WO2014112875A1

WO2014112875A1 - A new method to provide resistance to bacterial soft rot in plants

Info

Publication number: WO2014112875A1
Application number: PCT/NL2014/050021
Authority: WO
Inventors: Klaas BOUWMEESTER; Francine GOVERS
Original assignee: Wageningen Universiteit
Priority date: 2013-01-17
Filing date: 2014-01-17
Publication date: 2014-07-24

Abstract

The invention relates to a method for providing plants, preferably potato plants, with resistance to blackleg or soft rot caused by Pectobacterium carotovorum by providing them with a gene encoding the LecRK-I.9 protein.

Description

Title: A new method to provide resistance to bacterial soft rot in plants

FIELD OF THE INVENTION

The invention relates to a resistance gene isolated from Arabidopsis thaliana. Moreover, the invention relates to the use of said resistance gene, for example to clone functional homologues, and the use of said resistance gene in a method to increase or confer at least partial resistance to bacterial soft rot in plants. More in specific the invention provides a resistance gene that is capable of increasing or conferring at least partial resistance to Pectobacterium carotovorum (formerly known as Erwinia carotovora) through genetic engineering techniques or through marker-assisted breeding techniques.

BACKGROUND

Pectobacterium carotovorum (formerly known as Erwinia carotovora) causes economically important plant diseases. Pectobacterium carotovorum is most renowned to cause soft rot and blackleg of potato. Soft rot and blackleg are economically important as they reduce potato yield and cause downgrading or rejection of potato seeds in certification processes (Perombelon, M., OEPP/EPPO Bull. 30:413-420, 2000). Moreover, Pectobacterium carotovorum is capable to cause disease in vegetables, flowers and many different tree species.

P. carotovorum is a pectinolytic, Gram-negative, facultative anaerobic, non-sporulating, motile, straight rod bacterium with peritrichous flagellae

(Charkowsky, A., 2006. The soft rot Erwinia. In: Gnanamanickam S.S. ed. Plant- Associated Bacteria. Dordrecht, Netherlands: Springer, 423-505). It

characteristically produces a variety of cell wall degrading enzymes that allows maceration of the plant tissue on which they feed.

The most characteristic symptom of potato blackleg is a slimy, wet, black rot lesion which spreads, especially under wet conditions, from the rotting mother tuber up to the stem. Under persistent rainy conditions, extensive stem rot can develop as a result of infection with P. carotovorum, which starts from the top and progresses downwards to the base. Under dry conditions the symptoms are stunting, yellowing, wilting and desiccation of stems and leaves. In addition, extensive soft rot can develop when tubers with blackleg infection are stored. Tuber soft rot is initiated at lenticels, the stolon end, and/or in wounds under wet conditions. The lesion can spread to the whole tuber and thence to neighboring tubers in storage (Czajkowski, R., et al., Plant Pathol. 60:999-1013, 2011).

Several control strategies have been studied, but the degree of success has been variable (Czajkowski, R., et al., Plant Pathol. 60:999- 1013, 2011).

Methods based on avoiding contamination have partially been successful. Improved storage management can reduce bacterial load and contamination during storage. Physical (e.g. hot water treatment) and chemical methods have been explored, but with limited success. The use of biological control has been and still is being attempted, but it is too soon to say how successful it will be. Finally, breeding for resistance has so far failed, in particular since commercial potato varieties that are naturally immune to blackleg and soft rot do not exist. Several wild potato species are known to have a high resistance (Hijmans, R. and Spooner, D., Am. J. Botany 88:2101-2112, 2001), and offspring of crosses between these wild species and commercial potato varieties have been obtained (Fock, I. et al., Plant Physiol.

Biochem. 39:899-908, 2001), but this has not led to resistant potato lines.

Genetic engineering is a promising alternative to traditional plant breeding, but only a limited amount of research has been spent in this area with respect to blackleg and soft rot. Potato plants transformed with antimicrobial peptides, such as attacin and cecropin (Arce, P., et al., Amer. J. of Potato Res.

76; 169- 177, 1999), have been produced, but with variable success. Another example is given by C. Wegener, who showed that potato plants overexpressing pectate lyase have increased resistance to P. carotovorum in both in vitro and in planta tests (Wegener, C, Potato Res. 44:401-410, 2001).

