EXPRESSION OF BACTERIAL SIGNAL MOLECULES IN PLANTS

Abstract: The ability of a plant to defend against attack by bacteria, and any virus borne by the bacteria, is enhanced by transforming the plant genome with a gene of bacterial origin which enables the plant to produce a bacterial pheromone, N-acyl-L-homoserine lactone. Such plants also secrete the lactone into the soil enhancing the protective effect of antifungal rhozobacteria.

Description: EXPRESSION OF BACTERIAL SIGNAL MOLECULES IN PLANTS This invention relates to the expression of bacterial signal molecules in plants which allows, for example, modulation of the interaction between plants and infecting or symbiotic bacteria. The ability of bacteria to respond to environmental cues such as nutrient availability, temperature or pH is critical to microbe success. It is apparent that individual bacteria can also sense the density and state of the local bacterial population of which they are members. This sensing ability, referred to as "quorum sensing", allows a bacterial community to synchronise growth and development and, when the minimum population or "quorum" has been achieved, to initiate a concerted population response. Quorum sensing is thus an example of multicellular behaviour in prokaryotes and regulates diverse physiological processes including bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer and the production of virulence determinants in pathogens. The signalling pheromones upon which quorum sensing is based have been identified as N-acyl-L-homoserine lactones (reviewed by Swift et.al. "Quorum sensing: a population- density component in the determination of bacterial phenotype", Trends in Biochemical Science, 21, 214-219 (1996). N-acyl-L-homoserine lactones molecules comprise a homoserine lactone moiety (derived from amino acid metabolism, possibly via S-adenosyl methionine) linked to an acyl sidechain (probably derived from fatty acid synthesis). A number of N-acyl-L-homoserine lactones with different acyl side chains have been identified in different bacterial systems where they elicit a wide range of quorum-dependent responses such as swarming, pathogenicity, conjugation or production of colour, light or antibiotics. Several bacterial species produce the same N-acyl-L-homoserine lactone, although in some of the species it may regulate a different biological process. For example, the luxl gene product of Photobacterium fischeri synthesises N-(3-oxohexanoyl)-L -homoserine lactone which regulates bioluminescence in a cell density-dependent manner, whilst the carl gene product of Erwinia carotovora also produces N-(3-oxohexanoyl)-L-homoserine lactone which in this bacterium is responsible for the induction of secreted plant cell wall degrading exoenzymes and of the antibiotic carbapenem. The evil gene of Chromobacterium violaceum encodes the enzyme for synthesis of N-hexanoyl-L-homoserine lactone which is structurally very similar to the oxohexanoyl analogue and which induces production of the purple pigment violacein. Inactivation of luxl, carl ox evil results in loss of the density dependent bioluminescence, virulence or violacein production respectively. The relevant operons can, however, be induced by the addition of an exogenous supply of the N-acyl-L- homoserine lactone to the mutant bacteria. Carl mutants of Erwinia carotovora appear to be completely avirulent when tested on tobacco. They can neither macerate plant tissue nor multiply inplanta because they lack pectin lyase, pectate lyase, polygalacturonase, cellulase and protease. It is pertinent to ask how the expression of these exoenzymes only at high cell density in the wild-type cells may contribute to the success of Erwinia as a plant pathogen. It has been suggested that under aerobic conditions, a successful E. carotovora infection requires a relatively high inoculum (10 6 - 10 7 c.f.u.) and the progression of the disease is then a competition between bacterial multiplication and development of plant resistance. Thus, the production of macerating enzymes at low cell densities would not give rise to a successful