WO2019185129A1

WO2019185129A1 - Bacterial pathogen derived resistance in plants

Info

Publication number: WO2019185129A1
Application number: PCT/EP2018/057826
Authority: WO
Inventors: William Rooney; Daniel Walker; Joel MILNER; Rhys GRINTER
Original assignee: The University Court Of The University Of Glasgow
Priority date: 2018-03-27
Filing date: 2018-03-27
Publication date: 2019-10-03

Abstract

The present invention relates to bacterial pathogen derived resistance in plants and particularly, although not exclusively, to transgenic plants that are resistant to bacterial pathogens. This disclosure relates to transgenic plants with increased resistance to bacterial pathogens. More particularly, the disclosure relates to transgenic plants that produce a bacteriocin.

Description

Bacterial pathogen derived resistance in plants

Field of the Invention

The present invention relates to bacterial pathogen derived resistance in plants and particularly, although not exclusively, to transgenic plants that are resistant to bacterial pathogens.

Background

Worldwide losses as a result of plant diseases are conservatively estimated at approximately $150 bn.

Of these, about $50 bn are attributable to bacterial infections which cause diseases in plants in the field and spoilage (for example of fruit or tubers) in storage. In many cases, good sources of resistance for breeding are not available and the chemicals used to prevent spoilage are increasingly being deemed environmentally unacceptable.

Bacteria from the genuses Pectobacterium and Pseudomonas are amongst the most economically damaging bacterial pathogens. Pb atrosepticum causes soft rots of potato tubers in storage resulting in losses of up to £40m per annum in the UK alone and the related Pb carotovorum can lead to devastating losses of potato plants in the field. Ps syringae is one of the most economically important plant pathogens, infecting a very wide range of crop plants (including tomato, olive, kiwifruit, pears, apples, cherries, wheat etc.) and resulting in severe losses worldwide as a result of disease in the plants themselves or spoilage in storage.

One of the most severe bacterial plant pathogens is the rod-shaped gamma proteobacteria,

Pseudomonas syringae and related species of the genus Pseudomonas. P. syringae is a gram negative hemi-biotrophic plant pathogen. Plant pathogenic members of the genus Pseudomonas consists of over 50 different pathovars each of which cause different diseases fruit a large number of agronomically important crops such as bacterial speck, spot and blight disease on tomato, pepper, soybean and kiwi (O’Brien et al 2011 ). Moreover, P. syringae is considered the model organism for plant-pathogen interactions and its significance has been emphasized when it was voted as the number one most important plant pathogenic bacteria (Mansfield et al. 2012). Attempts to use chemical interventions aimed at attenuating P. syringae have ultimately failed.

One approach of engineering resistance which has proven successful is pathogen derived resistance (PDR), which allows plants to utilize pathogen-derived proteins to neuter virulence. Historically PDR has been used as a method to successfully control viral diseases by either expressing viral proteins (coat proteins and movement proteins) or viral nucleic acid sequences (DNA/RNA mediated resistance) in plants (Lomonossoff 1995; Baulcombe 1996).

Bacteriocins are novel proteinaceous antibiotics produced by bacteria and possess extremely specific killing spectra. All major bacterial lineages produce bacteriocins and bacteriocin production has also been characterised in some archaea (Riley and Wertz. 2002). The principle function of these bacteriocins is to kill closely related bacterial competitors to allow producing strains to establish dominance within a niche. Therefore, bacteriocins they play a pivotal role in bacterial population dynamics (Riley and Wertz. 2002).

Bacteriocins are modular proteins that consist of a C-terminal cytotoxic domain and the N-terminal receptor binding domain (Cascales et al 2007). This allows for the generation of chimeric bacteriocins which can be created by swapping the N-terminal domain to redefine the killing spectrum of bacteriocins (Gherique et al 2012). The cytotoxic C-terminal domains have a numerous functions such as the ability to degrade DNA, RNA and tRNA, to inhibit peptidoglycan synthesis, and form pores in the bacterial membranes which interfere with cell integrity (Cascales et al 2007). The N-terminal domains are involved with binding outer membrane proteins involved with functions such as nutrient uptake and allows bacteriocins to enter the bacterium via the periplasmic machinery (Cascales et al 2007).

The present invention has been devised in light of the above considerations.

Summary of the Invention

This disclosure relates to transgenic plants with increased resistance to bacterial pathogens which primarily pathogenise plants. More particularly, the disclosure relates to transgenic plants that produce a bacteriocin effective against a primarily plant pathogenic bacteria.

The disclosure also relates to methods of increasing tolerance to bacterial pathogens in a plant relative to a control plant, by expressing a nucleic acid encoding a bacteriocin polypeptide within one or more cells of the plant. Also provided are methods of producing plants with increased tolerance to bacterial pathogens relative to control plants, by incorporating a nucleic acid encoding a bacteriocin polypeptide into a cell by means of transformation, and regenerating the plant from one or more transformed cells.

Preferred/optional features of the plants and methods are set out below. These are combinable singly or in any combination, unless the context demands otherwise.

The bacteriocin is effective against one or more plant bacterial pathogens which are primarily plant pathogenic. Preferably, the one or more plant bacterial pathogens include Pseudomonas syringae, or one or more pathovars thereof. The bacteriocin may preferably be effective against the plant bacterial pathogen in the nanomolar range, i.e. has a minimum inhibitory concentration against the bacterial pathogen that is lower than 2000nM.

The bacteriocin may have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 1. In some instances, the bacteriocin comprises or consists of SEQ ID NO:1.

In other aspects disclosed herein, the bacteriocin has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO:2, 3, 4, 5 or 6. In some instances, the bacteriocin comprises or consists of SEQ ID NO:2, 3, 4, 5 or 6. In some instances, the bacteriocin comprises one or more carbohydrate binding domain motifs. For example a bacteriocin may comprise 1 , 2, 3, 4 or more carbohydrate binding domain motifs.

Carbohydrate binding domain motifs may comprise the motif laid out in SEQ ID NO:7. In some instances, the carbohydrate binding domain motifs have 80% sequence identity to SEQ ID NO:7.

In some instances, the carbohydrate binding domain motifs are located at an amino acid position relative to 41-49, 1 17-125, 171-179 and/or 202-210 of SEQ ID NO:1 . Preferably, there is a carbohydrate binding domain motif located at each of the positions relative to positions 41-49, 1 17-125, 171-179 and 202-210 of SEQ ID NO: 1.

The plants may produce or express bacteriocin at any effective level. In some instances, at least 0.1 % of the total protein produced by the plant is bacteriocin. In some instances, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6% of the total protein produced by the plant is bacteriocin.“Total protein” may relate to the protein content of the whole plant, or that of isolated cells, tissues, partial organs, or whole organs thereof.

The plants may have increased tolerance to one or more bacterial pathogens relative to a control plant. The plants may impair the growth of one or more bacterial pathogens relative to a control plant. Bacterial pathogens may include one or more pathogenic species from a genus selected from Pseudomonas, Xanthamonas, Pectobacterium and/or Ralstonia. In a preferred instance, the pathogenic species is selected from Pseudomonas syringae or related members of the genus Pseudomonas. In some instances, the plants may be tolerant to or impair the growth of one or more pathovars of a bacterial species, for example one or more Pseudomonas syringae pathovars selected from tomato, maculicola, persiae, ciccaronei, coronafaciens, morspurnorum, actinidiae, syringae, savastoni, glycinea and lachrymans. In a preferred instance, the one or more pathovars are selected from glycinea,

morspurnorum, actinidiae, syringae, savastoni, and lachrymans.

The disclosure is applicable to any suitable plant or plant cell derived therefrom. The plant is preferably a higher plant, for example an agricultural plant species selected from the group consisting of soybean, maize, Taxus spp, tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, rice, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, linseed, hemp and rye.

In some instances of the plants and methods, the transgenic plant transiently expresses the bacteriocin in one or more of its organs or tissues, such as leaves, roots, stems, seeds, fruits, and/or flowers. The bacteriocin may be targeted to cellular or tissue locations. Alternatively, transgenic plant may stably express the bacteriocin in one or more of its organs or tissues. Expression may be inducible, response to an internal or external stimulus, for example hormones, phytohormones, temperature, chemical agents, light, or stress signalling. Alternatively, expression of the bacteriocin may be constitutive. The transgenic plant may express the bacteriocin only in one or more specific tissues, cellular locations (for example, the apoplastic space) or organs. In some instances, the expression is systemic, i.e. bacteriocin is present in all or substantially all of the plant organs or tissues. Another aspect of the disclosure provides a transgenic plant comprising a DNA sequence encoding a polypeptide having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:1.

