WO2023073224A1 - Methods of increasing root endosymbiosis - Google Patents

Abstract

The present invention relates to genetically altered plants, parts thereof and plant cells that comprises one or more mutations in a conserved motif XDPX of nuclear localized cyclic nucleotide-gated ion channel (CNGC) channels, as well as methods of increasing yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses by introducing the one or more mutation into a CNGC gene.

Description

Methods of increasing root endosymbiosis

FIELD OF THE INVENTION

The present invention relates to genetically altered plants, parts thereof and plant cells that comprises one or more mutations in a conserved motif “XDPX” of nuclear localised cyclic nucleotide-gated ion channels (CNGC), as well as methods of increasing yield, nodulation and/or arbuscular mycorrhiza (AM) endosymbioses by introducing the one or more mutation into a nuclear-localised CNGC gene.

BACKGROUND OF THE INVENTION

Nitrogen-fixing bacteria and arbuscular mycorrhiza (AM) endosymbioses within plant roots provide a significant benefit to crops and wider agrosystems in many ways, including delivery of nutrients such as bioavailable nitrogen and phosphate as well as improving soil structure and resistance to pests and pathogens. In the 20^th century, despite the acknowledged benefit of endosymbioses, chemical fertilizers were instead extensively used to boost yield while concurrently leading to waterway pollution, chemical burn to crops, increased air pollution, acidification of the soil and depletion of global rock-phosphorus; ultimately challenging global food security. Climate change and increasing pressure to get more yield from less arable land is now accentuating this challenge. Globally sustainable agricultural systems require environmentally benign alternatives to chemical fertilizers including, increasing the contribution made by endosymbioses.

A challenge of crop breeding is to optimize net nitrogen acquisition via the soil from chemical fertilizers and/or symbiotic nitrogen fixation to increase yield. Therefore, developing new plant varieties with enhanced endosymbiotic benefit will result in increased yield, reduced reliance on chemical fertilizers and further contribute to improving sustainable agricultural practice and food production.

Until now, however, all identified hyper-nodulating and hyper-mycorrhizal mutants increase endosymbiosis but also impair growth with a negative impact on yield. There is therefore a pressing unmet need for plants with enhanced endosymbiosis but without any growth penalty. The present invention addresses this need. SUMMARY OF THE INVENTION

In one aspect of the invention, there is provided a genetically altered plant, plant part thereof or plant cell comprising at least one mutation in one or more gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC). Preferably, the CNGC is CNGC 15.

In another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a mutated nuclear-localised cyclic nucleotide-gated ion channel (CNGC) amino acid sequence operably linked to a regulatory sequence. Preferably the CNGC is CNGC 15.

In another aspect, there is provided a vector comprising the nucleic acid construct of the invention.

In another aspect, there is provided a host cell comprising the nucleic acid construct of the invention or the vector of the invention.

In another aspect, there is provided a genetically altered plant, plant part thereof or plant cell, wherein the plant, part thereof or plant cell comprises the nucleic acid construct of the invention, the vector of the invention or the host cell of the invention.

In another aspect of the invention, there is provided a method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant, the method comprising introducing and expressing the nucleic acid construct of the invention or the vector of the invention in the plant, part thereof or plant cell.

In another aspect of the invention, there is provided a method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant, the method comprising introducing at least one mutation in one or more (endogenous) gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC) such as CNGC 15. In another aspect of the invention, there is provided a method for maintaining yield while reducing the input of chemical fertilizer, the method comprising introducing at least one mutation in at one or more gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC), preferably CNGC 15.

In another aspect of the invention, there is provided a method for identifying and/or selecting a plant that will have an increase in at least one of yield, nodulation and/or AM endosymbiosis, the method comprising screening a population of plants and detecting in the plant or plant germplasm at least one polymorphism in at least one conserved domain of the nuclear localized cyclic nucleotide-gated ion channel (CNGC) gene, preferably a polymorphism in a XDPX (where X is any amino acid) domain, preferably a XDPL domain and more preferably a VDPL domain; and selecting said plant.

The plant may be a dicot or a monocot. Preferably the plant is selected from wheat, rice, potato, maize, soybean, tomato, barley, sugar cane, sorghum, sunflower, sugar beet, rye, cotton, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.

Preferably, the plant part is a seed. In another aspect of the invention, there is provided a seed obtained or obtainable from the genetically altered plant of the invention. In another aspect, there is provided a seed, wherein the seed expresses one or more CNGC amino acid sequence as defined in one of SEQ ID NO: 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof. In an alternative embodiment, the seed comprises one or more mutations in a gene encoding a nuclear-localised CNGC amino acid sequence, for example as defined herein.

DESCRIPTION OF THE FIGURES

The invention is further described in the following non-limiting figures:

Figure 1 : Point mutants in Medicago truncatula CNGC15c and CNGC15a exhibit spontaneous calcium oscillations and increased numbers of nodules and arbuscular mycorrhizal colonization.

Representative Ca²⁺ traces of WT, cngc15a-easy, and cngc15c-easy roots showing detrended YFP/CFP ratios in arbitrary units (a). Traces were recorded for approximately 30 minutes before and after Nod factor addition (n = 13, WT; 8, cngc15a-easy, and 8, cngc15c-easy).

Number of nodules per plant (b) and percentage mycorrhizal colonization (c) in WT, cngc15a-easy and cngc15c-easy. Plants were grown in nutrient-depleted conditions (sand and terragreen with water) and assessed at 14 dpi with Sinorhizobium meliloti strain 2011 or at 20% WT colonization percentages with arbuscular mycorrhizal fungi Rhizophagus irregularis, respectively.

Boxplots represent the medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from three experiments (b - cngc15a-easy) or two experiments (b - cngc15a-easy, c). Individual data points for the biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A Shapiro-Wilk test was used to test for normality, and statistical comparisons were made to the WT (Student's t-test (normal data), (Mann-Whitney (non-normal data): *, P < 0.05; **, P > 0.01 ; ***, P < 0.001 ; ****, P < 0.0001).

Figure 2: Increased nodulation in cngc15a-easy results in positive growth responses, nutrient status and seed yields.

WT and cngc15a-easy mutants were grown in nutrient-depleted conditions (sand and terragreen with water) and assessed after inoculation Sinorhizobium meiloti strain 2011. Rhizobial colonization structures (total nodules and pink nodules per plant) (a), plant dry weight (b), and nitrogen/carbon ratio of pooled shoot tissue (total number of plants are indicated in brackets) (c) were measured at 28 dpi. Seed yield (number of seeds per pod per plant) of plants was assessed at 16 weeks post-inoculation (wpi) (d). Plant dry weight of plants grown for 28 days in non-endosymbiotic nutrient-depleted conditions (sand and terragreen with water) (e).

Boxplots represent medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from three experiments (a, b). Lines represent the means from pooled samples from two experiments (c) or one experiment (d). Individual data points for biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A Shapiro- Wilk test was used to test for normality, and statistical comparisons were made to the WT (Student's t-test (normal data), Mann-Whitney test (non-normal data): ns; ****, P < 0.0001).

Figure 3: cngc15a-easy has increased mycorrhizal colonization and increased seed yield.

WT and cngc15a-easy mutants were grown in nutrient-depleted conditions (sand and terragreen with water) and assessed after inoculation with mycorrhizal fungi Rhizophagus irregularis. Mycorrhizal colonization structures (intraradical hyphae (IRH), arbuscules (A), and vesicles (V)) (a) and corresponding relative shoot dry weights (b) were quantified when WT arbuscule occurrence reached 20%. Occurrences of mycorrhizal structures are shown as a percentage of the total number of root sections assessed. Seed yield (number of seeds per pod per plant) of plants was assessed at 16 weeks post-inoculation (wpi) (c).

Boxplots represent medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from three experiments (a, b). Lines represent the means from pooled samples from one experiment (c). Individual data points for biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A Shapiro-Wilk test was used to test for normality, and statistical comparisons were made to the WT (Student's t-test (normal data, a IRH, b), Mann-Whitney test (non-normal data a A, a V, c): ns, P > 0.05; *, P < 0.05; ** P < 0.01 ; ****, P < 0.0001).

Figure 4: cngc15a-easy mutants have increased nodulation in the presence of nitrate but not ammonia.

WT and cngc15a-easy mutants were inoculated with Sinorhizobium meiloti strain 2011 and grown in the presence of different nutrients: control (BNM), + nitrate (BNM + 3 mM KNO₃-), + ammonia (BNM + 4.8 mM (NH₄)₂SO₄). Rhizobial colonization structures (total nodules and pink nodules per plant) (a), root and shoot dry weights (b), and nitrogen/carbon ratio of pooled shoot tissue (total number of plants indicated in brackets) (c) were measured at 28 dpi.

Boxplots represent medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from three experiments (a, b). Lines represent the means from pooled samples from two experiments (c). Individual data points for biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A two-way ANOVA was used to test for differences between genotypes, treatments, and interaction effects with Bonferroni correction for multiple comparisons (letters represent no significant difference at 0.05% level) (a). A Shapiro-Wilk test was used to test for normality, and statistical comparisons were made to the WT (b, c) (Student's t-test (normal data, b), Mann- Whitney test (non-normal data, c): ns, P > 0.05; *, P < 0.05; ***, P < 0.001).