infection, but would result in the induction of the local and systemic plant defence response, which in turn would hamper subsequent infections. Such resistance to E.carotovora infection is seen when the plant defence response is artificially induced by the application of salicylic acid. While not wishing to be bound by any theory as to the manner in which the invention proposed herein operates, the following explanation of the naturally occurring phenomenon of quorum sensing is offered. Using Photobacterium βscheri as a convenient example, the expression of two regulatory genes, luxl and luxR, is necessary for the expression of the genes necessary for bioluminescence. Expression of luxl leads to production of the pheromone N-(3- hydroxyl)hexanoyl-L-homoserine lactone, the mechanisms by which the lactone is synthesised being largely irrelevant to this discussion. A complex of the pheromone with the protein produced by the luxR gene gives a phenotypic response, in the case of P.βscheri, bioluminescence. At low population density of bacteria, luxl and luxR are transcribed at low level and there is insufficient accumulation of the pheromone (N-acyl-L-homoserine lactone) to elicit /zed-dependent transcription of the operon responsible for visible bioluminescence. It has been suggested that in the absence of sufficient pheromone, and/or a chaperonin known as GroESL, luxR is unstable and sensitive to degradation. As the population grows, however, the concentration of the pheromone increases gradually. At a critical level of the pheromone, which represents a critical population density, a complex between luxR and the pheromone is thought to bind to a palindromic sequence within the luxl operator thereby activating increased transcription of the operon necessary for increased production of the pheromone and for bioluminescence. The present invention seeks to provide a method and means of manipulating plant/microbe interactions. According to the present invention there is provided a method of protecting a plant against bacterial infection and/or virus infection transmitted by bacteria, comprising introducing into the genome of the plant by transformation the ability to synthesise a N-acyl- L-homoserine lactone. Further according to the invention there is provided a method of protecting a plant against bacterial infection and/or virus infection transmitted by bacteria, comprising introducing into the genome of the plant by transformation the ability to synthesise an analogue of N-acyl-L-homoserine lactone. The invention also provides a method of enhancing interaction between an antifungal rhizobacterium and a plant comprising introducing into the genome of the plant by transformation the ability to synthesise the N-acyl-L-homoserine lactone naturally produced by the rhizobacterium. The invention also provides a recombinant plant genome containing a gene construct for inplanta expression of an N-acyl-L-homoserine lactone. Preferably expression of introduced genes is targeted to plant chloroplasts. The gene specifying the Ν-acyl-L-homoserine lactone may be selected from the group consisting of, the yenl gene of Yersinia enterocolitica; the evil gene of Chromobacterium violaceum; the luxl gene, of Photobacterium βscheri; the carl gene of Erwinia carotovora; the tral gene of Agrobacterium tumefaciens and the lasl and vsml genes of Pseudomonas aeruginosa. Examples of suitable sources of DΝAs specifying N-acyl-L-homoserine lactones and the acyl group involved are as follows: Table 1 N-acyl-L-homoserine lactone These examples in Table 1 are quoted in Swift et.al, Trends in Biochemical Science, 21, 214-219 (1996). Table 2 below gives further examples along with references and the appropriate GenBank Accession Numbers. Table 2 Beck et.al. J.Bacteriol, 177, 5000-5008 (1995) Devine et.al. Biochemistry, 27,837-842 (1988) Fuqua etal. J.Bacteriol. 176, 269-275 (1994) Gray et.al. J.Bacteriol. 178, 372-376 (1996) Hwang et.al. J.Biotech. 177, 449-458 (1995) Latifi et.al. Molecular Microbiology, 17, 333-343(1995) McGowan c al.Microbiology, 141, 541-550 (1995) Meignhen wj. Rev. Genet. 