NZ789515A - Horizontal transfer of fungal genes into plants (3) - Google Patents

Abstract

The present invention relates to methods of creating or enhancing symbiotic relationships between plants and symbionts, in particular symbionts carrying genes encoding fungal transcriptional regulatory-like (FTR) proteins. 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. sis 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.

Description

Patent No. # - Complete Specification No. Date: HORIZONTAL TRANSFER OF FUNGAL GENES INTO PLANTS (3) We, Agriculture Victoria Services Pty Ltd, of AgriBio Centre for oscience, 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 described in and by the following statement NTAL TRANSFER OF FUNGAL GENES INTO PLANTS (3) Divisional ation status Pursuant to Section 34 of the New Zealand 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 Invention The present ion 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 nts, and new organisms and ta developed thereby.

The invention also relates to nucleic acids involved in the methods, vectors including the nucleic acids, plant cells, plants, seeds and other plant parts transformed with the nucleic acids and vectors, and methods of using the nucleic acids and vectors.

Background of the Invention Important plants, including forage grasses, legumes, trees, shrubs, and vines are commonly found in association with endophytes including 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 resistance to insect pests. For example, in grasses, insect resistance may be provided by ic metabolites produced 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 ore feeding. Considerable variation is known to exist in the metabolite e 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 symbiotic behaviour with endophytes. Difficulties in artificially breeding of these symbiota limit their usefulness. For e, 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 ng techniques, for example in forage s such as perennial ryegrass and tall fescue, grass varieties are bred using classic cross-breeding ques 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 y are then inoculated with a single endophyte and the ing grassendophyte associations are evaluated for any favourable characteristics such as insect resistance. The individual experimental synthetic varieties deploying a single endophyte in them are then evaluated for agronomic performance and resulting animal performance by grazing animals over a period of years. This evaluation process may reveal that the single endophyte being deployed in the different mental synthetic varieties may not show vegetative and/or intergenerational stability in some of these ies or the desired alkaloid profile conferred by the single yte may vary between different synthetic varieties failing to confer appropriate levels of insect resistance or g 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 cool-season turf and forage grass species, including those of sub-tribes Loliinae and Dactylidinae. Perennial ryegrass (Lolium perenne L.; ibe Loliinae) is one of the most important pasture crop species for the dairy industry, and it has consequently been a primary target for improvement using molecular biology and genetic technologies. Asexual fungal endophyte s of the genus Epichloë (syn. Neotyphodium) are symbionts of species belonging to the Poeae tribe genera Lolium and Festuca.

The fungal endophyte species rely on the plant host for ion, reproduction, and protection from abiotic and biotic stress. Benefits to the host plant e enhanced itive abilities, tolerance to pathogens, and resistance to animal and insect herbivory. Due to its agronomic ance, 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 novelty in both yotes and eukaryotes. In flowering plant species, organelle genomes have served as both donors and recipients of gene transfer events. In contrast to organellar genes, transfer of r genes to angiosperms appears to have been rare, and has been confined to date to genes originating from yotes or other plant species such as green algae, mosses and other angiosperms. A previous systematic in silico study, from igation of four completely sequenced angiosperm genomes (those of Arabidopsis thaliana L., rice [Oryza sativa L.], sorghum [Sorghum bicolor L.], and poplar us 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 t invention to me, or at least alleviate, one or more of the ulties or deficiencies associated with the prior art.

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

In a first aspect, the present ion provides a substantially purified 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), n the nucleic acid or c acid fragment is isolated from a plant s.

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

The Lolium or Festuca species may be of any le type, including Italian or annual ss, perennial ryegrass, 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 a arundinacea.

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

In a second aspect, the present invention provides a substantially purified or isolated nucleic 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 ae species. In a more preferred embodiment the plant is from a Lolium or a 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 ss or fescue species is a Lolium species such as Lolium perenne or Lolium multiflorum which is otherwise known as Festuca perennis.

In a third aspect, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a fungal transcriptional 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 ment the plant is from a Triticeae or e species.

The Triticeae or Aveneae 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 ed 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 present invention provides a substantially purified or isolated c acid or nucleic acid fragment from a plant species, the nucleic acid or nucleic acid fragment being horizontally transferred from a fungal s, n the c acid or nucleic acid fragment encodes a fungal cell wall degrading enzyme, is a DUF gene, or s 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, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.

The c acid or c 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, tural or altered nucleotide bases, and ations 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 nucleic acid fragment of the invention is derived, flank the nucleic acid or nucleic acid fragment. The term therefore includes, for e, a nucleic acid or nucleic acid fragment 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 genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a nucleic acid or nucleic acid fragment which is part of a hybrid gene. Preferably, the substantially 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 al is removed from its al environment (e.g. the natural environment if it is naturally occurring). For example, a naturally ing 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 nucleic 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 ments, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a fungal cell wall ing enzyme includes a tide sequence selected from the group consisting of the sequences shown in Sequence ID Nos: 1 to 4; and functionally 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 sequence selected from the group consisting of the sequence shown in Sequence ID No: 28 and sequences encoding the polypeptide shown in Sequence ID No: 29; and functionally active fragments and variants thereof.

Also in preferred embodiments, the substantially purified or isolated c acid or nucleic acid fragment encoding a FTR protein includes a nucleotide sequence selected from the group consisting of the sequence shown in ce ID No: 30 and sequences encoding the polypeptide shown in Sequence ID No: 31; and functionally active fragments and variants The substantially purified or ed nucleic acid or c acid of the present invention also includes sequences complementary or antisense to a sequence encoding a fungal cell wall degrading enzyme, to a DUF gene or to a sequence encoding a FTR protein, and preferably ces selected from the group consisting of sequences complementary or antisense to Sequence ID Nos: 1–4, 28 and 30; or mentary or nse to the sequences encoding the polypeptides shown in Sequence ID Nos: 29 and 31.

