AU2016259449B2 - Designer endophytes (2) - Google Patents

Abstract

The present invention relates to endophytic fungi (endophytes), including modified variants thereof, and to nucleic acids thereof. The present invention also relates to plants infected with endophytes and to related methods, including methods of 5 selecting, breeding, characterising and/or modifying endophytes. More particularly, the present invention provides endophyte variants having a desired genetic and metabolic profile, wherein said endophyte variants possess genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte variant.

Description

The present invention relates to endophytic fungi (endophytes), including modified variants thereof, and to nucleic acids thereof. The present invention also relates to plants infected with endophytes and to related methods, including methods of selecting, breeding, characterising and/or modifying endophytes. More particularly, the present invention provides endophyte variants having a desired genetic and metabolic profile, wherein said endophyte variants possess genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte variant.

P/00/001 Regulation 3.2

2016259449 18 Nov 2016

AUSTRALIA

Patents Act 1990

COMPLETE SPECIFICATION

STANDARD PATENT

Invention title: DESIGNER ENDOPHYTES (2)

The following statement is a full description of this invention, including the best method of performing it known to us:

-22016259449 18 Nov 2016

DESIGNER ENDOPHYTES (2)

This application is a divisional of Australian patent application no. 2013203584 which is an application for a patent of addition to Australian Patent Application No. 2011204749, the entire disclosures of which are incorporated herein by reference.

Field of the Invention

The present invention relates to endophytic fungi (endophytes), including modified variants thereof, and to nucleic acids thereof. The present invention also relates to plants infected with endophytes and to related methods, including methods of selecting, breeding, characterising and/or modifying endophytes.

Background of the Invention

Important forage grasses perennial ryegrass and tall fescue are commonly found in association with fungal endophytes.

Both beneficial and detrimental agronomic properties result from the association, including improved tolerance to water and nutrient stress and resistance to insect pests.

Insect resistance is provided by specific metabolites produced by the endophyte, in particular loline alkaloids and peramine. Other metabolites produced by the endophyte, lolitrems and ergot alkaloids, are toxic to grazing animals and reduce herbivore feeding.

Considerable variation is known to exist in the metabolite profile of endophytes. Endophyte strains that lack either or both of the animal toxins have been introduced into commercial cultivars.

Molecular genetic markers such as simple sequence repeat (SSR) markers have been developed as diagnostic tests to distinguish between endophyte taxa and detect genetic variation within taxa. The markers may be used to discriminate

-32016259449 18 Nov 2016 endophyte strains with different toxin profiles.

However, there remains a need for methods of identifying, isolating, characterising and/or modifying endophytes and a need for new endophyte strains having desired properties.

Neotyphodium endophytes are not only of interest in agriculture, as they are a potential source for bioactive molecules such as insecticides, fungicides, other biocides and bioprotectants, allelochemicals, medicines and nutraceuticals.

Difficulties in artificially breeding of these endophytes limit their usefulness. For example, many of the novel endophytes known to be beneficial to pasture-based agriculture exhibit low inoculation frequencies and are less stable in elite germplasm. Thus, there remains a need for methods of generating novel, highly compatible endophytes.

International patent application PCT/AU2011/000020 describes a method for identifying or characterising endophyte strains which involves subjecting multiple samples of endophytes to genetic and metabolic analyses, and optionally also assessing geographic origin. The application also identifies a number of endophytes which were isolated by this method, including E1, NEA10, NEA11, NEA12, NEA13and NEA14.

However, there remains a need for more endophyte strains with desirable properties and for more detailed characterisation of their toxin and metabolic profiles, antifungal activity, stable host associations and their genomes.

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

A large scale endophyte discovery program was undertaken to establish a ‘library’ of novel endophyte strains. A collection of perennial ryegrass and tall fescue accessions was established.

-42016259449 18 Nov 2016

Genetic analysis of endophytes in these accessions has lead to the identification of a number of novel endophyte strains. These novel endophyte strains are genetically distinct from known endophyte strains and are described in a copending Australian provisional patent application filed 1 June 2012 entitled ‘Novel

Endophytes’.

Phenotypic screens were established to select for novel ‘designer’ grass-endophyte associations. These screens were for desirable characteristics such as enhanced biotic stress tolerance, enhance drought tolerance and enhanced water use efficiency, and enhanced plant vigour.

Novel ‘designer’ endophytes were generated by targeted methods including polyploidisation and X-ray mutagenesis.

These endophytes may be characterised, for example using antifungal bioassays, in vitro growth rate assays and/or genome survey sequencing (GSS).

Metabolic profiling may also be undertaken to determine the toxin profile of these 15 strains grown in vitro and/or following inoculation in planta.

These endophytes may be delivered into plant germplasm to breed ‘designer’ grass endophyte associations.

Specific detection of endophytes in planta with SSR markers may be used to confirm the presence and identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars.

The endophytes may be subject to genetic analysis (genetically characterized) to demonstrate genetic distinction from known endophyte strains and to confirm the identity of endophyte strains artificially inoculated into, for example, grass plants, varieties and cultivars.

By ‘genetic analysis’ is meant analysing the nuclear and/or mitochondrial DNA of

-52016259449 18 Nov 2016 the endophyte.

This analysis may involve detecting the presence or absence of polymorphic markers, such as simple sequence repeats (SSRs) or mating-type markers. SSRs, also called microsatellites, are based on a 1-7 nucleotide core element, more typically a 1-4 nucleotide core element, that is tandemly repeated. The SSR array is embedded in complex flanking DNA sequences. Microsatellites are thought to arise due to the property of replication slippage, in which the DNA polymerase enzyme pauses and briefly slips in terms of its template, so that short adjacent sequences are repeated. Some sequence motifs are more slip-prone than others, giving rise to variations in the relative numbers of SSR loci based on different motif types. Once duplicated, the SSR array may further expand (or contract) due to further slippage and/or unequal sister chromatid exchange. The total number of SSR sites is high, such that in principle such loci are capable of providing tags for any linked gene.

SSRs are highly polymorphic due to variation in repeat number and are co15 dominantly inherited. Their detection is based on the polymerase chain reaction (PCR), requiring only small amounts of DNA and suitable for automation. They are ubiquitous in eukaryotic genomes, including fungal and plant genomes, and have been found to occur every 21 to 65 kb in plant genomes. Consequently, SSRs are ideal markers for a broad range of applications such as genetic diversity analysis, genotypic identification, genome mapping, trait mapping and marker-assisted selection.

Known SSR markers which may be used to investigate endophyte diversity in perennial ryegrass are described in van Zijll de Jong et al (2003) Genome 46 (2): 277-290.

Alternatively, or in addition, the genetic analysis may involve sequencing genomic and/or mitochondrial DNA and performing sequence comparisons to assess genetic variation between endophytes.

-62016259449 18 Nov 2016

The endophytes may be subject to metabolic analysis to identify the presence of desired metabolic traits.