Another aspect of the disclosure provides a transformation vector, comprising a DNA sequence encoding a bacteriocin, as well as transgenic plant cells comprising such vectors. The bacteriocin may have a polypeptide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with SEQ ID NO:1 :

MAGRTRIPFNGVGTSVLPAYQTLSAGQYLLSPNQRFKLLLQGDGNLVIQDNGATVWVANEQQP FSSTIPLRNKKAPLAFYVQYGAFLDDYSRRRVWLTDNSTFTSNDQWNRTHLVLQDDGNIVLVDS LALWNGTPAIPLVPGAIDSLLLAPGSELVQGVVYGAGASKLVFQGDGNLVAYGPNGAATWNAGT QGKGAVRAVFQGDGNLVVYGAGNAVLWHSHTGGHASAVLRLQANGSIAILDEKPVWARFGFQ PTYRHIRKINPDQKPIDIWTWHF (SEQ ID NO:1 )

Alternatively, the bacteriocin may have a polypeptide sequence having at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or more sequence identity with any one of SEQ ID NOs:2-6. The plant transformation vector may comprise additional polynucleotide sequences, such as a promoter sequence operatively linked to the bacteriocin. Preferably, the promoter is a plant promoter, such as a tissue or organ specific plant promoter enabling expression in a specific organ or tissue (for example, in leaf, root, stem, seed, fruits and/or flowers). A plant promoter may be an inducible promoter responsive to one or more stimuli such as hormones, phytohormones, temperature, chemical agents, light, or stress signalling. The vector may be comprised within a transgenic cell, such as a bacterial or plant cell. Suitable bacterial cells include cells of Agrobacterium spp., such as Agrobacterium tumefaciens and

Agrobacterium rhizogenes, and Escherichia coli. In a preferred embodiment, the vector is comprised within a transgenic plant cell.

The plants of the present disclosure may be plant seeds. In one instance, the application provides a transgenic plant seed comprising a polynucleotide sequence encoding a bacteriocin as previously defined. In a preferred instance, the transgenic plant seeds are seeds of soybean ( glycine max). Also contemplated are seed meals, feeds and/or food products produced from said transgenic plant seeds. These meals, feeds and/or food products may undergo additional downstream processing.

The methods of the disclosure may comprise the optional step of sexually or asexually propagating or growing off-spring or descendants of the plants having increased tolerance to a bacterial pathogen. Propagation may involve a selection step, for example selection for a nucleic acid encoding a bacteriocin polypeptide or for a selectable marker, in order to enrich the proportion of off-spring or descendants which possess increased tolerance to a bacterial pathogen.

In some instances, the methods further comprise the step of harvesting a plant product from the plant having increased tolerance to a bacterial pathogen. The method may involve the step of processing the plant product into a plant-derived product. Other optional features, aspects and embodiments of the invention are described in more detail below.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1. Transient expression of PL1 in N. benthamiana. A. western blot of c-myc PL1 3, 4 and 6 days post agrobacterium infiltratrion. B. quantification a, relative to the large RuBisCO subunit. C. PL1 activity from protein extracts. Error bars represent standard deviation to the mean, experiments were repeated 3 times with similar results (n=3).

Figure 2. PL1 expression reduces disease severity in N. benthamiana. A. index scores of 12 leaves. Disease index 1 - uninfected , 2 - chlorosis 3- black mottling on the leaf and 4- cell death. B. 3-week-old leaves expressing either GFP or PL1 were infiltrated with LMG5084 and symptoms were left to develop 10 days post infection.

Figure 3. Transient expression of PL1 in N. benthamiana impairs the growth of LMG5084 but not DC3000. 3-week-old N. benthamiana leaves transiently expressing either PL1 (triangle), GFP (square) or non-pre-infiltrated (diamond) were infected with either a. LMG5084 or b. DC3000. Bacterial counts were measured by grinding up leaf tissue and tittering out the bacterial CFU for 0, 1 and 3 days post infection. Error bars show ± standard error (n=8). An alternative qPCR based approach was used to measure c. LMG5084 and d. DC3000 genomic DNA (OPRF) relative to plant genomic DNA (18S rRNA). Error bars show ± standard error (n=3). Letters indicate groups of data which are significantly different within a time point according to a 1 way ANOVA Tukey t-test.

Figure 4. Expression of PL1 c-myc by T3 homozygous Arabidopsis lines a. anti-cmyc western blot of 10 pg of total protein extract b. Quantification of the western blot with the RuBisCO large subunit using Image J. Error bars show ± standard deviation (n=3). c. active % of PL1 in the total Arabidopsis protein extracts. Error bars show ± standard deviation (n=3).

Figure 5. Transgenic Arabidopsis expressing PL1 impairs the growth of LMG5084 but not DC3000. a. 6- week-old wild-type or transgenic lines were spray infected with 1x108 CFU mL ¹ of LMG5084 and bacterial counts were measured by grinding up leaf tissue and tittering out the bacterial CFU for 0, 1 and 3 days post infection. Error bars show ± standard error (n=6). b. wild-type and PL1 1-2 line were flood inoculated with 40 mL of 5 x 10⁷ Cfu mL ¹ of DC3000 and bacterial loads were measured using qPCR 0 and 3 days post infection c. WT, 1-2, 2-1 and 6-1 lines were flood inoculated with 40 mL of 5 x 10⁷ Cfu mL ¹ of LMG5084 and bacterial loads were measured using qPCR 0 and 3 days post infection. Error bars show ± standard error (n=3). Letters indicate groups of data which are significantly different within a time point according to a 1 way ANOVA Tukey t-test. Figure 6. Three independent PL1 expressing lines show qualitative disease resistance against LMG5084. Flood infection with a wild-type control and 3 different PL1 expressing lines 1-2), 2-1 and 6-1 3 days post infection with 40 mL of 5 x 10⁷ Cfu mL^-1. Pictures are representative of 3 independent replicates.

Figure 7. Example spot test of purified PL1 spotted onto LMG5084. Serial dilutions of purified PL1 was spotted onto soft agar plates containing LMG5084.

Figure 8. Protein extracts from PL1 producing N. benthamiana leaves kill LMG5084 in vitro. Protein extracts from leaves either expressing GFP or PL1 were spotted onto a soft agar lawn of LMG5084 and incubated overnight. Experiment has been repeated 3 times with similar results.

Figure 9. The effect of syringe infiltration and agro-infiltration on LMG5084 growth in N. benthamiana. 3- week-old N. benthamiana leaves were either infiltrated with MgCL, or Agrobacterium containing an empty pJO530 vector (EV) or GFP. Bacterial counts were measured by grinding up leaf tissue and tittering out the bacterial CFU for 0, 1 and 3 days post infection. Error bars indicate standard error of 3 independent replicates.

Figure 10. The effect of in planta PL1 production on LMG5084. 3-week-old N. benthamiana leaves expressing GFP (G) were infected with LMG5084 (1 x105 CFU mL ¹). One disc of the infected tissue was then mixed with either a leaf transiently expressing PL1 or a control (0). Bacterial counts were measured by grinding up leaf tissue and tittering out the bacterial CFU for 0, 1 and 3 days post infection. Error bars show ± standard error to the mean (n=3). Letters indicate groups of data which are significantly different within a time point according to a 1 way ANOVA Tukey t-test.

Figure 11. qPCR standard curve of P. syringae LMG5084 OPRF:18S rRNA levels a. DNA was extracted from N. benthamiana using the Plant DNAzol reagent and normalised to N. benthamiana 18S rRNA. Error bars show ± standard error to the mean (n=3). Melt curves for b. LMG5084 OPRF and c. N. benthamiana 18S rRNA primers used to make the standard curve.

Figure 12. qPCR standard curve of P. syringae LMG5084 OPRF:ACT2 levels a. DNA was extracted from Arabidopsis using the FastDNA™ SPIN Kit for Soil DNA Extraction (MP Biomedical) and normalised to Arabidopsis ACT2. Error bars show ± standard error to the mean (n=3). b. shows the melt curve for Arabidopsis ACT2 primers used to make the standard curve.

Figure 13. Alignment of bacteriocin polypeptide sequences. Black boxes show carbohydrate binding motifs.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Transgenic Plants In this specification,“plant” refers to any suitable member of the kingdom Plantae, and encompasses whole plants, as well as parts thereof, such as seeds, isolated organs and tissues, and/or cells. A plant is preferably a higher plant, for example an agricultural plant selected from the group consisting of

Lithospermum erythrorhizon, Taxus spp, tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soybeans, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, linseed, hemp and rye. In some instances, the plant may be a flowering plant (angiosperm). Flowering plants may include monocotyledons or dicotyledons, such as eudicots, in particular members of the Rosid clade, including Fabaceae, such as soybeans. Preferably, the plant is a soybean ( Glycine max), tomato ( Solanaceae lycospersicum), kiwi ( Actinidia deliciosa) or pepper ( Capsicum ) plant. The pepper may be a Capsicum annuum, Capsicum frutescenes Capsicum chinense, Capsciums pubescens, or Capsicum baccatum. Preferably, the pepper is a Capsicum annuum. Other plants disclosed herein include species of bean (plants of the family Fabaceae) and pea ( Pisum sativum).

Preferable plants are those susceptible to one or more bacterial diseases, particularly one or more diseases caused by Pseudomonas spp. These species include alfalfa/lucerne, papaya, potato, rapeseed/canola, apple, vegetable brassicas (such as beets, cabbages, cauliflower, broccoli, and others), rice, maize, soybean, tomato, grapes, rose, carnation, citrus (including lemons/limes, oranges, grapefruits, tangerines, and others), sorghum, sugarcane, beans, barley, banana, cassava, cane berries (rubus), chickpea, coffee, curcurbits (including cucumbers, melon, squashes, watermelon, and others), hazelnut, hop, lettuce, okra, olive, peanut, rye, strawberry, sweet potato, pear, cyclamen, impatiens, kalanchoe, geranium, gerbera, cattelya, chrysanthemum, cineraria, fennel, seed sprouts, avocado, kiwi, oats, pea, stone fruits (such as almond, plum, apricot, cherry, peach/nectarine, and others), passion fruit, pepper, celery, tobacco, wheat, and onion/garlic.