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment ng a FTR protein or a complementary or antisense ce to a nucleic acid or nucleic acid fragment encoding a FTR protein, 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: 30; (b) complement of the sequence d 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 active” is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of modifying pathogen resistance in a plant, or ng or enhancing a symbiotic relationship between a plant and a symbiont. Such variants include naturally occurring allelic ts 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 onal activity of the fragment or t.

Preferably the functionally active fragment or t has at least approximately 80% identity to the relevant part of the above-mentioned sequence to which the fragment or variant corresponds, more preferably at least imately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes. By ‘conservative nucleic 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 nucleic acid changes which result in vative amino acid tutions of one or more es in the corresponding amino acid sequence. By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another 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 ed a c construct or a vector including a nucleic acid or nucleic acid fragment according to the present ion.

In other words, the present invention provides a genetic construct or a vector ing a c 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 er, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.

By ‘genetic construct’ is meant a inant nucleic acid molecule.

By a ‘vector’ is meant a genetic construct used to er 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 ions, for example when appropriate molecules such as riptional activator proteins are bound to the regulatory sequence. Preferably an operatively linked promoter is upstream of the associated nucleic acid.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant s; ial plasmids; derivatives of the Ti d 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 element and ator may be of any le type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell. ably 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 er include the desired tissue specificity of the vector, and whether tutive or inducible expression is desired and the nature of the plant cell to be transformed (e.g. monocotyledon or dicotyledon). Particularly suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, the maize Ubiquitin promoter, the rice Actin promoter, and ryegrass endogenous OMT, 4CL, CCR or CAD promoters.

A y of terminators which may be employed in the vectors of the present invention 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 nopaline synthase (nos) and the octopine synthase (ocs) genes.

The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the t invention and the terminator, may include further elements necessary for sion of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of ation (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi ), antibiotic resistance genes and other able 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 (gusA)]. 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 selectable marker gene to e a phenotypic trait for selection of ormed 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 is, histochemical GUS assays, northern and Western blot hybridisation analyses.

Those d 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 ents 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 synthetic linkers, for example including one or more restriction enzyme sites.

The nucleic acids and s of the present invention may be used to up-regulate or gulate expression of ponding 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 l plant.

By ‘down-regulating’ expression of said gene is meant decreasing expression of said gene and, as a result, the protein d 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 incorporating additional copies of a sense copy of the gene. A gene may be down-regulated, for example, by incorporating an antisense nucleic acid, a frame-shifted or otherwise modified sense copy of the gene, or a nucleic acid encoding interfering RNA (RNAi).

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 encoding RNAi.

The vectors of the present invention may be incorporated into a variety of , including tyledons (such as grasses from the genera Lolium, Festuca, Dactylis, Cynodon, Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and other , turf and bioenergy grasses, corn, oat, sugarcane, rice, wheat and barley), dicotyledons (such as opsis, tobacco, legumes, Alfalfa, oak, Eucalyptus, maple, Populus, canola, soybean and chickpea) and gymnosperms (such as Pinus). In a preferred embodiment, the s are used to transform monocotyledons, preferably grass species 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 incorporating the vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art.

Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature s and high velocity projectile uction 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 present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as ature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be uced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

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

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

The nucleic acid, genetic construct or vector of the present invention 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, is, Cynodon, Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and other forage, turf and bioenergy grasses, corn, oat, sugarcane, rice, wheat and ), dicotyledons (such as Arabidopsis, o, legumes, Alfalfa, 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, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and other forage, turf and rgy grasses, more preferably a Lolium species such as Lolium perenne or Lolium arundinaceum or a Dactylis species.

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

By “modifying pathogen resistance” is meant modifying a plant’s ability 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 es generated by such fungal pathogens. Fungal es may include leaf spot, stalk rot, root rot, dying-off, choke disease and seedling wilt. ing pathogen resistance includes creating resistance to a pathogen in a plant, or sing or decreasing ng levels of resistance in a plant.

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

More particularly, nucleic acids or nucleic acid fragments according to the present invention, and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof, may be used as a lar genetic marker for qualitative trait loci (QTL) tagging, mapping, DNA printing and in marker assisted selection, and may be used as candidate genes or perfect markers, particularly in ryegrasses and s. 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 ibility, herbage quality, ical stress tolerance, disease resistance, insect pest resistance, 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 protein, said method ing introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment encoding the fungal cell wall ing enzyme, of the DUF gene or encoding the FTR protein, or a functionally active fragment or t thereof, wherein the c acid or c acid fragment is isolated from a plant species.

In a preferred embodiment, the present invention provides a method of creating or enhancing a symbiotic relationship between a plant and a symbiont carrying a fungal transcriptional regulatory-like (FTR) protein, said method including introducing into said plant an effective amount of a nucleic acid or c acid fragment encoding the FTR protein, wherein the nucleic acid or nucleic acid fragment is isolated 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 nucleic acid fragment, genetic construct and/or a vector according to the present invention and introducing said symbiont into said plant.

The present invention also es a plant endophyte symbiota produced by a method of the present invention. ably, the fungal cell wall ing enzyme is a glucanase, more preferably a BGNL as hereinbefore described.