By ‘metabolic analysis’ is meant analysing metabolites, in particular toxins, produced by the endophytes. Preferably, this is done by generation of inoculated plants for each of the endophytes and measurement of toxin levels in planta. More preferably, this is done by generation of isogenically inoculated plants for each of the endophytes and measurement of toxin levels in planta.

By a ‘desired genetic and metabolic profile’ is meant that the endophyte possesses genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte.

Such beneficial properties include improved tolerance to water and/or nutrient stress, improved resistance to pests and/or diseases, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency, reduced toxicity and enhanced vigour in the plant with which the endophyte is associated, relative to a control endophyte such as standard toxic (ST) endophyte or to a no endophyte control plant.

For example, tolerance to water, drought, nutrient and/or biotic stress may be increased by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as standard toxic (ST) endophyte or to no endophyte control plant. Preferably, tolerance to water and/or nutrient stress may be increased by between approximately 5% and approximately 50%, more preferably between approximately 10% and approximately 25%, relative to a control endophyte such as standard toxic (ST) endophyte or to a no endophyte control plant.

Such beneficial properties also include reduced toxicity of the associated plant to grazing animals.

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For example, toxicity may be reduced by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as ST endophyte. Preferably, toxicity may be reduced by between approximately 5% and approximately 100%, more preferably between approximately 50% and approximately 100% relative to a control endophyte such as ST endophyte.

In a preferred embodiment toxicity may be reduced to a negligible amount or substantially zero toxicity.

For example, water use efficiency and/or plant vigour may be increased by at least approximately 5%, more preferably at least approximately 10%, more preferably at least approximately 25%, more preferably at least approximately 50%, more preferably at least approximately 100%, relative to a control endophyte such as ST endophyte or to a no endophyte control plant. Preferably, tolerance to water and/or nutrient stress may be increased by between approximately 5% and approximately 50%, more preferably between approximately 10% and approximately 25%, relative to a control endophyte such as ST endophyte or to a no endophyte control plant.

In a first aspect the present invention provides an endophyte variant having a desired genetic and metabolic profile. Preferably the endophyte variant is generated by polyploidisation or induced chromosome doubling, for example by treating the endophyte with colchicine or a similar compound. Alternatively, the endophyte variant may be generated by X-ray mutagenesis or exposing the endophyte to ionising radiation, for example from a caesium source.

In a preferred embodiment, the present invention provides an endophyte variant having a desired genetic and metabolic profile, wherein said endophyte variant possesses genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with, the endophyte variant, when compared with a standard toxic control endophyte or with a no

-82016259449 18 Nov 2016 endophyte control plant, and wherein said endophyte variant is selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and

NEA12dh17 as deposited at the National Measurement Institute with accession numbers V12/001408, V12/001409, V12/001410, V12/001411 and V12/001412, respectively.

Preferably the endophyte which is treated to generate the endophyte variant is isolated from a Lolium species, preferably Lolium perenne. The endophyte may be of the genus Neotyphodium, for example from a species selected from the group consisting of N uncinatum, N coenophialum and N lolii,. The endophyte may also be from the genus Epichloe, including E typhina, E baconii and E festucae. The endophyte may also be of the non-Epichloe out-group. The endophyte may also be from a species selected from the group consisting of FaTG-3 and FaTG-3 like, and FaTG-2 and FaTG-2 like.

In a preferred embodiment, the endophyte variant may have a desired toxin profile.

By a ‘desired toxin profile’ is meant that the endophyte produces significantly less toxic alkaloids, such as ergovaline or Lolitrem B, compared with a plant inoculated with a control endophyte such as standard toxic (ST) endophyte; and/or significantly more alkaloids conferring beneficial properties such as improved resistance to pests and/or diseases in the plant with which the endophyte is associated, such as peramine, N-formylloline, N-acetylloline and norloline, again when compared with a plant inoculated with a control endophyte such as ST or with a no endophyte control plant.

For example, toxic alkaloids may be present in an amount less than approximately 1 pg/g dry weight, for example between approximately 1 and 0.001 pg/g dry weight, preferably less than approximately 0.5 pg/g dry weight, for example between approximately 0.5 and 0.001 pg/g dry weight, more preferably less than approximately 0.2 pg/g dry weight, for example between approximately 0.2 and 0.001 pg/g dry weight.

-92016259449 18 Nov 2016 ln a particularly preferred embodiment the endophyte may not produce Lolitrem B toxins.

For example, said alkaloids conferring beneficial properties may be present in an amount of between approximately 5 and 100 pg/g dry weight, preferably between approximately 10 and 50 pg/g dry weight, more preferably between approximately 15 and 30 pg/g dry weight.

In a particularly preferred embodiment, the present invention provides an endophyte variant selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17, which were deposited at The National

Measurement Institute on 3 April 2012 with accession numbers V12/001408, V12/001409, V12/001410, V12/001411 and V12/001412, respectively. Such endophytes may have a desired genetic and metabolic profile as hereinbefore described.

In a preferred embodiment, the endophyte may be substantially purified. By ‘substantially purified’ is meant that the endophyte is free of other organisms. The term therefore includes, for example, an endophyte in axenic culture. Preferably, the endophyte 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 endophyte is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring endophyte present in a living plant is not isolated, but the same endophyte separated from some or all of the coexisting materials in the natural system, is isolated.

On the basis of the deposits referred to above, the entire genome of an endophyte selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17, is incorporated herein by reference.

-102016259449 18 Nov 2016

Thus, in a further aspect, the present invention includes identifying and/or cloning nucleic acids including genes encoding polypeptides or transcription factors from said genome.

Methods for identifying and/or cloning nucleic acids encoding such genes are 5 known to those skilled in the art and include creating nucleic acid libraries, such as cDNA or genomic libraries, and screening such libraries, for example using probes for genes of the desired type; or mutating the genome of the endophyte of the present invention, for example using chemical or transposon mutagenesis, identifying changes in the production of polypeptides or transcription factors of interest, and thus identifying genes encoding such polypeptides or transcription factors.

Thus, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid encoding a polypeptide or transcription factor from the genome of an endophyte of the present invention.

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 double20 stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.

By a ‘nucleic acid encoding a polypeptide or transcription factor’ is meant a nucleic acid encoding an enzyme or transcription factor normally present in an endophyte of the present invention.

The present invention encompasses functionally active fragments and variants of the nucleic acids of the present invention. By ‘functionally active’ in relation to the nucleic acid is meant that the fragment or variant (such as an analogue, derivative

-11 2016259449 18 Nov 2016 or mutant) is capable of manipulating the function of the encoded polypeptide, for example by being translated into an enzyme or transcription factor that is able to catalyse or regulate a step involved in the relevant pathway, or otherwise regulate the pathway in the endophyte. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.

Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides.

Preferably, said fragments are able to produce the same activity as the original gene when expressed. Preferably, said fragments maintain conserved regions within consensus sequences of the original gene.