A transgenic plant is a plant that includes a heterologous nucleic acid. "Heterologous" indicates that the gene/sequence of nucleotides in question or a sequence regulating the gene/sequence in question, has been introduced into said cells of the plant or an ancestor thereof, using genetic engineering or recombinant means, i.e. by human intervention. Nucleotide sequences which are heterologous to a plant cell may be non-naturally occurring in cells of that type, variety or species (i.e. exogenous or foreign) or may be sequences which are non-naturally occurring in that sub-cellular or genomic environment of the cells or may be sequences which are non-naturally regulated in the cells i.e. operably linked to a nonnatural regulatory element. "Isolated" indicates that the isolated molecule (e.g. polypeptide or nucleic acid) exists in an environment which is distinct from the environment in which it occurs in nature. For example, an isolated nucleic acid may be substantially isolated with respect to the genomic environment in which it naturally occurs. An isolated nucleic acid may exist in an environment other than the environment in which it occurs in nature.

Techniques well known to those skilled in the art may be used to introduce nucleic acid constructs and vectors into plant cells to produce transgenic plants with the properties described herein. The transformation of plant cells by Agrobacterium mediated transfer is well known to those skilled in the art. Briefly, the Ti or Ri plasmids are typically used for transformation. In these instances at least the right border sequence, preferably both the right and the left border sequences, of the Ti or Ri plasmid T-DNA must be linked as a flanking region to the candidate sequence to be transformed. DNA for Agrobacterium mediated transfer must be transformed into suitable vectors, specifically either an intermediate or a binary vector. The sequences carried by an intermediate vector can be integrated into the Ti or Ri plasmid via by homologous recombination due to sequences which are homologous to sequences in the T-DNA. The Ti or Ri plasmid also contains the vir-region, which is necessary for T-DNA transfer. Whilst intermediate vectors cannot replicate in Agrobacteria, they can be transferred to Agrobacterium tumefaciens by means of a helper plasmid (conjugation). In contrast, binary vectors are able to replicate in E.coli as well as in Agrobacteria and can be transformed directly into Agrobacteria. They contain a selection marker, such as an antibiotic resistance marker gene, and a linker or polylinker framed by the right and left T-DNA border region. The agrobacterium acting as host cell to an intermediate vector must contain a plasmid carrying a vir-region, as this is required for the transfer of the T-DNA into the plant cell. This transformed agrobacterium may then be used for the transformation of plant cells. Such an agrobacterium Additional T-DNA may be present. T-DNA transformation of plant cells has been extensively studied and reported in review articles and manuals on plant transformation. Plant explants cultivated for this purpose with Agrobacterium tumefaciens or Agrobacterium rhizogenes can be used for the transfer of DNA into a plant cell.

Other methods, such as microprojectile or particle bombardment (US 5100792, EP-A-444882, EP-A- 434616), electroporation (EP 290395, WO 8706614), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987) Plant Tissue and Cell Culture, Academic Press), direct DNA uptake (DE 4005152, WO 9012096, US 468461 1 ), liposome mediated DNA uptake (e.g. Freeman et al. Plant Cell Physiol. 29: 1353 (1984)) or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (1990d)) may be preferred where Agrobacterium transformation is inefficient or ineffective, for example in some gymnosperm species. Physical methods for the transformation of plant cells are reviewed in Oard, 1991 , Biotech. Adv. 9: 1-1 1.

Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A- 486233).

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al., Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

A transgenic plant produced as described herein may be sexually or asexually propagated or grown to produce off-spring or descendants. Off-spring or descendants of the plant regenerated from the one or more cells may be sexually or asexually propagated or grown. The plant or its off-spring or descendants may be crossed with other plants or with itself.

In addition to a plants producing bacteriocins, and plants comprising nucleic acids encoding bacteriocins, such as those with homology to SEQ ID NOs:1 to 6, as described herein, the invention encompasses any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part or propagule of any of these, such as cuttings, cells and seed, which may be used in reproduction or propagation, sexual or asexual. Also encompassed by the invention is a plant which is a sexually or asexually propagated offspring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant.

“Plant material” refers to any part of a plant, including organs such as seeds, stems, flowers, roots, tubers, corms, leaves, rhizomes or fruits. Plant materials also include plant tissues such vascular tissue, seed coat, germplasm, endosperm, pollen, embryos, as well as isolated cells.

Plant materials include“plant products”, which refer to any products derived from plants, including whole organs such as seeds, fruits, leaves, roots, tubers, rhizomes and corms, as well as tissues derived therefrom. Plant products may be further processed to produce plant-derived products. Plant-derived products include seed meals, flours, slurries, plant extracts, oils (such as food oils, fuel oils and/or essential oils), pulps, industrial feedstocks (such as cellulose, lignin, starch and fatty acids), biofuels, secondary metabolites, small molecules, and large molecules. In particular, seed meal encompasses milled and/or ground seeds, as well as seed components remaining following the extraction of oil from the seed. Examples of components of meal include protein and fibre. Plant-derived products may be further processed or refined.

Bacteriocins

In this application,“bacteriocins” are proteinaceous antibiotics produced by bacteria and possess extremely specific killing spectra. All major bacterial lineages produce bacteriocins and bacteriocin production has also been characterised in some archaea (Riley and Wertz. 2002). The principle function of these bacteriocins is to kill closely related bacterial competitors, allowing the producing strains to establish dominance within a niche. Therefore, bacteriocins they play a pivotal role in bacterial population dynamics (Riley and Wertz. 2002).

Bacteriocins are modular proteins that consist of a C-terminal cytotoxic domain and the N-terminal receptor binding domain (Cascales et al 2007). The cytotoxic C-terminal domains have a numerous functions such as the ability to degrade DNA, RNA and tRNA, to inhibit peptidoglycan synthesis, and form pores in the bacterial membranes which interfere with cell integrity (Cascales et al 2007). The N-terminal domains are involved with binding outer membrane proteins involved with functions such as nutrient uptake and allows bacteriocins to enter the bacterium via the periplasmic machinery (Cascales et al 2007).

In this specification, a nucleic acid encoding a bacteriocin polypeptide may be any nucleic acid (DNA or RNA) having a nucleotide sequence having a specified degree of sequence identity to one of SEQ ID NOs: 8 to 13, to an RNA transcript of any one of these sequences, to a fragment of any one of the preceding sequences or to the complementary sequence of any one of these sequences or fragments. The specified degree of sequence identity may be from at least about 20% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

The bacteriocin polypeptide may additionally comprise one or more additional amino acids at one or both of the N and C terminals. For example, 1 , 2, 3, 4, or 5 amino acids at one or both of the N and C terminals. The bacteriocin may be expressed as a fusion protein, such as a fusion protein comprising a reporter such as a fluorescent protein or an antibody tag.

Alternatively a bacteriocin nucleic acid may be one that hybridises to one of these sequences under high or very high stringency conditions. Stringent conditions include, e.g. for hybridization of sequences that are about 80 to 90% identical, hybridization overnight at 42°C in 0.25M Na₂HP0₄, pH 7.2, 6.5% SDS,

10% dextran sulphate and a final wash at 55°C in 0.1X SSC, 0.1 % SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65°C in 0.25M Na2HP04, pH 7.2, 6.5% SDS, 10% dextran sulphate and a final wash at 60°C in 0.1X SSC, 0.1 % SDS.

An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M NaCI, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt’s solution, 0.5% SDS, at 50°C or 65°C overnight. Washes may be performed in 0.2x SSC/0.1 % SDS at 65°C or at 50-60°C in 1x SSC/0.1 % SDS, as required.

In this specification, a bacteriocin polypeptide may be any peptide, polypeptide or protein having an amino acid sequence having a specified degree of sequence identity to one SEQ ID NO.s 1 to 6 or to a fragment of one of these sequences. The specified degree of sequence identity may be from at least about 20% to 100% sequence identity. More preferably, the specified degree of sequence identity may be one of at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. Particular sequence variants may differ from the reference sequence, such as any one of SEQ ID NOS: 1 to 6, by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more than 50 amino acids.

Bacteriocin polypeptides may be any peptide, polypeptide or protein having one or more carbohydrate binding domain motifs of the sequence SEQ ID NO:7. In some instances, the carbohydrate binding domain motifs comprise up to one, or up to two, or up to three, or up to four, or up to five conservative amino acid substitutions relative to SEQ ID NO:7. In some instances, the carbohydrate binding domains comprise up to one, or up to two, or up to three, or up to four, or up to five non-conservative amino acid substitutions. In some instances, the carbohydrate binding domain motifs have 80% sequence identity to SEQ ID NO:7.

Carbohydrate binding domain motifs may be located at any position within the bacteriocin polypeptide, however in some instances the carbohydrate binding domain motifs are located at an amino acid position relative to 41-49, 1 17-125, 171-179 and/or 202-210 of SEQ ID NO: 1. In some instances, there are at least two carbohydrate binding domain motifs located at positions relative to positions 41-49 and 1 17-125, positions 41-49 and 171-179, positions 41-49 and 202-210, positions 1 17-125 and 171-179, positions 1 17-125 and 202-210, or positions 171-179 and 202-210 of SEQ ID NO: 1. Alternatively, there may be at least three carbohydrate binding domain motifs located at positions relative to positions 41-49, 171-179 and 202-210, positions 41-49, 1 17-125 and 202-210, positions 41-49, 1 17-125 and 171-179, or positions 1 17-125, 171-179 and 202-210 of SEQ ID NO: 1. Preferably, there is a carbohydrate binding domain motif located at each of the positions relative to positions 41-49, 1 17-125, 171-179 and 202-210 of SEQ ID NO:1. Carbohydrate binding domains may exhibit binding activity to mannose-containing carbohydrates, such as LPS-derived polysaccharides of susceptible P. syringae strains.