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

By “creating or enhancing a symbiotic relationship” is meant enabling a plant which otherwise would not form a symbiotic onship with a selected symbiont to form said symbiotic relationship or increasing the stability of the symbiotic relationship. For example, the nucleic acid or nucleic acid nt, genetic construct and/or a vector according to the t invention may be introduced into plants such as rice, wheat and barley, which do not naturally n an ortholog of the gene of the present invention, to ate establishment of symbiotic relationships between 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 therefrom. Such amounts can be readily determined by an appropriately skilled 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, lar 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 es 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) ining 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 tic relationship with said symbiont.

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

In a preferred embodiment, the present invention provides a method for selecting a plant for symbiosis with a symbiont carrying a FTR gene, said method comprising a) ining the presence of 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.

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

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

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

The Lolium or Festuca species may be of any le type, including Italian or annual ryegrass, perennial ryegrass, 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 a 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 plant from which the DUF protein is isolated is a Loliinae species. In a more preferred embodiment the plant is from a Lolium or a s.

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 multiflorum which is ise known as Festuca perennis.

In a preferred embodiment, the present ion provides a substantially purified or isolated FTR protein, wherein said FTR protein is isolated from a plant species.

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

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

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

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

\Thus in a preferred ment, the present invention provides a substantially purified or isolated FTR n, said protein including an amino acid sequence selected from the group consisting of: (a) the sequence shown in ce ID No: 31; and (b) functionally active fragments and variants of the sequences recited in (a).

By "functionally active" in this context is meant that the fragment or t is capable of modifying pathogen resistance in a plant, or creating or enhancing a symbiotic relationship between a plant and a symbiont. Preferably, the fragment or variant has one or more of the biological ties of the fungal cell wall degrading enzyme, DUF or FTR protein. Additions, ons, substitutions 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 nt 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 functionally active variants and fragments include, for example, those having vative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

Preferably the nt 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 ed a ptide recombinantly produced from a nucleic acid or nucleic acid nt according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.

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

Brief Description of the Drawings/Figures Figure 1: Amino acid sequence alignment of LpBGNL (ß-1,6-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), derma harzianum (Sequence ID No 8), Hypocrea 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 , asterisk (*), 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 ure of the LpBGNL and E. festucae ß-1,6-glucanase genes and genetic linkage analysis for LpBGNL. (a) Alignment of genome sequences from ial 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 vector. 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. Corresponding 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 genetic markers.

Figure 3: The expression levels of LpBGNL and the E. festucae var. lolii ß-1,6-glucanase gene. (a) The expression level of LpBGNL in each tissue of perennial ryegrass. The y-axis shows normalised read count number (CPM). (b and c) The expression 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- ase 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 s are indicated in a dashed box; the der are fungal species. Asterisk (*) denotes species from which gene ts have been med to have the ß-1,6-glucanase activity. For cocksfoot, three s (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 ce contig (scaffold) identifiers of the Genome Project at the University of Kentucky e are shown in brackets. For the other fungal sequences, NCBI UI is shown in brackets. The clade including the sequences from Trichoderma and Hypocreaas species were selected as an outer group to obtain a root of the enic 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 nt Technologies, CA, USA). The target fragments are indicated with the large arrows. 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 nt). 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 angiosperms, 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 positive and negative in the PCR screening step are indicated by the plus (+) and minus (-) signs, tively. The divergent points of plant species from other species (>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 s, 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 indicated with black arrows. The triangle ents the ve period of HGT in the evolutionary lineage.

Figure 6. DNA sequence alignment of the two ypes from the C3 pe (Sequence ID Nos 15 and 16). Dash (-) in the DNA sequence shows a gap, and asterisk (*) under the alignment denotes ‘conserved nucleotide’. DNA sequence corresponding 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 e gel including the SYBR™ Safe DNA Gel stain (Thermo Fisher Scientific, Waltham, Massachusetts, USA). EasyLadder I (BIOLINE) was used as a size rd, and the 250 and 500 bp fragments are ted with arrows. The genotype UI of the genetic mapping population is indicated in each lane. C3 and NTC stand for ‘the C3 genotype’ and mplate 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 ss-associated endophyte were visualised on the 2200 TapeStation instrument using the High Sensitivity D1000 Kit (Aglient). 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 Festuca species (sub-tribe Loliinae) 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 position of upper and lower markers, respectively, of the D1000 Kit. Two pes of coast tussock-grass were subjected, and the Impact04 genotype was used as a positive control.

Figure 9. DNA sequences of the conserved ca. 750-bp region from plant species (Sequence ID Nos 17-27). Putative ure 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, Dactylis , coast tussock-grass and harding grass were visualised on the 2200 TapeStation ment 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 ed arrows. The 04 genotype was used as a positive control, 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 sequence (Sequence ID No 4).

Figure 13. Taxonomic relationships of plant and fungal species and phylogenetic tree of Epichloë-Triticeae/Poeae HGT candidates and ponding fungal genes. (a) Taxonomic relationships of plant and fungal species and the presence/absence status of the LpFTRL and 632-like ces. The ent point of plant species from other species (fungi and animals) is indicated with the filled hexagon (>1,000 MYA), and the filled circle indicate the divergent point of species belonging to the Triticeae and Poeae tribes from other Poaceae species. The triangles represent species ing 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 results, respectively. The plus (+) and minus (-) te 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 ces. 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 Periglandula 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 fiers of the Genome Project at the University of Kentucky are shown in ts. For plant sequences, NCBI UI is shown in ts.