28,117-139(1994) Milton e al. J.Bacteriol. 179, 3004-3012 (1994) Ochsner and Reiser Proc.Natl.Acad.Sci. USA, 92, 6424-6428 (1995) Sitnikov et.al. Molecular Microbiology, 17, 801-812 (1995 Swift etal. Molecular Microbiology, 10, 511-520 (1993) Swift etal. J.Bacteriol. 179, 5271-5281 (1997) Throup etal. Molecular Microbiology, 17, 345-356 (1996) Winson et.al. Proc.Natl.Acad.Sci USA, 92, 9427-9431 (1995) Wood and Piersen, Gene 168, 49-53 (1996) Our invention is founded on our reasoning that if the inoculating bacteria were to encounter levels of N-acyl-L-homoserine lactone that gave a false indication of the local population size, the course of the ensuing infection would be drastically reduced. A second aspect of the invention concerns engineering the plant to take advantage of the potential protective effect of antifungal rhizobacteria. There exist in the rhizosphere certain bacteria which are capable of attacking potential pathogenic fungal microorganisms which are also present in the soil, perhaps the best known of which are certain strains of Pseudomonas fluorescens and P. aureofaciens. But the population of such antifungal bacterial strains in the soil will normally be low and their antifungal activity dependent on the quorum sensing phenomenon to be activated. By imparting to the plant the ability to produce the activator molecule, the N-acyl-L-homoserine lactone, appropriate to the antifungal bacteria the antifungal activity may be initiated at low colony size providing earlier than normal protection of the plant against the pathogenic fungi. The rhizosphere- expressed genes of the rhiABC operon of the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum, for example, are regulated by an AHL with a C14 side chain containing hydroxylated carbon in the 3 position and a single carbon-carbon double bond. Transgenic plants producing an AHL signal molecule enhance the establishment of an antifungal environment on the rhizosphere. This phenomenon would also enable the use of disarmed bacterial strains to be used as crop protection biocontrol agents in conjunction with the AHL-producing transgenic plants. The invention will now be described in the following Examples The ability of AHLs to induce changes in neighbouring bacteria was tested in four ways; (1) the ability of the AHLs to diffuse out of intact leaves was demonstrated by placing intact transgenic leaves on agar and subsequently removing it before overlaying with C. violaceum CV026 (see Example 4 below) and the outline of the whole leaf could be seen showing that the AHL diffused out of the leaf surface and not just the cut stem; (2) being interested in whether the AHLs were only produced in the chloroplasts or whether they could be found in other tissues such as roots, the ability of the AHLs to diffuse from the roots was demonstrated in that AHLs in the vicinity of the roots were able to induce bioluminescence in a recombinant E,coli strain carrying an AHL-inducible operon: this showed that the root plastids are competent to suynthesise the AHLs are, alternatively, that the AHLs synthesised in green tissue can be transported to the roots but in either case the roots were clearly capable of signalling to nearby bacteria. (3) AHL-producing plant tissue is capable of restoring G.graminis growth-inhibiting activity to the disarmed P. aureofaciens 30-84 phzl- strain (see Example 9 below) (4) Erwinia carotovora carl(expl) mutants, which have greatly reduced virulence in their natural host plants were shown to infect transgenic tobacco plants which are producing AHLs (see Example 10 below). Figure 1 herewith shows the components of the constructs pBDHEYI and pBDHERBYI described in the Examples. Example 1 Preparation of pBDHEYI pBDHEYI was constructed by fusing the alfalfa mosaic virus (AMV) translation enhancer sequence from pBI526 (Datla et.al., Plant Science 94, 139-149 (1993)) to the yenl coding sequence from Yersinia enter ocolitica. The yenl sequence had previously been amplified by PCR to create an Ncol site overlapping the translation initiation sequence. This changed the second amino acid from leucine