Figure 5: Increased nodulation in cngc15a-easy results in positive growth responses at high temperatures.

WT and cngc15a-easy mutants were inoculated with Sinorhizobium meiloti strain 2011 and grown in nutrient-depleted conditions (sand and terragreen with water) at 35 °C for one week followed by 30 °C for one week. The total number of nodules (a) and root and shoot dry weights (b) were assessed at 14 dpi.

Boxplots represent medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from two experiments (a, b). Individual data points for biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A Shapiro-Wilk test was used to test for normality, and statistical comparisons were made to the WT (Student's t- test (normal data, b), Mann-Whitney test (non-normal data, a): ns, P > 0.05; *, P < 0.05; ***, P < 0.001 ;).

Figure 6: Wheat cngc15-easy mutants have increased mycorrhizal colonization.

Wheat cv. Cadenza WT and cngc15a/c-easy (a) and cv. Kronos WT and cngc15a/c-easy (b) were grown in nutrient-depleted conditions (sand and terragreen with water) and assessed after inoculation with mycorrhizal fungus Rhizophagus irregularis. Mycorrhizal colonization structures (intraradical hyphae (I RH) , arbuscules (A), and vesicles (V)) were quantified when WT control colonization reached 10-20%. Occurrences of mycorrhizal structures are shown as a percentage of the total number of root sections assessed.

Boxplots represent medians (black lines), 25-75% quartile (box), and min to max (whiskers) of the biological replicates (plants) pooled from two experiments (c) or one experiment (b). Individual data points for biological replicates are shown, and the numbers of biological replicates analyzed are indicated at the bottom of each column. A Shapiro-Wilk test was used to test for normality, and statistical comparisons were made to the WT (Student's t-test (normal data, a IRH, a A, b IRH, b A), Mann-Whitney test (non-normal data, a V, b V): ns, P > 0.05; * P < 0.05; ** P < 0.01).

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics, which are within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the words "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene" or “gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.

The terms "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

One genetic pathway in plants is essential to the establishment of both AM and root nodule symbiosis. This genetic pathway is essential to activate and decode the nuclear calcium oscillation that is induced upon perception of bacterial and fungal elicitors by the plant. At the core of this pathway are nuclear localized ion channels. Among them, the cyclic nucleotide gated channels (CNGC) 15a, CNGC15b and CNGC15c are required to generate the nuclear calcium oscillation. However, their regulation and gating mechanism remains unknown. Here, we have identified a dominant mutation in a single residue of a highly conserved domain of the CNGCs family, and specifically that presents in particularly CNGC15a or CNGC15c (hereafter named cngc15a/c-easy). We have shown that this mutation results in the spontaneous activation (autoactivation) of nuclear calcium oscillations and increases endosymbiosis with arbuscular mycorrhizal fungi and with nitrogen-fixing bacteria.

Accordingly, in one aspect of the invention, there is provided a genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or cell comprises at least one mutation in one or more gene encoding a nuclear localized cyclic nucleotide-gated ion channel (CNGC).

For the purposes of the invention, a “genetically altered plant” is a plant that has been genetically altered compared to the naturally occurring wild type (WT) plant. In one embodiment, a genetically altered plant is a plant that has been altered compared to the naturally occurring wild type (WT) plant using a mutagenesis method, such as targeted genome modification or genome editing. In one embodiment, the plant genome has been altered compared to the wild-type using a mutagenesis method. Such plants have an altered phenotype as described herein, such as increased yield, increased nodulation and/or increased endosymbioses. Therefore, in this example, these phenotypes are conferred by the presence of an altered plant genome, for example the mutation of at least one gene encoding a CNCG15 gene. In particular, the aspects of the invention involve recombination DNA technology and exclude embodiments that are solely based on generating plants by traditional breeding methods.

Cyclic nucleotide-gated ion channels or CNGCs are calcium permeable cation transport channels. Plant CNGCs are tetrameric and have six transmembrane domains, with a cytosolic N-terminal (NT) and C-terminal (CT) region per subunit. Members of the CNGC1 5 family are localized to the nuclear envelope, where they participate in nuclear Ca²⁺ oscillations, which are crucial for root growth and symbiosis establishment. In one embodiment, the CNGC is selected from one or more of the CNGC15 sub-family/sub- type.

In one embodiment, there is provided a genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or cell comprises at least one mutation in at least one CNGC15 subtype.

For example, in Medicago truncatula, the CNGC15 family comprises MtCNGC15a, b and c. In one embodiment, the genetically altered plant comprises at least one mutation in MtCNGC15a or at least one mutation in MtCNGC15b or at least one mutation in MtCNGC15c. In one embodiment, the genetically altered plant comprises at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15b or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in MtCNGC15b and at least one mutation in MtCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in MtCNGC15a and at least one mutation in MtCNGC15b and at least one mutation in MtCNGC15c or homologues thereof. In one embodiment, the MtCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 12 or a functional variant or homologue thereof, MtCNGC15b comprises or consists of SEQ ID NO: 13 or a functional variant or homologue thereof and MtCNGC15c comprises or consists of SEQ ID NO: 14 or a functional variant or homologue thereof. In another embodiment, the MtCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 54 or a functional variant or homologue thereof, MtCNGC15b comprises or consists of SEQ ID NO: 55 or a functional variant or homologue thereof and MtCNGC15c comprises or consists of SEQ ID NO: 56 or a functional variant or homologue thereof.

In another example, in Arabidopsis the AtCNGC15 family comprises one member: AtCNGC15. In one embodiment, the genetically altered plant comprises at least one mutation in at least one gene encoding AtCNGC15. In one embodiment, the AtCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 1 or a functional variant or homologue thereof. In another embodiment, the AtCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 43 or a functional variant or homologue thereof. In another example, in soybean the GmCNGC15 family comprises five members, GmCNGC15 a, b, c, d and e. In one embodiment, the genetically altered plant comprises at least one mutation (in at least one gene of) at least one, two, three or four of GmCNGC15 a, b, c, d and e. In an alternative embodiment, the genetically altered plant comprises at least one mutation (in at least one gene of) all of GmCNGC15 a, b, c, d and e. In one embodiment, the GmCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 2 or a functional variant or homologue thereof, GmCNGC15b comprises or consists of SEQ ID NO: 3 or a functional variant or homologue thereof, GmCNGC15c comprises or consists of SEQ ID NO: 4 or a functional variant or homologue thereof, GmCNGC15d comprises or consists of SEQ ID NO: 5 or a functional variant or homologue thereof, and GmCNGC15e comprises or consists of SEQ ID NO: 6 or a functional variant or homologue thereof. In another embodiment, the GmCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 44 or a functional variant or homologue thereof, GmCNGC15b comprises or consists of SEQ ID NO: 45 or a functional variant or homologue thereof, GmCNGC15c comprises or consists of SEQ ID NO: 46 or a functional variant or homologue thereof, GmCNGC15d comprises or consists of SEQ ID NO: 47 or a functional variant or homologue thereof, and GmCNGC15e comprises or consists of SEQ ID NO: 48 or a functional variant or homologue thereof.

In another example, in tomato, the SICNGC15 family comprises three members; SICNGC15a, SICNGC15b and SICNGC15c. In one embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) SICNGC15a, SICNGC15b and SICNGC15c. In another embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15b. In another embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15c. In another embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) SICNGC15b and SICNGC15c. In a further embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) SICNGC15a and SICNGC15b and SICNGC15c. In one embodiment, the SICNGC15a amino acid sequence comprises or consists of SEQ ID NO: 29 or a functional variant or homologue thereof, SICNGC15b comprises or consists of SEQ ID NO: 30 or a functional variant or homologue thereof, and SICNGC15c comprises or consists of SEQ ID NO: 31 or a functional variant or homologue thereof. In another embodiment, the SICNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 71 or a functional variant or homologue thereof, SICNGC15b comprises or consists of SEQ ID NO: 72 or a functional variant or homologue thereof and SICNGC15c comprises or consists of SEQ ID NO: 73 or a functional variant or homologue thereof.

In another example, in maize the CNGC15 family comprises one member: ZmCNGC15. In one embodiment, the genetically altered plant comprises at least one mutation in at least one gene encoding ZmCNGC15. In one embodiment, the ZmCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 32 or a functional variant or homologue thereof. In another embodiment, the ZmCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 74 or a functional variant or homologue thereof.