Exemplary bacteriocins include Putidacin L1 (PL1 ; isolated from the banana rhizosphere isolate

Pseudomonas sp. BW1 1 M1 , Parret et al. 2003), Pyocin L1 (PL1’s ortholog in P. aeruginosa), LlpA1 (Pf-5; accession number AAY90516, isolated from Pseudomonas protegens), Syringicin L1 (accession number WP_010430508, isolated from Pseudomonas syringae), LlpA (AU1054; accession number ABF75998.1 , isolated from Burkholderia cenocepacia, and LlpAXcm761 (accession number AAM35756, isolated from Xanthomonas axonopodis pv. citri).

The peptide may be expressed from a nucleotide sequence. The nucleotide sequence may be contained in a vector present in one or more plant cell, or may be incorporated into the genome of the plant cell.

Other nucleic acid and polypeptide sequences which encode bacteriocins polypeptides are available on public databases.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps.

Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4.

Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and

Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981 ) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389- 3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequences described herein. A fragment or variant may comprise a sequence which encodes a functional bacteriocin polypeptide i.e. a polypeptide which retains one or more functional characteristics of the polypeptide encoded by the wild- type bacteriocin gene, for example, bactericidal properties. The bacteriocin polypeptide may be a fragment or derivative of a naturally occurring bacteriocin. For example, the polypeptide may be a fragment of having at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 30 amino acids. The fragment may be up to 150, 160, 170, 180, 190, 200, 210, 220, 230, 235, 240, 245, 250, 255, 265 or 270 amino acids in length. The polypeptide impairs the growth of P.syringae as compared to untreated P.syringae.

Plants producing bacteriocin polypeptides include those which contain, comprise or express a bacteriocin polypeptide within one or more of their cells. Bacteriocins may be intracellular, or extracellular, for example plants may secrete a bacteriocin polypeptide from one or more of their cells. Bacteriocins may be produced within a single, multiple, substantially all, or all cells, tissues or organs of a plant.

The plants may produce or express bacteriocin at any effective level. Bacteriocin production may be quantified as a proportion of total protein produced by the whole plant, or by one or more isolated cells, tissues, partial organs, or whole organs thereof. For example, at least about 0.01 % to about 1 % or more of the total protein produced may be bacteriocin. More preferably, the level of bacteriocin production may be one of at least about 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.95%, 1 %, 1 .05%, 1.10%, 1.15%, 1 .20%, 1.25%, 1.30%, 1.35%, 1.40%, 1 .45%, 1 .50%, 1.55%, 1.60%, 1.65%, 1.70%, 1.75%, 1 .80%, 1.85%, 1.90%, 1 .95%, 2%, 2.05%, 2.10%, 2.15%, 2.20%, 2.25%, 2.30%, 2.35%, 2.40%, 2.45%, 2.50%, 2.55%, 2.60%, 2.65%, 2.70%, 2.75%, 2.80%, 2.85%, 2.90%, 2.95%, 3%, 3.05%, 3.10%, 3.15%, 3.20%, 3.25%, 3.30%, 3.35%, 3.40%, 3.45%, 3.50%, 3.55%, 3.60%, 3.65%, 3.70%, 3.75%, 3.80%, 3.85%, 3.90%, 3.95%, 4%, 4.05%, 4.10%, 4.15%, 4.20%, 4.25%, 4.30%, 4.35%, 4.40%, 4.45%, 4.50%, 4.55%, 4.60%, 4.65%, 4.70%, 4.75%, 4.80%, 4.85%, 4.90%, 4.95%, or 4.99% of the total protein.

Alternatively or additionally, bacteriocin production may be quantified relative to the production of a reference protein, for example Ribulose-1 ,5-bisphosphate carboxylase/oxygenase (RuBisCO), by the whole plant, or by one or more isolated cells, tissues, partial organs, or whole organs thereof.. For example, production of a bacteriocin may be about 0.1 to about 5 times the production level of the reference protein. Production of a bacteriocin may be greater than 5 times the production level of the reference protein. More preferably, the level of bacteriocin production may be one of at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, or 4.9 times the production level of the reference protein.

Bacteriocins which are effective against one or more plant bacterial pathogens are those which show a minimum inhibitory concentration (MIC) against said pathogen in the nanomolar range. For example, a bacteriocin may have an MIC of less than about 2000nM, less than about 1900nM, less than about 1800nM, less than about 1700nM, less than about 1600nM, less than about 1500nM, less than about 1400nM, less than about 1300nM, less than about 1200nM, less than about 1 100nM, less than about 1000nM, less than about 900nM, less than about 800nM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, less than about 50nM, less than about 40nM, less than about 30nM, less than about 20nM, less than about 10nM, less than about 10nM, less than about 9nM, less than about 8nM, less than about 7nM, less than about 6nM, less than about 5nM, less than about 4nM, less than about 3nM, less than about 2nM, less than about 1 nM. Minimum inhibitory concentration may be measured in a soft agar overlay spot test, for example the test laid out in Example 2, although the skilled person will appreciate the various alternative methods which may be used to determine MIC.

Promoters

A nucleic acid encoding a bacteriocin polypeptide as described herein may be operably linked to a heterologous regulatory sequence, such as a signal peptide or promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter.

In this specification the term“operably linked” may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence (e.g. promoter and/or enhancer) are covalently linked in such a way as to place the expression of the nucleotide sequence under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus a regulatory sequence is operably linked to the selected nucleotide sequence if the regulatory sequence is capable of effecting transcription of the nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired protein or polypeptide.

A“promoter” refers to a natural, engineered or synthetic nucleotide sequence that directs the initiation and rate of transcription of a coding sequence (reviewed in Roeder, Trends Biochem Sci, 16: 402, 1991 ). Many suitable promoters are known in the art and may be used in accordance with the invention. The promoter contains the site at which RNA polymerase binds and also contains sites for the binding of other regulatory elements (such as transcription factors). See Datla et al. Biotech Ann. Rev 3:269, 1997 for review of plant promoters. Typically, a promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box or an Inr element, and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.

A tissue-specific promoter may be employed to express the bacteriocin in a specific tissue or organ. For example, a seed-, seed-coat- or integument-specific promoter may be used to express the bacteriocin in seeds. Suitable promoters include, for example Phaseolus vulgaris phas promoter, Vicia faba leB4-, usp- or sbp-promoters, Soybean b-conglycinin a-subunit promoter, Brassica FAE1 promoter and At4g12960 promoter (AtGILTpro) (Wu et al Plant Cell Rep (201 1 ) 30:75-80), as well as the napin, phaseolin, zein, globulin, dlec2, g-kafirin seed specific promoters.

Alternatively, or in addition, one might select an inducible promoter. In this way, for example, the bacteriocin may be expressed at specific times or places in order to obtain desired changes in organ growth. Inducible promoters include the alcohol inducible AlcA gene-expression system (Roslan et al., Plant Journal; 2001 Oct; 28(2):225-35), stilbene synthase promoter and promoters induced by light, heat, cold, drought, wounding, hormones, biotic stress, abiotic stress, and chemicals. Other examples include Flg22-induced Receptor-like Kinase 1 (FRK1 ) which is induced by biotic stress (Asai et al, 2002) and FLAG ELLIN SENSITIVE2 (FLS2) promoter which is expressed constitutively at low levels and is upregulated in response to biotic stress and hormones (Boutrot et al, 2010).

Alternatively, or in addition, a "constitutive promoter" may be selected, i.e. one which drives the expression of the downstream coding region in multiple, substantially all, or all tissues of a plant, irrespective of environmental or developmental factors. Exemplary constitutive promoters include the cauliflower mosaic virus 35S promoter; the Figwort mosaic virus promoter; and the nopaline synthase (nos) gene promoter (Singer et al., Plant Mol. Biol. 14:433 (1990); An, Plant Physiol. 81 :86 (1986)), maize ubiquitin, rice ubiquitin, rice actin, Arabidopsis actin, sugarcane bacilliform virus, CsVMV and CaMV 35S, Arabidopsis polyubiquitin, Solanum bulbocastanum polyubiquitin, octopine synthase, and mannopine synthase gene promoters.

Further, promoters may be species specific (for example, active only in B. napus); or developmentally specific (for example, active only during embryogenesis).

A promoter may also refer to a nucleotide sequence that includes a minimal promoter plus DNA elements that regulates the expression of a coding sequence, such as enhancers and silencers.

A“signal peptide” refers to a short peptide present on a newly synthesised protein that is destined towards the secretory pathway, and coordinated trafficking of the protein. This includes secretion into one or more organelles (for example, the endoplasmic reticulum, the chloroplast, golgi apparatus, or the endosomes), from the cell, or for insertion into a cellular membrane. In some cases, signal peptides mark the bacteriocin as destined for secretion into the apoplastic space (i.e. the space between the cell membrane and the cell wall). Exemplary signal peptides for trafficking to the apoplastic space include those from CLAVATA3 (Rojo et al, 2002) and Pathogenesis-related 1 (Pecenkova et al, 2017)

A construct or vector comprising nucleic acid as described above need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

Vectors

The nucleic acid encoding the bacteriocin may be contained on a nucleic acid construct or vector. A “vector” as used herein is an oligonucleotide molecule (DNA or RNA) used as a vehicle to transfer foreign genetic material into a cell. The vector may be an expression vector for expression of the foreign genetic material in the cell. Such vectors may include a promoter sequence operably linked to the nucleotide sequence encoding the gene sequence to be expressed. A vector may also include a termination codon and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express plant aspartic proteases from a vector according to the invention. Suitable vectors include plasmids, binary vectors, viral vectors and artificial chromosomes (e.g. yeast artificial chromosomes). An exemplary vector is pJ0530.