Figure 14. The expression levels of HGT candidates and corresponding fungal endophyte genes in perennial ryegrass. (a) The expression levels of LpFTRL and LpDUF3632 in each tissues of the perennial ryegrass Impact04 genotype. (b) The expression levels of the HGT candidates 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 ngs. The x-axis shows tissue types (c and d) or period after a germination treatment (e and f), and normalised read count numbers [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+ plants. Note that the scales of the y-axes are not Figure 15. PCR-based detection of the Epichloë-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 experiment, PCR primers for the florigen candidate gene (Hd3a in rice, and FT in wheat and barley) were used. For demonstration of absence of Epichloë and Claviceps species in the retail products, PCR primers ic to the fungal species were used. The gDNA samples from the perennial ryegrass genotype 04 and E. festuca were used, as ve controls for amplification with the plant and fungus-specific primers, respectively.

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

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

Figure 17. Amino acid ce of Lolium perenne DUF3632-like protein gene.

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

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

Detailed Description 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 ly of sequencing reads generated a 7.2 kb c DNA ce contig [NCBI GenBank unique identifier (UI): KY771173], which contained a ve ß-1,6-glucanase gene. The gene-like sequence was designated LpBGNL (Lolium perenne ß-1,6-Glucanase-Like). LpBGNL showed 74-90% and 72-82% identity at the DNA and amino acid ce levels, respectively, to ß-1,6-glucanase genes of fungal taxa, such as E. festucae, H. lixii, and T. harzianum (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 ces of plants such as A. thaliana, rice, Brachypodium 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 AF535131.1 EU747838 X79197.1 Species Perennial ss Table 1 Epichloe festucae Neotyphodium sp. Hypocrea lixii Trichoderma harzianum 5 Example 2: Genomic and genetic terisation of LpBGNL Methods Plant materials and DNA extraction s of plant genotypes are ised 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 ODE, 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 ation instrument.

Short-read sequencing of BAC clones and amplicons The BAC-based genomic library was screened through use of PCR. For the phylogenomic analysis, PCR primers were ed to obtain genomic fragments from Loliinae and Dactylidinae 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 MspJI-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 linkage analysis was performed through use of the p150/112 reference genetic mapping tion of perennial ryegrass using the JoinMAP 3.0 application.

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

Short-read sequencing of BAC clones and amplicons The BAC-based genomic library was screened through use of PCR. For the phylogenomic is, PCR primers were designed to obtain genomic fragments from Loliinae and Dactylidinae s (Table 4). Sequencing ies for the MiSeq platform (Illumina, San Diego, rnia, USA) were prepared from the BAC clones and PCR amplicons, ing 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 programs.

Genetic linkage analysis PCR primers were designed to detect the indel polymorphism within the LpBGNL sequence (Table 3). Genetic linkage analysis was performed through use of the p150/112 nce 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 ar 04 Genotype from Aberystwyth (Great Britain) Genotype from Tadham Moor (Great Britain) Demeter Quantum Genotype from wyd (Great Britain) Currie Wild genotype from Algeria Wild genotype from Australia Landmaster Huds. Schreb. Festuca arundinacea Schreb. Festuca ovina Dactylis glomerata L. Dactylis marina Borrill Scientific name Lolium perenne L. Lolium ntum L. Festuca pratensis Festuca arundinacea Poa poiformis (Labill.) Druce Phalaris aquatica L. ) Table 2 Species Common name Perennial ryegrass Darnel Meadow fescue Tall fescue Tall fescue Sheep fescue Cocksfoot (Orchard grass) lis marina Coast tussock- grass Harding grass (Phalaris) itted by UTE Auburn University Teagasc Auburn University Auburn University Subm Sichuan Agricultural University KOREA POLAR RESEARCH 31G G 9.3G 10.2G 8.9G bases bases M spots, 25.9 bases bases bases bases Data size 1 run, 162.8M spots, 32.6G 1 run, 153.38M spots, 1 run, 129.3 1 run, 46.3M spots, 1 run, 50.4M spots, 1 run, 44.4M spots, Instrument Illumina HiSeq 2000 Illumina HiSeq 2000 Illumina HiSeq Illumina HiSeq 2000 Illumina HiSeq 2000 2000 Illumina HiSeq 2000 UI SRX738187 SRX465632 SRX745831 SRX745855 858 SRX669405 Dactylis ata L. (orchardgrass) Deschampsia antarctica Poa annua Poa supina Poa infirma Species Phalaris aquatica Table 3 , , , Phalaris Phalaris iated DNA template perennial ryegrass, darnel, meadow fescue, tall fescue, sheep fescue, cocksfoot, is marina Phalaris aquatica, perennial darnel sheep fescue aquatica endophyte coast tussock- grass, ryegrass- associated endophyte, perennial ryegrass genomic library p150/112 genetic mapping population perennial ss, tall , meadow fescue perennial ryegrass, cocksfoot, is marina coast tussock- grass, cocksfoot, Dactylis marina ial ryegrass, cocksfoot, Dactylis marina coast tussock- grass, aquatica, perennial ryegrass Amplicon 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 GTTCACGGTGCGAT TTTGTCGTCCGGGCTCACGC TGGATGCGCYTCGTCATCC ATCCTCCTGGCAAGCTGAATG ATGATGGTGTTGGCCGCGTT TCGTCGCCGATGATGGTGTT GCCCGTCTGACGGGGCACAG CATCAACAAGATCAGGGGCG CACGACTTGGCTGCTTTCAA CTGCCTCCGAGTTCGACTG  3’) Sequence (5’ Forward GTCGGCATGATTGAGGTTCT AGGGCATCAACAAGATCAGG CGCGCCTAATCCTCTCCTCT TGCTTGCCCTTCAGGAGGCT ATCAGGAAGGAGAGCCTCCA TAACGCTCAACGGGGACG LpHistone** LpABCG5 LpABCG6 Table 4 Primer name LpBGNL_short LpBGNL_indel LpBGNL_long1 LpBGNL_long2 LpBGNL_long3 LpBGNL_long4 LpBGNL_cons * Length of DNA fragment based on the ial ryegrass genome sequence -Reductase Genes from Perennial Ryegrass -CoA . 2010 Functional Analyses of Caffeic Acid O- Methyltransferase and oyl (doi:10.1105/tpc.109.072827) –3373. ** Reference: Tu, Y. et al ). The Plant Cell 22, 3357 (Lolium perenne Results An se bacterial artificial chromosome (BAC)-based genomic library of perennial ryegrass had previously been constructed from endophyte-devoid (E-) individuals of the cultivar Grasslands Nui. PCR-based screening of the library fied two positive clones, ated 4-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 ) showing similarity at a DNA sequence identity of 82% to a Zea mays oson-related gene (NCBI UI: AF434192.1) (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 distance from the glucanase gene.