Hence, there is still a need to establish novel methods that provide resistance to blackleg and soft rot caused by P. carotovorum in plants, especially in Solanaceous plants such as potato.

SUMMARY OF THE INVENTION

The invention now relates to a method for inducing or increasing resistance in a plant against blackleg and/or soft rot caused by Pectobacterium carotovorum comprising providing a plant or a part thereof with a nucleic acid encoding the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment or a functional homologue thereof or a nucleic acid encoding a protein having an identity of 95% or more with the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof. Preferably said plant is a plant from the Solanaceae family, more preferably Solarium tuberosum.

In a further preferred embodiment the nucleic acid sequence as defined above comprises a nucleic acid sequence as depicted in Figure 1 or a nucleic acid sequence having an identity of 95% or more therewith.

Further part of the invention is a method for breeding a plant that is resistant to blackleg and/or soft rot caused by Pectobacterium carotovorum, or has an increased resistance as compared to a wild-type plant comprising

a. increasing the plo^'idy level of the gametes of a diploid plant that already contains a nucleic acid sequence as defined in claim 1;

b. using said gametes in a cross with gametes of a tetraploid plant; and

c. selecting the offspring of said cross for the presence of said nucleic acid sequence. Preferably, in such a method the diploid plant of step a) is plant from the genus S. chocaense, S. berthaultii, S. sucrense, or S. tarijense.

Also part of the invention is a method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to blackleg and/or soft rot caused by Pectobacterium carotovorum infection, said method comprising the steps of testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid encoding the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment or a functional homologue thereof or a nucleic acid encoding a protein having an identity of 95% or more with the

LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof..

Also comprised in the invention is the use of a nucleic acid encoding the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment thereof or encoding a protein having an identity of 95% or more therewith for providing plants with resistance to blackleg and/or soft rot caused by Pectobacterium carotovorum. LEGENDS TO THE FIGURES

Figure 1. Nucleotide and amino acid sequences of LecRK-1.9.

DETAILED DESCRIPTION

As used herein, the term "plant or part thereof means any complete or partial plant, single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which potato plants can be

regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, tubers, including potato tubers for consumption or 'seed tubers' for cultivation or clonal propagation, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like. As used herein, the term "variety" is as defined in the UPOV treaty and refers to any plant grouping within a single botanical taxon of the lowest known rank, which grouping can be: (a) defined by the expression of the characteristics that results from a given genotype or combination of genotypes, (b) distinguished from any other plant grouping by the expression of at least one of the said characteristics, and (c) considered as a unit with regard to its suitability for being propagated unchanged.

The term "cultivar" (for cultivated variety) as used herein is defined as a variety that is not normally found in nature but that has been cultivated by humans, i.e. having a biological status other than a "wild" status, which "wild" status indicates the original non-cultivated, or natural state of a plant or accession. The term "cultivar" specifically relates to a potato plant having a ploidy level that is tetraploid. The term "cultivar" further includes, but is not limited to, semi- natural, semi-wild, weedy, traditional cultivar, landrace, breeding material, research material, breeder's line, synthetic population, hybrid, founder stock/base population, inbred line (parent of hybrid cultivar), segregating population, mutant/genetic stock, and advanced/improved cultivar. As used herein, "crossing" means the fertilization of female plants (or gametes) by male plants (or gametes). The term "gamete" refers to the haploid or diploid reproductive cell (egg or sperm) produced in plants by meiosis, or by first or second restitution, or double reduction from a gametophyte and involved in sexual reproduction, during which two gametes of opposite sex fuse to form a diploid or polyploid zygote. The term generally includes reference to a pollen (including the sperm cell) and an ovule (including the ovum). "Crossing" therefore generally refers to the fertilization of ovules of one individual with pollen from another individual, whereas "selfing" refers to the fertilization of ovules of an individual with pollen from genetically the same individual.

The term "marker" as used herein means any indicator that is used in methods for inferring differences in characteristics of genomic sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), insertion mutations, micros atellite markers (SSRs), sequence-characterized amplified regions (SCARs), cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.