In another example, in wheat the CNGC15 family comprises seven members, TrCNGC15 a, b, c, d, e, f and g. In one embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) at least one, two, three, four, five or six of TrCNGC15. In an alternative embodiment, the genetically altered plant comprises at least one mutation in (in at least one gene of) all of TrCNGC15 a, b, c, d, e, f and g. In one embodiment, the TrCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 33 or a functional variant or homologue thereof, TrCNGC15b comprises or consists of SEQ ID NO: 34 or a functional variant or homologue thereof, TrCNGC15c comprises or consists of SEQ ID NO: 35 or a functional variant or homologue thereof, TrCNGC15d comprises or consists of SEQ ID NO: 36 or a functional variant or homologue thereof, TrCNGC15e comprises or consists of SEQ ID NO: 37 or a functional variant or homologue thereof, TrCNGC15f comprises or consists of SEQ ID NO: 38 or a functional variant or homologue thereof and TrCNGC15g comprises or consists of SEQ ID NO: 39 or a functional variant or homologue thereof. In one example, the genetically altered plant comprises at least one mutation in TrCNGC15a and at least one mutation in TrCNGC15c. In another embodiment, the TrCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 75 or a functional variant or homologue thereof, TrCNGC15b comprises or consists of SEQ ID NO: 76 or a functional variant or homologue thereof, TrCNGC15c comprises or consists of SEQ ID NO: 77 or a functional variant or homologue thereof, TrCNGC15d comprises or consists of SEQ ID NO: 78 or a functional variant or homologue thereof, TrCNGC15e comprises or consists of SEQ ID NO: 79 or a functional variant or homologue thereof, TrCNGC15f comprises or consists of SEQ ID NO: 80 or a functional variant or homologue thereof and TrCNGC15g comprises or consists of SEQ ID NO: 81 or a functional variant or homologue thereof. In one example, the genetically altered plant comprises at least one mutation in TrCNGC15a and at least one mutation in TrCNGC15c.

In another example, in rice the CNGC15 family comprises one member: OsCNGC15. In one embodiment, the genetically altered plant comprises at least one mutation in at least one gene encoding OsCNGC15. In one embodiment, the OsCNGC15 amino acid sequence comprises or consists of SEQ ID NO: 28 or a functional variant or homologue thereof. In another embodiment, the OsCNGC15 nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 70 or a functional variant or homologue thereof.

In another example, in Hordeum vulgare, the CNGC15 family comprises HvCNGC15a, b and c. In one embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15a or at least one mutation in HvCNGC15b or a homologue thereof or at least one mutation in HvCNGC15c or a homologue thereof. In one embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15b or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15b and at least one mutation in HvCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in HvCNGC15a and at least one mutation in HvCNGC15b and at least one mutation in HvCNGC15c or homologues thereof. In one embodiment, the HvCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 40 or a functional variant or homologue thereof, HvCNGC15b comprises or consists of SEQ ID NO: 41 or a functional variant or homologue thereof and HvCNGC15c comprises or consists of SEQ ID NO: 42 or a functional variant or homologue thereof. In another embodiment, the HvCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 82 or a functional variant or homologue thereof, HvCNGC15b comprises or consists of SEQ ID NO: 83 or a functional variant or homologue thereof and HvCNGC15c comprises or consists of SEQ ID NO: 84 or a functional variant or homologue thereof. In another example, in Arachis hypogaea, the CNGC15 family comprises AhCNGC15a, b and c. In one embodiment, the genetically altered plant comprises at least one mutation in AhCNGC15a or at least one mutation in AhCNGC15b or a homologue thereof or at least one mutation in AhCNGC15c or a homologue thereof. In one embodiment, the genetically altered plant comprises at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15b or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in AhCNGC15b and at least one mutation in AhCNGC15c or homologues thereof. In another embodiment, the genetically altered plant comprises at least one mutation in AhCNGC15a and at least one mutation in AhCNGC15b and at least one mutation in AhCNGC15c or homologues thereof. In one embodiment, the AhCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 232 or a functional variant or homologue thereof, AhCNGC15b comprises or consists of SEQ ID NO: 234 or a functional variant or homologue thereof and AhCNGC15c comprises or consists of SEQ ID NO: 233 or a functional variant or homologue thereof. In another embodiment, the AhCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 237 or a functional variant or homologue thereof, AhCNGC15b comprises or consists of SEQ ID NO: 239 or a functional variant or homologue thereof and AhCNGC15c comprises or consists of SEQ ID NO: 238 or a functional variant or homologue thereof.

In another example, in Linum usitatissimum, the CNGC family comprises LuCNGCa and c. In one embodiment, the genetically altered plant comprises at least one mutation in LuCNGCa or at least one mutation in LuCNGCc or a homologue thereof. In one embodiment, the genetically altered plant comprises at least one mutation in LuCNGCa and at least one mutation in LuCNGCc or homologues thereof. In one embodiment, the LuCNGCa amino acid sequence comprises or consists of SEQ ID NO: 235 or a functional variant or homologue thereof, and LuCNGCc comprises or consists of SEQ ID NO: 236 or a functional variant or homologue thereof. In another embodiment, the LuCNGCa nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 240 or a functional variant or homologue thereof and LuCNGCc comprises or consists of SEQ ID NO: 241 or a functional variant or homologue thereof. In another example, in Brassica napus, the CNGC family comprises BnCNGC15a. In one embodiment, the genetically altered plant comprises at least one mutation in BnCNGC15a or a homologue thereof. In one embodiment, the BnCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 247, 248 or a functional variant or homologue thereof. In another embodiment, the BnCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 249, 250 or a functional variant or homologue thereof.

In another example, in Fragaria vesca, the CNGC family comprises F.vCNGC15a. In one embodiment, the genetically altered plant comprises at least one mutation in F.vCNGC15a ora homologue thereof. In one embodiment, the F.vCNGC15a amino acid sequence comprises or consists of SEQ ID NO: 253 or a functional variant or homologue thereof. In another embodiment, the F.vCNGC15a nucleic acid sequence (or gene sequence) comprises or consists of SEQ ID NO: 254 or a functional variant or homologue thereof.

“By at least one mutation in at least one gene” is meant that where the gene of a CNGC15 subtype is present as more than one copy or homologue (with the same or slightly different sequence) there is at least one mutation in at least one (endogenous) gene. Preferably all genes are mutated. Preferably the mutation in the gene sequences leads to a mutation in the amino acid sequence of the CNGC.

In the above embodiments an ‘endogenous’ nucleic acid or gene may refer to the native or natural sequence in the plant genome - for example, one of the above-referenced nucleic acid or amino acid sequences (SEQ ID NO: 43 to 84, 237 to 241 , 249, 250, 254 and SEQ ID NO: 1 to 42, 232 to 236, 247, 248 and 253).

Accordingly, in one embodiment, there is provided a genetically altered plant, part thereof or plant cell, wherein the plant, part thereof or cell comprises at least one mutation in at least one CNGC, preferably a CNGC15 subtype, wherein the mutation is in one nucleic acid sequence encoding a CNGC15 subtype selected from one of the sequences defined in SEQ ID NO: 43 to 84, 237 to 241 , 249, 250, and 254, as described in detail above.

The term “variant” or “functional variant” as used herein with reference to any of the sequences defined herein refers to a variant gene sequence or part of the gene sequence which retains the biological function of the full non-variant (e.g. wild-type) sequence. In the context of CNGC, a functional variant is one that retains the wild-type function - i.e. acts as cyclic nucleotide gated channel and can generate nuclear calcium oscillations. In particular, a functional variant as used herein has spontaneous activation (autoactivation) of nuclear calcium oscillations

A functional variant also comprises a variant of the gene of interest, which has sequence alterations that do not affect function, for example in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, compared to the wild type sequences as shown herein and is biologically active. Alterations in a nucleic acid sequence that results in the production of a different amino acid at a given site that does not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

As used in any aspect of the invention described herein a “variant” or a “functional variant” has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %,

82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%,

97%, 98%, or at least 99% overall sequence identity to the non-variant nucleic acid or amino acid sequence.

The term homolog, as used herein, also designates a CNGC15 gene orthologue from other plant species. Suitable homologues can be identified by sequence comparisons and identifications of conserved domains as described above. There are predictors in the art that can be used to identify such sequences. The function of the homologue can be identified as described herein and a skilled person would thus be able to confirm the function, for example when overexpressed in a plant.

A homolog may also have, in increasing order of preference, at least 50%, 51 %, 52%,

53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%,

68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%,

83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%,

98%, or at least 99% overall sequence identity to the amino acid sequences represented by one of SEQ ID NO: 1 to 42 and 232 to 236, 247, 248, 253 or to the nucleic acid sequences shown in SEQ ID NOs:43 to 84 and 237 to 241 , 249, 250, 254. Functional variants of CNGC15 gene homologs as defined above are also within the scope of the invention.

In one embodiment, the homolog is Arabidopsis, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 1 or a variant thereof.

In another embodiment, the homolog is soybean, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 2, 3, 4, 5 or 6 or a variant thereof.

In another embodiment, the homolog is Medicago, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 7, 8, 9, 10, 11 ,12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26 or 27 or a variant thereof.

In another embodiment, the homolog is tomato, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 29, 30 or 31 or a variant thereof.

In another embodiment, the homolog is maize, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 32 or a variant thereof.

In another embodiment, the homolog is wheat, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 33, 34, 35, 36, 37, 38 or 39 or a variant thereof. In another embodiment, the homolog is rice, and the CNGC15 amino acid sequence comprises or consists of SEQ ID NO: 28 or a variant thereof.

