NZ789514A - Horizontal transfer of fungal genes into plants (2) - Google Patents

Abstract

The present invention relates to methods of creating or enhancing symbiotic relationships between plants and symbionts, in particular symbionts carrying domain-of-unknown-function (DUF) genes. The present invention also relates to methods for selecting plants for symbiosis with symbionts and methods of modifying pathogen resistance in plants. The present invention also relates to new organisms and symbiota developed by the methods of the invention and to related nucleic acids, polypeptides and constructs including vectors. of modifying pathogen resistance in plants. The present invention also relates to new organisms and symbiota developed by the methods of the invention and to related nucleic acids, polypeptides and constructs including vectors.

Description

Patent No. # - Complete Specification No. Date: HORIZONTAL TRANSFER OF FUNGAL GENES INTO PLANTS (2) We, Agriculture Victoria Services Pty Ltd, of AgriBio Centre for AgriBioscience, 5 Ring Road, Bundoora VIC 3083, Australia, hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly bed in and by the following statement HORIZONTAL TRANSFER OF FUNGAL GENES INTO PLANTS (2) Divisional Application status Pursuant to Section 34 of the New d Patents Act 2013, this application is intended to be filed as a divisional of New Zealand application no 760713 filed on 27 July 2018.

Pursuant to Regulation 52 of the New Zealand Patents Regulations 2014, it is requested that this divisional application is given the earlier filing date of 27 July 2018.

Field of the ion The present invention relates to methods of ing and breeding organisms, in particular organisms which exhibit symbiotic behaviour with symbionts such as fungal endophytes or epiphytes or bacterial microbiome in plants, methods of creating or enhancing symbiotic relationships between plants and symbionts, and new organisms and symbiota developed thereby.

The invention also relates to nucleic acids ed in the methods, vectors including the nucleic acids, plant cells, plants, seeds and other plant parts ormed with the nucleic acids and vectors, and methods of using the nucleic acids and vectors. ound of the Invention Important plants, including forage s, legumes, trees, shrubs, and vines are commonly found in association with endophytes ing fungi, bacteria, viruses and microbes. Both beneficial and detrimental horticultural and agronomic properties result from such associations, including improved tolerance to water and nt stress and ance to insect pests. For e, in grasses, insect resistance may be provided by specific metabolites ed by the endophyte, in particular loline alkaloids and peramine. Other metabolites produced by the endophyte, for example lolitrems and ergot alkaloids, may be toxic to grazing animals and reduce herbivore feeding. Considerable variation is known to exist in the metabolite profile of endophytes. Endophyte strains that lack either or both of the animal toxins have been introduced into commercial cultivars.

However, there remains a need for methods of using organisms which exhibit tic our with endophytes. Difficulties in artificially breeding of these symbiota limit their usefulness. For example, many of the endophytes known to be beneficial to pasture-based agriculture exhibit low inoculation frequencies and are less stable in elite germplasm.

Moreover, in traditional breeding techniques, for e in forage grasses such as perennial ryegrass and tall fescue, grass varieties are bred using classic cross-breeding techniques and grass genotypes are selected for their superior characteristics, after monitoring their performance over a period of multiple years. The selected grass genotypes that form the experimental variety are then inoculated with a single endophyte and the resulting grassendophyte associations are evaluated for any able characteristics such as insect resistance. The individual experimental synthetic varieties deploying a single yte in them are then evaluated for agronomic performance and resulting animal performance by grazing animals over a period of years. This tion process may reveal that the single endophyte being deployed in the ent experimental synthetic varieties may not show vegetative and/or intergenerational stability in some of these varieties or the desired alkaloid profile conferred by the single endophyte may vary n different synthetic varieties failing to confer riate levels of insect resistance or causing animal toxicoses. It would be a significant development in the art if this time-consuming process could be accelerated or otherwise improved.

The Poeae tribe of the Poaceae family is composed of a range of eason turf and forage grass species, including those of sub-tribes Loliinae and Dactylidinae. ial ryegrass (Lolium perenne L.; sub-tribe Loliinae) is one of the most ant pasture crop s for the dairy industry, and it has consequently been a primary target for improvement using molecular biology and genetic technologies. Asexual fungal endophyte species of the genus Epichloë (syn. Neotyphodium) are nts of species ing to the Poeae tribe genera Lolium and Festuca.

The fungal endophyte species rely on the plant host for nutrition, reproduction, and protection from abiotic and biotic stress. Benefits to the host plant include enhanced competitive abilities, tolerance to pathogens, and resistance to animal and insect herbivory. Due to its agronomic importance, the molecular basis of the symbiosis has been investigated, and deterrence of insect herbivory is largely due to the production of bioactive alkaloids, as well as makes caterpillars floppy-like (mcf-like) gene products.

Horizontal gene transfer (HGT) has been a source of evolutionary y in both prokaryotes and eukaryotes. In flowering plant species, organelle genomes have served as both donors and recipients of gene er events. In st to organellar genes, transfer of nuclear genes to perms appears to have been rare, and has been confined to date to genes originating from prokaryotes or other plant species such as green algae, mosses and other angiosperms. A previous systematic in silico study, from investigation of four completely sequenced angiosperm genomes (those of Arabidopsis thaliana L., rice [Oryza sativa L.], sorghum [Sorghum bicolor L.], and poplar [Populus trichocarpa Torr. & A.Gray ex. Hook.]), found no evidence for HGT from fungal species, despite two and three highly reliable events for moss and lycophyte lineages, respectively [Richards, T. A. et al.]. It was consequently concluded that gene transfer from fungi into angiosperms must be exceedingly infrequent.

It is accordingly an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

Summary of the Invention The applicant has found evidence for ancient horizontal transfer of a number of fungal genes into angiosperm lineages. In particular the applicant has found ce for multiple events of horizontal gene transfer (HGT) from ancestral species of fungal endophytes (of the Epichloë genus) into grass species. In ial ryegrass (Lolium perenne L.), three fungus- originating genes were identified. One was specific to the Poeae subtribes Loliinae, ae and Dactylidinae. The gene has an enzymatic activity for degradation of components commonly found in cell walls of fungi. The gene may be previously ered to be specific to fungi. For example, the gene may be a ß-1,6-glucanase gene. The ß-1,6-glucanase genes may be isolated from fungal species such as Epichloë festucae, for example strains such as Leuchtm., Schardl and M.R. Siegel (the sexual counterpart to the perennial ryegrass endophyte), ea lixii Pat., and Trichoderma harzianum Rifai. r gene was specific to the Loliinae subtribe, and the other was conserved within the Triticeae and Poeae tribes, including common wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and oat (Avena sativa L.). This is the first report of horizontal er of a nuclear gene from a taxonomically distant eukaryote to modern ing plants and provides evidence for a novel adaptation mechanism in angiosperms.

In a first aspect, the present invention provides a substantially ed or isolated nucleic acid or nucleic acid fragment encoding a fungal cell wall degrading enzyme, preferably a glucanase, more preferably a β-1, 6-glucanase (BGNL), wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

In a preferred embodiment, the plant is a Loliinae or Dactylidnae species. In a more red embodiment the plant is from a Lolium, Festuca or Dactylis species.

The Lolium or Festuca species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the ryegrass or fescue species is a Lolium s such as Lolium perenne or Lolium arundinaceum which is otherwise known as Festuca nacea.

The Dactylis species may be of any suitable type. Preferably the Dactylis species is a Dactylis marina or Dactylis glomerata.

In a second aspect, the present invention provides a substantially purified or isolated c acid or nucleic acid fragment of a domain-of-unknown-function (DUF) gene, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

In a preferred embodiment, the plant is a Loliinae species. In a more red embodiment the plant is from a Lolium or Festuca s.

The Lolium or Festuca species may be of any suitable type, including Italian or annual ss, perennial ss, tall fescue, meadow fescue and red fescue. Preferably the ryegrass or fescue species is a Lolium species such as Lolium e or Lolium multiflorum which is otherwise known as Festuca perennis.

In a third aspect, the present invention es a substantially purified or isolated nucleic acid or nucleic acid nt encoding a fungal riptional regulatory-like (FTR) protein, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

In a preferred embodiment, the plant is a Triticeae and Poeae species. In a more preferred embodiment the plant is from a Triticeae or Aveneae species.

The Triticeae or e species may be of any suitable type, including barley, wheat and oat. Preferably the ryegrass or fescue species is a Triticum, Hordeum or Avena species such as Triticum aestivum, Hordeum vulgare and Avena sativa.

Thus, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment: encoding a fungal cell wall degrading enzyme; of a DUF gene; or encoding a FTR protein, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

In other words, the t invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment from a plant species, the nucleic acid or nucleic acid fragment being horizontally transferred from a fungal species, wherein the nucleic acid or nucleic acid fragment encodes a fungal cell wall degrading , is a DUF gene, or encodes a FTR protein.

By ‘nucleic acid’ is meant a chain of nucleotides capable of carrying genetic information. The term generally refers to genes or functionally active fragments or variants thereof and or other sequences in the genome of the organism that influence its phenotype. The term ‘nucleic acid’ includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double-stranded, optionally containing synthetic, tural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.

The c acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or doublestranded , ally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.

By ‘substantially purified’ is meant that the nucleic acid or nucleic acid fragment is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid or c acid fragment of the ion is derived, flank the nucleic acid or nucleic acid fragment. The term therefore includes, for example, a nucleic acid or nucleic acid nt which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g. a cDNA or a c or cDNA fragment produced by PCR or restriction endonuclease ion) independent of other sequences. It also es a nucleic acid or nucleic acid fragment which is part of a hybrid gene. ably, the ntially purified nucleic acid or nucleic acid fragment is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.

The term “isolated” means that the material is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such c acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

In preferred embodiments, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a fungal cell wall degrading enzyme includes a nucleotide sequence selected from the group consisting of the sequences shown in Sequence ID Nos: 1 to 4; and onally active fragments and variants thereof.

Also in preferred embodiments, the substantially purified or isolated nucleic acid or nucleic acid fragment of the DUF gene includes a nucleotide ce selected from the group ting of the sequence shown in Sequence ID No: 28 and sequences encoding the ptide shown in Sequence ID No: 29; and onally active fragments and variants thereof.

Also in preferred embodiments, the substantially purified or ed nucleic acid or nucleic acid fragment encoding a FTR protein includes a nucleotide ce ed from the group consisting of the sequence shown in Sequence ID No: 30 and ces encoding the polypeptide shown in Sequence ID No: 31; and functionally active fragments and variants thereof.