The construct or vector is preferably suitable for transformation into and/or expression within a plant cell.

A vector is, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form, which may or may not be self-transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host, in particular a plant host, either by integration into the cellular genome or exist extrachromasomally (e.g. autonomous replicating plasmid with an origin of replication).

In particular, the vector may be an Agrobacteria tumefaciens binary vector.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different organisms, which may be selected from Actinomyces and related species, bacteria and eukaryotic (e.g. higher plant, mammalia, yeast or fungal) cells.

Constructs and vectors may further comprise selectable genetic markers consisting of genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones, glyphosate and d-amino acids.

Those skilled in the art can construct vectors and design protocols for recombinant gene expression, for example in a microbial or plant cell. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook et ai, 2001 , Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992. Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 871 1-8721 ), and Guerineau and Mullineaux, (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.

When introducing a chosen nucleic acid construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct that contains effective regulatory elements that will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, the target cell type is preferably such that cells can be regenerated into whole plants.

Those skilled in the art will also appreciate that in producing constructs for achieving production of the bacteriocins according to this invention, it is desirable to use a construct and transformation method which enhances expression of the nucleic acid encoding the bacteriocin. Integration of a single copy of the gene into the genome of the plant cell may be beneficial to minimize gene silencing effects. Likewise, control of the complexity of integration may be beneficial in this regard. Of particular interest in this regard is transformation of plant cells utilizing a minimal gene expression construct according to, for example, EP1407000B1 , herein incorporated by reference for this purpose.

Control plants

In this specification, "control plant" refers to a plant that serves as a standard of comparison for testing the results of a treatment or genetic alteration, or the degree of altered expression of a gene or gene product. Examples of control plants include plants that do not produce a bacteriocin, plant which do not comprise a polynucleotide which encodes a bacteriocin polypeptide, or plants which are genetically unaltered (i.e., wild-type).

“Wild type", as used herein, refers to a cell, tissue or plant that has not been genetically modified to knock out or overexpress one or more of the presently disclosed transcription factors. Wild-type cells, tissue or plants may be used as controls to compare tolerance to, or the ability to impair growth of, pathogenic bacteria with cells, tissue or plants in which bacteriocin production has been engineered or into which bacteriocin encoding polynucleotides have been introduced.

Preferably, growth of the transgenic plant is not impaired, as compared to a control or wild-type plant.

That is, the height, mass, leaf size or flowering time are approximately the same as, or not statistically different to, the height, mass, leaf size or flowering time of a control or wild-type plant.

Pathogenic bacteria

In this specification,“pathogenic bacteria” or“plant pathogenic bacteria” refers to any bacteria capable of infecting a plant and causing a plant disease, or any bacteria capable of infecting plant tissue and causing spoilage or damage for example during post-harvest storage. A“primarily plant pathogenic bacteria” is a pathogenic bacteria which pathogenises (i.e. infects and causes a disease, or infects a tissue and causes spoilage or damage) one or more plant species or tissues thereof as its primary host(s) under normal conditions, for example natural or field conditions. Such bacteria may be exclusively plant pathogenic (i.e. totally incapable of infecting and causing disease, spoilage and/or damage in non-plant species such as animals or humans), or may be mostly unable to infect and cause disease, spoilage and/or damage to non-plant species under normal conditions. Some primarily plant pathogenic bacteria may also be able to pathogenise non-plants, such as animals, under extreme, unnatural or unusual conditions, such as laboratory conditions. In some definitions, a small subset of strains of a primarily plant pathogenic bacteria may be capable of pathogenising animal or other non-plant species, so long as the majority of pathogenic strains pathogenise plants. Notably, a bacteria which generally pathogenises animal species and only rarely pathogenises plants under extreme, unnatural and/or abnormal (e.g. laboratory or other non-field conditions) conditions cannot be said to be primarily plant pathogenic. Exemplary pathogenic bacteria can be found in the phyla Proteobacteria, and Actinobacteria, as well as mollicutes such as Phytoplasma and Spiroplasma. Pathogenic bacteria include species of Xanthomonas, Agrobacterium, Burkholderia, Pseudomonas, Pectobacterium and Erwinia. In some embodiments, the pathogenic bacteria is a Pseudomonas species seleted from p.syringae and p.protegens. In some embodiments, the pathogenic species is not Pseudomonas aeruginosa. A particularly important pathogenic species is Pseudomonas syringae. In some embodiments, the pathogenic bacterium is a Pectobacterium selected from Pb atrosepticum and Pb carotovorum. The phrase“bacteria” may be substituted by bacterial genus, bacterial species, and/or bacterial pathovar.

“Pathovars” are bacterial strains or sets of strains that are differentiated at infrasubspecific level from other strains of the same species or subspecies on the basis of distinctive pathogenicity to one or more plant hosts For example, the P. syringae species consists of over 50 known pathovars which cause different diseases fruit a large number of agronomically important crops such as bacterial speck, spot and blight disease on tomato, pepper, soybean and kiwi (O’Brien et al 201 1 ). Exemplary pathovars of P. syringae include P. syringae pvr tomato, maculicola, persiae, ciccaronei, coronafaciens, morspurnorum, actinidiae, syringae, savastoni, glycinea and lachrymans.

Tolerance to pathogenic bacteria

The plants described herein may be tolerant, or show increased tolerance relative to a control plant, to one or more pathogenic bacteria. Tolerance is the ability to mitigate the negative fitness effects caused by pathogenesis when challenged with one or more pathogenic bacteria. Tolerant plants may exhibit a lower occurrence following challenge relative to control plants of one or more symptoms, such as chlorosis, black mottling, lesion formation, plant growth abnormality, canker formation, necrosis, decay, blight, atrophy, cell death, and plant death. Tolerant plants may show a 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 99%, or 100% reduction in the occurrence of one or more symptoms following challenge relative to control plants. Plants may also show an attenuated reduction in growth following challenge. Tolerance may be apparent immediately after challenge, and/or may become apparent after a period such as 1 , 2, 3, 4, 5, 6, 7 or more days following challenge with pathogenic bacteria.

Inhibiting growth of pathogenic bacteria

In some instances, the plants described inhibit the growth of one or more pathogenic bacteria relative to a control plant. For example, a plant producing bacteriocin may inhibit the growth of one or more challenging pathogenic bacteria so as to elicit a statistically significant difference in growth relative to control plants. The reduction in bacterial growth may be expressed as a log reduction, for example a 0.1 ,

1 , 2, 3, 4, 5 or more log reduction. In some instances, the reduction in growth is a 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .1 , 1.2, 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1 , 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 log reduction or greater. Alternatively, the reduction in bacterial growth may be expressed as a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, 100,000-fold or greater reduction relative to control plants. Rates of bacterial growth may be measured by any suitable technique, for example colony titre or qPCR of sampled plant tissue following challenge.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word“comprise” and“include”, and variations such as“comprises”,“comprising”, and“including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms“a,”“an,” and“the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from“about” one particular value, and/or to“about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent“about,” it will be understood that the particular value forms another embodiment. The term“about” in relation to a numerical value is optional and means for example +/- 10%.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example“A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention. Examples

Example 1: Materials and Methods

Plasmids

Putidacin L1 was cloned into pET21a according to (McCaughey et al. 2014). Putidacin L1 was cloned into the pBIN19 derivative vector pJO530 which contains a hygromycin resistance cassette (Cecchini et al 1997), with a N- terminal c-myc tag from pGWB16 (Nakagawa et al. 2007).

Plasmids used for aPCR (PCR2.1 OPRF+ ACT2+18S )

PCR products of standards used for qPCR were amplified via PCR and purified using the QIAquick PCR Purification Kit (Qiagen), and cloned into pCR2.1®-TOPO® vector (Invitrogen), according to the instructions of the manufacturer and yielding OPRF-TOPO and 18S-TOPO.

The resulting PCR products were purified using the Qiagen PCR cleanup protocol (Qiagen), and cloned into pCR2.1®-TOPO® vector (Invitrogen), according to the instructions of the manufacturer and yielding OPRF-TOPO and 18S-TOPO.

Growth conditions of plants

N. benthamiana seeds were sown onto compost containing Calypso (Bayer CropScience). Seeds were vernalized for 3 days and grown in short day condition (10 hours light: 14 hours dark; 120 pnnol nrr² sec ¹ , 24 °C).

Arabidopsis seeds were surfaced sterilised in 70% ethanol for 2 mins and followed by a 7 minute incubation in a 50% bleach solution. The seeds were then washed in water and re-suspended in 0.1 % agarose solution.

Agar grown seeds were spotted onto ½ MS media (pH 5.7) left to vernalize for 3 days and grown in short day condition (10 hours light: 14 hours dark; 120 pnnol nrr² sec ¹ , 24 °C).