Preferably said variants are variants of the original sequences that provide either conserved substitution, or limited modifications in consensus sequences to a level, for example, of no more than approximately 5%, more preferably no more than 1%, relative to the original gene.

For example, fragments and variants of a sequence encoding X may include a wild type sequence from species Z that encodes X, a fragment of a wild type sequence wherein the fragment encodes X, and that retains conserved regions within consensus sequences from species Z, and variants of the wild type sequence or fragments which encode X activity and have only conservative substitutions, a variant X’ that encodes X activity and in which sequence differs only by

-12 2016259449 18 Nov 2016 substitutions found in one or more contributing sequences used in formulating the consensus sequence, or a variant X that encodes X activity in which the variant has not more than approximately 95% amino acid variation, more preferably not more than approximately 99% amino acid variation from the wild type sequence or fragment.

By ‘conservative nucleic acid changes’ or ‘conserved substitution’ 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 conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another one of the same class, the classes being as follows:

Nonpolar: Ala, Val, Leu, lie, Pro, Met, Phe, Trp 15 Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gin

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, Gin, Lys, Arg, His, Trp

Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gin

In a further aspect of the present invention, there is provided a genetic construct including a nucleic acid according to the present invention.

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

-132016259449 18 Nov 2016 ln a preferred embodiment, the genetic construct according to the present invention may be a vector.

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

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, nonchromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens', derivatives of the Ri plasmid from Agrobacterium rhizogenes', 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 target cell.

In a preferred embodiment of this aspect of the invention, the genetic construct may further include a promoter and a terminator; said promoter, gene and terminator being operatively linked.

By a ‘promoter’ is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.

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

By ‘upstream’ is meant in the 3’->5’ direction along the nucleic acid.

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The promoter and terminator may be of any suitable type and may be endogenous to the target cell or may be exogenous, provided that they are functional in the target cell.

A variety of terminators which may be employed in the genetic constructs of the 5 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 genetic construct, in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns, antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptll) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes [such as betaglucuronidase (GUS) gene (gusA) and the green fluorescent protein (GFP) gene (gfp)]. The genetic construct may also contain a ribosome binding site for translation initiation. The genetic construct may also include appropriate sequences for amplifying expression.

Those skilled in the art will appreciate that the various components of the genetic construct are operably linked, so as to result in expression of said nucleic acid. Techniques for operably linking the components of the genetic construct of the present invention 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.

Preferably, the genetic construct is substantially purified or isolated.

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By ‘substantially purified’ is meant that the genetic construct is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter. The term therefore includes, for example, a genetic construct 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 (eg. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a genetic construct which is part of a hybrid gene encoding additional polypeptide sequence.

Preferably, the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.

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

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses.

The genetic constructs of the present invention may be introduced into plants or fungi by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells or fungal cells (for example by transduction,

-162016259449 18 Nov 2016 transfection, transformation or gene targeting) are well known to those skilled in the art. Such techniques include Agrobacterium-med\ated introduction, Rhizobiummediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof. The choice of technique will depend largely on the type of plant or fungus to be transformed, and may be readily determined by an appropriately skilled person. For transformation of protoplasts, PEG-mediated transformation is particularly preferred. For transformation of fungi PEG-mediated transformation and electroporation of protoplasts and Agrobacterium-med\ated transformation of hyphal explants are particularly preferred.

Cells incorporating the genetic constructs of the present invention may be selected, as described below, and then cultured in an appropriate medium to regenerate transformed plants or fungi, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants or fungi may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants or fungi.

In a further aspect, the present invention provides a plant inoculated with an endophyte variant as hereinbefore described, said plant comprising an endophytefree host plant stably infected with said endophyte variant.

Preferably, the plant is infected with the endophyte variant by a method selected from the group consisting of inoculation, breeding, crossing, hybridization and combinations thereof.

In a preferred embodiment, the plant may be infected by isogenic inoculation. This has the advantage that phenotypic effects of endophytes may be assessed in the absence of host-specific genetic effects. More particularly, multiple inoculations of

-17 2016259449 18 Nov 2016 endophytes may be made in plant germplasm, and plantlets regenerated in culture before transfer to soil.

The identification of an endophyte of the opposite mating-type that is highly compatible and stable in planta provides a means for molecular breeding of endophytes for perennial ryegrass. Preferably the plant may be infected by hyperinoculation.

Hyphal fusion between endophyte strains of the opposite mating-type provides a means for delivery of favourable traits into the host plant, preferably via hyperinoculation. Such strains are preferably selected from the group including an endophyte strain that exhibits the favourable characteristics of high inoculation frequency and high compatibility with a wide range of germplasm, preferably elite perennial ryegrass and/or tall fescue host germplasm and an endophyte that exhibits a low inoculation frequency and low compatibility, but has a highly favourable alkaloid toxin profile.

It has generally been assumed that interactions between endophyte taxa and host grasses will be species specific. Applicants have surprisingly found that endophyte from tall fescue may be used to deliver favourable traits to ryegrasses, such as perennial ryegrass.

In a further aspect of the present invention there is provided a method of analysing metabolites in a plurality of endophytes, said method including: providing:

a plurality of endophytes; and a plurality of isogenic plants;

inoculating each isogenic plant with an endophyte;

culturing the endophyte-infected plants; and analysing the metabolites produced by the endophyte-infected plants.

By ‘metabolites’ is meant chemical compounds, in particular toxins, produced by the

-182016259449 18 Nov 2016 endophyte-infected plant, including, but not limited to, lolines, peramine, ergovaline, lolitrem, and janthitrems, such as janthitrem I, janthitrem G and janthitem F.

By ‘isogenic plants’ is meant that the plants are genetically identical.

The endophyte-infected plants may be cultured by known techniques. The person 5 skilled in the art can readily determine appropriate culture conditions depending on the plant to be cultured.

The metabolites may be analysed by known techniques such as chromatographic techniques or mass spectrometry, for example LCMS or HPLC. In a particularly preferred embodiment, endophyte-infected plants may be analysed by reverse phase liquid chromatography mass spectrometry (LCMS). This reverse phase method may allow analysis of specific metabolites (including lolines, peramine, ergovaline, lolitrem, and janthitrems, such as janthitrem I, janthitrem G and janthitem F) in one LCMS chromatographic run from a single endophyte-infected plant extract.

In a particularly preferred embodiment, the endophyte variant may be selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17.

In another particularly preferred embodiment, LCMS including EIC (extracted ion chromatogram) analysis may allow detection of the alkaloid metabolites from small quantities of endophyte-infected plant material. Metabolite identity may be confirmed by comparison of retention time with that of pure toxins or extracts of endophyte-infected plants with a known toxin profile analysed under substantially the same conditions and/or by comparison of mass fragmentation patterns, for example generated by MS2 analysis in a linear ion trap mass spectrometer.

In a further aspect, the present invention provides a plant, plant seed or other plant part derived from a plant of the present invention and stably infected with an endophyte variant of the present invention.