As used herein, "locus" is defined as the genetic or physical position that a given gene occupies on a chromosome of a plant.

The term "allele(s)" as used herein means any of one or more alternative forms of a gene, all of which alleles relate to the presence or absence of a particular phenotypic trait or characteristic in a plant. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous

chromosomes. It is in some instance more accurate to refer to "haplotypes" (i.e. an allele of a chromosomal segment) instead of "allele", however, in these instances, the term "allele" should be understood to comprise the term "haplotype".

The term "heterozygous" as used herein, and confined to diploids, means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes. As used herein, and confined to diploids, "homozygous" is defined as a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.

As used herein, and confined to tetraploids, the term "nulliplex", "simplex", "duplex", "triplex" and "quadruplex", is defined as a genetic condition existing when a specific allele at a corresponding locus on corresponding

homologous chromosomes is present 0, 1, 2, 3 or 4 times, respectively. At the tetraploid level the phenotypic effect associated with a recessive allele is only observed when the allele is present in quadruplex condition, whereas the phenotypic effect associated with a dominant allele is already observed when the allele is present in a simplex or higher condition. The terms "haploid", "diploid" and "tetraploid" as used herein are defined as having respectively one, two and four pairs of each chromosome in each cell (excluding reproductive cells).

The term "haplotype" as used herein means a combination of alleles at multiple loci that are transmitted together on the same chromosome. This includes haplotypes referring to as few as two loci, and haplotypes referring to an entire chromosome depending on the number of recombination events that have occurred between a given set of loci.

As used herein, the term "infer" or "inferring", when used in reference to assessing the presence of the Pectobacterium resistance as related to the expression of the LecRK-1.9 gene, means drawing a conclusion about the presence of said gene in a plant or part thereof using a process of analysing individually or in

combination nucleotide occurrence(s) of said gene in a nucleic acid sample of the plant or part thereof. As disclosed herein, the nucleotide occurrence(s) can be identified directly by examining the qualitative differences or quantitative differences in expression levels of nucleic acid molecules, or indirectly by examining the presence of the LecRK-1.9 protein. As used herein, the term "increasing resistance" means that the resistance of a plant against blackleg and/or soft rot may be enhanced as compared to a wild-type plant of the same species. This increase in resistance may show because more of the pathogen would be needed to break through the resistance of a particular plant or because the number and severity of the pathogenic effects caused by the disease is less in a plant or because in a plot of plants of the same species the number of plants that has been infected and/or the number and severity of the pathogenic effects is less as compared to wild-type. The difference between the treated plant and the wild-type plant is that the treated plant is provided with a nucleic acid coding for the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment or a functional homologue thereof or a nucleic acid encoding a protein having an identity of 95% or more with the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof. The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of primer extension product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The (amplification) primer is preferably single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on many factors, including temperature and source of primer. A "pair of bidirectional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

As used herein, the term "probe" means a single-stranded

oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative. The terms "stringency" or "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O. lx SSC at 60°C. Hybridization procedures are well known in the art and are described in e.g. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., Struhl, K. eds. (1998) Current protocols in molecular biology. V.B. Chanda, series ed. New York: John Wiley & Sons.

The present invention describes a method for increasing resistance in a plant against blackleg and/or soft rot caused by Pectobacterium carotovorum comprising providing a plant or a part thereof with a nucleic acid encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment thereof or encoding a protein having an identity of 95% or more therewith.

It has been found that there are two variants of the gene, represented in Figure 1 by nucleic acid sequences designated At5g60300.1 and At5g60300.3. These sequences encode two proteins that only differ in length (718 and 756 amino acid residues, respectively). Both proteins contain identical lectin and kinase domains. When in this application is referred to the LecRK-1.9 gene or nucleotide sequence, or the LecRK-1.9 protein or amino acid sequence, both variants are comprised. Further also the percentage identity is related to both variants.

The term "nucleic acid" means a single or double stranded DNA or RNA molecule.

The term "functional fragment thereof is typically used to refer to a fragment of the LecRK-1.9 protein that is capable of providing at least partial resistance or increased resistance in a plant of the Solanaceae family against Pectobacterium carotovorum infection.