In another embodiment, the homolog is barley, and the CNGC15 comprises or consists of SEQ ID NO: 40, 41 , or 42 or a variant thereof.

In another embodiment, the homolog is peanut, and the CNGC15 comprises or consists of SEQ ID NO: 232, 233, 234 or a variant thereof.

In another embodiment, the homolog is flax (common flax or linseed), and the CNGC comprises or consists of SEQ ID NO: 235, 236 or a variant thereof.

In another embodiment, the homolog is strawberry, and the CNGC comprises or consists of SEQ ID NO: 253 or variant thereof.

In another embodiment, the homolog is oilseed rape, and the CNGC comprises or consists of SEQ ID NO: 247, 248 or a variant thereof.

In one embodiment, the mutation abolishes or reduces bending of the transmembrane domain of CNGC15. In a further embodiment, the mutation lead to the spontaneous generation of the calcium oscillation. The nuclear calcium oscillation generation can be easily determined by any routine method, for example, by performing calcium traces as demonstrated in Figure 1. As such, in a preferred embodiment, the mutation is a dominant mutation.

In one embodiment, CNGC15 or the CNGC15 subtype comprises at least one highly conserved motif. Accordingly, in one embodiment, the genetically altered plant of the invention comprises at least one mutation in at least one of conserved motifs. In one embodiment, the motif comprises the sequence XDPX, wherein X is any amino acid. More preferably, the motif comprises the sequence XDPL. Even more preferably, the conserved motif comprises the sequence VDPL.

In one embodiment, the mutation is a mutation at one or more positions in the XDPX motif. In a preferred embodiment, the mutation is a point mutation or a substitution mutation. That is, a mutation that exchanges one nucleotide base for another and that leads to a change in the codon causing the nucleic acid sequence to encode a different amino acid at that position.

In one embodiment, where the conserved motif is XDPX or XDPL or VDPL, the mutation is selected from a substitution of P for another amino acid, preferably a substitution of P for S.

In one embodiment, the mutation is selected from one or more of the following mutations in VDPL: - a substitution of V for another amino acid; and/or - a substitution of D for another amino acid; and/or - a substitution of P for another amino acid; preferably L or S and/or - a substitution of L for another amino acid, preferably F.

In a preferred embodiment, the mutation in the VDPL is a substitution of P for L.

In another embodiment, the mutation in the VDPL is a substitution of P for S.

In another embodiment, the mutation in the VDPL is a substitution of L for F.

In one embodiment VDPL is mutated to VDLL.

In another embodiment, VDPL is mutated to VDSL.

In another embodiment, VDPL is mutated to VDPF.

In another embodiment, VDPL is mutated to VDSF.

In another embodiment, VDPL is mutated to VDLF.

In one embodiment, where the conserved motif is XDPX or XDPL or IDPL or IDPM, the mutation is selected from a substitution of P for another amino acid, preferably a substitution of P for S.

In one embodiment, the mutation is selected from one or more of the following mutations in IDPL: - a substitution of I for another amino acid; and/or - a substitution of D for another amino acid; and/or - a substitution of P for another amino acid; preferably S; and/or - a substitution of L for another amino acid, preferably F or M. In a preferred embodiment, the mutation in the IDPL is a substitution of P for S.

In another embodiment, the mutation in the IDPL is a substitution of L for F.

In one embodiment, the mutation is selected from one or more of the following mutations in IDPM: - a substitution of I for another amino acid; and/or - a substitution of D for another amino acid; and/or - a substitution of P for another amino acid; preferably S; and/or - a substitution of M for another amino acid.

In a preferred embodiment, the mutation in the IDPM is a substitution of P for S.

In one embodiment IDPL is mutated to IDSL.

In another embodiment, IDPM is mutated to IDSM.

In another embodiment IDPL is mutated to IDSF.

In a further preferred embodiment, the mutation is at the following positions is selected from one of the following substitutions in Table 1 :

Table 1 : CNGC mutation positions

In one embodiment, there is provided a genetically altered plant, part thereof or plant cell comprising least one mutation in a CNGC15 nucleic acid sequence, wherein the CNGC15 nucleic acid sequence comprises or consists of: a. a nucleic acid sequence encoding a polypeptide comprising at least one XDPX, preferably a XDPL motif, and more preferably a VDPL motif or a variant thereof; b. a nucleic acid sequence encoding a polypeptide as defined in one of SEQ ID Nos 1 to 42, 232 to 236, 247, 248, 253, ; or c. a nucleic acid sequence as defined in one of SEQ ID Nos 43 to 84, 237 to 241 , 249, 250, 254; or d. a nucleic acid sequence with at least 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity to either (a) or (c); or e. a nucleic acid sequence encoding a CNGC15 polypeptide as defined herein that is capable of hybridising under stringent conditions as defined herein to the nucleic acid sequence of any of (a) to (d).

Hybridization of such sequences may be carried out under stringent conditions. Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12 hours. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

In one embodiment, the mutation is introduced using targeted genome editing. That is, in one embodiment, the invention relates to a method and plant that has been generated by genetic engineering methods as described above, and does not encompass naturally occurring varieties or generating plants by traditional breeding methods. Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events.

In a preferred embodiment, the genome editing method that is used according to the various aspects of the invention is CRISPR. Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand breaks in four sequential steps. First, two non-coding RNA, the CRISPR RNA (crRNA) and trans- activating crRNA (tracrRNA), are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre- crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.

One major advantage of the CRISPR-Cas9 system, as compared to conventional gene targeting and other programmable endonucleases is the ease of multiplexing, where multiple genes can be mutated simultaneously simply by using multiple sgRNAs each targeting a different gene. In addition, where two sgRNAs are used flanking a genomic region, the intervening section can be deleted or inverted (Wiles et al., 2015).

Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and is a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used. The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3. Accordingly, using techniques known in the art, such as http://chopchop.cbu.uib.no/, it is possible to design sgRNA molecules that targets a CNGC15 gene sequence as described herein.

In another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a sgRNA molecule, wherein the sgRNA molecule targets a nucleic acid sequence encoding the VDPL conserved motif.

By “sgRNA” (single-guide RNA) is meant the combination of tracrRNA and crRNA in a single RNA molecule, preferably also including a linker loop (that links the tracrRNA and crRNA into a single molecule). “sgRNA” may also be referred to as “gRNA" and in the present context, the terms are interchangeable. The sgRNA or gRNA provide both targeting specificity and scaffolding/binding ability for a Cas nuclease. A gRNA may refer to a dual RNA molecule comprising a crRNA molecule and a tracrRNA molecule.

In a further embodiment, the nucleic acid sequence encoding a sgRNA molecule is operable linked to a regulatory sequence, such as a plant promoter. A suitable plant promoter may be a constitutive or strong promoter or may be a tissue-specific promoter. In one embodiment, suitable plant promoters are selected from, but not limited to, cestrum yellow leaf curling virus (CmYLCV) promoter or switchgrass ubiquitin 1 promoter (Pvllbil), wheat U6 RNA polymerase III (TaU6), CaMV35S, wheat U6 or maize ubiquitin (e.g. Ubi1) promoters.

The nucleic acid construct of the present invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme. By “CRISPR enzyme” is meant an RNA- guided DNA endonuclease that can associate with the CRISPR system. Specifically, such an enzyme binds to the tracrRNA sequence. In one embodiment, the CRIPSR enzyme is a Cas protein (“CRISPR associated protein), preferably Cas 9 or Cpf1 , more preferably Cas9. The Cas9 enzyme may be modified as described below. In a specific embodiment Cas9 is codon-optimised Cas9. In another embodiment, the CRISPR enzyme is a protein from the family of Class 2 candidate x proteins, such as C2c1 , C2C2 and/or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In an alternative embodiment, the Cas protein may be from any one of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiles or Treponema denticola. In a preferred embodiment, the CRISPR enzyme is operably linked to a regulatory sequence - either the same or a different regulatory sequence as for the sgRNA sequence. Again, suitable regulatory sequences are described above.

In another aspect of the invention, there is provided a plant or part thereof or at least one isolated plant cell transfected with at least one nucleic acid construct as described herein. Cas9 and sgRNA may be combined or in separate expression vectors (or nucleic acid constructs, such terms are used interchangeably). In other words, in one embodiment, an isolated plant cell is transfected with a single nucleic acid construct comprising both sgRNA and a CRISPR enzyme as described in detail above. In an alternative embodiment, an isolated plant cell is transfected with two nucleic acid constructs, a first nucleic acid construct comprising at least one sgRNA as defined above and a second nucleic acid construct comprising a CRISPR enzyme or a functional variant or homolog thereof. The second nucleic acid construct may be transfected below, after or concurrently with the first nucleic acid construct. The advantage of a separate, second construct comprising a CRISPR enzyme is that the nucleic acid construct encoding at least one sgRNA can be paired with any type of CRISPR enzyme, as described herein, and therefore is not limited to a single CRISPR enzyme function (as would be the case when both the CRISPR enzyme and sgRNA are encoded on the same nucleic acid construct). In one embodiment, the nucleic acid construct comprising a CRISPR enzyme is transfected first and is stably incorporated into the genome, before the second transfection with a nucleic acid construct comprising at least one sgRNA nucleic acid. In an alternative embodiment, a plant or part thereof or at least one isolated plant cell is transfected with mRNA encoding a CRISPR enzyme and co-transfected with at least one nucleic acid construct as defined herein.