Transient expression in Nicotiana benthamiana

The leaves of N. benthamiana plants between 3 to 6 weeks of age were syringe-infiltrated with 0.4 OD600 A. tumefaciens GV3101 containing the desired binary vector in 10 mM sterile MgCI_å

supplemented with 100 mM acetosyringone 2 hours prior to infiltration.

Expression of bacteriocins was monitored from 3 and 6 days by grinding transformed leaf material, snap frozen in liquid nitrogen, in extraction buffer (50 mM Tris, 200 mM NaCI, pH 7.5, with protease inhibitor cocktail (Roche)) Buffer was then clarified and tested for expression using the soft agar overlay assay or western blotting. Generation of stable transgenics

For stable transformation of Arabidopsis with gene for putidacin L1 the vector pJO530 (pJOPLI ) was utilised. Stable transformations of Arabidopsis with c-myc PL1 were performed by floral dipping according to Zhang et. al. 2006. Transformed seedlings were sterilsed and plated onto 1/2 MS agar (4.3 g

Murashige & Skoog salts, 10 g sucrose, 0.5 g MES, 8 g agar per liter; pH 5.7) with 15 pg ml_ ¹ hygromycin B (the selection for pJO530). Transgene expression was tested by western blot and spotting protein extract onto lawns of susceptible P. syringae strains. T2 homozygous lines were with a single insertion were selected for.

Infection assays in olanta

P. syringae infection experiments in N. benthamiana were performed by re-suspending bacterial cultures in 10 mM MgC to an Oϋboo 0.0002 (1 x 10⁵ cfu mL ¹)and syringe-infiltrating the leaves with. The bacterial titre was measured by grinding 9.2 mm leaf disks in 10 mM MgC . Dilutions of these leaf extracts were spotted out onto Kings Agar B plates (20 g L¹ Peptone, 1.5 g L¹ MgS04, 1.5 g L¹ K2HP04, 10 g L¹ glycerol, pH 7.5, 0.8% Agar) and incubated at 28 °C overnight. Alternatively, leaf samples were flash frozen for DNA extraction.

P. syringae infection experiments in mature Arabidopsis plants were performed by re-suspending bacterial cultures in 10 mM MgC to an OD6oo 0.2 (1 x 10⁸ Cfu ml_ ¹), 0.05% Silwet L-77. The inoculum was sprayed onto the plants until saturated and humidified in autoclave bags for 24 hours. The bacterial titres were measured by grinding 9.2 mm leaf disks in 10 mM MgC . Dilutions of these leaf extracts were spotted out onto Kings Agar B plates and incubated at 28 °C overnight. Alternatively, leaf samples were flash frozen for DNA extraction.

P. syringae infection experiments in 14 day old seedlings were performed by re-suspending bacterial cultures in 10 mM MgC to an Oϋboo O. I (5 x 10^® Cfu ml_ ¹) for 1 minute and samples were taken 0, 1 and 3 days post flood-inncoulation (Ishiga Y et al. 201 1 ). The bacterial titres were measured by grinding 9.2 mm leaf disks in 10 mM MgC . Dilutions of these leaf extracts were spotted out onto Kings Agar B plates and incubated at 28 °C overnight. Alternatively, leaf samples were flash frozen for DNA extraction.

DNA extractions from infected tissue

For DNA extractions in N. benthamiana Plant DNAzol (Thermofisher Scientific) was used according to the manufacturers protocol. Briefly, 300 uL of plant DNAzol was added to 100 mg of macerated tissue and incubated for 5 minutes. Afterwards 300 uL of choloroform was added and samples were incubated for 5 minutes before being centrifuged for 10 minute spin at 14,000g. The supernatant was put into a new tube and 225 uL of 100% ethanol was added. This was followed by a 5 minute spin at 14,000 g. The supernatant was removed and the pellets were washed in a plant DNAzol-ethanol solution (1 : 0.75 vol). The samples were spun for 5 minute spin at 14,000 g and subsequently air dried and resuspended in TE buffer (pH8). For DNA extractions of Arabidopsis the FastDNA™ SPIN Kit for Soil (MP Biomedicals) according to manufacturer’s instructions. Briefly, 100 mg of infected tissue was macerated in liquid nitrogen with a pestle and mortar to a fine powder. Subsequently, the powder was suspended in sodium phosphate buffer and MT buffer and vortexed for 30 seconds. The supernatant was clarified by centrifugation for 10 min as 12, 000 g, the supernatant was then mixed with a protein precipitation solution, vortexed and clarified by centrifugation. The samples were then mixed with 1 ml_ of binding matrix and left to incubate with constant inversion for 5 minutes and left to settle for a further five minutes. 500 pl_ was removed and the binding matrix solution was centrifuged in a spin module. The binding matrix - now on the spin module was then re-suspended in 500 mI_ of SEWS-M, centrifuged and eluted in 100 mI_ of water.

QPCR

For the 20 mI_ qPCR reactions, the reaction mixture consisted of equal amounts of gDNA (2 mI_), primers (0.16 mM) and Fast SYBR™ Master Mix (Thermofisher Scientific). The qPCR was performed in an Applied Biosystems StepOnePlus Real-Time PCR System (Life Technologies).

To measure P. syringae levels in planta the primers for the OPRF gene were used (Ross and Sommisch. 2016). Primers designed specifically for the 18S rRNA (GenBank: KP824745.1 ) and ACT2 (AT3G18780) genes were used as an internal controls for N. benthamiana and Arabidopsis respectively.

Soot test

Soft agar overlay spot plates were performed using the method of Fyfe et. al.. 50 mI of test strain culture at OD600 nm = 0.4 - 0.6 was added to 10 mL of 0.8% soft agar and poured over a KB agar plate, as appropriate. 5 mI of neat or serially diluted bacteriocin solution/ plant protein extract was spotted onto the plates and incubated for 20 hours at 28 °C. After incubation plates were inspected for zones.

Western blotting

Plant tissue was homogenised in 50 mM Tris-HCL 7.5, 200 mM NaCI, 1x Complete Mini Protease Inhibitors (Roche) and clarified at 13,000 g for 10 min at 4°C. Equal amounts of homogenate were separated using SDS-PAGE and transferred onto a nitrocellulose membrane that was incubated, after blocking (8% milk in PBS-T (Tween20 (0.1 %))) in 1 : 5,000 anti c-Myc Antibody sc-40 (Santa Cruz Biotechnology) in PBST for 1 hour. The membranes were washed for 5 mins x 3 before adding the 1 : 10,000 anti-Mouse IgG (H+L), HRP Conjugate (Promega) for 1 hour. The membranes were then washed for 5 mins x5 in PBST.

Immunoblots were incubated with chemiluminescent ECL Plus Western Blotting Substrate (Pierce, 32132) as per manufacturer’s instructions. After ECL treatment the excess substrate was removed and the chemiluminescence was visualised using ChemiDoc™ MP Imaging System (Biorad).

Statistical analysis

All statistical analysis was done using minitab and Microsoft Excel. Example 2: Activity of PL1

Very many bacterial species produce proteinaceous antibiotics, bacteriocins, that can kill closely related bacterial species. By data mining genome sequences we identified a variety of putative bacteriocins encoded by plant pathogenic bacteria, in particular in the key genuses of plant pathogenic bacteria Pseudomonas and Pectobacterium.

Previous reports have identified a lectin-like bacteriocin PL1 which is present in the Pseudomonas species and has the ability to kill across the Pseudomonas genus (Parret et al. 2003; McCaughey et al. 2014; Ghequire et al. 2012, 2013). Consequently, PL1 represents a good candidate to establish whether PDR can be extended to bacterial as well as viral diseases.

To deepen our understanding of the specificity of PL1 activity we purified and tested PL1 protein (Parret et al. 2005) against a variety of different P. syringae pathovars (Table 1 and Figure 6). Of the strains tested 10/22, were susceptible including strains from pathovars morspurnorum, actinidae, syringae, savastoni, glycinea and lachrymans. Resistant strains from pathovars that were not susceptible to PL1 included maculicola, glycinea, tomato, savastoni, persiae, ciccaronei and coronafaciens. From the data in Table 1 , pathovars tomato, maculicola, persiae, ciccaronei and coronafaciens pathovars are all resistant to PL1 , however this could be due to small number of strains tested from each pathovar. This can also be applied to the pathovars syringae, actinidae and morspurnorum all of which are sensitive to PL1.

Interestingly, pv. glycinea sensitivity seems to be specific to North America as the Brazilian and Australian isolates are resistant. This could be due to the different selective pressures on pv. glycinea in these different geographical regions.

Table 1 : P.syringae pathovars tested for susceptibility to PL1

After establishing the killing spectrum of PL1 we then proceeded to test whether PL1 can be expressed heterologously in Nicotiniana benthamiana. C-myc tagged PL1 was expressed in N. benthamiana leaves via agroinfiltration and the protein levels were assayed 3, 4 and 6 days post infiltration (Figure 1A).

Protein levels peaked at 3 days and after 6 days the levels PL1 levels in the plant were reduced by 80% compared to day 3 (Figure 1 B). Protein extracts from PL1 expressing leaves and a GFP control were spotted onto lawns of LMG5084 to test for antibacterial activity. PL1 spots showed zones of bacterial growth inhibition whereas GFP showed no bacterial growth inhibition (Figure 7). The active concentration of PL1 in the N. benthamiana leaves was tittered out by diluting the protein extracts until the zones of bacterial growth inhibition were not present. These results showed that active concentration PL1 in the leaves decreased across the time course as well (Figure 1C).

