comparison with a wild-type non-transgenie plant, said process comprising the steps of:

(a) obtaining a nucleotide sequence coding for extensin expression; (b) inserting the coding nucleotide sequence in a DNA construct in anti-sense or sense orientation next to a promoter as a regulatory unit;

(c) transforming the plant with the DNA construct.

Suitably, the nucleotide sequence and promoter are as described for the earlier aspects of the invention, and the plant and plant cell are as described for the earlier aspects of the invention.

According to yet a further aspect of the present invention there is provided a transgenic seed obtainable from a transgenic plant of the earlier aspects of the invention.

Brief Description of the Drawings

The various aspects of the invention will now be described by way of example only, in the following examples, with reference to the accompanying drawings in which:

Figure 1 : Illustrates a plasmid map of the pGM2 clone, showing major restriction sites and features. The arrow indicates the 5' to 3' orientation. Plasmid map drawn using Plasmid Processor Version 1.02 (University of Kuopio, Finland) .

Figure 2 : Illustrates a plasmid map of the pBIN m-gfp5-ER expression vector, showing major restriction sites and features. The arrows indicate the 5' to 3' orientation. ^a Chimaeric kanamycin resistance gene - nopaline synthase promoter (NOS prom) : -.neomycin phosphotransferase (NPT JI) : :nopaline synthase terminator (NOS ter) .

Figure 3 : Illustrates a step-by-step cloning strategy for the construction of sense and antisense plant transformation vectors, containing the atExtl gene under the control of the constitutive CaMV 35S promoter.

Figure 4 : Illustrates a position of restriction sites in the promoter and coding sequence of the atExtl gene, used for the cloning strategy described in Figure 3.3. Restriction sites are labelled and indicated in blue. The two ATG start codons are underlined.

Figure 5 : Illustrates a 0.8% agarose gel showing dephosphorylated (lane 1) and non-dephosphorylated (lane 2) Sinai-cut pUC18 plasmid DNA following a test ligation reaction. Plasmid DNA was dephosphorylated to prevent self-ligation.

Figure 6a: Illustrates a autoradiograph of a colony hybridisation of E. coll DH5α cells transformed with the dephosphorylated pUC18 vector ligated to the 1.256kb and 1.538kb atExtl coding sequence and promoter sequence fragments, and probed with the 1.256kb atExtl coding sequence. 28 colonies hybridised strongly to the probe, out of 157 colonies on the membrane.

Figure 6b: Illustrates a autoradiograph of the colony hybridisation shown in Figure 3.6a re-hybridised to a radiolabeled probe made from the 1.538kb pGM2/ZhoI-BsUNI fragment containing the 3 '-end of the atExtl promoter. 97 colonies hybridised strongly to the new probe.

Figure 7 : Illustrates a diagram showing the different positions of the two Sail restriction sites in recombinant plasmids containing the atExtl coding sequence fragment in sense (a) orientation.

Figure 8a: Illustrates a 0.8% agarose TAE gel showing four putative [pUC18/SmaI: :1.256kb pGM2/ZήoI-BsUNI] clones digested with Sail (lanes 2-5) . Lanes 2-5 all contain 2.7kb and 1.3kb bands indicative of the presence of the atExtl coding sequence in the sense orientation. λ DNA digested with Pstl was used as a size marker (lane 1) .

Figure 8b: Illustrates a autoradiograph of the gel in 3.8a, hybridised to a radiolabelled probe made from the 1.256kb atExtl coding sequence. The probe hybridises strongly to the 1.3kb bands in lanes 2-5, indicating that they are all positive atExtl constructs. The relative position of the size marker is shown at the left of the photograph.

Figure 9 : Illustrates a 1.0% agarose 50ml TAE gel showing pUCl8 (lanes 2, 4 and 6) and the recombinant plasmid pKB3

(lanes 3, 5 and 7) cleaved with BamHl -Sad (lanes 2 and 3), Sail (lanes 4 and 5) and Xhol (lanes 6 and 7). λ DNA digested with Pstl was used as a size marker (lane 1) .

Figure IQa: Illustrates a plasmid map of sense construct pKB3 , showing major restriction sites and features. Arrows indicate the 5' to 3' orientation. The atExtl fragment interrupts the lacZ' and lacl sequences, giving white colonies on LB/amp/IPTG/X-Gal plates. Plasmid map drawn using Plasmid Processor Version 1.02 (University of Kuopio, Finland) .