In a preferred embodiment of any aspect of the invention described herein, sgRNA can be used with a modified Cas9 protein, such as nickase Cas9 or nCas9 or a “dead” Cas9 (dCas9) fused to a “Base Editor” - such as an enzyme, for example a deaminase such as cytidine deaminase, or TadA (tRNA adenosine deaminase) or ADAR or APOBEC. These enzymes are able to substitute one base for another. As a result no DNA is deleted, but a single substitution is made (Kim et al., 2017; Gaudelli et al. 2017). Alternatively, the method may use sgRNA together with a template or donor DNA constructs, to introduce a targeted SNP or mutation, in particular one of the substitutions described herein, into a CNGC gene. In this embodiment, the introduction of a template DNA strand, following a sgRNA-mediated snip in the double-stranded DNA, can be used to produce a specific targeted mutation (i.e. a SNP) in the gene using homology directed repair. As a further alternative, prime editing can be used to introduce the specific mutation (Anzalone et al., 2019). Here a catalytically impaired Cas9 endonuclease is fused to an engineered reverse transcriptase programmed with a prime editing guide RNA (pegRNA) that is both specific to the target site and encodes the desired edit. Using the above methods we can mutate the nucleotides that encode the conserved VDPL motif and therefore lead to one of the above-described substitutions in the amino acid sequence.

Once targeted genome editing has been performed, rapid high-throughput screening procedures can be used to analyse amplification products for the presence of a mutation in the CNGC15 gene, and in particular, in the VDPL motif. Once a mutation is identified, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the target gene CNGC15/easy. Mutants with one or more mutations in a CNGC, and in particular in the VDPL motif, and as a result, increased yield/endosymbioses compared to a control can thus be identified.

Plants obtained or obtainable and seeds obtained or obtainable from such plants by such method which carry a functional mutation or dominant mutation in at least one endogenous CNGC15 gene are also within the scope of the invention.

In one embodiment, the progeny plant is stably transformed with the CRISPR constructs, and comprises the exogenous polynucleotide which is heritably maintained in the plant cell. The method may include steps to verify that the construct is stably integrated. The method may also comprise the additional step of collecting seeds from the selected progeny plant. In another aspect of the invention, there is provided a method of making a genetically altered plant, the method comprising introducing at least one mutation into at least one nuclear-localised CNGC gene, preferably at least one gene encoding a CNGC15 sub- type.

In one embodiment, the method comprises a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one CRISPR construct or sgRNA molecule, wherein the CRISPR construct or sgRNA molecule targets the CNGC gene, preferably the CNGC15 gene and introduces at least one mutation into, preferably the VDPL motif as described above; c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that show at least one mutation in a CNGC gene, preferably a CNGC15 subtype, and preferably in the VDPL motif.

In one embodiment, the method may comprise obtaining a DNA sample from a transformed plant and carrying out DNA amplification to detect the at least one mutation in the CNGC gene, and preferably a substitution in the VDPL motif. In a further embodiment of any of the methods described herein, the method may further comprise at least one or more of the steps of assessing the phenotype of the genetically altered plant, measuring at least one of increased nodulation and/or arbuscular mycorrhiza (AM). In other words, the method may involve the step of screening the plants for the desired phenotype.

Alternatively, more conventional mutagenesis methods can be used to introduce at least one mutation into at least one CNGC gene. These methods include both physical and chemical mutagenesis. A skilled person will know further approaches can be used to generate such mutants, and methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488- 492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. In another embodiment, mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons. The targeted population can then be screened to identify a substitution mutation in a CNGC gene.

In another embodiment of the various aspects of the invention, the method comprises mutagenizing a plant population with a mutagen. The mutagen may be a fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N- nitrosurea (ENU), triethylmelamine (1'EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N'-nitro- Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl- benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy- 6-chloro-9 [3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) or formaldehyde. Again, the targeted population can then be screened to identify one of the above described mutations in the CNGC gene.

In another embodiment, the method used to create and analyse mutations is targeting induced local lesions in genomes (TILLING), reviewed in Henikoff et al, 2004. In this method, seeds are mutagenised with a chemical mutagen, for example EMS. The resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening. DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR. The PCR amplification products may be screened for mutations in the CNGC15 gene using any method that identifies heteroduplexes between wild type and mutant genes. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE), or by fragmentation using chemical cleavage. Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program. Any primer specific to a CNGC nucleic acid sequence may be utilized to amplify the CNGC nucleic acid sequence within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of a CNGC gene where useful mutations are most likely to arise e.g. in the XDPL motif that is highly conserved as explained elsewhere. To facilitate detection of PCR products on a gel, the PCR primer may be labelled using any conventional labelling method. In an alternative embodiment, the method used to create and analyse mutations is EcoTILLING. EcoTILLING is molecular technique that is similar to TILLING, except that its objective is to uncover natural variation in a given population as opposed to induced mutations. The first publication of the EcoTILLING method was described in Comai et al. (2004).

In another aspect of the invention there is provided a genetically altered plant, part thereof or plant cell, wherein the plant comprises a nucleic acid construct comprising and preferably expressing a nucleic acid sequence encoding a CNGC polypeptide, preferably a CNGC15 polypeptide.

In one embodiment, the nucleic acid sequence encodes a CNGC polypeptide as defined in one of SEQ ID NOs: 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.

In one embodiment, the nucleic acid encodes a CNGC polypeptide from the same plant - for example, a wheat CNGC polypeptide is expressed in wheat. In another embodiment, the nucleic acid encodes a CNGC polypeptide from a different plant - for example, a wheat CNGC polypeptide is expressed in rice.

In another embodiment, the nucleic acid construct is stably incorporated into the plant genome.

Accordingly, in another aspect of the invention, there is provided a nucleic acid construct comprising a nucleic acid sequence encoding a CNGC polypeptide as defined in any one of SEQ ID Nos: 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.

In another aspect, the invention relates to the use of a nucleic acid construct as described herein to increase yield, nodulation or AM endosymbioses as described herein. In another aspect of the invention, there is also provided a host cell that comprises, and preferably expresses the nucleic acid construct described herein. The host cell may be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or kit comprising a culture medium and an isolated host cell as described below.

In one embodiment of any of the aspects described herein, the nucleic acid sequence encoding a CNGC polypeptide is operably linked to a regulatory sequence. Preferably the regulatory sequence is a promoter.

The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.

A "plant promoter" comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The "plant promoter" can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other "plant" regulatory signals, such as "plant" terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3'- regulatory region such as terminators or other 3' regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest. In one embodiment, the promoter is a constitutive promoter. A "constitutive promoter" refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Examples of constitutive promoters include but are not limited to actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize H3 histone, alfalfa H3 histone, 34S FMV, rubisco small subunit, OCS, SAD1 , SAD2, nos, V-ATPase, super promoter, G-box proteins and synthetic promoters.

In another embodiment, the promoter is a tissue-specific promoter. Tissue specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development. In one embodiment, the tissue- specific promoter is a root-specific promoter. In one embodiment, the root-specific promoter is the P1534 promoter (as described in Li et al. 2019).

In another aspect of the invention there is provided a vector comprising the nucleic acid sequence described above. In one embodiment, the vector allows transient expression of the nucleic acid sequence expressing CNGC. Examples of suitable expression vectors include pB2GW7 (http://www.psb.ugent.be/gateway/). Preferably these sequences are operably linked to a regulatory sequence, wherein in one example, the regulatory sequence is 35S.

In another aspect of the invention, there is provided a method of making a genetically altered plant, the method comprising introducing and expressing a nucleic acid construct as described herein in a plant.

In one embodiment, the method comprises a. selecting a part of the plant; b. transfecting at least one cell of the part of the plant of paragraph (a) with at least one nucleic acid construct as described above; c. regenerating at least one plant derived from the transfected cell or cells; d. selecting one or more plants obtained according to paragraph (c) that express the CNGC polypeptide, for example a CNGC polypeptide as defined in SEQ ID NO: 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.

Transformation methods as used herein for generating a genetically altered plant of the invention are known in the art. Thus, according to the various aspects of the invention, a CRISPR or nucleic acid construct as described herein is introduced into a plant and expressed as a transgene. The construct is introduced into said plant through a process called transformation. The terms "introduction" or "transformation" or “transfection” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).

The CRISPR or nucleic acid construct may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.

Transformation of plants is now a routine technique in many species. Advantageously, any of several transformation methods may be used to introduce a CRISPR or nucleic acid construct into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microinjection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.

To select transformed plants, the plant material obtained in the transformation is subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility is growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker.

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).

The method may further comprise regenerating a genetically altered plant from the transformed plant or plant cell, and obtaining a progeny plant derived from the transgenic plant, wherein said progeny exhibits at least one mutation in a CNGC15 gene as described and shows an increase in nodulation and/or AM.