Consequently N. benthamiana leaves were infected with LMG5084 at 3 days post agro-infiltration to assess whether transient expression of PL1 resulted in increased resistance. To assess qualitative disease resistance we transiently expressed PL1 and a GFP control into leaves and left them to develop symptoms over 10 days. Disease symptoms were measured using a non-parametric disease index (Figure 2), which overall concluded that expressing PL1 resulted in leaves with an index of 1.2 (slightly chlorotic) whereas the control had an index of 3.6 (black mottling/ cell death). A reciprocal quantitative LMG5084 growth curve was performed over a 3 day time course. These results showed that leaves expressing GFP showed a 3 log reduction of growth compared to un-infiltrated leaves and expression of PL1 resulted in a further reduction of 2 logs (Figure 3A). Moreover, we looked at the effect of an empty vector and an infiltration buffer control. MgC showed the process of infiltration does not affect growth of LMG5084 whereas agrobacterium presence does effect growth (Figure 8). This is most likely due to the microbial-associated molecular patterns (MAMP) from the agrobacterium which can induce plant defences and therefore the reduction in growth is due to MAMP-induced defence priming (Zipfel et al. 2006; Rico et al. 2010).

Interestingly, it was observed that no bacteria were recovered immediately after infection with LMG5084 suggesting that PL1 was being released and subsequently killing LMG5084 which was likely to be happening during the process of bacterial extraction This was confirmed when GFP expressing N.

benthamiana leaves infected with LMG5084 were mixed with a sample of an uninfected PL1 expressing N. benthamiana leaf (Figure 9). To ensure that the resistance was PL1 specific bacteria DC3000 was used as a control and it showed a similar reduction when comparing pre-infiltrated and un-infiltrated plants. Moreover, the PL1 and GFP expressing plants showed no difference in growth (Figure 3B).

Due to the artefacts shown in Figure 3A and Figure 9 a new method to measure bacterial growth was required. We decided to pursue a qPCR based approach established by Ross and Somissch (2016), which measured the copies of OPRF gene from DC3000 relative to an in planta standard. We optimised this method for N. benthamiana (Figure 10) by creating a standard curve showing a relationship between OPRF: 18S rRNA gene sequences and CFU mg ¹. We then measured the OPRF: 18S levels in N.

benthamiana expressing either PL1 or GFP. After 3 days there was a significant difference of 0.4 logs between the PL1 and GFP expressing leaves (Figure 3C). The difference appears to be small however it was must be noted that the qPCR method does not discriminate between dead and alive bacteria therefore the differences are most likely to be conservative compared to other methods. The specificity of PL1 action in planta was repeated using DC3000. There was no significant difference detected between the GFP and PL1 expressing leaves (Figure 3D).

Example 3: Stable transgenic plants

After establishing the proof of principle transiently in N. benthamiana we made stable transgenic Arabidopsis lines constitutively expressing PL1 . We used 3 independent transgenic lines PL1 1 (2), 2 (1 ) and 6 (1 ) in our study all of which had varying amounts of protein and therefore varying amounts of bactericidal activity (Figure 4). Firstly we assayed for a qualitative difference in mature Arabidopsis plants we established that PL1 1 (2) lines did not form lesions compared to the wild-type when infiltrating high levels of LMG5084 into the leaves. Infections and subsequent bacterial counts showed promising results as lines 1 (2), 2 (1 ) and 6 (1 ) showed a log reduction of 2.5, 1.9 and 1.3 respectively, compared to wild- type (Figure 5A). However, due to the artefact identified in N. benthamiana the data was suggestive and not conclusive. Interestingly, the higher the PL1 expression the fewer CFU’s were recovered. Finally, we assayed disease resistance against LMG5084 in 14 day old Arabidopsis seedlings using a flood inoculation technique (Ishiga et al. 2011 ). In 1/2 MS plates we flooded seedlings for up to 1 minute with a bacterial suspension and the plates were left to develop symptoms over 3 days. Wild-type seedlings were severely chlorotic and dead whereas the PL1 expressing lines were mostly healthy, with a few plants showing some chlorosis (however this could be down to silencing) and this result was consistent for all 3 transgenic lines (Figure 6).

We then used this experimental set up to develop our qPCR approach optimised for Arabidopsis seedlings. We achieved this first by establishing a standard curve showing a relationship between OPRF:ACT2 and CFU mg ¹ (Figure 11 ). The measurements of bacterial genomic DNA showed that all 3 lines were all consistently 1.5 logs less than the wild-type (Figure 5B). Furthermore, there seems to be a relationship with PL1 protein levels and the standard error to the mean, the higher protein levels correlates intrinsically less variation. When the transgenic line PL1 1-2 was flooded with DC3000 there was no difference detected when compared to the wild-type (Figure 5C), therefore confirming that PL1 confers resistance/tolerance to PL1 -sensitive but not PL1 -insensitive pathovars of P. syrnigae.

In conclusion, these results demonstrate a proof of principle that bacteriocins active against gram negative plant pathogenic bacteria can be used as part of a transgenic solution towards obtaining global food security. The literature has already identified candidate bacteriocins active against agronomically important bacteria such as Xanthamonas, Pectobacterium and Ralstonia (Hert et al 2005; Bonini et al 2007; Chuang et al. 2007; Roh et al. 2010; Chan et al. 2011 ; Grinter et al. 2014; Huerta et al. 2015). Furthermore, due to the specificity of bacteriocins these in theory can be used in both leguminous and non-legumious crops as these bacteirocins should have no detrimental effect on the symbiotic relationships between plants and their symbiotic and commensal microbiota.

Moreover, Schulz and colleagues explored the use of bacteriocins active against foodborne pathogenic Escherichia coli as a food additive. Bacteriocins from E. coli would be generally recognised as safe according to US standards and therefore this could be extended to bacteriocins from plant pathogenic bacteria share homology to bacteriocins isolated from bacteria from the gastrointestinal tract. Finally, bacteriocin producing plant pathogenic bacteria are likely to be present on the surface of food we eat and are not classed as dangerous.

Our results demonstrate that we can efficiently express a bacteriocin, PL1 at high levels in planta using transient or permanent methods of transformation. We also show conclusively that two model plants, Arabidopsis and N. benthamiana engineered to express PL1 show highly significant reductions in susceptibility to a cognate natural isolate of Ps syringae as evidenced by greatly reduced bacterial growth and greatly reduced symptoms of infections. Genetic engineering of plants to produce bacteriocins represents a highly promising novel technology for plant protection against Ps. syringae and likely other bacterial pathogens. Example 4: Transgenic soybean

We will construct transgenic soybean plants expressing PL1 and carry out infection studies using Ps syringae.

Briefly, PL1 will be cloned into a tumor inducing (Ti) plasmid vector to generate a stable expression construct, with the bacteriocin expressed under the control of a plant promoter. This vector is transformed into Agrobacterium tumefaciens. Soybean tissue explants will be incubated with the transformed Agrobacterium. Tissue explants will then be cultured on selection media, to select for transformants. The transformants are then cultured to regenerate plants, using standard tissue culture techniques.

Table 2: Example Sequences

References

A number of publications are cited above in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. The entirety of each of these references is incorporated herein.

For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press

Asai T., Tena G., Plotnikova J., Willmann M. R., Chiu W. L., Gomez-Gomez L., Boiler T., Ausubel F. M., Sheen J. (2002). MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977-983 Baulcombe DC. (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell. 8(10): 1833-44.

Bonini M, Maringoni AC, Rodrigues Neto J. (2007). Characterization of Xanthomonas spp. strains by bacteriocins. Summa Phytopathologica 33:24-29

Boutrot F, Segonzac C, Chang KN, Qiao H, Ecker JR, Zipfel C, Rathjen JP. (2010) Direct transcriptional control of the Arabidopsis immune receptor FLS2 by the ethylene-dependent transcription factors EIN3 and EIL1. Proceedings of the National Academy of Sciences of the USA 107: 14502-14507

Cascales E., Buchanan S. K., Duche D., Kleanthous C., Lloubes R., Postle K. et al. (2007) Colicin biology. Microbiology and Molecular Biology Reviews 71 : 158-229

Chan, Y.-C., Wu, J.-L., Wu, H.-P., Tzeng, K.-C. and Chuang, D.-Y. (201 1 ). Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum. BMC Microbiology 1 1 , 99

Cecchini E, Gong ZH, Geri C, Covey SN, Milner JJ (1997). Transgenic Arabidopsis lines expressing gene VI from cauliflower mosaic virus variants exhibit a range of symptom-like phenotypes and accumulate inclusion bodies. Molecular Plant-Microbe Interactions 10: 1094-1 101 .

Chuang, D.-y., Chien, Y.-c. and Wu, H.-P. Cloning and expression of the Erwinia carotovora subsp. carotovora gene encoding the low-molecular-weight bacteriocin carocin S1 . (2007). Journal of

Bacteriology 189, 620-626.

Fyfe, J. A., Harris, G., and Govan, J. R. (1984). Revised pyocin typing method for Pseudomonas aeruginosa. Journal of Clinical Microbiology 20, 47-50Ghequire, M.G. K., Li, W., Proost, P., Loris, R. and De Mot, R. (2012) Plant lectin-like antibacterial proteins from phytopathogens Pseudomonas syringae and Xanthomonas citri. Environmental Microbiology. Rep. 4, 373-380

Ghequire M. G. et al. (2013) Structural determinants for activity and specificity of the bacterial toxin LlpA. PLoS Pathog. 9, e1003199.