Figure IQb: Illustrates a border sequences of the inserted atExtl fragment inside the recombinant vector pKB3, showing restriction sites and the orientation of the atExtl coding sequence in relation to the pUCl8 multiple cloning site. Both original Smal sites from the pUCl8 vector were lost during the ligation reaction. The BsMTI site from the atExtl fragment was also lost, whilst the Xhol site was reformed. The ATG start codon for the coding sequence is underlined.

Figure 11: Illustrates a 1% agarose gel showing the binary vector pBIN m-gfp5-ER (lanes 2, 4 and 6) and the atExtl-containing construct pKB5 (lanes 3, 5 and 7) digested with various restriction enzymes. 500ng DNA of each plasmid was digested with BaπMI-SacI to excise the m- gfp5-ER gene (lane 2) or atExtl coding sequence (lane 3);

Hindlll-Sacl excises the gene plus the CaMV 35S promoter (lanes 4 and 5) ; Pstl cuts the plasmids at three sites

(lanes 6 and 7) . λ DNA digested with Pstl was used as a size marker (lanes 1 and 8) . Expected fragment sizes are listed in Table 2, based on the plasmid map shown in

Figure 12a and 12b.

Figure 12a: Illustrates a plasmid map of plant transformation vector pKB5, showing major restriction sites and features. Arrows indicate the 5' to 3' orientation. ^a Chimaeric kanamycin resistance gene - nopaline synthase promoter (NOS prom) :.-neomycin phosphotransferase (NPT II) : :nopaline synthase terminator (NOS ter) .

Figure 12b: Illustrates a border sequences of the inserted atExtl fragment (green) inside the recombinant vector pKB5, showing restriction sites and the position of the atExtl coding sequence in relation to the CaMV 35S promoter and NOS terminator (blue) . The ATG start codon for the coding sequence is underlined.

Figure 13a: Illustrates a 1.0% agarose gel showing genomic DNA from transgenic Landsberg erecta plants 5.1, 5.2, 5.4 and 5.5 (containing the CaMV 35S: :atExtl fusion gene) digested with Hindlll-Sacl (lanes 4-7) . As a positive control, βOng pKB5 plasmid DNA was cut with BamHI-Hindlll-SacI (lane 2) . Wild-type Landsberg erecta genomic DNA was cut with Hindlll-Sacl and used as a negative control (lane 3) . λ DNA cut with Pstl was used as a size marker (lane 1) .

Figure 13b: Illustrates a autoradiograph of the gel in 3.18a, hybridised to a radiolabelled probe made from the 871bp CaMV 35S promoter sequence. Lanes 4-7 all show a single ~2.2kb band (1.275kb coding sequence plus 871bp CaMV 35S promoter) that hybridises strongly to the probe. Lane 1 shows the 1.25kb pKB5/BamHX-HindIII-SacI fragment hybridising strongly to the probe. The relative position of the size marker is shown to the left of the photograph.

Figure 14a: Illustrates a 1.0% agarose gel showing genomic DNA from transgenic Wassilewskija plants 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16 and 5.18 (containing the CaMV 35S: :atExtl fusion gene) digested with Hind Ill-Sac I (lanes 4-16) . As a positive control, 60ng pKB5 plasmid DNA was cut with HinduI-Sad (lane 2) . Wild-type Wassilewskija genomic DNA was cut with ffi-αdlll-≤'acl and used as a negative control (lane 3) . λ DNA cut with Pstl was used as a size marker (lane 1) .

Figure 14b: Illustrates a autoradiograph of the gel in 3.19a, hybridised to a radiolabelled probe made from the

871bp CaMV 35S promoter sequence. Lanes 4-16 all show a

«2.2kb band (1.275kb coding sequence plus 871bp CaMV 35S promoter) that hybridises strongly to the probe.

Transgenic lines pKB5.7 (lane 5) and pKB5.12 (lane 10) show extra bands that have undergone recombination. These lines were not used for further study. The relative position of the size marker is shown to the left of the photograph.

Figure 15 : Illustrates a graph showing bacterial pathogen enumeration of P.syringae in Arabidopsis thaliana wild- type and extensin over-expression transgenic plants .