A genetically altered plant of the present invention may also be obtained by transference of any of the sequences of the invention by crossing, e.g., using pollen of the genetically altered plant described herein to pollinate a wild-type or control plant, or pollinating the gynoecia of plants described herein with other pollen that is not transformed or genetically altered as described herein. The methods for obtaining the plant of the invention are not exclusively limited to those described in this paragraph; for example, genetic transformation of germ cells from the ear of wheat could also be carried out as mentioned, but without having to regenerate a plant afterwards.

In a further aspect of the invention there is provided a plant obtained or obtainable by the above-described methods. In a further aspect, there is provided a seed obtained or obtainable from the plant. Also included in the scope of the invention is progeny plants obtained from the seed and as well as seed obtained from the progeny plants. In another aspect of the invention, there is provided a method for identifying and/or selecting a plant that will have an increase in at least one of yield, nodulation and/or AM endosymbiosis, the method comprising screening a population of plants and detecting in the plant or plant germplasm at least one polymorphism in at least conserved domain of CNGC, preferably a conserved domain of a CNGC15 , preferably a polymorphism in the VDPL motif, as described above, compared to a control plant or a plant from the same or different plant population, and selecting said plant.

Suitable tests for assessing the presence of a polymorphism would be well known to the skilled person, and include but are not limited to, Isozyme Electrophoresis, Restriction Fragment Length Polymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats (SSRs-which are also referred to as Microsatellites), and Single Nucleotide Polymorphisms (SNPs). In one embodiment, Kompetitive Allele Specific PCR (KASP) genotyping is used.

In one embodiment, the method comprises a) obtaining a nucleic acid sample from a plant and b) carrying out nucleic acid amplification of CNGC alleles using one or more primer pairs.

In a further embodiment, the method may further comprise introgressing the chromosomal region comprising at least one of said CNGC polymorphisms as described above into a second plant or plant germplasm to produce an introgressed plant or plant germplasm.

In another aspect of the invention there is provided a plant obtained or obtainable by the method described herein. There is also provided a seed derived from a plant as described herein.

In another aspect of the invention, there is provided a method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant. The term "yield" in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight. The actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square metres.

Thus, according to the invention, yield comprises one or more of and can be measured by assessing one or more of: increased seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size or weight of seeds or pods or beans or grain, increased growth or increased branching, for example inflorescences with more branches, increased biomass, increased fresh weight, dry weight or grain fill. Preferably, increased yield comprises at least one of an increased number or weight of seeds, increased biomass, increased fresh weight and increased growth. Yield is increased relative to a control or wild-type plant. For example, the yield is increased by 40% or more compared to a control plant, for example by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

In a further embodiment or aspect of the invention there is provided a method of increasing at least one of nodulation. An increase in nodulation results in an increase in growth responses (root and shoot growth) and nutrient status (nitrogen/carbon ration), and ultimately, yield, in plants. In one embodiment, said increase is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120% or 130% compared to a control or wild-type plant. In one embodiment, the method comprises increasing at least one of nodulation in addition to increasing yield in a plant. In one embodiment, the increase is at least or about 50% dry weight. In another embodiment, the increase is at least or about 20% in the nutrient status. In another embodiment, there is an increase of about 10-20% in seed yield.

By “nodulation” is meant nodule development as a result of nitrogen-fixing rhizobial bacteria that colonise the roots of legumes, and an increase in nodulation can be reflected in an increase in the number and/or weight of nodules and/or pink nodules per plant. In one embodiment, nodulation is increased by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65% or 70% compared to a control or wild-type plant. In one embodiment, nodulation is increased by at least 5% compared to a control or wild-type plant. In another embodiment, nodulation is increased by at least 10% compared to a control or wild-type plant.

By “arbuscular mycorrhiza (AM) endosymbioses” is meant the symbiosis between plants and arbuscular mycorrhizal (AM) fungi. In AM endosymbiosis, the AM fungi penetrates the cortical cells of the roots of a vascular plant to form arbuscules. The level of AM endosymbiosis can be measured by determining the occurrence of mycorrhizal colonisation structures (as a %). Examples of mycorrhizal colonisation structures include intraradical hyphae, arbuscles and vesicles. In one embodiment, the % occurrence of AM structures is increased by at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65% or 70% compared to a control or wild-type plant. Preferably said increase is at least or around 5%. More preferably, said increase is is at least or around 10%.

In one embodiment, yield is increased through increasing at least one of nodulation and AM.

The terms “increase", "improve" or "enhance" according to the various aspects of the invention can be used interchangeably.

In another aspect of the invention, there is provided a method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant under low, normal or high nitrogen conditions. In one embodiment, an increase in any of the above- described phenotypes is observed under low nitrogen conditions. In one embodiment, low nitrogen conditions may be considered to be 120kg urea/ha or lower, preferably between 120 and 60 kg urea/ha, and even more preferably 60 kg urea/ha or lower (such as 120 kg urea/ha, 100 kg urea/ha, 60 kg urea/ha or 0 kg urea/ha). In an alternative embodiment, an increase in any of the above-described phenotypes is observed under normal (e.g. 240-300 kg urea/ha) or high N (above 300 kg urea/ha) conditions.

In another aspect of the invention, there is provided a method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant under high temperatures. In one embodiment, a high temperature may be considered between 28°C and 36°C, preferably above or around 30°C. In one embodiment, the method comprises introducing a nucleic acid construct comprising a nucleic acid sequence encoding a mutated CNGC polypeptide as described above, into a plant. Preferably the nucleic acid sequence encodes a mutated polypeptide selected from SEQ ID NOs 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.

In another embodiment, the method comprises introducing at least one mutation into one or more gene of a CNGC, preferably a CNGC15 sub-type as described above. Preferably the mutation is a substitution in a conserved motif, where that conserved motif is XDPX, preferably XDPL, and more preferably VDPL.

A plant according to all aspects of the invention described herein may be a monocot or a dicot plant.

In one embodiment, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In another embodiment the plant is Arabidopsis or Medicago truncatula.

The plant may be a dicot or a monocot. Preferably the plant is selected from wheat, rice, potatoes, maize, soybean, tomato, barley, sugar cane, sorghum, sunflower, sugar beet, rye, cotton, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.

The term "plant" as used herein encompasses whole plants and progeny of the plants and plant parts, including seeds, fruit, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the aforementioned expresses the nucleic acid construct of the invention. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores. The invention also extends to harvestable parts of a plant of the invention as described herein, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs.

In a most preferred embodiment, the plant part or harvestable product is a seed or grain. Therefore, in a further aspect of the invention, there is provided a seed or grain produced from a genetically altered plant as described herein. Accordingly, in one aspect of the invention there is provided seed, wherein the seed expresses the nucleic acid construct or CRISPR construct of the invention. Also provided is a progeny plant obtained from the seed as well as seed obtained from that progeny.

A control plant as used herein according to all of the aspects of the invention is a plant, which has not been modified according to the methods of the invention. Accordingly, in one embodiment the control plant does not express a nucleic acid construct of the invention or a CRSIPR construct or alternatively the plant does not have one or more mutations in the CNCG15 polypeptide as described herein. In one embodiment, the control plant is a wild type plant. The control plant is typically of the same plant species, preferably having the same genetic background as the modified plant.

While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments, which are described.

The invention is now described in the following non-limiting examples.

Example I: Quantifying the nutritional status and yield of cngc15a-easy.

1.1 Analysis of nutrient status and yield under symbiotic (nutrient-depleted) conditions

The analyses of the calcium oscillation in both cncg15c-easy and cngc15a-easy demonstrate that the mutation P>S in the conserved domain is sufficient to autoactivate nuclear calcium oscillation in the Medicago truncatula root in the absence of a nodulation factor (Fig. 1a). Upon inoculation with Sinorizobium meliloti 2011 , in nutrient depleted condition (sand, terragreen, water), both cncg15c-easy and cngc15a-easy mutant allele exhibit an increase in root nodule symbiosis after 14 days post inoculation (Fig. 1 b). Under low nutrient (Plant supply with BNM solution (Ehrhardt et al., 1992)), this increase of nodulation of the cngc15a-easy is translated after 28 dpi, in an enhanced number of pink nodule (nitrogen fixing nodule) compared to WT (Fig. 2a), increase root and shoot dry weight (Fig. 2b), and increase leaf nitrate/carbon ratio (Fig. 2c). In addition, we assessed the seed yield of cngc15a-easy 16 weeks post-inoculation (wpi) with S. meiloti and observed a slight but increase in seed yield (Fig. 2d).