The substantially purified or isolated nucleic acid or nucleic acid of the present invention also es sequences complementary or nse to a sequence encoding a fungal cell wall degrading enzyme, to a DUF gene or to a sequence ng a FTR protein, and preferably sequences selected from the group consisting of sequences complementary or antisense to Sequence ID Nos: 1–4, 28 and 30; or complementary or antisense to the sequences encoding the polypeptides shown in Sequence ID Nos: 29 and 31.

In a preferred embodiment, the t invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment of a DUF gene or a mentary or antisense sequence to a nucleic acid or nucleic acid fragment of a DUF gene, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of: (a) the sequence shown in Sequence ID No: 28; (b) complement of the ce recited in (a); (c) sequences antisense to the sequence recited in (a); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).

By “functionally ” is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of modifying pathogen resistance in a plant, or creating or enhancing a symbiotic relationship between a plant and a symbiont. Such variants e naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant.

Preferably the functionally active fragment or variant has at least imately 80% identity to the relevant part of the above-mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% ty, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid s. By ‘conservative c acid changes’ is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having c acid changes which result in vative amino acid substitutions of one or more residues in the corresponding amino acid sequence. By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by r one of the same class, the classes being as follows: Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp ged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic: Asp, Glu Basic: Lys, Arg, His Other conservative amino acid substitutions may also be made as follows: Aromatic: Phe, Tyr, His Proton Donor: Asn, Gln, Lys, Arg, His, Trp Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.

In a fourth aspect of the present invention there is provided a c uct or a vector including a nucleic acid or c acid fragment according to the present invention.

In other words, the present invention provides a genetic construct or a vector including a nucleic acid or nucleic acid fragment according to the present invention, or sequences complementary or antisense thereto. Preferably the nucleic acid or nucleic acid fragment has a sequence selected from the group consisting of Sequence ID Nos: 1–4, 28, 30, and nucleic acids encoding the polypeptides shown in Sequence ID Nos: 29 and 31.

In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the t ion and a ator; said regulatory t, nucleic acid or nucleic acid fragment and terminator being operatively linked.

By ‘genetic construct’ is meant a recombinant c acid molecule.

By a ‘vector’ is meant a genetic construct used to transfer genetic material to a target cell.

By ‘operatively linked’ is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence. Preferably an operatively linked er is upstream of the associated nucleic acid.

The vector may be of any suitable type and may be viral or ral. The vector may be an expression . Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium enes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the plant cell.

The regulatory t and ator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.

Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art.

Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether tutive or ble expression is desired and the nature of the plant cell to be transformed (e.g. monocotyledon or ledon). Particularly le promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, the rice Actin er, and ryegrass endogenous OMT, 4CL, CCR or CAD promoters.

A variety of terminators which may be employed in the vectors of the present ion are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the ne synthase (nos) and the octopine synthase (ocs) genes.

The vector, in addition to the tory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different ations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene ]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.

As an alternative to use of a able marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS , northern and Western blot hybridisation analyses.

Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment.

Techniques for operatively linking the components of the vector of the present ion are well known to those skilled in the art. Such techniques include the use of linkers, such as tic linkers, for example including one or more restriction enzyme sites.

The nucleic acids and vectors of the present invention may be used to up-regulate or downregulate expression of corresponding genes in plants.

By ‘up-regulating’ expression of said gene is meant increasing expression of said gene and, as a result, the protein encoded by the gene, in a plant relative to a control plant.

By regulating’ expression of said gene is meant decreasing expression of said gene and, as a result, the protein encoded by the gene, in a plant relative to a control plant.

The up-regulation or down-regulation may be carried out by methods known to those skilled in the art. For example, a gene may be up-regulated by orating additional copies of a sense copy of the gene. A gene may be down-regulated, for example, by incorporating an antisense c acid, a frame-shifted or otherwise modified sense copy of the gene, or a nucleic acid encoding ering RNA .

The up- or down-regulation may be carried out by introducing into said plant an effective amount of a genetic construct including the gene or a modified form thereof, such as an antisense nucleic acid, a frame shifted copy of the gene or a nucleic acid ng RNAi.

The vectors of the present invention may be orated into a variety of plants, including monocotyledons (such as s from the genera Lolium, Festuca, Dactylis, Cynodon, Bracharia, Paspalum, Panicum, thus, Pennisetum, Phalaris, and other forage, turf and bioenergy grasses, corn, oat, sugarcane, rice, wheat and barley), dicotyledons (such as Arabidopsis, tobacco, legumes, Alfalfa, oak, ptus, maple, Populus, canola, soybean and chickpea) and gymnosperms (such as Pinus). In a preferred embodiment, the vectors are used to transform monocotyledons, preferably grass s such as Lolium, Festuca, Dactylis, Cynodon, Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and other forage, turf and bioenergy grasses, more preferably a Lolium species such as Lolium perenne or Lolium arundinaceum, including cultivars for forage and turf applications, or a Dactylis species.

Techniques for orating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those d in the art.

Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, re and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.

Cells incorporating the vector of the t ion may be selected, as described above, and then ed in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using s well known in the art, to produce successive generations of transformed plants.

In a fifth aspect of the present ion there is provided a transformed plant cell, plant, plant seed or other plant part, or plant biomass, ing digestible biomass such as hay, including, e.g. transformed with, a nucleic acid, genetic construct or vector of the present invention. ably the transgenic plant cell, plant, plant seed or other plant part is produced by a method according to the present invention.

The present invention also provides a transgenic plant, plant seed or other plant part, or plant biomass, derived from a plant cell of the present ion and including a nucleic acid, genetic construct or vector of the t invention.

The present invention also provides a transgenic plant, plant seed or other plant, part or plant biomass, derived from a plant of the present invention and including a nucleic acid, genetic construct or vector of the present invention.

The nucleic acid, genetic construct or vector of the present ion may be stably integrated into the genome of the plant, plant seed, other plant part or plant biomass.

The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons (such as grasses from the genera Lolium, Festuca, Dactylis, Cynodon, Bracharia, Paspalum, m, Miscanthus, Pennisetum, Phalaris, and other forage, turf and bioenergy grasses, corn, oat, sugarcane, rice, wheat and ), dicotyledons (such as Arabidopsis, tobacco, legumes, a, oak, Eucalyptus, maple, Populus, canola, soybean and chickpea) and gymnosperms (such as Pinus). In a preferred embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably a grass species, such as Lolium, Festuca, Dactylis, Cynodon, Bracharia, um, Panicum, Miscanthus, Pennisetum, Phalaris, and other , turf and bioenergy grasses, more preferably a Lolium species such as Lolium perenne or Lolium arundinaceum or a Dactylis species.

In a sixth aspect of the present ion there is ed a method of modifying pathogen resistance in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, c construct and/or a vector according to the present invention.

By “modifying pathogen resistance” is meant ing a plant’s y to protect itself from, or reduce the extent of, pathogen growth on or in the plant. Such pathogen resistance may subsequently protect the plant from, or reduce the extent of, fungal diseases generated by such fungal pathogens. Fungal diseases may include leaf spot, stalk rot, root rot, dying-off, choke disease and seedling wilt. Modifying pathogen resistance includes creating resistance to a pathogen in a plant, or sing or decreasing existing levels of resistance in a plant.

In a seventh aspect of the t invention there is provided use of a nucleic acid or nucleic acid fragment according to the t invention, and/or nucleotide ce information thereof, and/or single nucleotide polymorphisms thereof, as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention, and/or nucleotide sequence information thereof, and/or single nucleotide rphisms thereof, may be used as a molecular c marker for qualitative trait loci (QTL) tagging, mapping, DNA fingerprinting and in marker assisted selection, and may be used as candidate genes or perfect markers, particularly in ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention, and/or nucleotide sequence information thereof, may be used as molecular genetic markers in forage and turf grass improvement, e.g. tagging QTLs for dry matter digestibility, e quality, mechanical stress tolerance, disease resistance, insect pest ance, plant stature and leaf and stem colour.

In an eighth aspect of the present invention there is provided a method of creating or enhancing a symbiotic relationship between a plant and a symbiont carrying: a fungal cell wall degrading enzyme a DUF gene; or a FTR n, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment encoding the fungal cell wall degrading enzyme, of the DUF gene or encoding the FTR protein, or a functionally active fragment or variant thereof, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

In a preferred embodiment, the t invention es a method of ng or enhancing a symbiotic relationship between a plant and a symbiont carrying a domain-of-unknownfunction (DUF) gene; said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment of the DUF gene, wherein the nucleic acid or nucleic acid fragment is ed from a plant species.

In a ninth aspect of the present invention there is provided a method of creating or enhancing a symbiotic relationship between a plant and a symbiont carrying: a fungal cell wall degrading enzyme; a DUF gene; or a FTR protein, said method including introducing into said plant an effective amount of a nucleic acid or c acid fragment, genetic construct and/or a vector according to the present invention and introducing said symbiont into said plant.

The present invention also provides a plant endophyte symbiota ed by a method of the present invention.

Preferably, the fungal cell wall degrading enzyme is a ase, more ably a BGNL as hereinbefore described.

By “symbiont” is meant one or more sms that live within the body or cells of another organism, such as fungal endophytes or epiphytes or a ial microbiome in plants.

By “creating or enhancing a symbiotic relationship” is meant enabling a plant which otherwise would not form a tic relationship with a selected symbiont to form said symbiotic relationship or increasing the stability of the symbiotic relationship. For e, the nucleic acid or nucleic acid fragment, genetic construct and/or a vector according to the present ion may be introduced into plants such as rice, wheat and barley, which do not naturally contain an ortholog of the gene of the present invention, to faciliate establishment of symbiotic relationships n these plants and symbionts.

By “an effective amount” is meant an amount sufficient to result in an identifiable phenotypic change in said plant cells or a plant, plant seed or other plant part derived rom. Such amounts can be readily determined by an appropriately d person, taking into account the type of plant cell, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

In a tenth aspect, the present invention provides a method for selecting a plant for symbiosis with a symbiont carrying a fungal cell wall degrading gene, a DUF gene or a FTR gene, said method comprising a) determining the presence of a fungal cell wall degrading gene, a DUF gene or a FTR gene in said plant, b) measuring the level of expression of the gene identified in a), and c) using the expression level measured in b) to determine if the plant will form a symbiotic relationship with said symbiont. ably, the fungal cell wall degrading enzyme is a glucanase, more ably a BGNL as hereinbefore described.

