Patent Application
20240229060
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 794542000700SUBSEQLIST.TXT, date recorded: Feb. 9, 2023, size: 620,044).
The present disclosure relates to genetically altered plants. In particular, the present disclosure relates to genetically altered plants with increased activity of one or more of a NODULATION SIGNALING PATHWAY 1 (NSP1) protein, a NODULATION SIGNALING PATHWAY 2 (NSP2) protein, or a C-TERMINALLY ENCODED PEPTIDE (CEP peptide) that have increased mycorrhization and/or promoted symbiotic responses under high phosphate and/or high nitrate conditions. Further, the present disclosure relates to methods of cultivating plants with exogenous butenolide agents or CEP peptides that have increased mycorrhization and/or promoted symbiotic responses under high phosphate and/or high nitrate conditions, which may be in combination with the genetically altered plants of the present application.
Plant growth and development depends on carbon dioxide and sunlight above ground, and water and mineral nutrients in the soil. The accessibility of nutrients in the soil depends on many factors, and nutrient availability varies spatially and temporally. Local nutrient sensing, as well as the perception of overall nutrient status, shape the plant's response to its nutrient environment, and act to coordinate plant development with microbial engagement to optimize nutrient capture and regulate plant growth.
The principle nutrients that limit plant productivity are nitrogen (N) and phosphorus (P). In soils where these nutrients are ample, shoot biomass can exceed root biomass, because minimal root systems are able to capture sufficient nutrients. Vegetative growth is also promoted, allowing plants to accumulate resources and invest in seed production. In soils where these nutrients are limiting, overall plant growth is reduced to optimize productivity, while root systems are expanded to facilitate nutrient capture. In addition to the expansion of root systems, colonization by microorganisms is promoted, to further facilitate nutrient capture.
The mutualistic association with arbuscular mycorrhizal fungi dates to the earliest land plants, and is thought to have facilitated the transition from an aquatic to a terrestrial lifestyle (M. Parniske, Arbuscular mycorrhiza: the mother of plant root endosymbioses. Nature Reviews Microbiology 6, 763-775 (2008)). Because of the evolutionarily early establishment of this association, the arbuscular mycorrhizal association is both extremely widespread in the plant kingdom and intricately networked with plant nutrient capture physiology. Mutualistic mycorrhizal associations increase the surface area for nitrogen and phosphorus capture and make additional nutrients in the soil more available to the plant. While these associations would seem to be uniformly beneficial to plants, studies have shown that they can have substantial energetic costs for plants (L. H. Luginbuehl et al., Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant. Science 356, 1175-1178 (2017)), and so are inhibited when sufficient nutrients are present in the soil.
In high-intensity agriculture, nitrogen and phosphorus are typically applied at high concentrations in the form of inorganic fertilizers, in order to promote crop productivity. The concentrations used are generally in excess of the amounts needed by plants or the amounts able to be stored in soil, and so the nutrients are often released into the environment, where they reduce biodiversity and contribute to climate change (C. J. Stevens, Nitrogen in the environment. Science 363, 578-580 (2019); J. A. Foley et al., Solutions for a cultivated planet. Nature 478, 337-342 (2011); J. Rockstrom et al., A safe operating space for humanity. Nature 461, 472-475 (2009)). Similarly, the manufacture of inorganic fertilizers is costly in terms of resources and energy (W. F. Zhang et al., New technologies reduce greenhouse gas emissions from nitrogenous fertilizer in China. Proc Natl Acad Sci USA 110, 8375-8380 (2013)).
There exists a need for a system by which nitrogen and phosphorus can be made more available to plants across agricultural systems. Preferably, this system would function in conditions where there are high levels of nutrients (e.g., nitrogen, phosphorus) in the environment surrounding the plant roots, whether natural or due to application of fertilizers.
In order to meet these needs, the present disclosure provides methods of cultivation that increase mycorrhization and/or promote symbiotic responses under nutrient conditions that suppress mycorrhization and genetically altered plants for use of such methods, whereby the increased mycorrhization and/or promoted symbiotic responses allows the plant to obtain greater nutrients from the environment around the plant roots. The present disclosure provides genetically altered plants with increased activity of one or more of a NODULATION SIGNALING PATHWAY 1 (NSP1) protein, or a NODULATION SIGNALING PATHWAY 2 (NSP2) protein that have increased mycorrhization and/or promoted symbiotic responses under high phosphate and/or high nitrate conditions. The present disclosure further provides genetically altered plants with increased activity of a C-TERMINALLY ENCODED PEPTIDE (CEP peptide) that have increased mycorrhization and/or promoted symbiotic responses under high phosphate and/or high nitrate conditions. In addition, the present disclosure provides methods of cultivating these plants that include exogenous application of strigolactones, karrikins, and/or CEP peptides to increase mycorrhization and/or promote symbiotic responses under specific nutrient conditions.