The term "functional homologue" is typically used to refer to a protein sequence that is highly homologous to or has a high identity with the herein described LecRK-1.9 protein, which protein is capable of providing at least partial resistance or increasing resistance in a plant of the Solanaceae family against

Pectobacterium carotovorum infection. Included are artificial changes or amino acid residue substitutions that at least partly maintain the effect of the LecRK-1.9 protein. For example, certain amino acid residues can conventionally be replaced by others of comparable nature, e.g. a basic residue by another basic residue, an acidic residue by another acidic residue, a hydrophobic residue by another hydrophobic residue, and so on. Examples of hydrophobic amino acids are valine, leucine and isoleucine. Phenylalanine, tyrosine and tryptophan are examples of amino acids with an aromatic side chain, and cysteine and methionine are examples of amino acids with sulphur-containing side chains. Serine and threonine contain aliphatic hydroxyl groups and are considered to be hydrophilic. Aspartic acid and glutamic acid are examples of amino acids with an acidic side chain. In short, the term "functional homologue thereof includes variants of the LecRK-1.9 protein in which amino acids have been inserted, replaced or deleted and which at least partly maintain the effect of the LecRK-1.9 protein (i.e. at least partly providing or increasing resistance in a plant of the Solanaceae family against Pectobacterium carotovorum infection). Preferred variants are variants which only contain conventional amino acid replacements as described above. A high identity in the definition as mentioned above means an identity of at least 80, 85 or 90%. Even more preferred are amino acids that have an identity of 91, 92, 93, 94 or 95%. Most preferred are amino acids that have an identity of 96, 97, 98 or 99% with the amino acid sequence of LecRK-1.9.

A functional homologous nucleic acid sequence is a nucleic acid sequence that encodes a functional homologous protein as described above.

Homology and/or identity percentages can for example be determined by using computer programs such as BLAST, ClustalW or ClustalX.

Many nucleic acid sequences code for a protein that is 100% identical to the LecRK-1.9 protein as presented in Figure 1. This is because nucleotides in a nucleotide triplet may vary without changing the corresponding amino acid (wobble in the nucleotide triplets). Thus, without having an effect on the amino acid sequence of a protein the nucleotide sequence coding for this protein can be varied. However, in a preferred embodiment, the invention provides a method comprising transforming plants with a nucleic acid sequence as depicted in Figure 1.

Fragments as well as homologues of the herein described LecRK-1.9 gene and the LecRK-1.9 protein can for example be tested for their functionality by using Agrobacterium tumefaciens transient transformation assays (agro-infiltration) and/or by using a detached leaf assay.

Typically, in such an agro-infiltration assay 4 week old wild type Nicotiana benthamiana plants are infiltrated with Agrobacterium strains containing the candidate LecRK-1.9 homologues. The infiltrated leaves are subsequently challenged one day after infiltration with a P. carotovorum strain that is virulent on N. benthamiana. This system is equally suitable for testing candidate homologous fragments of LecRK-1.9. A person skilled in the art thus can easily determine whether or not an LecRK-1.9 homolog or fragment can be considered to be a functional homolog or fragment. Transient gene expression, as is achieved through agro-infiltration, is a fast, flexible and reproducible approach to high-level expression of useful proteins. In plants, recombinant strains of Agrobacterium tumefaciens can be used for transient expression of genes that have been inserted into the T-DNA region of the bacterial Ti plasmid. A bacterial culture is infiltrated into leaves, and upon T-DNA transfer, there is ectopic expression of the gene of interest in the plant cells.

However, the utility of the system is limited because the ectopic RNA expression ceases after 2-3 days. It is shown that post-transcriptional gene silencing (PTGS) is a major cause for this lack of efficiency. A system based on co-expression of a viral - encoded suppressor of gene silencing, the pl9 protein of tomato bushy stunt virus (TBSV), prevents the onset of PTGS in the infiltrated tissues and allows high level of transient expression. Gene expression has been reported to be enhanced over 50- fold or more in the presence of p l9, and hence simplified protein purification could be achieved from as little as 100 mg of infiltrated leaf material. Although it is clear that the use of pl9 has advantages, an agroinfiltration without pl9 can also be used to test the functionality of candidate fragments and functional homologues.