Description of the Preferred Embodiments

1.1 Strategy for the construction of the sense CaMV 35S: :atExtl gene fusions

The A. thaliana extensin gene, atExtl, was initially isolated and cloned into the Lambda FIX II replacement vector (Merkouropoulos efc al . , 1999). A 4.4kb Sail fragment from this vector containing both the atExtl promoter and coding sequence, was then sub-cloned by Merkouropoulos (2000) into the pBluescript^® II SK⁺ (pSK II) phagemid vector from Stratagene (Stratagene Limited, Cambridge, Cambridgeshire, U.K.). This construct, pGM2, shown in Figure 1, contains a 3.288kb promoter fragment, 1.122kb coding sequence and a putative 95bp intron, and provided the basis for further manipulation of the atExtl gene. The full nucleotide sequence of atExtl is shown in SEQ ID 1.

The generation of Arabidopsis plants expressing atExtl to different levels can be achieved by transforming plants with binary vectors constitutively expressing the atExtl coding sequence in the sense direction. The cloning strategy involved excising the atExtl coding sequence from pGM2 and inserting it in the sense orientation into a plant expression vector containing the CaMV 35S promoter, NOS terminator sequence, and the nos promoter: -.nptll gene (conferring kanamycin resistance) as a plant selection marker. A suitable vector containing all of these features was the plasmid pBIN m-gfp5-ER (Figure 2), kindly provided by J. Haseloff (MRC Laboratory of Molecular Biology, Cambridge, U.K.). This construct was developed from the binary vector pBI121 (Jefferson et al., 1987) by replacing the original 1.87kb GUS reporter gene with the 816bp m-gfp5-ER gene (Siemering et al., 1996).

Cloning strategy for making sense and antisense atExtl constructs

Figure 3 outlines the strategy used to create the sense and antisense atExtl constructs. This involved excising the m-gfp5-ER gene from pBIN m-gfp5-ER with a BamHI-SacI digest, and replacing it with the coding sequence of atExtl. As the extensin coding sequence lacks BamΑT and SaCl sites, it was first inserted into the high-copy 2.686kb plasmid pUCl8 (MBI Fermentas) . As both sense and antisense constructs were required, a blunt-end ligation into the pUC18/S-riaI site provided an opportunity for sub- cloning of the insert in both orientations. The sub- cloned insert could then be excised from pUCl8 with a Ba-T-HI-SaCl digest and ligated into pBIN m-gfp5-ER.

(a) The 5'-end of the atExtl cloning fragment

The atExtl sequence downstream of the promoter fragment includes a 28 amino acid signal peptide and a 350 amino acid protein (SEQ ID 2) . The signal peptide is required for transport of the immature extensin protein into the endoplasmic reticulum for post-translational modification Although the sequence in atExtl includes two in-frame ATG codons, the BsUNI site in-between them was used to create the 5 '-end of the cloned fragment as no other unique restriction sites within 64bp of the first ATG were available. Signal peptide sequence analysis using the TargetP Server vl.01 (Emanuelsson et al., 2000) shows that the shortened signal peptide still remains sufficient for targeting proteins to the secretory pathway. A full analysis of 5' deletions of the atExtl signal peptide is shown in SEQ ID 2.

(b) The 3 '-end of the atExtl cloning fragment

Comparison of atExtl expressed sequence tags (ESTs available from the Arabidopsis Genome Initiative at http : / /www, arabidopsis . org/info/agi .html ) with the atExtl genomic sequence highlights 95 extra base pairs located in a 168bp 3' transcribed but un-translated region. Sequences either side of this region are identical to consensus intron-splicing sequences in other dicot extensins (Merkouropoulos, 2000) . As the presence of an intron sequence is believed to affect the efficient processing of pre-mRNA (Simpson and Filipowicz, 1996) , this 3 ' region was included in the cloned insert by using an Xhol site, downstream of the putative intron in pGM2. This also retained a unique internal Sail site that, in combination with a Sail site located in the multiple cloning site of pUClδ, would provide a useful method for determining the orientation of the insert.