In another red embodiment, the present invention provides a method for selecting a plant for symbiosis with a symbiont carrying a DUF gene; said method comprising a) determining the presence of a DUF gene in said plant, b) measuring the level of expression of the gene identified in a), and c) using the expression level measured in b) to determine if the plant will form a symbiotic relationship with said symbiont.

Techniques for ing the presence of a specific gene in a plant are well known to those skilled in the art. Such techniques may involve one or more of rase chain reaction (PCR), sequencing of PCR products, sequencing of c and/or mitochondrial DNA, and performing sequence analysis and comparisons to assess genetic variation.

Techniques for ing the level of gene expression in plant tissues are well known to those skilled in the art. Such techniques may involve one or more of e transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), northern ng, DNA microarray, Western blotting, 2D-Gel Electrophoresis, and Immunoassays.

In an eleventh aspect of the present invention there is provided a substantially purified or isolated fungal cell wall degrading enzyme, DUF or FTR protein, n said fungal cell wall degrading enzyme, DUF or FTR protein is isolated from a plant species.

In a red embodiment the fungal cell wall degrading enzyme is a glucanase, more ably a BGNL, and the plant is a Loliinae or Dactylidnae species. In a more preferred embodiment the plant is from the Lolium, Festuca or Dactylis s.

The Lolium or Festuca s may be of any suitable type, including Italian or annual ryegrass, perennial ss, tall fescue, meadow fescue and red fescue. Preferably the ryegrass or fescue species is a Lolium species such as Lolium perenne or Lolium arundinaceum which is otherwise known as Festuca arundinacea.

The Dactylis species may be of any suitable type. Preferably the Dactylis species is a Dactylis marina or Dactylis glomerata.

In a preferred embodiment, the present invention provides a substantially purified or isolated DUF protein, n said DUF protein is isolated from a plant species.

In a preferred embodiment, the plant from which the DUF protein is isolated is a Loliinae species. In a more preferred embodiment the plant is from a Lolium or Festuca species.

The Lolium or Festuca species may be of any suitable type, including Italian or annual ryegrass, perennial ss, tall fescue, meadow fescue and red fescue. Preferably the ryegrass or fescue species is a Lolium species such as Lolium perenne or Lolium multiflorum which is otherwise known as Festuca perennis.

In a preferred embodiment, the plant from which the FTR protein is isolated is a Triticeae, or e species, preferably Triticum aestivum, Hordeum vulgare or Avena sativa.

In a preferred embodiment, the substantially purified or isolated fungal cell wall degrading enzyme includes an amino acid sequence shown in Sequence ID No: 7; or a functionally active fragment or variant f.

In a preferred embodiment, the ntially purified or isolated DUF protein includes an amino acid ce shown in Sequence ID No: 29; or a functionally active fragment or variant thereof.

Thus, in a preferred embodiment, the present invention provides a substantially purified or isolated DUF protein, said protein including an amino acid sequence selected from the group consisting of: (a) the sequence shown in Sequence ID No: 29; and (b) functionally active fragments and variants of the sequence recited in (a).

In a preferred embodiment, the substantially purified or isolated FTR protein includes an amino acid ce shown in Sequence ID No: 31; or a functionally active fragment or variant thereof.

By ionally " in this context is meant that the fragment or variant is capable of modifying pathogen resistance in a plant, or ng or enhancing a tic relationship between a plant and a symbiont. Preferably, the fragment or variant has one or more of the biological properties of the fungal cell wall degrading , DUF or FTR protein. Additions, deletions, tutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the fragment or variant has at least approximately 60% identity to the relevant part of the above-mentioned sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. Such onally active variants and nts include, for example, those having conservative amino acid tutions of one or more residues in the ponding amino acid sequence.

Preferably the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.

In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing ptides are well known to those skilled in the art.

The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the ption following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Brief ption of the Drawings/Figures Figure 1: Amino acid sequence alignment of LpBGNL -glucanase gene; Sequence ID No 7) and fungus ß-1,6-glucanase gene products [Neotyphodium sp. (Sequence ID No 5), Epichloë festucae (Sequence ID No 6), Trichoderma harzianum (Sequence ID No 8), ea lixii (Sequence ID No 9)]. The aryl-phospho-beta-D-glucosidase domain is shown in boxes (Sequence ID Nos 10-14). Dash (-) in the amino acid sequences shows a gap.

Following the CLUSTAL W format, sk (*), colon (:) and dot (.) under the alignment denote ‘conserved amino acid residues’, ‘including conserved substitution(s)’ and ‘including semi-conserved substitution(s)’. NCBI UI is shown at the end of each sequence.

Figure 2: Genome structure of the LpBGNL and E. festucae ß-1,6-glucanase genes and genetic linkage analysis for LpBGNL. (a) Alignment of genome sequences from perennial ryegrass and Epichloë species. The filled and striped boxes in the upper part of Fig 2 (a) show the location of LpBGNL and the plant genome-related sequence, respectively. The black-filled box represents the BAC . The filled and empty boxes in the lower part of Fig 2 (a) show the location of the ß-1,6-glucanase gene and flanking gene, respectively. The transcription direction of the genes is indicated with the arrow. ponding gene sequences are connected with black dashed lines. (b) Alignment of coding regions of the LpBGNL and Epichloë ß-1,6-glucanase genes. The solid lines represent non-gene coding region of perennial ryegrass (upper part of Fig 2 (b)) and Epichloë (lower part of Fig 2 (b)) species. The location of the intron and aryl-phospho-beta-D-glucosidase domain (breaking line) are shown. (c) Genetic linkage map of perennial ryegrass LG5 with the LpBGNL-related locus. The LpBGNL-related marker locus is indicated with the arrow. Genetic distance (cM) is shown on the right side of the c markers.

Figure 3: The expression levels of LpBGNL and the E. festucae var. lolii ß-1,6-glucanase gene. (a) The sion level of LpBGNL in each tissue of perennial ryegrass. The y-axis shows normalised read count number (CPM). (b and c) The sion levels of the genes in E- and E+ perennial ryegrass genotypes. The x-axis shows time since the germination treatment, and y-axis shows CPM for LpBGNL (b) and the E. festucae var. lolii ß-1,6- glucanase gene (c).

Figure 4. Phylogenetic tree of LpBGNL orthologues and fungal ß-1,6-glucanase genes. The phylogram was generated based on amino acid sequence of the aryl-phospho-beta-D- glucosidase domain. S equences from angiosperm species are indicated in a dashed box; the remainder are fungal species. Asterisk (*) denotes species from which gene products have been med to have the ß-1,6-glucanase activity. For cocksfoot, three contigs (haplotypes 1-3) were generated, and amino acid sequences from those contigs were used.

For Dactylis marina, the contig without a putative premature stop codon was used. Strain and sequence contig (scaffold) fiers of the Genome Project at the University of ky website are shown in brackets. For the other fungal ces, NCBI UI is shown in brackets. The clade including the sequences from derma and eaas species were selected as an outer group to obtain a root of the phylogenic tree.

Figure 5. PCR screening for the LpBGNL sequence, and taxonomic classification of plant species described in the current study (a) PCR amplicons from perennial ryegrass genotype Impact04, cooksfoot, Dactylis marina, coast tussock-grass and harding grass, and a strain of E. festucae are visualised on the 2200 TapeStation instrument (Agilent Technologies, CA, USA). The target fragments are indicated with the large . Two genotypes of coast tussock-grass were subjected to the screening. The uppermost and lowermost bands show the position of upper and lower markers, respectively, of the D1000 Kit (Agilent). NTC denotes no-template control for the PCR assay. The small arrows indicate the positions of 400 and 500-bp fragments of the ladder. (b) Species classified into perms, the tribe Poeae, and sub-tribes Loliinae and Dactylidinae are indicated by the boxes on the right side of the phylogenetic tree. The species which were ve and negative in the PCR screening step are indicated by the plus (+) and minus (-) signs, respectively. The divergent points of plant species from other s (>1,000 MYA) and the clade monocotyledon from other plant species (ca. 150 MYA) are indicated with dashed arrows. The divergent points of sub-tribes Loliinae and Dactylidinae from the remaining Poeae species, especially from genus Poa, which is a closely related taxon (ca. 13 MYA), and fine-leaved fescue (Sheep fescue) from broad-leaved fescues (tall fescue and meadow fescue) (ca. 9 MYA) are ted with black . The triangle represents the putative period of HGT in the evolutionary lineage.

Figure 6. DNA sequence alignment of the two ypes from the C3 genotype (Sequence ID Nos 15 and 16). Dash (-) in the DNA sequence shows a gap, and asterisk (*) under the alignment denotes ‘conserved nucleotide’. DNA sequence ponding to the PCR primers for the indel-based genotyping assay is underlined.

Figure 7. Result of the indel-based genotyping assay. The PCR amplicons were visualised on an agarose gel including the SYBR™ Safe DNA Gel stain (Thermo Fisher Scientific, Waltham, Massachusetts, USA). EasyLadder I NE) was used as a size standard, and the 250 and 500 bp fragments are indicated with arrows. The genotype UI of the genetic mapping population is indicated in each lane. C3 and NTC stand for ‘the C3 genotype’ and ‘no-template control’.

Figure 8. PCR screening for the LpBGNL sequence using the LpBGNL locus-specific primers (LpBGNL_short). (a) PCR amplicons from perennial ryegrass genotypes Impact04 and C3, and perennial ryegrass-associated endophyte were ised on the 2200 TapeStation instrument using the High Sensitivity D1000 Kit nt). The uppermost and lowermost bands show the position of upper and lower markers, respectively, of the High Sensitivity D1000 Kit. (b) PCR amplicons from Lolium and a species (sub-tribe ae) were visualised on an agarose gel containing the SYBR™ Safe stain. Tall fescue genotypes from two cultivars (Demeter and Quantum) were subjected to the screening. NTC denotes ‘notemplate control’ for the PCR assay. (c) PCR amplicons from cocksfoot, Dactylis marina, coast tussock-grass and harding grass were visualised on the 2200 TapeStation instrument using the D1000 Kit. The uppermost and lowermost bands show the on of upper and lower markers, respectively, of the D1000 Kit. Two genotypes of coast tussock-grass were subjected, and the Impact04 genotype was used as a ve control.

Figure 9. DNA ces of the conserved ca. 750-bp region from plant species (Sequence ID Nos 17-27). Putative premature stop codons were found in is marina haplotypes 2 and 3.