to valine but did not affect the ability of the encoded enzyme to synthesise N-acyl-L-homoserine lactones in a bacterial assay. The AMV /yenl fusion was cloned on a BgRl/BamHT fragment into the BamRl of pDH51 (Pietrzak et.al, Nucl Acids Res. 14, 5857-5868(1986)) to give pDHEYI. An EcoRI fragment of pDHEYI was cloned into the EcoRI site of pB IN 19 (Bevan, Nucl. Acids Res. 12, 8711-8721 (1984)) to give pBDHEYI. Example 2 Preparation of pBDHERBYI pBDHERBYI was constructed by fusing the petunia SSU611 ribulose bisphosphate carboxylase small subunit (rbcS) chloroplast targeting sequence (Dean et.al. Mol, Gen. Genet., 206, 465-474 (1987)) to the AMV translation enhancer sequence of pBI526. An Ncol site was engineered to overlap the initiating ATG codon of rbcS. An Sphϊ site was engineered to overlap the initiating ATG codon of yenl and the yenl coding sequence cloned into the SphT site of the SSU611 fragment. This site spans the cleavage site of the encoded chloroplast transit peptide. The AMV/rbcS/yenl fusion was cloned on a BglϊT/BamRT fragment into the Bam L site of pDH51 to give pDHERBYL An EcoRI fragment from pDHΕRBYI was cloned into the EcoRI site of pBIΝ19 to give pBDHERBYI. The rationale for producing pBDHERBYI and believing that it would be active in chloroplasts was as follows: in E.coli homoserine lactone is not produced by mutants of the threonine biosynthetic pathway that are blocked prior to homoserine synthesis but is produced by those mutants when supplied with an exogenous source of homoserine. However, Tral, the N-acyl- L-homoserine lactone biosynthetic enzyme in Agrobacterium tumefaciens, has been found to utilise S-adenosylmethionine and not homoserine as a substrate in vitro. There is also evidence for the acyl moiety being derived from fatty acid biosynthetic intermediates. In plants the enzymes of the threonine biosynthetic pathway are located in the chloroplast and this organelle is also active in fatty acid metabolism. Therefore the chloroplasts may be expected to contain the necessary precursors for N-acyl-L-homoserine lactone synthesis by yenl and more closely approximate to the environment in which yenl is normally active than would be the cytoplasm. Example 3 Generation of Transgenic Plants Construct pBDHEYI. for Example 1 and pBDHERBYI from Example 2 were transferred to the Agrobacterium tumefaciens strain LB A 4404 and used to transform tobacco leaf discs according to standard protocol (Draper et.al., pages 69-160, In Plant Genetic Transformation and gene expression: a laboratory manual; Draper et.al. (Eds) Blackwell Scientific Publications, London (1988)) . The transgenic status of the resulting kanamycin positive explants was confirmed by Southern analysis (data not given) Example 4 Complementation of Violacein Production Leaf segments of the transgenic plants produced in Example 3 were tested for their ability to synthesise N(3-oxohexanoyl)-L-homoserine lactone or a related analogue. A transgenic tobacco leaf was placed in an agar plate overnight. The leaf was then removed and the evil mutant of Chromobacterium violaceum spread over the plate. Violacein production by the bacteria could be seen where the N-(3-oxohexanoyl)-L- homoserine lactone had diffused out of the leaf and into the agar. Two leaf segments tested positive as indicated by the ability of a diffusible product to complement C.violaceum, inducing the production of the purple pigment violacein by the bacteria. Example 5 Complementation of carl Construct pBDHERBYI (Example 2) was transferred to the Agrobacterium tumefaciens strain LBA 4404 and transformed into tobacco. Leaf segments were tested for their ability to synthesise N(3-oxohexanoyl)-L-homoserine lactone or a related analogue. An untransformed control and a transgenic BDHERBYI tobacco leaf were inoculated with Erwinia carotovora mutant for carl. The bacteria were applied at a high culture density (OD600 of 2.5) in a volume of 10 μl to a small wound site made with a hypodermic needle. A second BDHERBYI leaf was mock inoculated with bacterial culture medium alone. The leaves were inspected after four days. The