1.2 Sub-cloning the coding sequence of the atiExtlgene

Escherichia coli DH5α cells containing the plasmid pGM2 were obtained from G. Merkouropoulos (University of Wales, Bangor) . The presence of the atExtl insert within the plasmid was verified by restricting a mini plasmid preparation (using an alkaline lysis method) with Sail, which yielded the expected 4.45kb and 3.0kb fragments (see plasmid map in Figure 1) .

A large-scale plasmid preparation using a QIAGEN^® Plasmid Midi Kit yielded purified plasmid DNA for subsequent cloning reactions. pGM2 plasmid DNA was digested with Xhol and the two resultant fragments were separated by gel electrophoresis. The 2.794kb fragment (containing the 3'- end of the promoter, coding sequence and intron) was isolated from the gel and digested with BsUNI (Helena Biosciences) to separate the coding sequence and intron (1.256kb fragment) from the 3' end of the promoter (1.538kb fragment). This is shown diagrammatically in Figure 4.

The Xhol and BshNI 5 '-overhangs of the 1.256kb and 1.538kb fragments were filled-in by a blunt-ending reaction. Following the reaction, the Klenow enzyme was inactivated and removed immediately by extraction with phenol . The cloning vector pUC18 was first digested with Smal and, to prevent the plasmid religating to itself during the ligation reaction, pUC18/Smal DNA was then dephosphorylated using CIAP. Test ligation reactions, containing only pUCl8/SmaI DNA and examined by gel electrophoresis (Figure 5) , showed that whereas untreated pUC18/SmaI DNA produced numerous self-ligation products, the dephosphorylated pUC18/SznaI DNA showed no detectable self-ligation.

The blunt-ended 1.256kb and 1.538kb pGM2/Xhol-BsftNI fragments were ligated into the dephosphorylated pUC18/,SznaI vector in a blunt-ended ligation reaction using a 1:10 vector: insert ratio (50ng vector:233ng insert). Ligation products were electroporated into competent E. coll DH5α cells that were then plated onto LB/amp/IPTG/X- GaI plates for screening potential recombinant plasmids by blue-white screening. From a single 50ng vector: 233ng insert ligation reaction, a total of 197 white and pale- blue colonies developed.

Individual colonies were screened for the presence of the 1.256kb pGM2/XhoI-BshNI insert by colony hybridisation using the 1.256kb fragment as a probe. The autoradiograph in Figure 6a shows 28 colonies that hybridised strongly to the probe. Some of the remaining colonies were likely to be pUCl8/5iΩaI ligated to the 1.538kb fragment (containing the 3 '-end of the atExtl promoter) and this was confirmed by hybridising the membrane a second time to a probe made from the 1.538kb fragment (Figure 6b).

Colonies that appeared positive for the presence of pUCl8 with the atExtl coding sequence were analysed further by digestion with PvuII . With one PvuII site either side of the MCS in pUCl8, this digest would yield fragments of 2.37kb and 322bp if no insert was present, or 2.37kb and 1.578kb if the atExtl coding sequence was present. Out of the 28 original positive colonies, 14 showed a 1.6kb insert whilst the others displayed multiple bands or bands of an incorrect size. To ascertain the orientation of the insert these 14 clones were then cleaved with Sail yielding either 1.264kb + 2.682kb fragments (to indicate the insert in a sense direction), or 3.926kb + 20bp fragments (indicating the insert in an antisense direction) . This is shown diagrammatically in Figure 7.

Digestion products were electrophoresed on an agarose gel, Southern-blotted onto nylon membrane and then hybridised with the radiolabelled 1.256kb coding sequence probe. Figure 8a shows the results from 4 of the 14 clones. Lanes 2-5 in Figure 8b show the 1.3kb Sail band hybridising strongly to the probe, indicating successful cloning of the atExtl coding sequence in the sense orientation. From the 14 clones examined, 11 appeared to be successful sense transformants .

Analysis of plasmid DNA from bacterial colonies that hybridised to the 1.538kb probe (the 3 '-end of the atExtl promoter) showed that this fragment had been successfully cloned in both orientations. These constructs were named pKBl (plasmid Karen Brown number 1) for the 5' to 3' 1.538kb-insert recombinant plasmid, and pKB2 for the 3' to 5' .