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Preferably, the plant cell, plant, plant seed or other plant part is a grass, more preferably a forage, turf or bioenergy grass, such as those of the genera Lolium and

Festuca, including L. perenne and L. arundinaceum.

By ‘plant cell’ is meant any self-propagating cell bounded by a semi-permeable 5 membrane and containing plastid. Such a cell also required a cell wall if further propagation is desired. Plant cell, as used herein includes, without limitation, seeds suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.

In a further aspect, the present invention provides use of an endophyte variant as 10 hereinbefore described to produce a plant stably infected with said endophyte variant.

In a still further aspect, the present invention provides a method of quantifying endophyte content of a plant, said method including measuring copies of a target sequence by quantitative PCR.

In a preferred embodiment, the method may be performed using an electronic device, such as a computer.

Preferably, quantitative PCR may be used to measure endophyte colonisation in planta, for example using a nucleic acid dye, such as SYBR Green chemistry, and qPCR-specific primer sets. The primer sets may be directed to a target sequence such as an endophyte gene, for example the peramine biosynthesis perA gene.

The development of a high-throughput PCR-based assay to measure endophyte biomass in planta may enable efficient screening of large numbers of plants to study endophyte-host plant biomass associations.

As used herein, except where the context requires otherwise, the term comprise 25 and variations of the term, such as comprising, comprises and comprised, are not intended to exclude further additives, components, integers or steps.

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Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

Detailed Description of the Embodiments

In the figures:

Figure 1 shows the structures of Lolitrem B, Erogvaline and Peramine, with desirable toxin profiles indicated.

Figure 2 shows in vitro bioassays to assess antifungal activity of Neotyphodium endophytes.

Figure 3 shows a detached leaf assay to assess resistance to crown rust (Puccinia coronata f. sp. Lolii) of perennial ryegrass plants with and without Neotyphodium endophytes.

Figure 4 shows glasshouse and field trial screens for drought tolerance and water use efficiency of perennial ryegrass plants with and without Neotyphodium endophytes.

Figure 5 shows the steps involved in cell division.

Figure 6 shows experimental work flow for chromosome doubling of endophyte cells.

Figure 7 shows flow cytometry calibrations for DNA content assessment in Neotyphodium endophyte strains. Peaks indicate relative nuclear DNA content.

Figure 8 shows flow cytometry analysis of NEA12^dh Neotyphodium endophyte strains.

-21 2016259449 18 Nov 2016

Figure 9 shows analysis of growth rate in culture after 8 weeks of NEA12^dh Neotyphodium endophyte strains compared to control endophyte strains.

Figure 10 shows analysis of growth rate in culture over 5 weeks of NEA12^dh Neotyphodium endophyte strains compared to control endophyte strains.

Figure 11 shows antifungal bioassays of NEA12^dh Neotyphodium endophyte strains.

Figure 12 shows antifungal bioassays of NEA12^dh Neotyphodium endophyte strains.

Figure 13 shows analysis of genome survey sequencing read depth of colchicine10 treated Neotyphodium endophyte strains.

Figure 14 shows analysis of genome survey sequencing reads mapping to NEA12 genome survey sequence assembly.

Figure 15 shows experimental work flow for X-ray mutagenesis.

Figure 16 shows the indole-diterpene biosynthetic pathway of Neotyphodium 15 endophytes.

Figure 17 shows in vitro growth of X-ray irradiated Neotyphodium endophyte strains.

Figure 18 shows Itm gene clusters of Neotyphodium endophytes.

Figure 19 shows determination of genome sequence variation in X-ray irradiated 20 Neotyphodium endophyte strains.

Figure 20 shows single nucleotide polymorphisms (SNPs) in genome sequences of X-ray irradiated Neotyphodium endophyte strains.

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Figure 21 shows small insertions/deletions (INDELs) in genome sequences of X-ray irradiated Neotyphodium endophyte strains.

Figure 22 shows deletions in genome sequences of X-ray irradiated Neotyphodium endophyte strains.

Figure 23 shows numbers of SNPs in genic regions of genome sequences of X-ray irradiated Neotyphodium endophyte strains.

Figure 24 shows numbers of INDELs in genic regions of genome sequences of Xray irradiated Neotyphodium endophyte strains.

Figure 25 shows the spectrum of genome sequence changes (deletions) in genome 10 sequences of X-ray irradiated Neotyphodium endophyte strains.

Figure 26 shows mutagenesis index of X-ray irradiated strains based on number of genome sequence changes observed in genome sequences of X-ray irradiated Neotyphodium endophyte strains.

Figure 27 shows metabolic profiling of NEA12^dh Neotyphodium endophyte strains.

Figure 28 shows metabolic profiling of X-ray irradiated Neotyphodium endophyte strains.

The invention will now be described with reference to the following non-limiting examples.

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Example 1 - Overview of generation of novel designer Neotyphodium endophyte variant strains through mutagenesis

The objective of this work was to create novel variants of a perennial ryegrass Neotyphodium endophyte, through induced polyploidisation and mutagenesis, with desirable properties such as enhanced bioactivities (e.g. antifungal activity), and/or altered plant colonization ability and stability of grass host - endophyte variant associations (e.g. altered in vitro growth), and/or altered growth performance (e.g. enhanced plant vigour, enhanced drought tolerance, enhanced water use efficiency) of corresponding grass host - endophyte variant associations. These grass host - endophyte variant associations are referred to as novel ‘designer’ grass-endophyte associations.

Experimental strategies for the generation and characterisation of novel designer Neotyphodium endophyte variant strains through mutagenesis

The experimental activities thus included:

1. Establishment of phenotypic screens for novel ‘designer’ grass-endophyte associations such as:

• Enhanced biotic stress tolerance • Enhanced drought tolerance and enhanced water use efficiency • Enhanced plant vigour

2. Targeted generation (i.e. polyploidisation and X-ray mutagenesis) and characterisation (i.e. antifungal bioassays, in vitro growth rate, genome survey sequencing [GSS]) of novel ‘designer’ endophytes

3. Breeding of ‘designer’ grass-endophyte associations • Delivery of ‘designer’ endophytes into grass (e.g. perennial ryegrass) germplasm development process.

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Example 2 - Establishment of phenotypic screens for novel ‘designer’ grassendophyte associations

Assessment of enhanced biotic stress tolerance using NEA12 is shown in Figures 2 and 3. Figure 2 shows in vitro bioassays to assess antifungal activity of

Neotyphodium endophytes. Figure 3 shows a detached leaf assay to assess resistance to crown rust (Puccinia coronata f.sp. to///).

Assessment of enhanced drought tolerance and enhanced water use efficiency is shown in Figure 4. This involved glasshouse and field trial screens for drought tolerance, survival and recovery, regrowth after drought, metabolic profiling and detailed phenotypic characterisation including multiple trait dissection (based on assessments and measurements associated with plant morphology, plant physiology, plant biochemistry).