Figure 10. PCR assay for confirmation of a cross-species amplification ty. PCR amplicons from cooksfoot, is marina, coast tussock-grass and harding grass were visualised on the 2200 TapeStation instrument using the D1000 Kit. The uppermost and lowermost bands show the position of upper and lower markers, respectively, of the D1000 Kit. The position of the target amplicons is indicated with a solid arrow. Size markers are indicated with outlined arrows. The Impact04 genotype was used as a positive l, and NTC denotes ‘no-template control’ for the PCR assay.

Figure 11. Genomic sequence of LpBGNL gene (Sequence ID No 1). The coding sequence is underlined (Sequence ID No 2), and the region in bold is a single intron (Sequence ID No Figure 12. LpBGNL cDNA ce (Sequence ID No 4).

Figure 13. Taxonomic relationships of plant and fungal species and phylogenetic tree of Epichloë-Triticeae/Poeae HGT candidates and corresponding fungal genes. (a) mic relationships of plant and fungal species and the presence/absence status of the LpFTRL and 632-like sequences. The ent point of plant species from other species (fungi and animals) is ted with the filled hexagon (>1,000 MYA), and the filled circle indicate the divergent point of s belonging to the Triticeae and Poeae tribes from other Poaceae species. The triangles ent species belonging to the Loliinae subtribe, or Epichloë or Claviceps genera. The presence/absence status of putative homologous (orthologous) sequence for each gene is shown on the right side of species (subtribe/genius) name, in which ‘In silico’ and ‘PCR’ denote the database and PCR-based screening s, tively. The plus (+) and minus (-) indicate presence and absence, respectively, and ‘N/A’ stands for ‘not analyzed’. The ancient Epichloë-Poeae/Triticeae HGT events are shown with curved arrows. The phylograms for LpFTRL(b) and LpDUF3632(c) generated based on predicted amino acid sequences. Sequences from plant species are Barley, Tausch’s goatgrass and perennial ryegrass. For LpFTRL, the sequence from Trichoderma was selected as an outer group to obtain a root of the phylogenic tree (b). The DUF3632-like sequence was specific to Epichloë and Claviceps species, and no corresponding sequence was found in other related species of the Clavicipitaceae family, except for andula ipomoeae, which is shown with an asterisk. No sequence was, therefore, selected as an outer group in the phylogram for LpDUF3632 (c). For fungus sequences, strain and sequence identifiers of the Genome Project at the University of Kentucky are shown in brackets. For plant sequences, NCBI UI is shown in brackets.

Figure 14. The expression levels of HGT candidates and corresponding fungal endophyte genes in perennial ryegrass. (a) The sion levels of LpFTRL and LpDUF3632 in each tissues of the perennial ryegrass Impact04 genotype. (b) The expression levels of the HGT ates and corresponding fungal genes in E+ and E- perennial ryegrass genotypes. (c) A time course gene expression analysis for the HGT candidates and corresponding fungal genes in perennial ryegrass seeds and young seedlings. The x-axis shows tissue types (c and d) or period after a germination treatment (e and f), and normalised read count s [counts per million (CPM)] are indicated on the y-axis. The bars or lines show the expression levels of the HGT candidates in E- and E+ plants, respectively, and the expression levels of corresponding fungal genes in E+ . Note that the scales of the y-axes are not uniformed.

Figure 15. PCR-based detection of the oë-Poeae/Triticeae HGT candidate from retail products. PCR assay results using the primers designed for LpFTRL. The PCR products were visualised on an agarose gel, using the SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific). The size (bp) of PCR amplicons is indicated on the right side of each image. As a control ment, PCR primers for the florigen candidate gene (Hd3a in rice, and FT in wheat and barley) were used. For tration of e of Epichloë and Claviceps species in the retail products, PCR primers specific to the fungal species were used. The gDNA samples from the perennial ryegrass genotype Impact04 and E. festuca were used, as positive controls for amplification with the plant and fungus-specific primers, respectively.

With the PCR primers specific to Claviceps species, amplification from E. festucae gDNA template was observed, presumably due to sequence similarity between Epichloë and eps species. ‘NTC’ stands for ‘no DNA template control’.

Figure 16. Nucleotide sequence of Lolium perenne DUF3632-like protein gene.

Figure 17. Amino acid sequence of Lolium e DUF3632-like protein gene.

Figure 18. Nucleotide sequence of Lolium e fungal transcriptional regulatory (FTR) gene.

Figure 19. Amino acid sequence of Lolium e fungal transcriptional regulatory (FTR) gene.

Detailed ption of the Embodiments Example 1: Identification of a putative plant ß-1,6-glucanase gene A single genotype of perennial ryegrass (Impact04) was subjected to whole-genome shotgun and transcriptome sequencing using the Illumina HiSeq platform (NCBI BioProject Accession: PRJNA379202) . De novo assembly of sequencing reads generated a 7.2 kb genomic DNA sequence contig [NCBI GenBank unique identifier (UI): KY771173], which contained a putative ß-1,6-glucanase gene. The gene-like sequence was designated LpBGNL m perenne ß-1,6-Glucanase-Like). LpBGNL showed 74-90% and 72-82% identity at the DNA and amino acid sequence levels, respectively, to ß-1,6-glucanase genes of fungal taxa, such as E. festucae, H. lixii, and T. num (Fig. 1, Table 1). The full genomic sequence of LpBGNL is shown in Figure 11. No matching sequence, however, was identified in the full genome sequences of plants such as A. na, rice, podium distachyon (L.) P. Beauv., barley (Hordeum vulgare L.) or wheat (Triticum aestivum L.), based on database searches.

T. harzianum (1103 bp) - 74% 73% (1112 bp) 73% (1288 bp) 96% (1320 bp) ce identity (length of homologus sequence) H. lixii 75% (1101 bp) 74% (1101 bp) 74% (1283 bp) - - Neotyphodium 89% (1140 bp) 98% (1164 bp) - - - E. festucae 90% (1140 bp) - - - - NCB UI Lp BGNL) KY771173 ( EF015481.1 31.1 EU747838 X79197.1 Species Perennial ryegrass Table 1 Epichloe festucae Neotyphodium sp. Hypocrea lixii Trichoderma harzianum 5 Example 2: Genomic and genetic characterisation of LpBGNL Methods Plant materials and DNA extraction Details of plant genotypes are summarised in Table 2. Genomic DNA was extracted from young leaves of plants and fungal endophyte mycelium using the DNeasy plant mini kit (QIAGEN, Hilden, Germany).

PCR amplification Locus-specific primers were designed using the Sequencher software (GENECODE, MI, USA) and the PCR primers are listed in Table 4. PCR amplification was performed with MyFi polymerase kit (BIOLINE, London, UK). PCR amplicons were visualised on the 2200 TapeStation ment.

Short-read sequencing of BAC clones and amplicons The BAC-based genomic library was screened h use of PCR. For the phylogenomic is, PCR s were designed to obtain genomic nts from Loliinae and idinae species (Table 4). Sequencing libraries for the MiSeq platform (Illumina, San Diego, California, USA) were prepared from the BAC clones and PCR amplicons, following the previously described based method [Shinozuka et al]. The library was characterised with the TapeStation and Qubit instruments (Thermo Fisher Scientific). The outcome reads were assembled with the Sequencher and SOAPdenovo programs.

Genetic linkage analysis PCR primers were designed to detect the indel polymorphism within the LpBGNL sequence (Table 3). Genetic e analysis was performed through use of the p150/112 reference genetic mapping population of perennial ss using the P 3.0 ation.

PCR amplification Locus-specific primers were designed using the Sequencher software (GENECODE, MI, USA) and the PCR primers are listed in Table 4 PCR amplification was performed with MyFi polymerase kit (BIOLINE, London, UK). PCR amplicons were visualised on the 2200 TapeStation instrument.

Short-read cing of BAC clones and amplicons The BAC-based genomic library was screened through use of PCR. For the phylogenomic analysis, PCR primers were designed to obtain genomic fragments from Loliinae and Dactylidinae species (Table 4). cing libraries for the MiSeq platform (Illumina, San Diego, California, USA) were prepared from the BAC clones and PCR amplicons, following the previously described MspJI-based method. The library was characterised with the TapeStation and Qubit instruments (Thermo Fisher Scientific). The outcome reads were assembled with the Sequencher and SOAPdenovo ms.

Genetic linkage analysis PCR primers were ed to detect the indel polymorphism within the LpBGNL sequence (Table 3). c linkage analysis was performed through use of the p150/112 reference genetic mapping population of perennial ryegrass using the JoinMAP 3.0 application.

., Reference Shinozuka et al 2010 2010 2010 2010 2017 Hand et al., Hand et al., Hand et al., Hand et al., UI IBERS: BA13157 IBERS: BF1199 IBERS: BL2643 SARDI: 778 SARDI: 38013 SARDI: 41525 Genotype or cultivar Impact04 Genotype from Aberystwyth (Great Britain) Genotype from Tadham Moor (Great Britain) Demeter m Genotype from Ponterwyd (Great n) Currie Wild genotype from Algeria Wild genotype from Australia Landmaster Huds.