To assess the mycorrhizal colonization phenotypes and growth responses of cngc15a- easy under nutrient-depleted conditions (sand, terragreen, water), we inoculated WT,cngc15a-easy, cngc15c-easy plants with Rhizophagus irregularis. We performed a detailed analysis of mycorrhizal structures (intraradical hyphae, arbuscules, and vesicles) (Fig. 1c, Fig. 3a) and quantified shoot dry weight (Fig. 3b) once WT roots had reached 20% arbuscule colonization levels. Compared to WT, cngc15a-easy and cngc15c-easy showed an increase in intraradical hyphae and arbuscules; however, this did not correspond to an increase in shoot dry weight for cngc15a-easy. We assessed the seed yield of cngc15a-easy 16 weeks post-inoculation (wpi) with R. irregularis and observed a significant increase in the number of seeds per plant (Fig. 3c). Altogether our data indicate that the “easy" mutation in CNGC15a and CNGC15c is sufficient to autoactivate nuclear calcium oscillation and increase endosymbioses. In addition, the increase in endosymbiosis in cngc 15a-easy was accompanied by positive plant growth responses: an improvement of plant weight and nutrient status (N/C ratio) in the presence of nitrogen-fixing rhizobia and an increased seed yield in the presence of arbuscular mycorrhizal fungi (Fig. 2b, c, d). Until now, all identified hyper-nodulating and hyper-mycorrhizal mutants display impaired growth; thus, the improved plant performance of cngc15a-easy mutants during elevated endosymbiosis is a unique discovery with vast agricultural potential.

1.2 Analysis of endosymbiosis and growth responses under nutrient-rich conditions.

Many agricultural soils are enriched in nitrogen and phosphorus due to excessive fertilizer usage. High soil nitrate and ammonia levels inhibit nodule formation and nitrogen fixation by rhizobia, and high soil phosphate levels inhibit the establishment of mycorrhizal symbiosis.

To determine whether cngc15a-easy has increased endosymbiosis compared to WT in the presence of fertilizers (sand, terragreen, nutrient solutions), we grew WT and cngc15- easy plants in the presence of nitrate, ammonia, or phosphate and assessed nodulation and mycorrhizal phenotypes and corresponding growth responses.

We found an increase in the number of total nodules and pink nodules in cngc15a-easy compared to WT in presence of control and nitrate-treated samples (Fig.4a). In control samples, this increase in nodules corresponded to an increase in root dry weight, shoot dry weight (Fig. 4b), and leaf nitrate/carbon ratio (Fig. 4c) in the CNGC15a-easy. The increased number of nodules in cngc15a-easy compared to WT was maintained during nitrate treatment but not ammonia treatment suggesting that cngc15a-easy is resistant to nitrate-inhibition of nodulation.

We also observed an increase in mycorrhizal colonization in cngc15a-easy compared to WT in control samples (Fig. 3a), corresponding to an increase in shoot dry weight (Fig. 3b).

In summary, our data suggest that nitrate fertilizer does not inhibit the increased (compared to WT) formation of nodules and the development of pink (nitrogen-fixing) nodules in cngc15a-easy. This discovery is attractive from an agricultural perspective — since allowing nitrogen acquisition via the soil while maximizing symbiotic nitrogen fixation remains a challenge for crop breeding.

Example II: Analyse the cngc15a-easy nodulation phenotype in detrimental environments

2.1 Analyses of cngc 15a-easy at different temperatures

Climate models predict that plants will be exposed to higher average temperatures and more frequent heat stress in the future. Thus, we investigated the effect of the cngc15a- easy mutation at high temperatures.

To assess the performance of cngc 15a-easy at high temperatures, plants were grown at 35 °C for one week followed by 30 °C for one week under nutrient-depleted conditions (sand, terragreen, water). We observed an increase in total nodules (Fig. 5a) and root and shoot dry weight (Fig. 5b) in cngc15a-easy compared to WT. These data suggest that the increased nodulation and positive growth responses in cngc15a-easy are maintained at high temperatures.

Example III: Application of the point mutation to wheat

Our data show that the cngc15a-easy mutation in M. truncatula results in activation of nuclear calcium oscillations and increased endosymbiosis, resulting in positive growth responses. It was important to determine if the effects of cngc15a/c-easy can be translated to other species — particularly those of agricultural value. Wheat is an economically important crop species that can form symbioses with arbuscular mycorrhizal fungi; thus, increasing endosymbiosis in wheat could be an advantageous resource for sustainable agriculture. We have identified two mutants from two different spring cultivars — hexapioid cv. Cadenza and tetrapioid cv. Kronos — with (Kronos 111 P>L) or identical point mutations to cngc15a/c-easy (Cadenza 121 P>S). To assess the mycorrhizal colonization phenotypes of wheat cngc15a/c-easy under nutrient- depleted conditions (sand, terragreen, water), we inoculated cv. Cadenza and Kronos WT and cngc15a/c-easy plants with Rhizophgus irregularis. We performed a detailed analysis of mycorrhizal structures (intraradical hyphae, arbuscules, and vesicles) (Fig. 6a, b) once WT roots had reached 10-20% arbuscule colonization levels. Compared to WT, cngc15a-easy showed an increase in arbuscules and vesicles.

In summary, we were able to translate the cngc15a/c mutation and corresponding increased mycorrhizal colonization phenotype from model species M. truncatula to important crop species, such as wheat. The successful transfer to a polyploid crop variety such as wheat suggest that the discovery could be transferred to any crop.

Materials and Methods

Plant material seed sterilization and germination

Medicago truncatula WT (A17 x YC3.6), cngc15a-easy (JI20275 x YC3.6), and cngc15c- easy (JI22876 x YC3.6) seeds were scarified using sandpaper, treated with 10% sodium hypochlorite for 4 min, rinsed 5 x sterile dH₂O, then imbibed in sterile dH₂O containing Nystatin (5 pg/ml) and Augmentin (50 pg/ml) for 5 h. Seeds were placed onto water agar plates containing Nystatin (2 pg/ml) and Augmentin (50 pg/ml) and stratified for 6 days in darkness at 4 °C before germination.

Wheat cv. Cadenza WT and cngc15a/c-easy and cv. Kronos WT and cngc15a/c-easy seeds were treated with 2% sodium hypochlorite for 4 min, rinsed 5 x sterile dH₂O, then spread onto plates containing sterilized filter paper. Seeds were stratified for 7 days in darkness.

Calcium imaging

M. truncatula seeds (see seed sterilization and germination) were germinated overnight in darkness at 23 °C then transferred to Buffered Nodulation Medium (BNM) [1] plates containing 0.1 mM ami noethoxy vinyl glycine (AVG) for 1-2 days. Calcium oscillation measurements were performed using a Nikon ECLIPSE FN1 equipped with an emission image splitter (OptoSplit II, Cairn Research) and an electron multiplying cooled charge coupled (Rolera™ Thunder EMCDD) CaMera (Qlmaging). ECFP was excited using light emitting diode (OptoLED, Cairn) at 436±20 nm and emitted fluorescence detected at 535±30 nm (cpVenus) and 480±40 nm (ECFP). Images were collected in 3s intervals for 1 h30 using MetaFluor software.

Nodulation and arbuscular mycorrhizal assays

M. truncatula seeds (see seed sterilization and germination) were germinated overnight in darkness at 23 °C, then transferred to plates containing modified Fahraeus plant agar medium (modFP) and grown in a controlled-environment room (CER) for 7 days (23 °C, 16-h photoperiod, and 300 mmol m^-2 s^-1). Wheat seeds were germinated for 2 days in darkness at 23 °C, then transferred to modFP plates and grown in a CER for 5 days (23 °C, 16-h photoperiod, and 300 mmol m^-2 s^-1).

For M. truncatula nodulation assays, 7-day-old WT and cngc15-easy seedlings were transferred to 1 :1 Sand/Terragreen mix (Oil-Dri Company, Wisbech, UK) and inoculated with 5 mL Sinorhizobium meliloti 2011 (OD₆₀₀=0.001). Plants were grown in a controlled environment room at 22 °C (80% humidity, 16-h photoperiod, and 300 mmol m^-2 s^-1) or 35 °C for 1 week followed by 30 °C for 1 week (35% RH day, 50% RH night; 16-h photoperiod, and 500 pmol m^-2 s^-1). Plants were watered with either sterile water, Buffered Nodulation Medium (BNM), BNM containing 3 mM KNO₃, or BNM containing 4.8 mM (NH₄)₂SO₄. Nodules (total nodule number and number of pink nodules) were quantified at either 14 days post-inoculation (dpi) or 28 dpi using an M80 microscope (Leica). The roots and shoots were separated and dried for 7 days at 28 °C before measuring dry weights.

For M. truncatula mycorrhization experiments, 7-day-old WT and cngc15-easy seedlings were transferred in 90% 1 :1 Sand/Terragreen mix and 10% mycorrhizal inoculum (homogenized soil substrate containing Allium schoenoprasum roots colonized by Rhizophagus irregularis DAOM 197198). M. truncatula plants were grown in a controlled environment room at 22 °C (80% humidity, 16-h photoperiod, and 300 mmol m^-2 s^-1) and watered with either sterile water, modified BNM solution (without phosphate, KH₂PO₄), or modified BNM containing 0.5 mM KH₂PO₄. Shoots were dried for 7 days at 28 °C before measuring dry weights. For wheat mycorrhization experiments, 5-day-old WT and cngc15a/c-easy seedlings were transferred in 80% 1 :1 Sand/Terragreen mix and 20% mycorrhizal inoculum (as above). Wheat plants were grown in a controlled environment room at 22 °C (35% RH day, 50% RH night; 16-h photoperiod, and 500 pmol m^-2 s^-1 and watered with sterile water.