Example 3 - Generation of designer Neotyphodium genotypes by polyploidisation

This involved creation of novel variation in Neotyphodium endophytes without the use of transgenic technology. Colchicine has been widely and successfully used for chromosome doubling in plants, e.g. perennial ryegrass. It inhibits chromosome segregation during mitosis inducing autopolyploidisation (chromosome doubling; see Figure 5). This enables the generation of novel endophytes through induced chromosome doubling and may be applicable to the production of artificial polyploid endophytes.

The experimental work flow for chromosome doubling is shown in Figure 6.

Flow cytometry calibrations to assess DNA content in Neotyphodium endophytes are shown in Figure 7. Peaks indicate relative nuclear DNA content.

Flow cytometry analysis of NEA12^dh strains is shown in Figure 8 and Table 1.

1. ST endophyte strain is highly stable, broadly compatible and produces lolitrems, peramine and ergovaline. 2. NEA12 endophyte strain produces janthitrem only. 3.

AR1 produces peramine only.

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Endophyte	Oo1chicirt&	it of Colonies	MCMmripd
N. Lolii ST	0.2	12	12
NEA12	0.1	60	2
NEA12	0.2	60	18
N. Lolii AR1	0.1	60	0
N. Lolii AR1	0.2	60	0

Table 1: Colchicine treated endophyte strains (ST, NEA12 and AR1 endophyte strains) subjected to colchicine treatments (at different colchicine concentrations in %) leading to the recovery of endophyte colonies (# of colonies) used for flow cytometry analysis

Example 4 - Analysis of in vitro growth of NEA12^dh Neotyphodium variant 10 endophyte strains

Analysis of growth rate of NEA12^dh Neotyphodium variant endophyte strains in in vitro culture after 8 weeks is shown in Figure 9. In an initial screen, analysis of variance identified two NEA12^dh Neotyphodium variant endophyte strains (NEA12^dh17 and NEA12^dh4) showing significantly different in vitro growth rate to the control NEA12 endophyte:

NEA12^dh17 grows significantly faster (p<0.01**)

NEA12^dh4 grows significantly slower (p<0.05*)

Analysis of growth rate of NEA12^dh Neotyphodium variant endophyte strains in in vitro culture over 5 weeks is shown in Figure 10. In a validation screen, Student’s t20 tests identified two NEA12^dh Neotyphodium variant endophyte strains (NEA12^dh17 and NEA12^dh15) showing significantly different in vitro growth rate to the control NEA12 endophyte:

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NEA12^dh17 grows significantly faster (p<0.01**)

NEA12^dh15grows significantly slower (p<0.01**)

Example 5 - Antifungal bioassays of NEA12^dh Neotyphodium variant endophyte strains

A list of fungal pathogens (causing a range of fungal diseases and infecting a range of different plant hosts) that were included in antifungal bioassays used to analyse NEA12^dh Neotyphodium variant endophyte strains to assess their spectrum of antifungal activities is shown in Table 2.

Fungus	Disease	Hosts
Alternaria alternata	leaf spot, rot, blight	Numerous (dead plant materials)
Bipolaris portulacae	Damping-off	Asteraceae (daisies), Portulacaceae (purslane)
Botrytis cinerea	Stem rot, mould, seedling wilt	Many dicots, few monocots
Colletotrichum graminicola	Leaf spot, stalk rot	Poaceae (especially Zea mays)
Drechslera brizae	Leaf blight	Poaceae (Briza spp.)
Phoma sorghina	Spot (leaf, glume, seed), Root rot, Dying-off	Poaceae (grasses)
Rhizoctonia cerealis	Spot (wheat) Yellow patch (turfgrass)	Poaceae (grasses)
Trichoderma harzianum	Green mould, Parasite of other fungi	Many dicots, few monocots, Fungi

Table 2: Fungal pathogens (causing a range of fungal diseases and infecting 10 a range of different plant hosts) included in antifungal bioassays to analyse

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NEA12^dh Neotyphodium variant endophyte strains to assess their spectrum of antifungal activities

Antifungal bioassays of NEA12^dh Neotyphodium variant endophyte strains are shown in Figures 11 and 12. Twenty NEA12^dh strains were screened for changes in antifungal activity. Four NEA12^dh strains (i.e. dh5, dh6, dh13 and dh14) were identified as having greater antifungal activity compared to NEA12.

Example 6 - Genome survey sequencing and sequence analysis of NEA12^dh Neotyphodium variant endophyte strains

NEA12^dh Neotyphodium variant endophyte strains with enhanced antifungal activity, 10 showing faster in vitro growth rate and higher DNA content were subjected to genome survey sequencing (GSS). Sequence data was generated for 10 NEA12^dh strains and control NEA12 strain (highlighted in blue on Table 3).

Endophyte	Antifungai	Growth
NEA12	Std	Std
NEA12dh1	Std	Std
NEA12dh2	Std	Std
NEA12dh3	Std	Std
NEA12dh4	Std	Slower
NEA12dh5	Higher	Std
NEA12dh6	Higher	Std
NEA12dh7	Std	Std
NEA12dhS	Std	Std
NEA12dh9	Std	Std
NEA12dh10	Std	Std
NEA12dh11	Std	Std
NEA12dh12	Std	Std
NEA12dh13	Higher	Std
NEA12dh14	Higher	Std
NEA12dh15	Std	Slower
NEA12dh16	Std	Std
NEA12dh17	Std	Fester
NEA12dh1S	Std	Std
NEA12dh19	Std	Std
NEA12dh20	Std	Std

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Table 3: List of NEA12^dh Neotyphodium variant endophyte strains showing different antifungal activity [higher than control or equal to control (standard, Std)] and different in vitro growth [slower than control, faster than conrol or equal to control (standard, Std)] compared to control NEA12 strain

Genome survey sequencing (GSS) data obtained for NEA12^dh Neotyphodium variant endophyte strains derived from colchicine treated NEA12 control strain (highlighted in blue on Table 3) were analysed as follows:

• De-novo assembly of the GSS data from NEA12 control strain - to act as a reference genome sequence for the analysis of the NEA12^dh Neotyphodium variant endophyte strains • Map the GSS data sequence reads from the NEA12^dh Neotyphodium variant endophyte strains to the NEA12 reference genome sequence • Identify potentially duplicated regions, i.e. regions with higher than expected sequence coverage · Identify gene sequences that may have been duplicated

Analysis of GSS read depth of NEA12^dh Neotyphodium variant endophyte strains is shown in Figure 13. Analysis of sequence contigs that appeared to have higher than expected read depth indicates that no major duplication event has occurred (excepting whole genome events). The patterns of read depth across these contigs are not identical between strains. This suggests there are differences between the NEA12^dh Neotyphodium variant endophyte strains and the control NEA12 strain.

Analysis of GSS sequence assemblies for the NEA12^dh Neotyphodium variant endophyte strains and the control NEA12 strain is shown in Table 4.