Alternatively, each candidate gene (for example being a fragment or homologue) construct is targeted for transformation to a P. carotovorum susceptible potato cultivar, for example Desiree. Primary transformants are challenged in detached leaf assays as described in the examples. Transformants that are resistant to these isolates harbor for example functional fragments or homologues of LecRK-1.9.

As will be outlined below there are multiple ways in which a nucleic acid of the invention can be transferred to a plant. One suitable means of transfer is mediated by Agrobacterium in which the nucleic acid to be transferred is part of a binary vector and hence it is preferred that the plant transformation vector is a binary vector. Another suitable means is by crossing a plant which contains the LecRK-1.9 gene or a homologue thereof with a plant that does not contain such a gene and to identify those progeny of the cross that have inherited the LecRK-1.9 gene or homologue thereof.

In the present invention advantageously a vector comprising an isolated, synthetic or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the amino acid sequence of LecRK-1.9 of Figure 1 or a functional fragment or a functional homologue thereof is used. Examples of a suitable vector are pBeloBACII, pBINplus, pKGW-MG or any commercially available cloning vector.

The invention also provides the means to introduce or increase resistance against soft rot or blackleg in a plant. The invention therefore also provides a method for providing at least partial resistance or increasing resistance in a plant against P. carotovorum comprising providing a plant or a part thereof with:

- an isolated or recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof, or

- an isolated or recombinant nucleic acid sequence as depicted in Figure 1, or

- a vector comprising the herein described nucleic acid sequences, or

- a bacterial, preferably Agrobacterium cell comprising a nucleic acid sequence encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof.

Such a method for providing at least partial resistance or increasing resistance in a plant against blackleg or soft rot may be based on classical breeding, departing from a parent plant that already contains the LecRK-1.9 gene or a functional homolog thereof, or it involves the transfer of DNA into a plant, i.e., involves a method for transforming a plant cell comprising providing said plant cell with a nucleic acid as described herein or a vector as described herein or a bacterial host cell.

There are multiple ways in which a recombinant nucleic acid can be transferred to a plant cell, for example via Agrobacterium-mediated

transformation. However, besides by Agrobacterium-mediated transformation, there are other means to effectively deliver DNA to recipient plant cells when one wishes to practice the invention. Suitable methods for delivering DNA to plant cells are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts, by desiccation/inhibition-mediated DNA uptake (Potrykus, I., et al., Mol. Gen. Genet., 199: 183-188, 1985), by electroporation (U.S. Pat. No. 5,384,253), by agitation with silicon carbide fibers (Kaeppler, H.F., et al., Plant Cell Reports, 9:415-418, 1990; U.S. Pat. No. 5,302,523; U.S. Pat. No. 5,464,765), and by acceleration of DNA coated particles (U.S. Pat. No. 5,550,318; U.S. Pat. No.

5,538,877; U.S. Pat. No. 5,538,880). Through the application of techniques such as these, cells from virtually any plant species may be stably transformed, and these cells may be developed into transgenic plants.

In case Agrobacterium-meAiateA transfer is used, it is preferred to use a substantially virulent Agrobacterium species, such as A. tumefaciens, as exemplified by strain A281 or a strain derived thereof or another virulent strain available in the art. These Agrobacterium strains carry a DNA region originating from the virulence region of the Ti plasmid pTiBo542, which coordinates the processing of the T-DNA and its transfer into plant cells. Agrobacterium-baseA plant transformation is well known in the art (as e.g. described in, for example by Komari, T., et al., Plant Transformation Technology: Agrobacterium-Mediated Transformation, in: Handbook of Plant Biotechnology, Eds. Christou, P., and Klee,

H. , John Wiley & Sons, Ltd, Chichester, UK 2004, pp. 233-262). Preferably a marker-free transformation protocol is used, such as described in WO 03/010319.