Scientific name Lolium perenne L. Lolium temulentum L. Festuca sis a arundinacea Schreb. Festuca arundinacea . Festuca ovina Dactylis glomerata L. Dactylis marina Borrill Poa poiformis (Labill.) Druce Phalaris aquatica L. ) Species Table 2 Common name Perennial ryegrass Darnel Meadow fescue Tall fescue Tall fescue Sheep fescue Cocksfoot (Orchard grass) (Dactylis marina Coast tussock- grass Harding grass (Phalaris) Sichuan Agricultural University INSTITUTE Auburn University Teagasc Auburn University Auburn University Subm itted by KOREA POLAR CH 1 run, 162.8M spots, 32.6G 31G G 10.2G 25.9G 8.9G bases bases bases M spots, 9.3 bases bases bases Data size 1 run, 153.38M spots, 1 run, 129.3M spots, 1 run, 46.3 1 run, 50.4M spots, 1 run, 44.4M spots, Illumina HiSeq Instrument 2000 Illumina HiSeq 2000 Illumina HiSeq Illumina HiSeq 2000 Illumina HiSeq 2000 2000 na HiSeq 2000 UI SRX738187 SRX465632 SRX745831 SRX745855 SRX745858 SRX669405 Dactylis ata L. (orchardgrass) Deschampsia antarctica Poa annua Poa supina Poa infirma Phalaris aquatica Table 3 Species , , , is Phalaris DNA template perennial ryegrass, darnel, meadow fescue, tall fescue, sheep fescue, cocksfoot, Dactylis marina Phalaris aquatica, perennial darnel sheep fescue aquatica ryegrass- associated endophyte coast tussock- grass, ryegrass- associated endophyte, perennial ryegrass genomic library p150/112 genetic mapping population perennial ryegrass, tall fescue, meadow fescue ial ryegrass, cocksfoot, Dactylis marina coast tussock- grass, cocksfoot, Dactylis marina perennial ryegrass, cocksfoot, Dactylis marina coast tussock- grass, aquatica, perennial on size* 178 bp 253/306 bp 1452 bp 1590 bp 1067 bp 248 bp 235 bp 1147 bp 415 bp 126 bp** Reverse ACTGCACATGGAGCTTGTTG CGTCGCTCATCATCCATGGC CAGATATCTTGATACACATTCC CAGATATCTTGATACACATTCC CTGCCCGTTCACGGTGCGAT TTTGTCGTCCGGGCTCACGC TGGATGCGCYTCGTCATCC ATCCTCCTGGCAAGCTGAATG ATGATGGTGTTGGCCGCGTT CCGATGATGGTGTT GCCCGTCTGACGGGGCACAG CATCAACAAGATCAGGGGCG CACGACTTGGCTGCTTTCAA CCGAGTTCGACTG  3’) Sequence (5’ Forward GTCGGCATGATTGAGGTTCT AGGGCATCAACAAGATCAGG CGCGCCTAATCCTCTCCTCT TGCTTGCCCTTCAGGAGGCT AAGGAGAGCCTCCA TAACGCTCAACGGGGACG LpHistone** LpABCG5 LpABCG6 Table 4 Primer name LpBGNL_short LpBGNL_indel _long1 LpBGNL_long2 LpBGNL_long3 LpBGNL_long4 LpBGNL_cons * Length of DNA fragment based on the perennial ryegrass genome sequence -Reductase Genes from ial Ryegrass Cinnamoyl- CoA . 2010 Functional Analyses of Caffeic Acid O- Methyltransferase and –3373. (doi:10.1105/tpc.109.072827) ** Reference: Tu, Y. et al ). The Plant Cell 22, 3357 (Lolium perenne An in-house bacterial artificial chromosome (BAC)-based c library of ial ryegrass had previously been constructed from endophyte-devoid (E-) individuals of the cultivar Grasslands Nui. sed screening of the y identified two positive clones, designated LpBAC94-B20 and LpBAC125-N24. De novo sequence analysis and assembly identified the presence of LpBGNL in both clones. The gene was located within 39 kb- and 24 kb-contigs (NCBI UI: KY771171 and KY771172) of LpBAC94-B20 and LpBAC125-N24, respectively, along with a sequence (ca. 2 kb in length) showing similarity at a DNA sequence identity of 82% to a Zea mays transposon-related gene (NCBI UI: 92.1) (Fig. 2a). A 11 kb-contig of E. festucae genome sequence (NCBI UI: EF015481), which includes the corresponding ß-1,6-glucanase gene, was obtained from the NCBI database and was shown to contain three other genes located within a 5 kb ce from the ß-1,6-glucanase gene.

In the BAC clone-derived contigs, however, no sequences similar to these flanking genes were identified. Putative coding regions for the E. festucae ß-1,6-glucanase and LpBGNL genes were identified (Fig. 2b). A single intron was found in both ces, and comparison of the exonic and ic regions identified 4 insertion-deletion ) variations between them. Although the position of the intron was conserved, it seemed that almost all intron sequence was replaced in perennial ryegrass, due to insertion and deletion events. Although the coding regions were relatively highly conserved, no sequence similarity was found in the flanking sequence of the coding regions. A BLAST search of 1.5-kb upstream and downstream sequences of LpBGNL identified partial sequence similarity to the genomes of wheat and rice, while the ponding upstream and downstream sequences of the E. festucae ß-1,6-glucanase gene included partial sequences of the flanking genes (Fig. 2b).

Sequencing of PCR products generated using -specific s identified a 51-bp intron-located rphism between haplotypes of the heterozygous parent (C3 genotype) of the perennial ryegrass p150/112 genetic linkage g population (Figure 6), which facilitated development of an indel-based DNA marker. From the p150/112 population, 48 individuals were genotyped (Figure 7), and the LpBGNL-related marker locus was assigned to a distal region of perennial ryegrass linkage group (LG) 5 (Fig. 2c).

Example 3: LpBGNL gene expression analysis Methods Gene sion analysis The transcriptome sequencing reads from Impact04 tissues were mapped against Impact04 genome contigs (>999 bp) for filtering. The number of reads which contained LpBGNL ce (no sequence mismatch for 60 bp or longer) were counted as LpBGNL-derived reads. For gene expression in seedlings, E+ and E- seeds of perennial ryegrass cultivar Alto were subjected to germination ent by placement on wet filter paper in the dark for 2 days followed by seedling growth under full-light conditions [44]. RNA was extracted with a CTAB extraction method, and sequencing libraries were prepared using the SureSelect -specific RNA library preparation kit (Agilent). Sequencing analysis was performed on the Illumina HiSeq 3000 platform. The full LpBGNL cDNA sequence is shown in Figure 12.

Results Expression of LpBGNL was determined using data from the transcriptome sequence of the Impact04 genotype (NCBI BioProject Accession: PRJNA379202) . Sequencing reads ponding to LpBGNL were identified from leaf, root and flower s, and higher sion levels [based on counts per million reads (CPM)] were detected in root and flower than in leaf (Fig. 3a, Table 6). Specificity of gene expression was examined using endophyteinfected (E+) and E- perennial ryegrass seeds and seedlings. Due to sequence divergence, sequencing reads corresponding to the plant and fungal gene could be reliably discriminated (Table 7). Read counts were very low for both E+ and E- seeds immediately after the germination treatment (Fig. 3b). Although no scale morphological change was ed during the following two days, the read counts substantially increased in both s. The counts remained at relatively high levels in young seedlings at 5 and 10 days after treatment. As similar trends were observed for both E+ and E- genotypes, presence of yte did not significantly affect LpBGNL expression pattern. Expression of the endogenous E. festucae var. lolii ß-1,6-glucanase gene was observed in E+ seedlings, but the read count approach revealed relatively low levels throughout the 10 days, in contrast to LpBGNL (Fig. 3c, Table 7). glucanase gene CPM 0.14 0.17 0.44 0.43 0.16 0.41 - β 1,6 - Epichloe Counts 5 7 28 25 9 22 CPM 0.11 2.59 12.47 49.38 32.26 72.58 CPM 32.7 55.0 3.7 2.5 3.7 12.1 5.9 9.2 66.5 Lp BGNL Counts 4 109 802 2838 1847 3901 Counts 479 798 87 49 54 272 113 154 767 E+ individual 36,389,285 42,014,889 64,295,493 57,472,563 57,259,134 53,748,829 Total reads ,730 14,515,287 23,613,687 19,292,712 14,752,797 22,525,848 13,271,284 16,738,932 11,536,030 Total reads glucanase gene CPM 0.00 0.02 0.04 0.01 0.02 0.00 Library Root tip Root e) Leaf middle 1 Leaf middle 2 Leaf middle 3 Leaf tip 1 Leaf tip 2 Leaf tip 3 1,6 Flower Epichloe - β Counts 0 1 3 1 1 0 BGNL CPM 0.35 0.48 36.77 87.56 57.32 64.31 Lp Counts 19 30 2751 6285 3391 6565 - individual E Total reads 54,109,844 62,109,304 74,819,980 71,779,944 59,159,973 102,085,102 0 h 4 h 1 day Table 6 Table 7 2 days 5 days 10 days [Link] http://www.megasoftware.net/ Example 4: Phylogenetic analysis of plant and fungal ß-1,6-glucanase(-like) genes Method In silico analysis The DNA sequences of fungal ß-1,6-glucanase genes were obtained from the NCBI (http://www.ncbi.nlm.nih.gov/) database and the Genome Project at the University of Kentucky e. Putative ogous sequences were sought in the NCBI, Brachypodium hyon (http://www.brachypodium.org/) and Ensembl (http://plants.ensembl.org/index.html) databases. Non-synonymous and synonymous nucleotide substitution rates (Ka and Ks, respectively) were calculated using the Synonymous Non-synonymous Analysis Program (SNAP; http://www.hiv.lanl.gov/).

Alignment of DNA sequences was performed with the CLUSTALW program (http://www.genome.jp/tools/clustalw/) with the default parameters. eny was generated with the MEGA7 program (http://www.megasoftware.net/).

The presence of LpBGNL orthologues in other Poeae species was determined by PCR-based screening. LpBGNL-specific primers were designed and short DNA fragments (178 bp in length) were amplified from genomic DNA templates of darnel (Lolium temulentum L.), meadow fescue (Festuca pratensis Huds.), tall fescue (Festuca arundinacea Schreb.), sheep fescue ca ovina) (Table 2, Figure 8). Products were also obtained from those of oot/orchard grass (Dactylis glomerata L.) and is marina Borrill, but not from those of coast tussock-grass [Poa poiformis (Labill.) Druce] or harding grass/phalaris (Phalaris aquatica L.). DNA fragments were not amplified from genomic DNA templates of E. festucae var. lolii., confirming the specificity of the oligonucleotide primers that were used . Alignment of LpBGNL and the fungal genes identified a conserved region ca. 750 bp in length, corresponding to the hospho-beta-D-glucosidase domain. For a phylogenetic analysis, DNA fragments, ing the aryl-phospho-beta-D-glucosidase , were ied from the selected Loliinae and Dactylidinae species. De novo amplicon sequence analysis and ly obtained a single sequence contig of the 750 bp region for each Lolium and Festuca species (Figure 9). For each Dactylis species, three contigs (haplotypes) were generated. A putative premature stop codon was found in two haplotypes of is marina, and the haplotypes with the premature stop codon were excluded from the further analysis.

Fungal ß-1,6-glucanase gene-like sequences were ed from the NCBI database and the Genome Project at the University of Kentucky website. Phylogenetic analysis with the m likelihood method was performed, and the plant species-derived sequences were found to be clustered in the phylogram with those from Epichloë (Neotyphodium) species (Fig 4). The sequences from other fungi were more distantly related to the plant sequences.

Synonymous and non-synonymous nucleotide substitution (Ks/Ka) ratios were calculated using the 750 bp sequences. Both Ks and Ka values between the oë and plant species were substantially lower than those between the Epichloë species and other fungi (H. lixii and T. harzianum) (Table 2). The Ks/Ka ratios for candidate LpBGNL orthologues were between 0.027-0.221, lower or equivalent to values from the fungal ß-1,6-glucanase genes (0.057- 0.275).