Strain

# contigs

N50

Max contig

# bases

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NEA12	143202	28621	181461	32734984
NEA12dh5	305031	29444	191191	30994592
NEA12dh17	274394	37802	209957	30777017
NEA12dh18	282692	30717	177813	30889903

Table 4: Analysis of GSS sequence assemblies for the NEA12^dh

Neotyphodium variant endophyte strains and the control NEA12 strain

Independent de novo sequence assemblies were performed using parameters identical to those used in assembling the genome sequence for the control NEA12 endophyte strain. Differences in sequence assembly statistics may indicate genomic differences between strains. GSS data obtained for the NEA12^dh Neotyphodium variant endophyte strains and used in the sequence assemblies reveal fewer bases incorporated into the sequence assembly and produce more sequence contigs. Increased numbers of smaller sequence contigs may be caused by transposon movement/replication.

Analysis of sequence reads mapping to the NEA12 genome sequence assembly is shown in Figure 14. While we do not wish to be restricted by theory, if the genomes were the same no difference in the number of sequence reads mapping to the reference genome sequence would be expected. NEA12^dh Neotyphodium variant endophyte strains range from 35-70% sequence reads mapping to NEA12 sequence contigs > 5kb in size. There are differences between the genome sequences of the NEA12^dh Neotyphodium variant endophyte strains and the control NEA12 strain.

Summary of results on generation and characterisation of novel designer

Neotyphodium variant endophyte strains through colchicine treatment based mutagenesis

Sequence read depth changes were analysed in NEA12^dh Neotyphodium variant endophyte strains compared with the control NEA12 strain. Whilst no large partial

-302016259449 18 Nov 2016 genome sequence duplication events were detected, the occurrence of full genome duplication events in the NEA12^dh Neotyphodium variant endophyte strains cannot be excluded based on the GSS sequence analysis.

De novo sequence assemblies were independently performed on GSS data 5 obtained from the NEA12^dh Neotyphodium variant endophyte strains. Differences in sequence assembly statistics indicate that genomic changes were caused by the colchicine-treatment in the NEA12^dh Neotyphodium variant endophyte strains. The number of sequence reads from NEA12^dh Neotyphodium variant endophyte strains mapping to the NEA12 reference genome sequence varies between strains. All

GSS data analyses performed on the NEA12^dh Neotyphodium variant endophyte strains indicate genomic differences.

In summary, the following novel designer endophytes were generated by colchicine treatment of NEA12 endophytes:

• Four NEA12^dh Neotyphodium variant endophyte strains (dh5, dh6, dh13 and dh14) with enhanced bioprotective properties (i.e. antifungal bioactivities);

• One NEA12^dh Neotyphodium variant endophyte strain (dh17) with higher in vitro growth rate than control NEA12 strain (i.e. potentially with enhanced stability/host colonization ability);

• Ten NEA12^dh Neotyphodium variant endophyte strains (including dh5, dh6, dh13, dh14 and dh17) and control NEA12 strain subjected to genome survey sequencing; and • Five NEA12^dh Neotyphodium variant endophyte strains (including dh5, dh13 and dh17) selected and subjected to isogenic inoculation in planta.

Example 7 - In planta isogenic inoculation in perennial ryegrass with NEA12^dh Neotyphodium variant endophyte strains

The following NEA12^dh Neotyphodium variant endophyte strains and control NEA12

-31 2016259449 18 Nov 2016 strain were used for in planta isogenic inoculation in perennial ryegrass:

NEA12

NEA12dh5

NEA12dh13

NEA12dh4

NEA12dh15

NEA12dh17 showing higher antifungal activity than control NEA12 showing higher antifungal activity than control NEA12 showing slower in vitro growth rate than control NEA12 showing slower in vitro growth rate than control NEA12 showing faster in vitro growth rate than control NEA12

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Plant Genotype	NEA12 dh4	NEA12 dh5	NEA12 dh13	NEA12 dh15	NEA12 dh17	NEA12
IMP04	30	30	30	30	32	30
TOL03	25	30	30	20	30	20

Table 5: Isogenic inoculation of perennial ryegrass genotypes (IMP04 and TOL03) with NEA12dh Neotyphodium variant endophyte strains. Numbers indicate number of perennial ryegrass plants of the two genotypes subjected to isogenic inoculation with the different NEA12dh Neotyphodium variant endophyte strains.

Example 8 - Generation of designer Neotyphodium genotypes by X-ray mutagenesis

The generation of designer Neotyphodium endophytes genotypes by X-ray mutagenesis offers the opportunity to create novel endophyte variant strains with enhanced properties, such as enhanced stability in grass hosts, broader host compatibility as well as improved toxin profiles e.g. following elimination of the production of the detrimental alkaloid lolitrem B in the highly stable and broadly compatible ST endophyte.

Such an novel designer endophyte would be advantageous over existing commercial endophytes, such as AR1 and AR37, as it would be highly stable and broadly compatible and with optimal toxin profile.

Figure 15 shows an experimental work flow for X-ray mutagenesis of endophyte strains.

Figure 16 shows the indole-diterpene biosynthetic pathway. Lolitrem B is the major toxin that causes ryegrass staggers, a disease of grazing animals. Ten genes in 3 gene clusters are required for lolitrem biosynthesis. We focused initial analysis on 3 Ltm genes, one from each gene cluster. Optimised multiplex PCR analysis was designed and implemented.

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Example 9 - Screening of X-ray irradiated N. ZoZZZ strains

In a preliminary primary screen >5,000 colonies of X-ray irradiated N. lolii established as an initial resource of novel variation of N. lolii endoophytes induced through X-ray mutagenesis and representing a mutagenised N. lolii endophyte strain collection - of were screened by multiplex PCR analysis for the presence of targeted Ltm genes leading to a preliminary identification of ~140 putative lolitrem B gene cluster PCR-negative colonies (~2.5% of 5,000 colonies screened). In a secondary screen high quality DNA was extracted (140 liquid cultures) and PCR analysis conducted. This identified 2 putative deletion mutants for one of the lolitrem B genes (ltm J).

Dose (Gy)	Colony	ltm J	ltm C |	ltm M |
30 Gy (1 irradation)	139-6
30 Gy (1 irradation)	145-15

Table 6: Putative X-ray irradiation-induced ltm gene deletion mutants of N. lolii derived from irradiation with 30 Gy dose. The colony number represents the unique identifier of the putative X-ray irradiation-induced ltm gene deletion mutant (i.e. 139-6 and 145-15). Black represents PCR-negative result for respective ltm gene analysis, grey represents PCR-positive result for respective ltm gene analysis.

Example 10 - Antifungal bioassays of designer X-ray irradiated N. ZoZZZ variant strains

There were eight X-ray irradiated N. lolii variant strains (i.e. X-ray mutagenesis 20 derived variant strains 1-35, 4-7, 7-22, 7-47, 123-20, 124-6, 139-6, 144-16 and 14515) and one control N. lolii strain (i.e. ST endophyte strain).