An aspect of the disclosure includes methods of cultivating a genetically altered plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a phosphate level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) providing the genetically altered plant, wherein the plant or a part thereof includes one or more genetic alterations, wherein the one or more genetic alterations reduce the phosphate level suppression of mycorrhization and/or symbiotic responses; and (b) cultivating the genetically altered plant under the phosphate level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a wild type (WT) plant grown under the same conditions. An additional embodiment of this aspect includes the one or more genetic alterations resulting in increased activity of one or more of a NODULATION SIGNALING PATHWAY 1 (NSP1) protein or a NODULATION SIGNALING PATHWAY 2 (NSP2) protein. Yet another embodiment of this aspect includes the increased activity being at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 150% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. A further embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the increased activity being no greater than 500%, no greater than 400%, no greater than 300%, no greater than 200%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the NSP1 protein including an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207. In an additional embodiment of this aspect, the NSP1 protein includes SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207. Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the NSP2 protein including an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208. In a further embodiment of this aspect, the NSP2 protein includes SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes one or more of the NSP1 protein and the NSP2 protein being endogenous. A further embodiment of this aspect includes increased activity of the one or more endogenous NSP1 protein and the endogenous NSP2 protein being achieved using a gene editing technique to introduce the one or more genetic alterations. Still another embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter, modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein.
Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the increased activity being due to heterologous expression of one or more of the NSP1 protein and the NSP2 protein. A further embodiment of this aspect includes increased activity of the one or more of the heterologous NSP1 protein and the heterologous NSP2 protein being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. An additional embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In an additional embodiment of this aspect, the nitrogen around the plant roots is present in the form of nitrate, and wherein the nitrate level around the plant roots is less than 2.5 mM, less than 2 mM, less than 1.5 mM, less than 1 mM, less than 0.75 mM, less than 0.5 mM, or less than 0.25 mM. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots includes at least 100 μM phosphate, at least 200 μM phosphate, at least 300 μM phosphate, at least 400 μM phosphate, at least 500 μM phosphate, at least 600 μM phosphate, at least 800 μM phosphate, at least 1000 μM phosphate, at least 2000 μM phosphate, at least 3000 μM phosphate, at least 3750 μM phosphate, at least 4000 μM phosphate, or at least 5000 μM phosphate. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley, maize, rice, wheat, another cereal crop, cassava, potato, soy, or a legume crop. Yet another embodiment of this aspect includes the plant being barley.
In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein the genetically altered plant of step a) further includes one or more genetic alterations that reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses, and wherein step b) further includes cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In a further embodiment of this aspect, the one or more genetic alterations result in increased activity of a C-TERMINALLY ENCODED PEPTIDE (CEP peptide). In still another embodiment of this aspect, the increased activity is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 150% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is no greater than 500%, no greater than 400%, no greater than 300%, no greater than 200%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide is endogenous. Yet another embodiment of this aspect includes increased activity of the endogenous CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In a further embodiment of this aspect, which may be combined with any of the preceding aspects that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter; modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is due to heterologous expression of the CEP peptide. An additional embodiment of this aspect includes increased activity of the heterologous CEP peptide being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. A further embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein step a) further includes cultivating the plant under conditions including the nitrogen level around the plant roots, and wherein step b) further includes exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In still another embodiment of this aspect, the effective amount of the CEP peptide includes at least 0.1 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.75 μM CEP peptide, at least 1 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.75 μM CEP peptide, or at least 2 μM CEP peptide. Further embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, include the plant or the part thereof being exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In additional embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. An additional embodiment of this aspect includes the nitrogen around the plant roots being present in the form of nitrate, and the nitrate level around the plant roots being greater than 2.75 mM, greater than 3 mM, greater than 3.25 mM, greater than 3.5 mM, greater than 3.75 mM, greater than 4 mM, greater than 4.25 mM, greater than 4.5 mM, greater than 4.75 mM, greater than 5 mM, or greater than 5.5 mM.
An additional aspect of the disclosure includes methods of cultivating a plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a phosphate level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) cultivating the plant under conditions including the phosphate level around the plant roots; and (b) exposing the plant or a part thereof to an effective amount of a butenolide agent, wherein the effective amount of the butenolide agent increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the butenolide agent. In yet another embodiment of this aspect, the effective amount of the butenolide agent includes at least 0.1 μM butenolide agent, at least 0.25 μM butenolide agent, at least 0.5 μM butenolide agent, at least 0.75 μM butenolide agent, at least 1 μM butenolide agent, at least 1.25 μM butenolide agent, at least 1.5 μM butenolide agent, at least 1.75 μM butenolide agent, or at least 2 μM butenolide agent. A further embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the plant or the part thereof being exposed to the butenolide agent by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the butenolide agent being a strigolactone. Still another embodiment of this aspect includes the strigolactone being selected from the group of 5-deoxystrigol, strigol, sorgomol, sorgolactone, other strigol-like compounds, 4-deoxyorobanchol, orobanchol, fabacyl acetate, solanocol, other orobanchol-like compounds, GR24, or any combination thereof. An additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has a butenolide agent, includes the butenolide agent being a karrikin. Yet another embodiment of this aspect includes the karrikin being selected from the group of karrikin1, karrikin2, karrikin3, karrikin4, karrikin5, karrikin6, a mixture of karrikin1 and karrikin2, GR24, karrikin contained in liquid smoke, or any combination thereof.