untransformed control and the mock inoculated leaf remained substantially unchanged. The sample inoculated with E. carotovora displayed advanced disease symptoms demonstrating that the pathogen can perceive and respond to the N-acyl-L-homoserine lactone being made by the transgenic plant. Example 6 Complementation of luxl Following a similar protocol as described above, the luxR N-acyl-L-homoserine lactone response regulator and the lux operon (minus luxl) of Pseudomonas βscheri was inserted into E.coli. When transgenic tobacco carrying the BDHERBYI construct was challenged with the E.coli, bioluminescence was induced in the bacteria demonstrating that the luxR gene was able to respond to the N-acyl-L-homoserine lactone produced by the plant. Twenty-nine tobacco plants that were independently transformed with either BDHERBYI or BDHEYI were challenged with C.violaceum mutant for evil (Example 3) and E.coli carrying an N-acyl-L-homoserine lactone-inducible lux operon. Table I summarises the results. Example 7 Extraction and TLC analysis of AHLs For thin-layer chromatographic analysis, transgenic plant extracts were made by grinding two grams of plant tissue to a fine powder in liquid nitrogen and mixing the frozen powder with 200ml of warn distilled water. After five minutes, solid matter was filtered off and the filtrate extracted with an equal volume of ethyl acetate. The ethyl acetate layer was then dried over anhydrous magnesium sulphate, filtered and evaporated to dryness. The residue was taken up in 500 μl of acetonitrile and 20μl of this was applied to a C18 reverse phase TLC plate (Merck). A similar extract from an untranformed control plant was also spotted on to the plate. N-hexanoyl-L- homoserine lactone (HHL) (lxlO'^g) and N-(3-oxohexanoyl) -L homoserine lactone (OHHL) (5xl0 _ 7g) were applied as standards and the chromatogram developed with methanol/water (60:40 vol/vol) as running solvent (Shaw P.D. et.al. Proc.Νatl.Acad,Sci. USA 94: 6036-6041 (1997)). After drying, AHLs were located on the TLC plate by overlaying C.violaceum strain CV026 in top agar as described by McClean et.al. (Microbiology-UK, 143: 3703-3711 (1997). After 16 hours growth at 28°C the presence of AHLs was indicated by localised violacein production. SUBSTITUTE SHEET RULE W Two different molecules with R f values identical to the synthetic HHL and OHHL standards were observed. Example 8 HPLC and LC-MS Analyses For HPLC and LC-MS analyses, transgenic plant extracts were made by grinding the issue in ethyl acetate. The supernatant was taken and the plant residue re-extracted with ethyl acetate, the supernatants pooled and the process repeated until the plant residue was white/brown n colour and free of chlorophyll. The ethyl acetate layer was separated from a small plant-derived aqueous layer and dried over anhydrous magnesium sulphate, filtered, and evaporated to dryness. The residue was resuspended in 500 μl of methanol, this was brought to 60% methanol with sterile distilled water and placed at -20°C overnight to precipitate out the majority of the chlorophyll. After pelleting any solid matter by centrifugation in a bench-top microfuge, the AHL-containing supernatant was partitioned against 10 volumes of ethyla acetate and the organic phase evaporated to dryness. The residue was taken up into 500μl of acetonitrile. For both LC-MS and HPLC analyses linear gradients of acetonitrile in water were run (20-100% over 32 minutes) as described by Camara et.al In Methods in Microbiology: Bacterial Pathogenesis Vol. 27: 319-330, Williams et.al. (Eds) (1998). OHHL and HHL eluted at 9 minutes and 13.5 minutes respectively. The presence of HHL and OHHL, detected in TLC analysis, were confirmed. Example 9 Assay for restoration of activity to P.aureofaciens mutant Leaf material from transgenic and non-transformed control plants were placed in wells cut in a potato dextrose agar plate (Oxoid). P.aureofaciens strain 80-841 (phzP) was inoculated adjacent to the wells and the plates incubated for 24 hours at 22°C. The G.graminis var. tritici was then introduced on the opposite side of the