In the BAC clone-derived contigs, however, no ces similar to these flanking genes were identified. Putative coding regions for the E. ae ß-1,6-glucanase and LpBGNL genes were identified (Fig. 2b). A single intron was found in both sequences, and comparison of the exonic and intronic regions identified 4 insertion-deletion (indel) 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 ce 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 s of wheat and rice, while the corresponding upstream and downstream sequences of the E. festucae ß-1,6-glucanase gene ed partial sequences of the flanking genes (Fig. 2b).

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

Example 3: LpBGNL gene sion analysis Methods Gene expression analysis The transcriptome sequencing reads from Impact04 tissues were mapped against Impact04 genome contigs (>999 bp) for filtering. The number of reads which ned LpBGNL sequence (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 ss cultivar Alto were subjected to germination treatment 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 strand-specific RNA library ation kit (Agilent). Sequencing is was performed on the na 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 corresponding to LpBGNL were identified from leaf, root and flower samples, and higher expression levels [based on counts per million reads (CPM)] were detected in root and flower than in leaf (Fig. 3a, Table 6). Specificity of gene sion was examined using yteinfected (E+) and E- perennial ss 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 large-scale morphological change was observed during the ing two days, the read counts substantially increased in both samples. 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 endophyte 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 ed 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 14,656,730 14,515,287 23,613,687 19,292,712 ,797 22,525,848 13,271,284 ,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 (middle) 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 website. ve orthologous sequences were sought in the NCBI, podium distachyon (http://www.brachypodium.org/) and Ensembl (http://plants.ensembl.org/index.html) databases. Non-synonymous and synonymous nucleotide tution 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. Phylogeny was generated with the MEGA7 program (http://www.megasoftware.net/).

The presence of LpBGNL orthologues in other Poeae species was determined by PCR-based ing. LpBGNL-specific primers were designed and short DNA nts (178 bp in length) were ied from genomic DNA templates of darnel (Lolium temulentum L.), meadow fescue ca pratensis Huds.), tall fescue (Festuca nacea Schreb.), sheep fescue (Festuca ovina) (Table 2, Figure 8). Products were also obtained from those of cocksfoot/orchard grass (Dactylis glomerata L.) and Dactylis marina Borrill, but not from those of coast k-grass [Poa poiformis (Labill.) Druce] or g grass/phalaris (Phalaris aquatica L.). DNA fragments were not amplified from genomic DNA templates of E. festucae var. lolii., ming 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 aryl-phospho-beta-D-glucosidase domain. For a phylogenetic analysis, DNA fragments, including the aryl-phospho-beta-D-glucosidase domain, were amplified from the selected Loliinae and Dactylidinae species. De novo on sequence analysis and assembly obtained a single ce 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 ure stop codon was found in two haplotypes of Dactylis marina, and the haplotypes with the premature stop codon were excluded from the further analysis.

Fungal glucanase gene-like sequences were obtained from the NCBI database and the Genome Project at the University of Kentucky website. Phylogenetic analysis with the maximum 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 ces.

Synonymous and non-synonymous nucleotide substitution (Ks/Ka) ratios were calculated using the 750 bp sequences. Both Ks and Ka values between the Epichloë and plant species were substantially lower than those between the Epichloë s 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 s 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 absence of the gene in the Poa and is species samples (Fig. 5). As a control experiment for capacity to y cross- species, PCR was performed with primers specific to perennial ryegrass histone H3 and candidate plant architecture genes, such as the ATP -binding cassette protein sub-family G 5 and 6 genes (LpABCG5 and LpABCG6, respectively). Although all primers were designed based on ial 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 icantly matching the LpBGNL and E. festucae ß-1,6-glucanase genes were found from Dactylis species, 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 mically closer than Poa species to s of the sub-tribes Loliinae and Dactylidinae (Fig 5b). As a l analysis, sequences similar to the LpABCG 5 and 6 genes were sought, leading 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 , 0.1453 (0.2276) 0.6563, 0.1432 (0.2182) , 0.1449 (0.2231) 0.6805, 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 8) 0.6285, 0.1470 (0.2339) 0.6093, 0.1487 (0.2441) , 0.1442 0.6363, 0.1488 (0.2339) 0.6546, 0.1533 (0.2186) Neotyphodium sp. 0.2539, 0.0629 (0.2477) - 0.2533, 0.0646 (0.2550) 0.2369, 0.0655 (0.2766) 0.2305, 0.0638 0) 0.2969, 0.051 0.0896, 0.0247 (0.2751) - (0.1717) -glucanase gene ß- 1,6 E. festucae 0.1961, 0.0501 (0.2553) - , 0.0518 (0.2647) 0.1887, 0.0535 (0.2838) 0.1823, 0.0519 (0.2845) 0.2207, - - 0.0433 (0.1961) Sheep fescue 0.3333, 0.0416 (0.1247) 0.3575, 0.0433 (0.1211) 0.3313, 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 , 0.0102 (0.0783) 0.1670, 0.0085 (0.0509) - - - - - - ryegrass Darnel 0.0622, 0.0017 (0.0273) - - - - - - - Perennial ss 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 similarity to the fungal genes, and absence of LpBGNL-like sequences from other representative angiosperm species suggested that LpBGNL was ed from a fungal species through HGT. Genomic and genetic characterisation was uently performed in order to demonstrate that LpBGNL is d 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 ct, even though LpBGNL shows unusually high DNA sequence rity (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 otes.