In order to verify presence/absence boundaries, a set of PCR primers was designed to amplify 415 bp fragments within a region highly conserved between plant and fungal ß-1,6- glucanase(-like) genes. This assay confirmed the e of the gene in the Poa and Phalaris species samples (Fig. 5). As a control experiment for capacity to amplify cross- species, PCR was med with primers specific to perennial ryegrass histone H3 and ate plant architecture genes, such as the ATP -binding cassette protein mily G 5 and 6 genes (LpABCG5 and LpABCG6, respectively). Although all primers were ed based on perennial ryegrass sequence, PCR amplification from coast k-grass and harding grass/phalaris was observed, except for the combination of ABCG 5 primers and one of the Poa genotypes (Figure 10). Database searches were performed using published shortread sequencing data on the NCBI Sequence Read Archive (SRA; https://www.ncbi.nlm.nih.gov/sra). Sequences significantly matching the LpBGNL and E. festucae ß-1,6-glucanase genes were found from Dactylis s, but not from Poa or Phalaris species (Table 9, Figure 9). No significantly matching sequence was obtained from Antarctic hair grass (Deschampsia antarctica É. Desv.), which is believed to be taxonomically closer than Poa species to members of the sub-tribes Loliinae and Dactylidinae (Fig 5b). As a control analysis, sequences similar to the LpABCG 5 and 6 genes were , g to identification of matching sequences from all tested Poeae species, including those belonging to both Poa and Phalaris. um ) ia n 2 7 T. r z 5 h a 0.6396, 0.1469 (0.2297) (0.2304) 0.1186, 0.0068 . 0 ( 0 0.6383, 0.1453 (0.2276) 0.6563, 0.1432 (0.2182) 0.6494, 0.1449 (0.2231) , 0.1407 0.6424, 0.1492 (0.2323) 0.6670, 0.1537 (0.2068) H. lixii 0.6116, 0.1507 (0.2341) - (0.2464) 0.6041, 0.1491 (0.2468) , 0.1470 (0.2339) 0.6093, 0.1487 (0.2441) 0.6598, 0.1442 0.6363, 0.1488 (0.2339) 0.6546, 0.1533 (0.2186) hodium sp. 0.2539, 0.0629 (0.2477) - 0.2533, 0.0646 0) 0.2369, 0.0655 (0.2766) 0.2305, 0.0638 (0.2770) 0.2969, 0.051 0.0896, 0.0247 (0.2751) - (0.1717) nase gene ß- 1,6 E. festucae 0.1961, 0.0501 (0.2553) - 0.1957, 0.0518 (0.2647) 0.1887, 0.0535 (0.2838) , 0.0519 5) 0.2207, - - 0.0433 (0.1961) Sheep fescue 0.3333, 0.0416 (0.1247) 0.3575, 0.0433 (0.1211) , 0.0467 (0.1411) 0.3247, 0.045 (0.1387) - - - - BGNL Meadow Fescue 0.0991, 0.0102 (0.1029) 0.1359, 0.0085 (0.0625) 0.0308, 0.0068 (0.2213) - - - - - Candidate orthologue of Lp Tall fescue 0.1302, 0.0102 (0.0783) 0.1670, 0.0085 (0.0509) - - - - - - ryegrass Darnel 0.0622, 0.0017 (0.0273) - - - - - - - Perennial ryegrass Darnel ryegrass Tall fescue Meadow Fescue Sheep Fescue Epichloe festucae Neotyphodium sp. Hypocrea lixii Table 8 Ks, Ka (Ka/Ks) Results The high level of DNA sequence rity to the fungal genes, and absence of LpBGNL-like sequences from other representative perm species ted that LpBGNL was obtained from a fungal species through HGT. Genomic and genetic characterisation was consequently performed in order to demonstrate that LpBGNL is located on a perennial ryegrass chromosome. Furthermore, LpBGNL orthologues were found to be present in other Loliinae and Dactylidinae species. It is hence unlikely that LpBGNL was an assembly or annotation artefact, even though LpBGNL shows unusually high DNA sequence similarity (ca. 90%) to the ß-1,6-glucanase genes of contemporary species descended from the putative donor, when compared to other horizontally transferred genes in eukaryotes.

The PCR-based screening and database searches suggested that the ß-1,6-glucanase-like gene is present in only a limited number of Poeae s including the genera Lolium, Festuca and Dactylis, which are confined to the sub-tribes Loliinae and Dactylidinae. The phylogenetic analysis suggested a common origin of the oë-derived ß-1,6-glucanase genes and LpBGNL orthologues, and the close relationship between the LpBGNL orthologues of contemporary Loliinae and Dactylidinae grasses suggests that the gene may have been introduced into the genome of a common ancestor of the sub-tribes by a single transfer event. The HGT event may consequently have occurred between ca. 9 to 13 n years ago (MYA), based on the ted time of divergence of the two ibes from other Poeae lineages (Fig. 5b).

The Ks/Ka ratios between plant ß-1,6-glucanase-like genes (0.027-0.221) were not substantially different from those of the fungal ß-1,6-glucanase genes, and those of LpABCG5 and LpABCG6 (0.166 and 0.238). This suggests that LpBGNL may have been subjected to selection pressures. Although similar Ks/Ka ratios were obtained from the perm and fungus groups, DNA mutation rates between those two groups may not be equivalent. From cocksfoot/orchard grass and is marina, three haplotypes of the aryl- phospho-beta-D-glucosidase domain were identified, and two haplotypes obtained from Dactylis marina contained a putative premature stop codon. As these Dactylis species have autopolyploid s, the ß-1,6-glucanase-like genes in these species may have been subjected to unique selection pressures due to c redundancy, when compared with genes in the other plant species.

As asexual symbiotic Epichloë species grow as hyphae between cells of vegetative aerial tissues in Poeae species, it is likely that the close physical proximity of both partners in the sis may have facilitated an HGT event, similar to the physical contacts n tic and host plants. The conservation of the intron position between the LpBGNL and Epichloë ß-1,6-glucanase genes suggests that a part of the endophyte genome including the ß-1,6-glucanase gene, rather than a reverse-transcription product of yte gene mRNA, was incorporated into the recipient genome. In prokaryotes, transformation is a prevalent mechanism of gene exchange, in which HGT occurs through uptake of exogenous doublestranded DNA by the recipient cell. 4) G6 -38) -26) C e- 3 (4 pA B % L 94% (5e- 32) 97% (1e 95% (1e- 33) 90% (6e- 27) 92% (2e 4 9 LpABCG5 99% (3e- 41) 99% (7e- 42) 100% (5e- 43) 99% (8e- 42) 100% (2e- 43) 97% (5e- 39) -glucanase gene E. festucae ß -1,6 99% (6e- 41) N.S. N.S. N.S. N.S. N.S. LpBGNL 95% (8e- 35) N.S. N.S. N.S. N.S. N.S. Data size 32.6G bases 31G bases 25.9G bases 8.9G bases 9.3G bases 10.2G bases Source Transcriptome Genome Transcriptome Transcriptome Transcriptome Transcriptome UI SRX738187 632 SRX745831 SRX745855 SRX745858 SRX669405 Table 9 s Dactylis glomerata (orchardgrass) Deschampsia antarctica Poa annua Poa supina Poa infirma Phalaris aquatica Example 5: Loss of function of LpBGNL Given the role of plant-encoded ß-1,6-glucanase enzyme in the establishment of a stable symbiotic relationship with an endophyte, a loss of function is is carried out. A LpBGNL-like gene is removed from the genome of a ryegrass plant which normally forms a stable onship with an Epichloë endophyte. The stability of the plant-endophyte association is then evaluated in such plants compared with unmodified control plants.

Example 6: Gain of on of LpBGNL Given the role of plant-encoded ß-1,6-glucanase enzyme in the establishment of a stable tic relationship with an endophyte, a gain of function analysis is carried out. A LpBGNL-like gene is introduced into plants such as rice and wheat that do not normally form stable symbiotic relationship with Epichloë endophytes. The stability of the plant-endophyte association is then evaluated in such plants compared with unmodified control plants.

Example 7: LpBGNL as a promoter of natural stable associations with asexual Epichloë Fungal glucanases have been reported to be specifically secreted into plant apoplasts during endophyte ion, and may play a role in provision of ion to the infecting endophyte, l of branching of the endophyte hyphae, and protection of plant tissues from infection of other fungal pathogens. The plant-encoded enzyme may participate in one or more of these processes, and so contribute to establishment of a stable symbiotic relationship.

Although oë endophytes do not colonise root tissues, a relatively high level of expression of LpBGNL was observed in root tissues. Hence, the plant-encoded enzyme may function to protect against infection by orne fungal pathogens. Similarly, active expression in flowers may suggest the capacity to protect against fungal pathogens such as Epichloë a, which causes choke disease. However, some Festuca and Dactylis species are relatively susceptible to infection by E. typhina, even though those species presumably also possess the ß-1,6-glucanase-like gene. As natural stable associations with asexual Epichloë ytes are confined to the Poeae lineages that possess LpBGNL-like genes, the ancestral HGT event (which might have occurred from a sexual pathogenic Epichloë-like s) may have provided pre-adaptive conditions for the contemporary symbiosis. The evidence for selective pressure on the gene is suggestive.

As major symbionts, the asexual Epichloë species provide abiotic and biotic stress tolerance to grass species of the Poeae tribe. Tolerance to ebrate herbivory is a wellcharacterised benefit to the host, partially attributable to the effects of a makes caterpillers floppy-like (mcf-like) gene. The ke gene was horizontally transferred into the endophyte genome from a bacterial species 7.2-58.8 MYA. Hence, multiple horizontal transfer events, including transfer of the LpBGNL-like gene as described in the present study, may have been involved in the establishment of the t stable symbiotic relationship.

Given the role of plant-encoded LpBGNL enzyme in the establishment of a stable symbiotic relationship with an endophyte, a stable symbiotic onship with a plant and an yte may be created, which otherwise may not occur. The introduction of BGNL into plants which do not contain BGNL and are thus not able to form a stable symbiotic onship with endophytes, may enable the establishment of such a relationship.

Example 8: A role for LpBGNL in pathogenic resistance The potential role of a ß-1,6-glucanase-like gene in protection against other, pathogenic, fungal species is of particular interest. Species such as T. num are mycoparasites of fungal phytopathogens, and this property is d to glucanase activity. Fungal-derived genes for anti-fungal enzymes such as endochitinases and glucanases have also been used for generation of transgenic plants with enhanced pathogen resistance.