Alternatively, the nucleic acid of the LecRK-1.9 gene or a functional homolog thereof may be introduced into a plant by crossing. Such a crossing scheme starts off with the selection of a suitable parent plant. It has been established (Hijmans, R.J., Spooner, D.M., Am. J. Botany 88:2101-2112, 2001) that many wild, diploid potato species demonstrate a high level of resistance against black leg or soft rot. It is contemplated herein that such high levels of resistance may be due to the presence of the LecRK-1.9 gene, or an orthologue thereof. Such an orthologue would fall within the definitions of functional homologue of LecRK-

I.9. Selection of suitable parent plants that can be used for breeding new potato varieties is based on the herein described nucleic acid sequences. Selection then can be performed with probes and primers (i.e.

oligonucleotide sequences complementary to one of the (complementary) DNA strands of the nucleotide sequence encoding the LecRK-1.9 gene or a functional homolog thereof). Probes are for example useful in Southern or northern analysis and primers are for example useful in PCR analysis. Primers based on the herein described nucleic acid sequences are very useful to assist plant breeders active in the field of classical breeding and/or breeding by genetic modification of the nucleic acid content of a plant (preferably said plant is a Solanum tuberosum, Solanum lycopersicum, formerly known as Lycopersicon esculentum), pepper (Capsicum spp.) or eggplant (Solanum melongena)) in selecting a plant that is capable of expressing for example LecRK-1.9 or a functional fragment or functional homolog thereof.

Hence, in a further embodiment, the invention provides a method for selecting a plant or plant material or progeny thereof for its

susceptibility or resistance to blackleg and/or soft rot caused by Pectobacterium carotovorum infection, said method comprising the steps of testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid as defined herein. One can for example use a PCR analysis to test plants for the presence of absence of LecRK-1.9 in the plant genome. Such a method would be especially preferable in marker-free transformation protocols, such as described in WO 03/010319. Any suitable method known in the art for crossing selected plants may be applied in the method according to the invention. This includes both in vivo and in vitro methods. A person skilled in the art will appreciate that in vitro techniques such as protoplast fusion or embryo rescue may be applied when deemed suitable.

Selected plants that are used for crossing purposes in the methods according to the invention may have any type of plo^'idy. For example, selected plants may be haploid, diploid or tetraploid. However, crossing diploid plants, such as Solanum canasense, S. tarijense, S. phureja or S. commersonnii will only provide diploid offspring. Crossing a diploid plant with a tetraploid plant will result in triploid offspring that is sterile.

Thus, when plants are selected that are diploid, their plo^'idy must be increased to tetraploid level before they can be crossed with another tetraploid plant in the methods according to the invention. Methods for increasing the plo^'idy of a plant are well known in the art and can be readily applied by a person skilled in the art. For example, plo^'idy of a diploid plant for crossing purposes can be increased by using 2N gametes of said diploid plant. Plo^'idy can also be increased by inhibiting chromosome segregation during meiosis, for example by treating a diploid plant with colchicine. By applying such methods on a diploid plant, embryos or gametes are obtained that comprise double the usual number of chromosomes. Such embryos or gametes can then be used for crossing purposes. For potatoes a resistant tetraploid plant is preferred, since tetraploid plants are known to have higher yields of tubers.

Preferably, selected plants are crossed with each other using classical in vivo crossing methods that comprise one or more crossing steps including selfing. By applying such classical crossing steps characteristics of both the parents can be combined in the progeny. For example, a plant that provides a high yield can be crossed with a plant that contains large amounts of a certain nutrient. Such a crossing would provide progeny comprising both characteristics, i.e. plants that not only comprise large amounts of the nutrient but also provide high yields.

When applying backcrossing, Fl progeny is crossed with one of its high- yielding parents P to ensure that the characteristics of the F2 progeny resemble those of the high-yielding parent. For example, a selected diploid potato with bacterial resistance is made tetraploid by using colchicine and then crossed with a selected high-yielding tetraploid potato cultivar, with the purpose of ultimately providing a high-yielding tetraploid progeny having bacterial resistance. Also selfing may be applied. Selected plants, either parent or progeny, are then crossed with themselves to produce inbred varieties for breeding. For example, selected specimens from the above mentioned Fl progeny are crossed with themselves to provide an F2 progeny from which specimens can be selected that have an increased level of resistance.