The PCR-based screening and database searches ted that the ß-1,6-glucanase-like gene is present in only a limited number of Poeae species including the genera Lolium, Festuca and Dactylis, which are confined to the sub-tribes Loliinae and Dactylidinae. The enetic analysis suggested a common origin of the Epichloë-derived 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 ibes by a single transfer event. The HGT event may consequently have occurred between ca. 9 to 13 million years ago (MYA), based on the predicted time of divergence of the two sub-tribes 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 . This suggests that LpBGNL may have been subjected to selection pressures. Although similar Ks/Ka ratios were obtained from the angiosperm and fungus groups, DNA mutation rates between those two groups may not be equivalent. From cocksfoot/orchard grass and Dactylis 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 genomes, the ß-1,6-glucanase-like genes in these species may have been ted 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 s in Poeae species, it is likely that the close physical proximity of both partners in the symbiosis may have facilitated an HGT event, r to the physical contacts between parasitic and host plants. The conservation of the intron position n 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 endophyte 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 187 SRX465632 SRX745831 SRX745855 SRX745858 SRX669405 Table 9 Species Dactylis glomerata (orchardgrass) Deschampsia tica Poa annua Poa supina Poa infirma Phalaris aquatica e 5: Loss of function of LpBGNL Given the role of plant-encoded glucanase enzyme in the establishment of a stable symbiotic relationship with an endophyte, a loss of function analysis is carried out. A LpBGNL-like gene is removed from the genome of a ryegrass plant which ly forms a stable relationship with an Epichloë endophyte. The stability of the plant-endophyte association is then evaluated in such plants compared with unmodified control plants. e 6: Gain 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 gain of function analysis is carried out. A LpBGNL-like gene is uced 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 ed with unmodified control plants.

Example 7: LpBGNL as a promoter of natural stable associations with asexual Epichloë Fungal ß-1,6-glucanases have been ed to be specifically ed into plant apoplasts during endophyte ion, and may play a role in provision of nutrition to the infecting endophyte, control of branching of the yte 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 tic relationship.

Although Epichloë ytes 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 soil-borne fungal pathogens. Similarly, active expression in flowers may suggest the capacity to protect against fungal pathogens such as Epichloë typhina, which causes choke disease. However, some Festuca and Dactylis species are relatively susceptible to infection by E. a, even though those species presumably also possess the ß-1,6-glucanase-like gene. As natural stable associations with asexual Epichloë endophytes are confined to the Poeae lineages that possess LpBGNL-like genes, the ancestral HGT event (which might have occurred from a sexual pathogenic oë-like species) may have provided pre-adaptive conditions for the contemporary symbiosis. The evidence for selective pressure on the gene is suggestive.

As major symbionts, the l Epichloë species provide abiotic and biotic stress nce to grass species of the Poeae tribe. Tolerance to invertebrate ory is a wellcharacterised benefit to the host, partially attributable to the effects of a makes caterpillers floppy-like ike) gene. The mcf-like gene was horizontally erred into the endophyte genome from a bacterial species 7.2-58.8 MYA. Hence, multiple horizontal transfer events, ing 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 relationship with a plant and an endophyte 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 relationship 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 st. Species such as T. harzianum are mycoparasites of fungal phytopathogens, and this property is related 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 ed pathogen resistance.

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

The method involves selecting a plant for ing for symbiosis with a symbiont such as an Epichloë endophyte. The first step es determining r 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 ed 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-) ial ryegrass individual, a sequencing library was ed. A single Illumina MiSeq run generated a total of 8,216,014 reads from the library. A dataset of unique RNA reads, including 274 ces, was generated. A BLAST search of the perennial ryegrass (E-) transcriptome shotgun assembly (TSA) and unique RNA read datasets against the oë festucae transcriptome data identified 88 and 123 sequence similarity hits, respectively. Sequences putatively derived from microbiome, highly conserved genes between fungi and plants, such as actin and ubiquitin genes, and low confident 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 n 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) n (Genbank UI: CRL18938; identity: 48%, e-value: 2e-150) of Penicillium camemberti (Ascomycota, comaceae), one identified gene was designed LpFTRL (FTR-like).

The other candidate, designated 632, 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 (Sequence UI: 734684-1).

Genome sequence contigs containing LpFTRL and LpDUF3632 from an in-house shotgun sequencing assembly data of the ial ryegrass Impact04 genotype (E-) were subject to a BLASTN search against nucleotide sequences catalogued in the NCBI GenBank database.

Relatively high ce similarity matches against LpFTRL were identified from Tausch's goatgrass (Aegilops tauschii L., UI: XM_020322891.1) and barley (UI: AK375773.1), ed by ascomycota species, 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 HORVU7Hr1G108080, respectively), while the corresponding sequences were only found from chromosome 7 of each sub-genome of hexaploid wheat bl UI: TRIAE_CS42_7AL_TGACv1_557369_AA1780460, _AA1881100, and _AA1997520), and from cereal rye (Secale cereal L.) chromosome 7 (NCBI short read archive (SRA) UI: ERX140518), suggesting that the fungal gene was transferred into the chromosome 7 of a common or of Triticeae and Poeae species.