To generate transgenic plants with enhanced en resistance using the LpBGNL-like gene the LpBGNL-like gene is transferred into the genomes of crop plants such as rice and wheat and then the resistance of the transformed plants to a range of fungal diseases is evaluated.

Example 9: ß-1,6-glucanase as an indicator of stable associations with asexual Epichloë Given the role of the encoded ß-1,6-glucanase enzyme in the ishment of a stable symbiotic relationship with the endophyte, the presence and level of expression of a plant- encoded glucanase may be used to predict the likelihood of the plant forming a stable association with an endophyte carrying the ß-1,6-glucanase, such as Epichloë.

The method involves ing a plant for screening for symbiosis with a symbiont such as an Epichloë yte. The first step requires determining whether the plant to be screened contains the BGNL gene, through genetic analysis. If the plant selected for screening contains the BGNL gene, the second step is to determine the level and location of expression of the gene. The expression level of BGNL measured is then used to determine if the said plant screened would form a symbiotic relationship with the Epichloë endophyte.

High levels of expression in the root tissues and in the flowers of the plant are indicative of the plant screened being able to form a stable association with the endophyte.

Example 10: Identification of putative plant FTRL and DUF genes Using non-polyadenylated RNA from an endophyte-absent (E-) perennial ryegrass individual, a sequencing library was ed. A single Illumina MiSeq run generated a total of 014 reads from the library. A dataset of unique RNA reads, ing 1,424,274 sequences, was generated. A BLAST search of the perennial ryegrass (E-) transcriptome shotgun assembly (TSA) and unique RNA read datasets against the Epichloë festucae transcriptome data identified 88 and 123 sequence rity hits, respectively. Sequences putatively derived from microbiome, highly conserved genes between fungi and plants, such as actin and ubiquitin genes, and low ent matches were excluded through a manual examination, and 2 novel HGT candidates were identified.

From the TSA data, a sequence [unique identifier (UI): ID_150936_C1449060_17.0] showing a relatively high similarity (85%) to an E. festucae unknown protein gene (UI: EfM3.066060.partial-2.mRNA-1) was identified. Due to a similarity of predicted amino acid sequence to a fungal transcriptional regulatory (FTR) protein nk UI: CRL18938; identity: 48%, e-value: 2e-150) of Penicillium camemberti ycota, Trichocomaceae), one identified gene was designed LpFTRL (FTR-like).

The other candidate, ated LpDUF3632, showing a higher similarity (96%) to the E. festucae DUF3632 (domain of unknown function 3632)-like gene (UI: EfM3.028800. mRNA- 1) was identified from the unique reads nce UI: 734684-1).

Genome sequence contigs containing LpFTRL and LpDUF3632 from an in-house shotgun sequencing assembly data of the perennial ryegrass 04 genotype (E-) were subject to a BLASTN search t nucleotide sequences catalogued in the NCBI GenBank database.

Relatively high sequence similarity matches against LpFTRL were identified from Tausch's goatgrass (Aegilops tauschii L., UI: XM_020322891.1) and barley (UI: AK375773.1), followed by ascomycota s, while only fungal genes showed significant sequence similarity to LpDUF3632. Putative orthologues of LpFTRL were identified in barley chromosomes 1H and 7H (Ensembl UI: HORVU1Hr1G009870 and Hr1G108080, respectively), while the corresponding ces were only found from chromosome 7 of each sub-genome of oid wheat (Ensembl UI: TRIAE_CS42_7AL_TGACv1_557369_AA1780460, _AA1881100, and _AA1997520), and from cereal rye (Secale cereal L.) chromosome 7 (NCBI short read e (SRA) UI: ERX140518), suggesting that the fungal gene was transferred into the chromosome 7 of a common ancestor of Triticeae and Poeae species.

No significant match was obtained from the genome sequences of Brachypodium [B. distachyon (L.) P.Beauv.], rice (Oryza sativa L.), sorghum [Sorghum bicolor (L.) Conrad ], Zea Mays (L.) and Arabidopsis thaliana (L.).

The LpDUF3632 sequence was subjected to a BLASTN search against NCBI SRA data from eason grass species, to find significant hits from Italian ryegrass (L. multiflorum L.) and tall fescue (Festuca arundinacea L.), but not from orchard grass (Dactylis ata L.) or tic hairgrass (Deschampsia Antarctica É.Desv.). The gene presence/absence status of the DUF3632-like sequence in Poeae species was confirmed with a PCR-based assay (Fig. 13a).

The presence of LpFTRL-like sequence in all tested Poeae species was confirmed. A phylogenetic analysis revealed close relationships of LpFTRL and LpDUF3632 between corresponding oë sequences (Fig. 13b and c), suggesting that the two genes were transferred from the fungus lineage into plant species, but not vice versa, after ification [59 n years ago (MYA)] of ancestral Epichloë species from the Claviceps lineage. Based on the ence ages of the Brachypodieae tribe from other Pooideae species, the FTRL gene was predicted to have been transferred into plants after 32-39 MYA. An LpDUF3632- derived DNA-based marker was assigned into perennial ryegrass linkage group (LG) 3, which ponds to the chromosome 3 of Triticeae species. The DUF3632-like sequence is likely to have been transferred independently of the β-glucanase(-like) gene, of which DNA-based marker has been assigned into perennial ryegrass LG 5, indicating a possibility of at least three independent HGT events since 32-39 MYA. Comparison of genomic sequences ing DUF3632-like genes suggested transformation-like ancestral HGT events, due to conservation of the position of the putative intron between plant and Epichloë species, similar to LpBGNL. No intron was predicted for LpFTRL and corresponding sequences in Epichloë species.

From the E- perennial ryegrass transcriptome data, a leaf ecific expression n of LpFTRL was proposed (Fig. 14a). Although the counts per n reads (CPM) values were relatively low, LpDUF3632 was ubiquitously expressed (Fig. 14b). An analysis of the SRA data from Massay University, which did not include a leaf tip sample, indicated that the expression levels of LpFTRL and the corresponding Epichloë gene (EfM3.066060) were below the limit of detection in most tested tissues, regardless of the endophyte-infected (E+) /E- plants (Fig. 14c). Expression patterns of LpDUF3632 were similar between E+ and E- plants, and the Epichloë DUF3632 gene (EfM3.028800) was expressed in all tested tissues of the E+ plant (Fig. 14d). From the E+ and E- young seedlings, an ent of LpDUF3632 expression level was observed in E+ seeds after a germination-treatment, although the expression levels of LpFTRL, EfM3.066060, and EfM3.028800 stayed low throughout the tested 10 days (Fig. 14e and f). Although the LpDUF3632 sequence was identified from the non-polyadenylated RNA sequencing library, it is likely that the gene is also expressed as polyadenylated RNA, due to identification of corresponding reads from the SRA data.

Identification of LpFTRL provided a unique unity to compare nucleotide tution rates n fungus and plant lineages, using the sequences diverged only less than 40 MYA. The synonymous (Ks) substitution rate of the plant FTRL sequences was not substantially different from those from fungus sequences. Although some variation was observed in the non-synonymous (Ka) substitution rates, the Ka/MYA rate from the plant sequences was between those from Claviceps-Epichloë and E. gansuensis-other Epichloë combinations (Table 1).

Ka/Ks 0.2174 0.1657 0.3760 9 1 . 3 6 0 Rate (Ka/MYA) Claviceps were Claviceps and 0.0040 0.0024 0.0050 ations are shown as 0.0072 SDV 0.0064 0.0073 Epichloë Epichloë and 0.0008 0.0055 Non- synonymous Ka (average) 0.2307 0.1412 0.1041 0.0519 Rate (Ks/MYA) and the rest of lineage (from 58.8 MYA to the HGT event), while the ecies. Epichloë, respectively. As 0.0183 0.0147 E. gansuensis Epichloë 0.0132 0.0199 Claviceps genes. The ratios calculated from the and Synonymous SDV 0.2560 0.1745 E. gansuensis- other 0.0231 0.0112 Ks (AVE) 1.0612 0.8517 (plants), and Poeae, and Poeae 0.2767 0.1435 Diverged age (MYA) and 58 58 Triticeae- 21 7.2 FTRL orthologues and corresponding Epichloë and Plants,Triticeae Epichloë -Plants, , Claviceps Claviceps and Plants combination include the diversification period in the Poeae combination merely represent ification between plant sp Table 10. Claviceps- plants Claviceps- oë Triticeae- Poeae E. gansuensis- other Ka and Ks rates for Lp Epichloë, Claviceps- Epichloë ed around 58.8 MYA followed by horizontal transfer of the FTRL sequence into plant species after 39 MYA, the ison of the Claviceps Triticeae and 5 The Ka/Ks ratio for the plant FTRL and corresponding E. gansuensis-other Epichloë ces (0.376 and 0.3619, respectively) were relatively close, suggesting that the plant genes have been subjected to selection pressures since HGT. A comparison with E. ae 2 suggested a similar level (Ka/Ks = 0.31) of ion pressures for LpDUF3632.

These results proposed that plant FTRL and DUF3632 genes retain functionalities at the protein level, and have contributed to natural adaptation of the plant s. Similar to LpBGNL, the intron sequences of LpDUF3632 were less conserved than the exon sequences, likely due to lower levels of the pressure, which implies that the selective res have been an important factor for retaining of ce similarity to the corresponding Epichloë sequences. The close physical contacts between the host plants and symbiotic and/or pathogenic fungi could have facilitated the relatively frequent transfer events. As both Epichloë and Claviceps species infect to plant reproductive tissues, the presence close to germ cells might have been an important factor of the multiple HGT .

Comparison of codon usage of LpFTRL orthologues from diploid plant species and corresponding Epichloë sequences revealed a similar usage ratio for each amino acid, except for the termination codons. Only ‘TGA’ was used in the plant genes as termination codon, the ratio of ‘TGA’ was only 38.5% in the Epichloë sequences.

Pearl barley, wheat plain flour, rolled oat, and rice flour products were ed from retail shops, and DNA was extracted from the retail products. PCR primers for the LpFTRL sequence within a conserved region between plant and Epichloë species were designed.

Through PCR, the sequences ponding to LpFTRL were amplified from barley, wheat, oat, and E. festuca gDNA templates, but not from rice, confirming the presence of the ‘natural’ transgene in Triticeae and Poeae cereal species (Fig. 15). A control experiment with fungus- specific primers confirmed absence of gDNA of Epichloë and Claviceps species, especially the rye ergot fungus, C. ea, in the retail products.

Materials and Methods sRNA sequencing Fresh young leaves of an individual of perennial ryegrass cultivar Trojan were ted.