Five fungal pathogens (causing a range of fungal diseases and infecting a range of different plant hosts) were included in antifungal bioassays used to analyse the Xray irradiated N. lolii variant strains, as follows:

Bipolaris portulacae

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Colletotrichum graminicola

Drechslera brizae

Phoma sorghina

Rhizoctonia cerealis

No significant difference in antifungal activities of X-ray irradiated N. lolii variant strains tested was observed compared to the spectrum of antifungal activities observed for the control ST endophyte strain.

Example 11 - In vitro growth of designer X-ray irradiated N. lolii variant strains

Results from the analysis of in vitro growth rate of designer X-ray irradiated N. lolii variant strains are shown in Figure 17, with a statistical analysis of in vitro growth undertaken at week 5 for the X-irradiated N. lolii variant strains compared to the control ST strain, revealing significant differences in in vitro growth rates as follows: p< 0.05* (for X-irradiated N. lolii variant strain 139-6) p<0.01** (for all other mutants)

Example 12 - Genome survey sequencing of designer X-ray irradiated N. lolii variant strains

Eight X-ray irradiated N. lolii ST variant strains and corresponding control ST strain were subjected to genome survey sequencing (GSS), leading to 46-fold to 79-fold genome sequence coverage for the different strains as shown in Table 7.

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Strain	Description	Coverage
ST	ST	23x
139-6	ST irradiated	61x
145-15	ST irradiated	52x
144-16	ST irradiated	46x
1__35	ST irradiated	79x
U	ST irradiated	46x
7~22	ST irradiated	53x
7_47	ST irradiated	3Sx
123-20	ST irradiated	54x
124-6	ST irradiated	75x

Table 7: Genome sequence coverage obtained in genome survey sequencing for for 8 X-ray irradiated N. lolii ST variant strains and corresponding control ST strain

Example 13 - Detecting genome sequence variation in designer X-ray irradiated N. lolii variant strains

Results from the analysis to detect genome sequence variation in X-ray irradiated N. lolii variant strains are shown in Figure 19. Corresponding results on the detection of single nucleotide polymorphisms (SNPs) are shown in Figure 20 and results on the detection of small insertions/deletions (INDELs) are shown in Figure

21. Differences in sequence read depth and pair insert size in X-ray irradiated N. lolii variant deletion mutant strains are shown in Figure 22.

Results on sequence analysis for Ltm gene clusters are shown in Figure 18. No deletions, large or small, were found in the coding or regulatory sequences of ltm gene clusters. No SNPs, insertions or translocations were found in the coding or regulatory sequences of ltm gene clusters.

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Example 14 - Spectrum of genome sequence changes detected in the X-ray irradiated N. lolii variant strains

Figure 23 shows numbers of SNPs detected in genic regions of X-ray irradiated N. lolii variant deletion mutant strains. There are large differences in the number of

SNPs detected in the X-ray irradiated N. lolii variant deletion mutant strains and compared to the control ST strain. All X-ray irradiated N. lolii variant deletion mutant strains have over double the number of SNPs per Mb across genic regions compared to the control ST strain. X-ray irradiated N. lolii variant deletion mutant strains have on average 6 SNPs per Mb, where the control ST strain has 2 SNPs per Mb.

Figure 24 shows numbers of INDELs in genic regions of X-ray irradiated N. lolii variant deletion mutant strains. All X-ray irradiated N. lolii variant deletion mutant strains contain more indels in genic regions than the control ST strain. The difference in indel numbers between the X-ray irradiated N. lolii variant deletion mutant strains and the control ST strain is on average 134 indels per Mb. When grouped by irradiation treatment (i.e. irradiation dose applied and number of repeat irradiations) there appears to be a peak in number of indels at 10Gy*2 treatment, consistent with the results obtained in the SNP detection analysis.

Figure 25 shows the spectrum of genome sequence changes in the form of deletions detected in X-ray irradiated N. lolii variant deletion mutant strains.

Table 8 shows examples of some of these genome sequence deletions detected in X-ray irradiated N. lolii variant deletion mutant strains.

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Strain	Radiation Treatment	Deletion
123_20	30Gy*2	Contig00915 (268bp)
124_6	30Gy*2	Partial duplication
139_6	30Gy	Partial duplication
144_16	30Gy
145_15	30Gy	Partial duplication
1_35	10Gy	Contig00831 (3.6kb)
4_7	10Gy
7_22	10Gy*2
7_47	10Gy*2	Contig01131 (0.6kb), contig01082 (4.2kb), contig02985 (1kb), contig02725 (83bp), contig01095 (130bp)

Table 8: Deletions detected in genome sequences of X-ray irradiated N. lolii variant deletion mutant strains. Bold indicates deletions confirmed by changes in sequence read coverage. The remainder are potential transposon deletions.

The X-ray irradiated N. lolii variant deletion mutant strain # 7_47, which was generated following two X-irradiation treatments at 10 Gy dose (10Gy*2) of N. lolii ST endophyte, had the greatest number of large deletions.

Example 15 - Annotation of deleted sequences in the genomes of X-ray irradiated N. lolii variant deletion mutant strains

X-Ray Irradiated N. lolii Variant Mutant Strain 1_35:

For the X-ray irradiated N. lolii variant mutant strain 1_35 the following deleted sequences in ST454Contig00831 contig with a ~ 4,400-8,000 bp length was detected, with this genome sequence region containing the following two predicted genes:

ST454contig00831_AUGUSTUS_gene_3318:6018 (847 letters)

1) ref |XP_386347.11 hypothetical protein FG06171.1 [Gibberella 660x0.0

-382016259449 18 Nov 2016 gb|EAW12630.11 DUF500 domain protein [Aspergillus NRRL 1]; 253 x 9e-66, and ST454contig00831_AUGUSTUS_gene_3958:4728 (183 letters); and 2) gb|EAW13545.11 2,3-cyclic-nucleotide 2-phosphodiesterase [Aspergillus 32 x 2.4

X-Ray Irradiated N. /o///Variant Mutant Strain 7_47:

For the X-ray irradiated N. lolii variant mutant strain 7_47 the following deleted sequences in ST454Contig01082, ST454Contig01131 and ST454Contig02985, with these genome sequence regions containing no predicted genes:

Query= ST454contig01082 length=9120 numreads=287 gb|AAA21442.11 putative pol polyprotein [Magnaporthe grisea] 145 1e-32 Query= ST454contig02985 length=2414 numreads=99 gb|AAA21442.11 putative pol polyprotein [Magnaporthe grisea] 92 2e-17

Example 16 - Mutagenesis index of X-ray irradiated N. lolii variant deletion mutant strains

Figure 26 shows SNPs and Indels per Mb in genic regions of X-ray irradiated N. lolii variant deletion mutant strains derived from X-ray irradiation of N. lolii at different levels of irradiation. Strain 1_35 has a 3.6 kb deletion; Strain 7_47 has 3 deletions (4.2 kb, 1 kb, 0.6 kb in length). Strain 124_6 has a partial duplication. Strains 139_6 and 145_15 have partial duplications.