After transfer of a nucleic acid into a plant or plant cell, it must be determined which plants or plant cells have been provided with said nucleic acid. When selecting and crossing a parental genotype in a method according to the invention, a marker is used to assist selection in at least one selection step. It is known in the art that markers, indicative for a certain trait or condition, can be found in vivo and in vitro at different biological levels. For example, markers can be found at peptide level or at gene level. At gene level, a marker can be detected at RNA level or DNA level. Preferably, in the present invention the presence of such a marker is detected at DNA level, using the above described primers and/or probes. Alternatively, presence of LecRK-1.9 or a functional homolog thereof can be assessed in plant parts by performing an immunoassay with an antibody that specifically binds the protein. Next to the primers and probes, use can also be made of specific markers that are to be found in the vicinity of the coding sequence. In case of transgenic approaches selecting a transformed plant may be accomplished by using a selectable marker or a reporter gene. Among the selective markers or selection genes that are most widely used in plant transformation are the bacterial neomycin phosphotransferase genes (nptl, nptll and nptlll) conferring resistance to the selective agent kanamycin, suggested in EP131623 and the bacterial aphlV gene suggested in EP186425 conferring resistance to hygromycin. EP 275957 discloses the use of an acetyl transferase gene from Streptomyces

viridochromogenes that confers resistance to the herbicide phosphinotricin. Plant genes conferring relative resistance to the herbicide glyphosate are suggested in EP218571. Suitable examples of reporter genes are beta-glucuronidase (GUS), beta-galactosidase, luciferase and green fluorescent protein (GFP). The invention also provides a plant that is obtainable by using a method for providing at least partial resistance or increasing resistance in a plant to Pectobacterium as described above. A preferred plant is a plant from the Solanaceae family and even more preferred said plant is a Solarium tuberosum or a Solanum lycopersicum, formerly known as Lycopersicon esculentum, Solanum melononga, Capsicum spp., such as C. annuum, C. baccatum, C. chinense, C.

frutescens and C. pubescens. The invention thus also provides a plant that has been provided with a nucleic acid encoding the LecRK-1.9 protein or a functional fragment or a functional homologue thereof.

The invention further provides a plant part or progeny of a plant according to the invention comprising a nucleic acid encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof. A further advantage of the present invention is that it has been shown that providing plants with a nucleic acid encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof provides resistance to Phytophthora infestans in potato and Nicotiana plants (Bouwmeester, K., 2010, PhD-thesis Wageningen University, dissertation no. 4824, 1-224; Han, M., Blanco-Portales, R., van der Vossen, E., Covers, F., Bouwmeester, K., 2012. The Arabidopsis lectin receptor kinase LecRK-1.9 confers late blight resistance in Solanaceous plants. In: Book of abstracts of the 30th New Phytologist Symposium, Fallen Leaf Lake, California, USA). Thus providing plants with this gene provides resistance to two major pathogens of Solanaceous plants.

The invention further provides use of an isolated or

recombinant nucleic acid sequence comprising a nucleic acid sequence encoding the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof or use of a vector comprising any of said nucleic acid sequences or use of a bacterial host cell comprising any of said nucleic acid sequences for providing a plant with at least partial resistance against soft rot or blackleg. In a preferred embodiment, said soft rot or blackleg is caused by infection of

Pectobacterium carotovorum and even more preferably Pectobacterium carotovorum spp. carotovorum. In yet another preferred embodiment said plant comprises

Solarium tuberosum, Nicotiana tabacum or Solanum lycopersicum, formerly known as Lycopersicon esculentum.

EXPERIMENTAL PART Example 1: Plasmid construction

The complete coding sequence of LecRK-1.9 was PCR-amplified from the BAC clone F15L12 (GenBank accession AB026632.1, clone available via TAIR) with primers pK60300s and pK60300as; [pK60300s:

CACCATGGCTCGTTGGTTGCTTCA; pK60300as

TTACCTCTGACTGCTGATGC. (Bouwmeester, K. et al., PLoS Pathog. 7:el001327, 2011). PCR amplicons were cloned via a BP recombination reaction into pENTR/D-TOPO vector (Invitrogen), and the resultant plasmid was transformed to E. coli DH5a. Transform ants were checked for the presence of the relevant inserts by colony PCR. Plasmids were purified and checked by sequence analysis and used to subclone the PCR fragment via LR reaction in the Gateway plant transformation vector pK2GW7 (http://gateway.psb.ugent.be; Karimi, M., et al., Trends Plant Sci. 7: 193-195, 2002). The resulting construct enables

constitutive expression of LecRK-1.9 under control of the 35S-CaMV promoter and upstream of the 35S-CaMV terminator, and was named pK-35S-LecRK-I.9