Total RNA including small molecules was extracted using the RNeasy Plant mini kit (QIAGEN) following the ed protocol of related products (Purification of miRNA from animal cells using the RNeasy® Plus Mini Kit and RNeasy MinElute® p Kit). A sequencing library was prepared using the Small RNA sequencing library preparation kit (NEB), and a fraction containing non-coding RNA molecules (bp in length) was purified using the BluePippin platform (Sage Science). The products were characterised on the 2200 TapeStation instrument using the D1000 kit (Agilent). The library was loaded on the MiSeq platform (Illumina), following the manufacture’s ction, and a sequencing analysis was performed with the MiSeq Reagent Kit v3 (150-cycle) kit (Illumina). The outcome data was processed with the FastX-tool-kit package, to remove r sequence and generate a unique read dataset. based screening The transcriptome dataset of E. festucae (file name: M3 transcript ces) was obtained from the website of Kentucky university //www.endophyte.uky.edu). The TSA 1000001-GFSR01044773) and sRNA unique read datasets of perennial ryegrass were prepared for a DNA sequence homology search. The homology search was performed with the megablast function of the BLAST+ package, and the threshold E value was set at 1e-10. The resulting data was imported into the Microsoft Excel software for a manual examination. The manual examination was performed using the NCBI BLAST tools.

The homologues sequence in representative flowering plant species were performed on the Ensembl Plants website (http://plants.ensembl.org/index.html). The LpFTRL ce was subjected to BLAST-based search against cereal rye SRA dataset (SRA UI: DRP000390).

The LpDUF3632 sequence were subjected to the search against the following SRA datasets; Italian ryegrass (SRX1604870 and SRX1604871), tall fescue (SRX1056957), orchard grass (ERX1842528), and Antarctic hairgrass 5632), from which significant homology hits against the perennial ss architecture candidate genes, LpABCG5 and 6 (GenBank UIs: JN051254.1 and JN051255.1) were obtained, but no significantly similar sequence to an oë-specific gene, makes caterpillars floppy (mcf)-like gene (GenBank UI: KJ502561.1), was identified. This screening method was validated through a BLAST search of the LpFTRL, LpDUF3632, and Epichloë mcf sequences against E+ and E- ial ryegrass SRA datasets (SRA UIs: SRX1167577-SRX1167590).

DNA sample preparation Plant seeds were obtained from the Genetic Resources Unit of Institute for Biological Environmental and Rural Studies (IBERS; Aberystwyth, Wales, UK) and the South Australian Research and Development Institute (SARDI). The plants were germinated on a filter paper in a petri dish. Total DNA was extracted from fresh leaf of each plant genotype using the DNeasy Plant mini kit (QIAGEN) ing the cture’s instruction. Total DNA was also extracted from barley (pearled grains), wheat (plain , oat (rolled grains) and rice (flour) using the DNeasy Plant mini kit.

PCR-based screening PCR s were designed using the Oligo Calc tools. For cross-species amplification, primers were designed to generate short DNA fragments (< 251 bp in length) within highly conserved regions of the target sequences. The PCR was performed on the CFX Real-Time PCR Detection Systems (Bio-Rad), with the Luna® Universal qPCR Master Mix (NEB), and data analysis was performed using the CFX Manager™ Software (Bio-Rad). isation of PCR products was performed on the 2200 TapeStation instrument or on an agarose gel (2.0% w/v) stained with SYBR Green (Thermo Fisher) through electrophoresis.

Validation of the PCR-based screening method PCR primers were designed based on perennial ryegrass sequences to amplify larger fragments (> 900 bp), for use as DNA tes of a standard curve assay (SCA). The PCR amplicons were amplified from the Impact04 genotype, using the MyTaq™ DNA Polymerase kit, and the ons were cleaned using the Monarch® PCR & DNA Cleanup Kit (NEB).

DNA concentration was adjusted to 1 pg/μl, and a dilution series was prepared. For fungus- specific primers, a dilution series of E. festucas gDNA was prepared. The SCA was performed on the CFX Real-Time PCR Detection Systems, with the Luna® Universal qPCR Master Mix, followed by data analysis with the CFX Manager™ Software.

In silico analysis Amino acid and DNA ces were prepared for in silico analysis. Gene structure tion was performed using the FGENESH program of the Softberry website using the ‘Monocot plants’ parameter. Alignments of amino acid sequences were generated with the CLUSTALW program with the default parameters. Sequence homology search was performed with the NCBI and PredictProtein websites. Phylogenetic analysis was med with the MEGA7 program. Non-synonymous and synonymous nucleotide substitution rate (Ka and Ks, respectively) was ated using the Synonymous nonymous Analysis Program . For gene expression analysis, an in-house transcriptome read dataset was prepared through filtering of the transcriptome sequencing reads from Impact04 tissues using Impact04 genome s (>999 bp). The number of reads which contained LpFTRL or LpDUF3632 sequences (no sequence mismatch for 60 bp or ) were counted.

Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.

References Hand, M. L., Cogan, N. O., Stewart, A. V. & Forster, J. W. Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the Lolium-Festuca s complex.

BMC Evolutionary Biology 10, 303 (2010).

Richards, T. A. et al. Phylogenomic analysis trates a pattern of rare and ancient horizontal gene transfer between plants and fungi. The Plant Cell 21, 1897–1911 (2009). uka, H. et al. A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI. BMC Biotechnology 15, 25 (2015).

Claims (24)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of creating or ing a symbiotic onship between a plant and a symbiont carrying a domain-of-unknown-function (DUF) gene; said method including 5 introducing into said plant an effective amount of a nucleic acid or nucleic acid nt of the DUF gene, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.

2. A method according to claim 1, wherein said plant is from a Lolium or Festuca species.

3. A method according to claim 2, wherein said plant species is Lolium perenne or Lolium multiflorum. 15

4. A plant endophyte symbiota produced by a method according to any one of claims 1 to 3.

5. A method for selecting a plant for symbiosis with a symbiont carrying a DUF gene; said method comprising 20 a) determining the presence of a DUF gene in said plant, b) measuring the level of expression of the gene identified in a), and c) using the expression level measured in b) to determine if the plant will form a symbiotic relationship with said symbiont. 25

6. A method according to claim 5, wherein the symbiont is an yte from an Epichloe species.

7. A ntially purified or isolated nucleic acid or nucleic acid fragment of a DUF gene; wherein the c acid or nucleic acid fragment is isolated from a plant species.

8. A nucleic acid or nucleic acid fragment according to claim 7, n said plant is from a Lolium or a species.

9. A nucleic acid or nucleic acid fragment according to claim 8, wherein said plant 35 species is Lolium perenne or Lolium multiflorum.

10. A ntially purified or isolated nucleic acid or nucleic acid fragment of a DUF gene or a complementary or antisense sequence to a c acid or nucleic acid fragment of a DUF gene, said nucleic acid or nucleic acid fragment including a nucleotide 5 ce selected from the group consisting of: (a) the sequence shown in ce ID No: 28; (b) complement of the sequence recited in (a); (c) sequences antisense to the sequence recited in (a); and (d) functionally active fragments and variants of the ces recited in (a), (b) 10 and (c).

11. A nucleic acid or nucleic acid fragment according to claim 10, wherein one of the following applies i) said functionally active fragment or variant has a size of at least 20 nucleotides 15 and has at least imately 90% identity to the relevant part of the sequence recited in (a), (b) or (c), upon which the fragment or variant is based; or ii) said functionally active fragment or variant has a size of at least 100 nucleotides and has at least approximately 95% identity to the relevant part of the sequence recited in (a), (b) or (c), upon which the fragment or variant is based.

12. A vector ing a nucleic acid or c acid fragment according to any one of claims 7 to 11.

13. A vector according to claim 12 further including a promoter and a terminator, said 25 er, nucleic acid or nucleic acid fragment and terminator being operatively linked.

14. A plant cell, plant, plant seed or other plant part, including vector according to claim 12 or 13. 30

15. A method of modifying pathogen resistance in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to any one of claims 7 to 11, or a vector according to claim 12 or 13.

16. Use of a nucleic acid or nucleic acid fragment according to any one of claims 7 to 11 for modifying pathogen ance in a plant.

17. Use of a nucleic acid or nucleic acid fragment according to any one of claims 7 to 11, 5 or nucleotide sequence information thereof, or single nucleotide polymorphisms thereof, as a molecular genetic marker.

18. A method of creating or enhancing a symbiotic relationship between a plant and a symbiont carrying a DUF gene; said method including introducing into said plant an effective 10 amount of a nucleic acid or nucleic acid fragment according to any one of claims 7 to 11, or a vector according to claim 12 or 13.

19. A plant endophyte symbiota produced by a method according to claim 18. 15

20. A substantially purified or isolated DUF protein, wherein said DUF protein is isolated from a plant species.

21. A DUF protein ing to claim 20, wherein said plant from which the DUF protein is isolated is a Lolium or Festuca species.

22. A DUF protein according to claim 21, wherein the plant species is Lolium perenne or Lolium multiflorum.

23. A substantially purified or isolated DUF n, said protein including an amino acid 25 sequence ed from the group consisting of: (b) the sequence shown in Sequence ID No: 29; and (c) functionally active fragments and ts of the sequence recited in (a).

24. A protein according to claim 23, n one of the following applies: 30 i) the functionally active fragment or variant has a size of at least 20 amino acids and has at least approximately 90% identity to the nt part of the sequence recited in (a) upon which the nt or variant is based; ii) the onally active fragment or variant has a size of at least 100 amino acids and has at least approximately 95% identity to the relevant part of the sequence recited in (a) upon which the fragment or variant is based. EPWMMSDEWNNVMGCNGAPSEFDCMQNIYGGSKR NFGGWLICEPWMMSDEWNNVRGCNGAASEFDCMRNNYGGSKR KGINKIRGVNFGGWLICEPWMMSDEWNNVMGCNGAASEFDCMLNNYMGSNR EPWMMSNEWNNNMGCNNAASEFDCMRNNYMGSKR ALNVPSNKKLHVQFMSSKWDSGDPR SPGMDGWIYWTWKTELNDPR .*:.************.******:**** ***.*.****** * * **:* PGALKAVRDAEASLGVADGKKLHVQFMSQKWDSGNPR ADFFKKFFTAQQQLYEAPGMSGWVYWTWKTQLNDPR ***** :******:***** D DKDFFKKFFTAQQQLYEAPGMSGWVYWTWKTQLNDPR KFFTAQQQLYEEPGMSGWIYWTWKTQLNDPR DGNFFTKFFTAQQQLYESPGMDGWIYWTWKTELNDPR