Given that ST endophyte has approximately 443.5 genes per Mb, using 10Gy*2 treatment, the expected rate of SNP/INDEL occurrence is 0.33 per gene in the genome.

Summary

X-ray irradiated N. lolii variant deletion mutant strains were analysed for many types of genome sequence variation i.e. deletions, SNPs, INDELs, inversions and translocations. SNPs, INDELs, deletions and duplications were identified in the genome survey sequences of X-ray irradiated N. lolii variant deletion mutant strains. There was an apparent peak in number of SNPs and INDELs in X-ray irradiated N.

-392016259449 18 Nov 2016 lolii variant deletion mutant strains recovered from administering 10Gy*2 X-ray irradiation treatment to N. lolii ST endophyte. The X-ray irradiated N. lolii variant deletion mutant strain 7_47 had 3 large deletions. It was demonstrated that this mutagenesis method based on X-ray irradiation can be used to create novel designer Neotyphodium endophyte strains, and enabled:

• 5,000 X-ray irradiated N. lolii variant endophyte strains derived from X-ray irradiation of ST N lolii endophyte were screened;

• 140 putative X-ray irradiated N. lolii variant endophyte mutant strains were identified;

• 9 X-ray irradiated N. lolii variant endophyte mutant strains were subjected to antifungal bioassays;

• 9 X-ray X-ray irradiated N. lolii variant endophyte mutant strains were subjected to in vitro growth assays;

«9 X-ray irradiated N lolii variant endophyte mutant strains were subjected to genome survey sequencing;

• 2 X-ray irradiated N lolii variant endophyte mutant strains with gene deletions (1_35 and 7_47) were identified; and • 3 X-ray irradiated N lolii variant endophyte mutant strains with gene duplications (124_6, 139_6 and 145_15) were identified.

Example 17 - In planta isogenic inoculation in perennial ryegrass with X-ray irradiated N. /o///variant endophyte mutant strains

-402016259449 18 Nov 2016

Plant Genotype	ST-IRM 139-6	ST-IRM 145-15	ST-IRM 144-16	ST-IRM 1-35	ST-IRM 7-47	ST
IMP04	30	25	30	30	30	25
TOL03	25	0	25	30	30	20

Table 9: Isogenic inoculation of perennial ryegrass genotypes (IMP04 and TOL03) with X-ray irradiated N. lolii variant endophyte mutant strains.

Numbers indicate number of perennial ryegrass plants of the two genotypes subjected to isogenic inoculation with the different X-ray irradiated N. lolii variant endophyte mutant strains (i.e. ST-IRM 139-6, ST-IRM 145-15, ST-IRM 144-16, STIRM 1-35 and ST-IRM 7-47) and control ST endophyte strain.

Example 18 - Metabolic profiling of colchicine treatment-derived NEA12dh 10 and X-ray irradiation-derived Neotyphodium variant endophyte strains

Results from metabolic profiling of colchicine treatment derived NEA12dh endophyte variant strains is shown in Figure 27.

Results from metabolic profiling of X-ray irradiation treatment derived N. lolii ST endophyte variant strains is shown in Figure 28.

The following endophytes were grown on PDB for 3 weeks:

Control N. lolii ST endophyte strain

X-ray irradiation treatment derived N. lolii ST endophyte variant strain 4-7

X-ray irradiation treatment derived N. lolii ST endophyte variant strain 139-6

X-ray irradiation treatment derived N. lolii ST endophyte variant strain 144-16

X-ray irradiation treatment derived N. lolii ST endophyte variant strain 145-15 and subjected to metabolic profiling using LCMS on corresponding

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1. Liquid filtrate

2. Mycelial extract

The X-ray irradiation treatment derived N. lolii ST endophyte variant strains could be readily distinguished from control N. lolii ST strain using mycelia extracts or filtrates alone.

-42 2016259449 24 Jan 2017

Claims (21)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. An endophyte variant having a desired genetic and metabolic profile, wherein said endophyte variant possesses genetic and/or metabolic characteristics that result in a beneficial phenotype in a plant harbouring, or otherwise associated with,

   5 the endophyte variant, when compared with a standard toxic control endophyte or with a no endophyte control plant, and wherein said endophyte variant is selected from the group consisting of NEA12dh5, NEA12dh6, NEA12dh13, NEA12dh14, and NEA12dh17 as deposited at the National Measurement Institute with accession numbers V12/001408, V12/001409, V12/001410, V12/001411 and V12/001412,

   10 respectively.
2. 2. An endophyte variant according to claim 1, wherein said beneficial phenotype is selected from the group consisting of improved tolerance to water and/or nutrient stress, improved resistance to pests and/or diseases, enhanced biotic stress tolerance, enhanced drought tolerance, enhanced water use efficiency,

   15 reduced toxicity and enhanced vigour.
3. 3. An endophyte variant according to claim 1 or 2, wherein said endophyte variant is generated by polyploidisation or induced chromosome doubling.
4. 4. An endophyte variant according to claim 3, wherein said endophyte variant is generated by treating an endophyte with colchicine or a similar compound that

   20 induces polyploidisation or induced chromosome doubling.
5. 5. An endophyte variant according to claim 1 or 2, wherein said endophyte variant is generated by subjecting an endophyte to X-ray mutagenesis or exposing an endophyte to ionising radiation.
6. 6. An endophyte variant according to any one of claims 1 to 5, wherein said 25 endophyte variant is generated from an endophyte which is isolated from a Lolium species.

   -432016259449 24 Jan 2017
7. 7. An endophyte according to claim 6, wherein said Lolium species is Lolium perenne.
8. 8. An endophyte variant according to any one of claims 1 to 7, wherein said endophyte variant has a desired toxin profile, wherein the endophyte variant

   5 produces significantly less toxic alkaloids and/or significantly more alkaloids conferring beneficial properties, in the plant with which the endophyte is associated, when compared with a standard toxic (ST) control endophyte or with a no endophyte control plant.
9. 9. An endophyte variant according to claim 8, wherein said toxic alkaloids are 10 present in an amount less than approximately 1 pg/g dry weight.
10. 10. An endophyte variant according to claim 8 or 9, wherein said alkaloids conferring beneficial properties are present in an amount of between approximately 5 and 100 pg/g dry weight.
11. 11. A plant inoculated with an endophyte variant according to any one of claims

    15 1 to 10, said plant comprising an endophyte-free host plant stably infected with said endophyte variant.
12. 12. A plant, plant seed or other plant part derived from a plant according to claim 11 and stably infected with an endophyte variant according to any one of claims 1 to 10.

    20 13. Use of an endophyte variant according to any one of claims 1 to 10 to produce a plant stably infected with said endophyte variant.

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    2016259449 18 Nov 2016

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    2016259449 18 Nov 2016

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    2016259449 18 Nov 2016

    Fig. 14

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    2016259449 18 Nov 2016

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