Grinter R., Josts I., Zeth K., Roszak A. W., McCaughey L. C., Cogdell R. J., Milner J. J., Kelly S. M.,

Byron O., Walker D. (2014). Structure of the atypical bacteriocin pectocin M2 implies a novel mechanism of protein uptake. Molecular Microbiology 93, 234-246

Hann DR, Rathjen JP. (2007). Early events in the pathogenicity of Pseudomonas syringae on Nicotiana benthamiana. The Plant Journal; 49 (4):607-18.

Huerta Al, Milling A, Allen C. Tropical strains of Ralstonia solanacearum outcompete race 3 biovar 2 strains at lowland tropical temperatures. (2015). Applied Environmental Microbiology 81 :3542-3551. Ishiga, Y., Ishiga, T., Uppalapati, S. R., and Mysore, K. S. (201 1 ) Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions. Plant methods 7, 32 Lomonossoff G.P. (1995) Pathogen-Derived Resistance to Plant Viruses. Annual Review of

Phytopathology. Vol 33; 323-343

Mansfield J, et al. (2012)Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology 13; 614-629

McCaughey L. C., Grinter R., Josts I., Roszak A. W., Waloen K. I., Cogdell R. J., et al. (2014). Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor. PLoS Pathogens. 10:e1003898. 10.1371/journal. ppat.1003898

Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M.,Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Journal of Bioscience and Bioengineering 104: 34-41.

O’Brien HE, Thakur S. Guttman DS. (201 1 ). Evolution of plant pathogenesis in Pseudomonas syringae : a genomics perspective. Annual Review of Phytopathology ;49:269-289

Parret AH, Schoofs G, Proost P, De Mot R. (2003). Plant lectin-like bacteriocin from a rhizosphere- colonizing Pseudomonas isolate. Journal of Bacteriol 185:897-908.

Parret AH, Temmerman K., De Mot R. (2005). Novel lectin-like bacteriocins of biocontrol strain

Pseudomonas fluorescens Pf-5. Applied Environmental Microbiology 2005;71 :5197-5207.

Pecenkova T.,Pleskot R., and Zarsky V. (2017) Subcellular Localization of Arabidopsis Pathogenesis- Related 1 (PR1 ) Protein. International Journal of Molecular Sciences 18(4): 825.

Rico A, Bennett MH, Forcat S, Huang WE, Preston GM (2010) Agroinfiltration Reduces ABA Levels and Suppresses Pseudomonas symgae-Elicited Salicylic Acid Production in Nicotiana tabacum. PLOS ONE 5(1 ): e8977.

Riley M. A., Wertz J. E. (2002). Bacteriocins: evolution, ecology, and application. Annual Reviews in Microbiology 56, 1 17-137

Roh, E., Park, T.-H., Kim, M.-i., Lee, S., Ryu, S., Oh, C.-S., Rhee, S., Kim, D.-H., Park, B.-S. and Heu, S. (2010). Characterization of a new bacteriocin, carocin D, from Pectobacterium carotovorum subsp. carotovorum Pcc21. Applied Environmental Microbiology 76, 7541-7549

Rojo E, Sharma VK, Kovaleva V, Raikhel NV, Fletcher JC. (2002) CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway. Plant Cell 14: 969-977 Ross A., Somssich I. E. (2016). A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana. Plant Methods 12:48

10.1 186/s 13007-016-0149-z

Schulz S., Stephan A., Hahn S, Bortesi L., Jarczowski F., Bettmann U., et al. (2015). Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins. Proceedings of the National Academy of Sciences USA 1 12: E5454-60.

Zipfel C., Kunze G., Chinchilla D., Caniard A., Jones J. D. G., Boiler T., et al. (2006). Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749-760

Claims

CLAIMS:

1. A transgenic plant that produces a bacteriocin effective against a primarily plant pathogenic

bacteria.

2. The transgenic plant according to claim 1 , wherein the bacteriocin is a polypeptide having at least 80% sequence identity to SEQ ID NO: 1-5.

3. The transgenic plant according to any previous claim, wherein the primarily plant pathogenic bacteria is Pseudomonas syringae, optionally wherein the Pseudomonas syringae is one or more pathovar selected from P. syringae pv. glycinea, morspurnorum, actinidiae, syringae, savastoni, and/or lachrymans.

4. The transgenic plant according to any previous claim, wherein at least 0.1 % of the total protein produced by said plant is bacteriocin.

5. The transgenic plant according to any previous claim, wherein the plant has increased tolerance to the primarily plant pathogenic bacteria as compared to control plants.

6. The transgenic plant according to any previous claim, wherein the plant impairs growth of the primarily plant pathogenic bacteria as compared to control plants.

7. The transgenic plant according to any of claims 1 to 5, wherein the transgenic plant stably

expresses the bacteriocin.

8. The transgenic plant according to any previous claim, wherein the bacteriocin comprises one or more carbohydrate binding domain motifs having at least 80% identity to SEQ ID NO:7.

9. The transgenic plant according to claim 3, wherein the one or more carbohydrate binding domain motifs are located at an amino acid position relative to positions 41-49, 1 17-125, 171-179 and/or 202-210 of SEQ ID NO: 1.

10. The transgenic plant according to any previous claim, wherein the bacteriocin has a minimum inhibitory concentration of equal to or less than about 2000nM against the primarily plant pathogenic bacteria .

1 1. The transgenic plant according to any previous claim, wherein the plant is a higher plant.

12. The transgenic plant according to claim 1 1 , wherein the plant is an agricultural plant selected from the group consisting of soybean ( Glycine max), tomato ( Solanaceae lycospersicum), kiwi Actinidia deliciosa) or pepper ( Capsicum ) plant.

13. A transgenic plant comprising a DNA sequence encoding a polypeptide having at least 80% sequence identity with SEQ ID NO: 1.

14. A plant transformation vector comprising a polynucleotide sequence encoding a bacteriocin having a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO: 1.

15. The plant transformation vector according to claim 14, wherein the polynucleotide sequence encoding the bacteriocin is operatively linked to a promoter.

16. The plant transformation vector according to claim 15, wherein the promoter is a plant promoter, for example a tissue specific plant promoter and/or a plant inducible promoter.

17. An Agrobacterium comprising the plant transformation vector according to any one of claims 14 to 16.

18. A transgenic cell comprising a plant transformation vector according to any one of claims 14 to 16.

19. A transgenic cell according to claim 18, wherein the cell is a plant cell.

20. A plant comprising a transgenic cell according to claim 19.

21. Progeny, seed or plant material from a plant according to claim any one of claims 1 to 13, which optionally comprises a plant cell according to claim 19.

22. A plant cell that produces a bacteriocin effective against a primarily plant pathogenic bacteria, preferably wherein the bacteriocin is a polypeptide having at least 80% sequence identity to SEQ ID NO: 1.

23. A transgenic plant seed comprising a polynucleotide sequence encoding a bacteriocin having a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO: 1.

24. Meal, feed or food produced from the transgenic plant seed of claim 23.

25. A method of increasing tolerance to a primarily plant pathogenic bacteria in a plant relative to a control plant comprising expressing a nucleic acid encoding a bacteriocin polypeptide within one or more cells of said plant, wherein the bacteriocin is effective against the primarily plant pathogenic bacteria.

26. A method of producing a plant with increased tolerance to a primarily plant pathogenic bacteria relative to a control plant comprising: incorporating a nucleic acid encoding a bacteriocin polypeptide into a plant cell by means of transformation, and;

regenerating the plant from one or more transformed cells;

wherein the plant produces a bacteriocin effective against a primarily plant pathogenic bacteria.

27. A method comprising producing a bacteriocin effective against a primarily plant pathogenic

bacteria within one or more cells of a plant.

28. The method according to any one of claims 25 to 27, wherein the primarily plant pathogenic bacteria is a pathogenic Pseudomonas, preferably Pseudomonas syringae, optionally one or more P. syringae pathovar selected from glycinea, morspurnorum, actinidiae, syringae, savastoni, and/or lachrymans.

29. The method according to any one of claims 25 to 28, wherein the bacteriocin polypeptide

comprises at least 80% sequence identity to any one of SEQ ID NO: 1-6.

30. The method according to any one of claims 25 to 29, wherein the bacteriocin comprises one or more carbohydrate binding domain motifs having at least 80% identity to SEQ ID NO:7, preferably at an amino acid position selected from a position relative to positions 41-49, 1 17-125, 171-179 and/or 202-210 of SEQ ID NO: 1.

31. The method according to any one of claims 25 to 30, wherein the plant is a higher plant.

32. The method according to claim 31 , wherein the plant is an agricultural plant selected from the group consisting of soybean ( Glycine max), tomato ( Solanaceae lycospersicum), kiwi ( Actinidia deliciosa) or pepper ( Capsicum ) plant.

33. The method according to any one of claims 25 to 32, further comprising the step of:

sexually or asexually propagating or growing off-spring or descendants of the plant having increased tolerance to a bacterial pathogen.

34. The method according to any one of claims 25 to 33, further comprising the steps of:

harvesting one or more plant products, such as seeds, from the plants; and optionally processing the plant products into a plant-derived product, such as a seed meal.

35. A plant according to any one of claims 1 to 13, produced by a method according to any one of claims 25 to 33.