No significant match was obtained from the genome ces of Brachypodium [B. hyon (L.) v.], rice (Oryza sativa L.), sorghum [Sorghum bicolor (L.) Conrad Moench], Zea Mays (L.) and Arabidopsis thaliana (L.).

The LpDUF3632 sequence was subjected to a BLASTN search against NCBI SRA data from cool-season grass species, to find significant hits from Italian ryegrass (L. multiflorum L.) and tall fescue ca arundinacea L.), but not from orchard grass (Dactylis glomerata L.) or Antarctic 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 ce of LpFTRL-like sequence in all tested Poeae species was confirmed. A phylogenetic analysis revealed close relationships of LpFTRL and LpDUF3632 n corresponding Epichloë 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 million years ago (MYA)] of ancestral Epichloë species from the Claviceps lineage. Based on the divergence 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 sed marker was assigned into perennial ss linkage group (LG) 3, which corresponds to the chromosome 3 of eae 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 ility of at least three independent HGT events since 32-39 MYA. Comparison of genomic sequences including DUF3632-like genes ted transformation-like ancestral HGT events, due to conservation of the position of the putative intron between plant and Epichloë s, r to LpBGNL. No intron was predicted for LpFTRL and corresponding sequences in Epichloë species.

From the E- perennial ryegrass transcriptome data, a leaf tip-specific expression pattern of LpFTRL was proposed (Fig. 14a). Although the counts per million reads (CPM) values were vely 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 ponding 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 ns of LpDUF3632 were similar between E+ and E- plants, and the Epichloë DUF3632 gene (EfM3.028800) was expressed in all tested s of the E+ plant (Fig. 14d). From the E+ and E- seeds/young seedlings, an increment 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 opportunity to compare nucleotide tution rates between fungus and plant lineages, using the sequences ed 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 n those from Claviceps-Epichloë and E. gansuensis-other Epichloë combinations (Table 1).

Ka/Ks 0.2174 0.1657 0.3760 9 1 6 . 3 of the 0 Rate (Ka/MYA) Claviceps and Claviceps were 0.0040 0.0024 0.0050 0.0072 combinations are shown as SDV 0.0064 0.0073 0.0008 0.0055 Epichloë Epichloë and Non- synonymous Ka (average) 0.2307 0.1412 0.1041 0.0519 Rate (Ks/MYA) and the rest of Epichloë, respectively. As lineage (from 58.8 MYA to the HGT event), while the 0.0183 0.0147 0.0132 0.0199 Claviceps genes. The ratios calculated from the Epichloë and E. gansuensis mous SDV 0.2560 0.1745 0.0231 0.0112 E. nsis- other Ks (AVE) 1.0612 0.8517 0.2767 0.1435 Poeae (plants), and Poeae, and Diverged and age (MYA) 58 58 21 orthologues and corresponding Epichloë Triticeae- 7.2 Triticeae oë FTRL s, Claviceps and Plants, , eps Poeae combination merely represent diversification between plant species. and Plants combination include the diversification period in the Table 10. Claviceps- plants Claviceps- Epichloë Triticeae- Poeae E. gansuensis- other Ka and Ks rates for Lp Epichloë, Claviceps- Epichloë diverged around 58.8 MYA followed by horizontal transfer of the FTRL sequence into plant species after 39 MYA, the comparison Claviceps Triticeae and 5 The Ka/Ks ratio for the plant FTRL and corresponding E. gansuensis-other Epichloë sequences (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. festucae DUF3632 suggested a similar level (Ka/Ks = 0.31) of selection pressures for LpDUF3632.

These results proposed that plant FTRL and DUF3632 genes retain functionalities at the protein level, and have contributed to natural tion of the plant species. Similar to LpBGNL, the intron sequences of LpDUF3632 were less conserved than the exon ces, likely due to lower levels of the pressure, which implies that the selective pressures have been an important factor for retaining of sequence 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 eps 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 ces 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 obtained 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. h PCR, the sequences corresponding 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 , C. purpurea, in the retail products.

Materials and Methods sRNA cing Fresh young leaves of an individual of perennial ss cultivar Trojan were harvested.

Total RNA ing small molecules was ted using the RNeasy Plant mini kit N) following the modified protocol of related products ication of miRNA from animal cells using the RNeasy® Plus Mini Kit and RNeasy MinElute® Cleanup Kit). A sequencing library was prepared using the Small RNA cing library preparation kit (NEB), and a fraction containing non-coding RNA molecules (bp in ) 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 cture’s instruction, and a sequencing analysis was performed with the MiSeq Reagent Kit v3 ycle) kit (Illumina). The outcome data was sed with the FastX-tool-kit package, to remove adapter ce and generate a unique read dataset.

BLAST-based screening The transcriptome dataset of E. festucae (file name: M3 ript sequences) was obtained from the website of Kentucky university (http://www.endophyte.uky.edu). The TSA (GFSR01000001-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 old E value was set at 1e-10. The resulting data was ed into the Microsoft Excel software for a manual examination. The manual examination was performed using the NCBI BLAST tools.

The gues sequence in representative flowering plant species were performed on the Ensembl Plants website (http://plants.ensembl.org/index.html). The LpFTRL sequence 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 56957), orchard grass 42528), and Antarctic hairgrass (SRX465632), from which significant homology hits against the perennial ryegrass architecture candidate genes, LpABCG5 and 6 (GenBank UIs: 54.1 and JN051255.1) were obtained, but no significantly similar sequence to an Epichloë-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 t E+ and E- perennial ryegrass SRA datasets (SRA UIs: SRX1167577-SRX1167590).