1.3 The atExtl-coding sequence sense construct, ρKB3

Purified plasmid DNA from a single colony that contained the sense construct was isolated from liquid culture using a QIAGEN^® Plasmid Midi Kit. Restriction digests of the clone with Baπϋil-Sacl, Sail and Xhol were performed to fully characterise the recombinant sense construct and to check the integrity of the reformed Xhol restriction site. Figure 9 shows restriction products from the recombinant clone as well as from intact pUClδ plasmid DNA. The fragment sizes obtained from the recombinant clone match those expected from the correct insertion of the atExtl fragment into pUC18 (Table 1) . A plasmid map of the new construct, pKB3 , is shown in Figure 10a, with the atExtl- pUC18 border sequences detailed in Figure 10b. Table 1 : Expected DNA fragment sizes from the restriction digests of the sub-cloning vector pUCl8 and the recombinant atExtl-containing vector pKB3. Expected sizes are based on the plasmid map shown in Figure 10a.

1.4 Construction of CaMV 35S: zatExtl gene fusions

1.4.1 Construction of the atExtl sense binary vector, pKB5

Purified plasmid DNA from pKB3 was digested with BaπH.1 and Sad to excise a 1.275kb fragment containing the atExtl coding sequence. This fragment was then inserted into the pBIN m-gfp5-ER plasmid (with the m-gfp5-ER gene removed in a BanBJ.-SacX digest) in a cohesive-end ligation reaction using a 1: 3 vector : insert ratio (100ng vector: 30ng insert) . Ligation products were electroporated into E. coli DH5α cells and screened on LB media plates containing

SSμg.ml^"1 kanamycin (kan) . Recovered colonies were then analysed for the presence of the recombinant binary vector by colony hybridisation, using the 1.275kb pKB3/SamHI-Sad coding sequence fragment as a probe. The twenty colonies that gave positive signals were further analysed by restriction digests of plasmid DNA with BamRl-SacI, yielding fragments of 12.9kb and 1.3kb for recombinant expression vectors successfully containing the atExtl fragment. Eleven colonies gave the correctly-sized restriction fragments. Plasmid DNA from a single colony was then fully characterised by digestion with BairiΑI-Sacl, Hindlll-Sacl and Pstl (Figure 11) , yielding the expected sizes listed in Table 2.

A plasmid map of the new construct, pKB5, is shown in Figure 3.14a, with the atJSxfcl-pBIN border sequences detailed in Figure 12b.

Table 2 : Expected DNA fragment sizes from restriction digests of the binary vector pBIN m-gfp5-ER and the recombinant CaMV 35S: : atExtl vector pKB5. Expected sizes are based on the plasmid maps of pBIN m-gfp5-ER (Figure 2) and pKB5 (Figure 12a) .

1.5 Arabidopsis transformation with the pKB5 binary vector The Agrobacterium tumefaciens strain LBA4404 was transformed with the pKB5 plasmid by triparental mating. Genomic DNA was extracted from seven of the A. tumefaciens colonies recovered after tri-parental mating and digested with BaitiΑX-Sacϊ to excise the inserted atExtl coding sequence. Digested DNA was separated on a 1.0% agarose gel, Southern blotted to nylon membranes and then hybridised to a radiolabelled probe made from the 1.275kb pKB5/BamH.I-Sacl fragment (containing the atExtl coding sequence) . The autoradiograph showed single hybridising bands in all seven lanes, indicating successful transformation.

1.5.1 Arabidopsis transformation and screening

Arabidopsis thaliana ecotype Wassilewskija (Ws) plants were transformed with the CaMV 35S: : atExtl construct using the floral-dip method. The Ws ecotype was used for this work to continue the analysis of atExtl in Ws started by Merkouropoulos (2000) . Seeds obtained from the treated (T₀) plants were screened on 0.8% agar selection plates containing SSμg.ml^"1 kanamycin. Resistant (Ti) seedlings were allowed to mature and set seed. Characterisation of transgenics by DNA analysis was performed on tissue from T₂ plants . An explanation of the nomenclature for each generation is shown in Table 3. Table 3 : Explanation of transformant generation designations for transgenic plants, and the nomenclature used in this study.

Note: N/A = not applicable.