plate and the whole incubated for a further four days. The antifungal activity of the P.aureofaciens phzp strain against the G.graminis was found to have been restored. Example 10 Assay for restoration of virulence to Erwinia carotovora avirulent mutant Untransformed and control BDHERBYI tobacco leaves were inoculated with the avirulent E.carotovora mutant PNP22 (Bainton et.al, Biochem.Journal, 288: 997-1004 (1992) and also Jones et.al, The EMBO Journal, 12: 2477-2482 (1993)) The bacteria were applied at high culture density (OD600=2.5) in a volume of lOμl to a small wound site made with a hypodermic needle. Normally these Erwinia mutants are avirulent in the tobacco system, in which they can neither macerate plant tissue nor multiply in planta because they are defective in the production of plant cell-wall-degrading enzymes pectin lyase, pectate lyase, polygalacturonase, cellulase and protease. The regulated expression of plant cell wall- degrading enzymes only at high density in wild-type bacteria may contribute to the success of Erwinia as a plant pathogen. Under aerobic conditions, E. carotovora infection only occurs when the bacteria has reached sufficiently high population densitiy such that disease progression depends on competition between bacterial multiplication and the plant host defences. Thus the production of macerating enzymes at low cell densities would not give rise to a successful infection, but would result in the premature induction of the local and systemic plant defence response, which in turn would hamper subsequent infection. Thus, if the infecting pathogen were to encounter AHL levels that gave a false indication of the local bacterial population size the course of the ensuing infection will be substantially reduced as the plant is able to mount a successful defence to a weak attack. Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

Claims: CLAIMS 1. A method of protecting a plant against bacterial infection and/or virus infection transmitted by bacteria, comprising introducing into the genome of the plant by transformation the ability to synthesise a N-acyl-L-homoserine lactone. 2. A method of protecting a plant against bacterial infection and/or virus infection transmitted by bacteria, comprising introducing into the genome of the plant by transformation the ability to synthesise an analogue of N-acyl-L-homoserine lactone capable of competing with the N-acyl-L-homoserine lactone secreted by infecting bacteria for N-acyl-L-homoserine lactone receptor sites therein. 3. A method of enhancing interaction between a rhizobacterium and a plant comprising introducing into the genome of the plant by transformation the ability to synthesise the N-acyl-L-homoserine lactone naturally produced by the rhizobacterium. 4. A method as claimed in any of claims 1 to 3 in which the gene expressing the Ν-acyl- L-homoserine lactone is selected from the group consisting of, the yenl gene of Yersinia enterocolitica; the evil gene of Chromobacterium violaceum; the luxl gene of Photobacterium fischeri; the carl gene of Erwinia carotovora; the tral gene of Agrobacterium tumefaciens and the lasland vsml genes of Pseudomonas aeruginosa. 5. A recombinant plant genome containing a gene construct for in planta expression of an N-acyl-L-homoserine lactone and/or the response regulator thereof. 6. A genome as claimed in claim 5 in which expression of the said N-acyl-L- homoserine lactone is targeted to plant chloroplasts.

Inventors: FRAY RUPERT GEORGE, GRIERSON DONALD, STEWART GORDON SIDNEY ANDERSON, THROUP JOHN PETER, WALLACE ANDREW DAVID, FRAY, RUPERT, GEORGE, GRIERSON, DONALD, STEWART, GORDON, SIDNEY, ANDERSON, BERNIE, THROUP, JOHN, PETER, WALLACE, ANDREW, DAVID
Applicants: FRAY RUPERT GEORGE, GRIERSON DONALD, STEWART LESLEY PERSONAL REPRES, THROUP JOHN PETER, WALLACE ANDREW DAVID, ZENECA LTD, FRAY, RUPERT, GEORGE, GRIERSON, DONALD, STEWART, LESLEY (PERSONAL REPRESENTATIVE OF STEWART, GORDON, SIDNEY, ANDERSON, BERNIE), THROUP, JOHN, PETER, WALLACE, ANDREW, DAVID, ZENECA LIMITED
Application year: 1999
Main classification: C12N15
IPC codes: C07K14, C12N15