DNA sample preparation Plant seeds were obtained from the Genetic Resources Unit of ute 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 N) following the manufacture’s instruction. Total DNA was also extracted from barley (pearled grains), wheat (plain flour), oat (rolled grains) and rice (flour) using the DNeasy Plant mini kit.

PCR-based screening PCR primers 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 med using the CFX Manager™ Software (Bio-Rad). Visualisation of PCR products was performed on the 2200 TapeStation instrument or on an agarose gel (2.0% w/v) stained with SYBR Green o 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 rd curve assay (SCA). The PCR amplicons were amplified from the 04 genotype, using the MyTaq™ DNA rase kit, and the amplicons were d 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 med on the CFX Real-Time PCR Detection s, with the Luna® Universal qPCR Master Mix, followed by data analysis with the CFX Manager™ Software.

In silico analysis Amino acid and DNA sequences were prepared for in silico analysis. Gene structure prediction was performed using the FGENESH program of the Softberry e using the ‘Monocot plants’ parameter. Alignments of amino acid sequences were ted with the CLUSTALW program with the default parameters. Sequence homology search was performed with the NCBI and PredictProtein websites. Phylogenetic analysis was performed with the MEGA7 m. Non-synonymous and synonymous nucleotide substitution rate (Ka and Ks, respectively) was calculated using the Synonymous Non-synonymous Analysis Program (SNAP). For gene expression analysis, an in-house riptome read dataset was prepared through filtering of the riptome sequencing reads from Impact04 tissues using Impact04 genome contigs (>999 bp). The number of reads which contained LpFTRL or LpDUF3632 sequences (no sequence mismatch for 60 bp or longer) 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 y 10, 303 (2010).

Richards, T. A. et al. Phylogenomic analysis demonstrates a pattern of rare and ancient ntal gene transfer between plants and fungi. The Plant Cell 21, 1897–1911 (2009).

Shinozuka, H. et al. A simple method for semi-random DNA on fragmentation using the methylation-dependent restriction enzyme MspJI. BMC Biotechnology 15, 25 (2015).

Claims (24)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method of creating or ing a symbiotic relationship between a plant and a symbiont carrying a fungal transcriptional regulatory-like (FTR) protein, said method including 5 introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment encoding the FTR protein, wherein the nucleic acid or nucleic acid fragment is isolated from a plant species.
2. 2. A method according to claim 1, wherein said plant is from a Triticeae,or Aveneae 10 species.
3. 3. A method according to claim 2, wherein said plant s is ed from the group ting of Triticum aestivum, Hordeum vulgare and Avena sativa. 15
4. 4. A plant endophyte symbiota produced by a method according to any one of claims 1 to 3.
5. 5. A method for selecting a plant for symbiosis with a symbiont carrying a FTR gene, said method comprising 20 a) determining the presence of 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. 25
6. 6. A method according to claim 5, wherein the symbiont is an endophyte from an Epichloe species.
7. 7. A substantially purified or ed nucleic acid or nucleic acid nt encoding a FTR protein; n the nucleic acid or c acid fragment is isolated from a plant species.
8. 8. A nucleic acid or nucleic acid nt according to claim 7, wherein said plant is from a from a Triticeae or Aveneae species.
9. 9. A nucleic acid or nucleic acid fragment ing to claim 8, wherein said plant s is selected from the group consisting of um aestivum, Hordeum vulgare and 11. Avena sativa. 5
10. 10. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding a FTR protein or a complementary or antisense sequence to a nucleic acid or c acid fragment encoding a FTR protein, said nucleic acid or nucleic acid fragment including a tide sequence selected from the group ting of: (a) the sequence shown in ce ID No: 30; 10 (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 sequences recited in (a), (b) and (c). 15
11. 11. A nucleic acid or nucleic acid fragment according to claim 10, wherein one of the following applies: i) said onally active fragment or variant has a size of at least 20 nucleotides and has at least imately 90% identity to the relevant part of the ce recited in (a), (b) or (c), upon which the fragment or variant is based; or 20 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. 12. A vector including a nucleic acid or nucleic acid fragment according to any one of 25 claims 7 to 11.
13. 13. A vector according to claim 12 further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked. 30
14. 14. A plant cell, plant, plant seed or other plant part, including vector according to claim 12 or 13.
15. 15. A method of modifying pathogen ance in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment 35 according to any one of claims 7 to 11, or a vector according to claim 12 or 13.
16. 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. 5
17. 17. Use of a nucleic acid or nucleic acid fragment according to any one of claims 7 to 11, or nucleotide ce ation thereof, or single nucleotide polymorphisms thereof, as a molecular genetic marker.
18. 18. A method of creating or ing a symbiotic relationship between a plant and a 10 symbiont carrying a FTR protein, 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.
19. 19. A plant endophyte symbiota produced by a method according to claim 18.
20. 20. A substantially purified or isolated FTR protein, wherein said FTR protein is isolated from a plant species.
21. 21. A FTR protein according to claim 20, wherein said plant from which the FTR protein 20 is isolated is a Triticeae or Aveneae species.
22. 22. A FTR protein according to claim 21, wherein said plant species is selected from the group ting of um aestivum, Hordeum vulgare and Avena sativa. 25
23. 23. A substantially ed or isolated FTR protein, said protein including an amino acid sequence selected from the group consisting of: (b) the sequence shown in Sequence ID No: 31; and (c) functionally active fragments and ts of the sequences recited in (a). 30
24. 24. A protein according to claim 23, wherein one of the following applies: 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 fragment or variant is based; or ii) the functionally active fragment or variant has a size of at least 100 amino acids and has at least approximately 95% ty 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