1.5.2 Analysis of transgenic plants by Southern hybridisation

Antibiotic-resistance selection of transformed A. thaliana seedlings carrying the CaMV 35S: -.atExtl transgene yielded five transgenic Ti plants with a Ler background (termed 5.1 to 5.5) and thirteen transgenic Ti plants with a Ws background (termed 5.6 to 5.18). Plants 5.2 (Ler) and 5.17 (Ws) produced non-viable seed and therefore were not included in further analysis . Southern analysis was used to confirm the presence of the CaMV 35S: : atExtl transgene in transformed A. thaliana plants and to determine whether truncation of the transgene had occurred during the T-DNA integration process. Genomic DNA was isolated from each line, digested with Hindlll-Sacl and restriction fragments separated by gel electrophoresis. The DNA was then transferred to nylon membranes and hybridised with a radiolabelled probe made from the 871bp CaMV 35S promoter sequence. Figures 13a and 13b, and 14a and 14b, show the Southern hybridisation results from Ler-derived lines 5.1 to 5.5, and Ws-derived lines 5.6 to 5.18, respectively.

The signal intensity of the hybridised bands is not constant between the transgenics and, as this is not entirely explained by differences in DNA concentration between gel lanes, it may be explained by the presence of more than one transgene in the genome. The her lines 5.1 and 5.4 have stronger intensity signals than lines 5.2 and 5.5 and therefore may have additional copies of the CaMV 35S: -.atExtl transgene. Ws lines 5.8 and 5.13 also appear to have multi-copy insertions, with far more intense signals than any of the other Ws lines (Figure 14b) . As the faintest signal (e.g. line 5.16) may be expected to be a single-copy insertion, additional Ws lines may also have more than one copy of the transgene (e.g. lines 5.6, 5.11, 5.12, 5.14 and 5.18) . However, in order to determine the exact number of insertions, additional restriction digests need to be performed.

Ws lines 5.7 and 5.12 displayed extra bands indicating rearrangement of at least one copy of the transgene and, therefore, were not used for further study. 1.6 Infection of transgenic Arabidopsis thaliana and wild- type non-transgenic Arab±dopsxs thaliana by infection

Transgenic Arabidopsis thaliana plants which over-express extensin and wild-type non-transgenic Arabidopsis thaliana plants were infected with p.syringae by syringe infiltration of the plant leaves with the pathogen.

Comparison of symptoms of pathogen infection in the infected leaves was made one day to five days after infection, and the results collated.

The results are shown as follows in Table 4 below:

Table 4 : Summary of phenotypes of wild type and extensin over-expression transgenic arabidopsis thaliana after bacterial infection

Bacterial pathogen enumeration was determined using the following procedure:

1. 6-week old Arabidopsis leaves were infiltrated with 10 micro-litres of a pseudomonas syringae suspension containing 10⁶ cfu/ml using a 1 ml syringe without needle.

2. At regular intervals, 0.5 cm² leaf discs at the site of infection were harvested and surface sterilised. 3. The bacteria and the leaf tissue was extracted by macerating the discs with a plastic pestle in 0.2 ml of 1OmMgCl₂.

4. Serial dilutions were plated on nutrient broth plates containing rifampicin.

5. The plates were placed at 30 "C for 2 days and then the colony-forming units for each dilution of each sample was counted.

The results of the bacterial pathogen enumeration are shown in Figure 16.

In other embodiments, single plant cells can be transformed by using the procedures described hereinbefore and cultivated to produce transgenic plants over- expressing the atExtl gene.

1.7 Conclusions

In the first three days of infection, the extensin over- expressing transgenic plants accumulate 100-fold lower levels of bacteria compared to wild-type (10⁶ compared to 10⁸) . Lesion development in the wild-type is very different to lesions seen in the transgenic plants; wild- type lesions are large, wet chlorotic lesions compared to small, dry, grey lesions on the transgenic plants. After five days, lesions on the transgenics are one-third the area of lesions on the wild-type.

Therefore, applicant has shown that extensin over- expression limits pathogen infectivity in plant cells .

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference .

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features .

The invention is not restricted to the details of the foregoing embodiment (s ). The invention extend to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings) , or to any novel one, or any novel combination, of the steps of any method or process so disclosed. REFERENCES

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Claims

1. A transgenic plant cell, wherein the genome of said cell is modified to over-express production ^■ of extensin when compared to a non-transgenic reference cell of the same species .