Mycorrhizal fungal structures were first cleared by incubating roots in 10% KOH for 5 min at 96 °C, rinsing 3 times in dH₂O, and then stained using 5% black ink (Waterman, France) and 5% acetic acid for 3 min at 96 °C. Next, roots were de-stained in 70% chloral hydrate for 10 min then stored in dH₂O. Mycorrhizal colonization structures (intraradical hyphae, arbuscules, and vesicles) were visualized using an M80 microscope (Leica) and quantified using the gridline intersect method [6],

For M. truncatula seed yield analysis, 7-day-old WT and cngc15-easy plants were transferred to 9 cm pots containing 1 :1 Sand/Terragreen mix inoculated with 10 mL S. meliloti 2011 (OD₆₀₀=0.001) or containing 10% mycorrhizal inoculum (R. irregularis, Endorize; Agrauxine, France). Seed pods were harvested after 16 weeks, and the number of pods and the total number of seeds were quantified.

Nitrogen/carbon ratio

Dried leaf tissue was ground in liquid nitrogen, and CHN elemental microanalysis was performed by Butterworth Laboratories Ltd. (UK) using CHN analyser. References

1. Gibert, A. et al. (2019) Plant performance response to eight different types of symbiosis. New Phytol. 222, 526-542

2. Van Noorden, G.E. et al. (2016) Molecular signals controlling the inhibition of nodulation by nitrate in Medicago truncatula. International Journal of Molecular Sciences 17, 1060

3. Dusha, I. and Kondorosi, A. (1993) Genes at different regulatory levels are required for the ammonia control of nodulation in Rhizobium meliloti. Molecular and General Genetics MGG 240, 435-444

4. Lin, J.-s. et al. (2018) NIN interacts with NLPs to mediate nitrate inhibition of nodulation in Medicago truncatula. Nature plants 4, 942-952

5. Wang, X. et al. (2021) A transceptor-channel complex couples nitrate sensing to calcium signaling in Arabidopsis. Molecular Plant 14, 774-786

6. Lobell, D.B. et al. (2011) Climate trends and global crop production since 1980. Science 333, 616-620

7. Delaux, P.-M. et al. (2013) Evolution of the plant-microbe symbiotic ‘toolkit’. Trends Plant Sci. 18, 298-304

8. Leitao, N. et al. (2019) Nuclear calcium signatures are associated with root development. Nature communications 10, 1-9

9. Dalmais, M. et al. (2008) UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome biology 9, 1-12

10. Ehrhardt D W, Atkinson E M, Long S R(1992) Science 256:998-1000, pmid: 10744524.

11. Anzalone, A.V., Randolph, P.B., Davis, J.R. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019). https://doi.org/10.1038/s41586-019-1711-4

12. Li, Y., Liu, X., Chen, R. et al. Genome-scale mining of root-preferential genes from maize and characterization of their promoter activity. BMC Plant Biol 19, 584 (2019). https://doi.org/10.1186/s12870-019-2198-8

SEQUENCE LISTING

The conserved domain “VDPL” is bold and highlighted as VDPL

Ill

Mutant Polypeptide Sequences in Arabadopsis thaliana; Mutations are Bold and Underlined

Mutant Polypeptide Sequences in Glycine Max; Mutations are in Bold and Underlined

Mutant Polypeptide Sequences in Medicago truncatula; Mutations are in Bold and Underlined

Mutant Polypeptide Sequences in Oryza sativa var. japonica; Mutations are Bold and Underlined

Mutant Polypeptide Sequences in Solanum lycopersicum; Mutations are in Bold and Underlined

10

15 Mutant Polypeptide Sequences in Zea mays; Mutations are Bold and Underlined

Mutant Polypeptide Sequences in Triticum aestivum; Mutations are in Bold and Underlined

Mutant Polypeptide Sequences in Hordeum vulgare; Mutations are Bold and Underlined

Polypeptide Sequences in Arachis hypogaea

Polypeptide Sequences in Linum usitatissimum

Nucleic Acid Sequences for Arachis hypogaea

Nucleic Acid Sequences for Linum usitatissimum

Mutant Polypeptide Sequences in Arachis hypogaea; Mutations are in Bold and

5 Underlined

Mutant Polypeptide Sequences in Linum usitatissimum; Mutations are in Bold and Underlined

5

Polypeptide Sequences in Brassica napus

Nucleic Acid Sequences for Brassica napus

Mutant Polypeptide Sequences in Brassica napus; Mutations are in Bold and Underlined

5

Polypeptide Sequences in Fragaria vesca

Nucleic Acid Sequences for Fragaria vesca

Mutant Polypeptide Sequences in Fragaria vesca; Mutations are in Bold and Underlined

Claims

CLAIMS:

1 . A genetically altered plant, plant part thereof or plant cell comprising at least one mutation in at least one gene encoding at least one nuclear localized cyclic nucleotide-gated ion channel (CNGC), wherein the at least one mutation is in at least one conserved motif, wherein the motif comprises the residues XDPX,

2. The genetically altered plant of claim 1 , wherein the CNCG gene encodes an amino acid sequence as defined in any one of SEQ ID NOs 1 to 42, 232 to 236, 247, 248, 253 or a functional variant or homologue thereof.

3. The genetically altered plant of any preceding claim, wherein the motif comprises XDPL, more preferably VDPL.

4. The genetically altered plant of claim 3, wherein the mutation comprises at least one substitution at one more positions in XDPX, preferably VDPL, wherein preferably the substitution is a P for L or S and/or a L for F.

5. The genetically altered plant of any preceding claim, wherein the plant is selected from wheat, rice, maize, soybean, tomato, barley, sugar cane, sorghum, sunflowers, sugar beet, rye, cotton, potato, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.

6. The genetically altered plant part of any preceding claim, wherein the plant part is a seed.

7. A nucleic acid construct comprising a nucleic acid sequence encoding a mutated nuclear-localised cyclic nucleotide-gated ion channel (CNGC) amino acid sequence, preferably a mutated CNGC15 amino acid sequence, wherein the nucleic acid sequence is operably linked to a regulatory sequence.

8. The nucleic acid construct of claim 7, wherein the nucleic acid sequence comprises one or more mutations in a conserved motif, wherein the conserved motif is XDPX, preferably VDPL, more preferably VDPL.

9. The nucleic acid construct of claim 7 or 8, wherein the nucleic acid sequence encodes a CNGC polypeptide as defined in any of SEQ ID Nos 85 to 231 , 242 to 246, 251 , 252, 255 or a functional variant thereof.

10. A vector comprising the nucleic acid construct of any of claims 7 to 9.

11 . A host cell comprising the nucleic acid construct of any of claims 7 to 9 or the vector of claim 10.

12. A genetically altered plant, plant part thereof or plant cell, wherein the plant, part thereof or plant cell comprises the nucleic acid construct of any of claims 7 to 9, the vector of claim 10 or the host cell of claim 11.

13. A method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant, the method comprising introducing and expressing the nucleic acid construct of any of claims 7 to 9 or the vector of claim 10 in the plant.

14. A method of increasing at least one of yield, nodulation and arbuscular mycorrhiza (AM) endosymbioses in a plant, the method comprising introducing at least one mutation in at least one gene encoding at least one nuclear localized cyclic nucleotide-gated ion channel (CNGC), preferably CNGC 15 into the plant.

15. A method for maintaining yield while reducing the input of chemical fertilizer, the method comprising introducing at least one mutation in at least one gene encoding at least one nuclear localized cyclic nucleotide-gated ion channel (CNGC), preferably CNGC 15 into the plant.

16. The method of claim 14 or 15, wherein the CNCG gene encodes an amino acid sequence as defined in any one of SEQ ID NOs 1 to 42, 232 to 236, 247, 248, 253 or a functional variant or homologue thereof.

17. The method of any of claims 14 to 16, wherein the at least one mutation is in at least one conserved motif, wherein the motif comprises the residues XDPX, preferably XDPL, more preferably VDPL.

18. The method of claim 17, wherein the mutation comprises at least one substitution at one more positions in VDPL, wherein preferably the substitution is a P for L or S and/or a L for F.

19. The method of any of claims 13 to 18, wherein the plant is selected from wheat, rice, maize, soybean, tomato, barley, sugar cane, sorghum, sunflower, sugar beet, rye, cotton, potato, peanut, flax (common flax or linseed), strawberry, oilseed rape and any leguminous plant.

20. The method of any of claims 13 to 19, wherein the plant is grown under high N conditions.

21. The method of any of claims 13 to 20, wherein the plant is grown under high temperatures.

22. A method for identifying and/or selecting a plant that will have an increase in at least one of yield, nodulation and/or AM endosymbiosis, the method comprising screening a population of plants and detecting in the plant or plant germplasm at least one polymorphism in at least conserved domain of a nuclear localized cyclic nucleotide-gated ion channel (CNGC) gene, preferably a polymorphism in the VDPL motif, and selecting said plant.