(Bouwmeester, K. et al., PLoS Pathog. 7:el001327, 2011). Example 2: Potato transformation

Binary plasmid pK-35S-LecRK-I.9 was transferred to A. tumefaciens strain COR308 through electroporation. An overnight culture of a positive clone, selected on LB medium containing 5 μg ml ¹ tetracycline and 100 μg ml ¹ spectinomycin, was used to transform potato tuber discs of cv. Desiree according to Hoekema et al. (Hoekema, A., et al., Biotechnology 7:273-278, 1989). Resultant transgenics were selected for kanamycin resistance on selective medium and via PCR and Southern hybridization. LecRK-1.9 expression levels were determined by Q-RT-PCR using primers RT-LecRK-I.9-F

(TTTGCCAGATTTCTCACCATACAC) and RT-LecRK-I.9-R

(TCTGTTGACTGCCAAGCGTAAG) normalized with respect to actin gene expression. Transgenic potato lines were maintained in vitro in climate chambers at 20°C and a 16 h photoperiod on MS30 medium, and were propagated by transplanting vegetative tissue to fresh medium with regular intervals. Potato tubers and in vitro material was used for long-term storage. To obtain fresh plant material for infection assays in vitro plantlets or tubers were transferred to soil.

Example 3: Plant growth and Pectobacterium carotovorum inoculation

Potato plants were grown in potting soil under standard greenhouse conditions. Pectobacterium carotovorum isolate PD1302 (syn. Erwinia carotovora) was routinely grown in LB medium at 28°C. Pectobacterium carotovorum cells were collected by centrifugation, washed and resuspended in 10 mM MgS04, and subsequently diluted to a concentration of 10⁷ cfu ml ¹ in lOmM MgS04 containing 0.01% Silwet L-77 (Lehle Seeds). Detached potato leaves were dipped in the inoculum suspension for 2 min. Inoculated potato leaves were kept at 23°C in closed trays at high humidity. Disease symptoms were scored at 3 and 5 dpi, respectively.

Claims

1. A method for inducing or increasing resistance in a plant against

blackleg and/or soft rot caused by Pectobacterium carotovorum

comprising providing a plant or a part thereof with a nucleic acid encoding the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment or a functional homologue thereof or a nucleic acid encoding a protein having an identity of 95% or more with the LecRK-1.9 amino acid sequence of Figure 1 or a functional fragment or a functional homologue thereof. 2. Method according to claim 1, wherein said plant is a plant from the

Solanaceae family.

3. Method according to claim 2, wherein said plant is Solarium tuberosum. 4. Method according to any of claim 1 - 3, wherein the nucleic acid

sequence as defined in claim 1 comprises a nucleic acid sequence as depicted in Figure 1 or a nucleic acid sequence having an identity of 95% or more therewith

A method for breeding a plant that is resistant to blackleg and/or soft rot caused by Pectobacterium carotovorum, comprising

b. using said gametes in a cross with gametes of a tetraploid plant; and c. selecting the offspring of said cross for the presence of said nucleic acid sequence.

6. Method according to claim 5, wherein the diploid plant of step a) is plant from the genus S. chocaense, S. berthaultii, S. sucrense, or S. tarijense.

7. A method for selecting a plant or plant material or progeny thereof for its susceptibility or resistance to blackleg and/or soft rot caused by Pectobacterium carotovorum infection, said method comprising the steps of testing at least part of said plant or plant material or progeny thereof for the presence or absence of a nucleic acid as defined in claim 1.

8. Use of a nucleic acid encoding the amino acid sequence LecRK-1.9 of Figure 1 or a functional fragment thereof or encoding a protein having an identity of 95% or more therewith for providing plants with resistance to blackleg and/or soft rot caused by Pectobacterium carotovorum.