Still another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the phosphate level around the plant roots completely suppressing mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the butenolide agent. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the butenolide agent. In yet another embodiment of this aspect, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is less than 2.5 mM, less than 2 mM, less than 1.5 mM, less than 1 mM, less than 0.75 mM, less than 0.5 mM, or less than 0.25 mM. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots includes at least 100 μM phosphate, at least 200 μM phosphate, at least 300 μM phosphate, at least 400 μM phosphate, at least 500 μM phosphate, at least 600 μM phosphate, at least 800 μM phosphate, at least 1000 μM phosphate, at least 2000 μM phosphate, at least 3000 μM phosphate, at least 3750 μM phosphate, at least 4000 μM phosphate, or at least 5000 μM phosphate. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley, maize, rice, wheat, another cereal crop, cassava, potato, soy, or a legume crop. Still another embodiment of this aspect includes the plant being barley.
In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein the genetically altered plant of step a) further includes one or more genetic alterations that reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses, and wherein step b) further includes cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In a further embodiment of this aspect, the one or more genetic alterations result in increased activity of a CEP peptide. In still another embodiment of this aspect, the increased activity is at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 150% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is no greater than 500%, no greater than 400%, no greater than 300%, no greater than 200%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide is endogenous. Yet another embodiment of this aspect includes increased activity of the endogenous CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In a further embodiment of this aspect, which may be combined with any of the preceding aspects that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter; modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is due to heterologous expression of the CEP peptide. An additional embodiment of this aspect includes increased activity of the heterologous CEP peptide being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. A further embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein step a) further includes cultivating the plant under conditions including the nitrogen level around the plant roots, and wherein step b) further includes exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In still another embodiment of this aspect, the effective amount of the CEP peptide includes at least 0.1 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.75 μM CEP peptide, at least 1 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.75 μM CEP peptide, or at least 2 μM CEP peptide. Further embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, include the plant or the part thereof being exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In additional embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to an effective amount of a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. An additional embodiment of this aspect includes the nitrogen around the plant roots being present in the form of nitrate, and the nitrate level around the plant roots being greater than 2.75 mM, greater than 3 mM, greater than 3.25 mM, greater than 3.5 mM, greater than 3.75 mM, greater than 4 mM, greater than 4.25 mM, greater than 4.5 mM, greater than 4.75 mM, greater than 5 mM, or greater than 5.5 mM.
A further aspect of the disclosure includes methods of cultivating a genetically altered plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) providing the genetically altered plant, wherein the plant or a part thereof includes one or more genetic alterations, wherein the one or more genetic alterations reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses; and (b) cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In an additional embodiment of this aspect, the one or more genetic alterations result in increased activity of a CEP peptide. Yet another embodiment of this aspect includes the increased activity being at least 10% greater, at least 20% greater, at least 30% greater, at least 40% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 150% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the increased activity is no greater than 500%, no greater than 400%, no greater than 300%, no greater than 200%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the CEP peptide is endogenous. A further embodiment of this aspect includes increased activity of the CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter, modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein.
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the increased activity is due to heterologous expression of the CEP peptide. In a further embodiment of this aspect, increased activity of the heterologous CEP peptide is achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. An additional embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots includes less than 1000 μM phosphate, less than 800 μM phosphate, less than 600 μM phosphate, less than 500 μM phosphate, less than 400 μM phosphate, less than 300 μM phosphate, less than 200 μM phosphate, or less than 100 μM phosphate. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is greater than 2.75 mM, greater than 3 mM, greater than 3.25 mM, greater than 3.5 mM, greater than 3.75 mM, greater than 4 mM, greater than 4.25 mM, greater than 4.5 mM, greater than 4.75 mM, greater than 5 mM, or greater than 5.5 mM. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley, maize, rice, wheat, another cereal crop, cassava, potato, soy, or a legume crop. Yet another embodiment of this aspect includes the plant being barley.
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
A further aspect of this disclosure includes methods of cultivating a plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) cultivating the plant under conditions including the nitrogen level around the plant roots; and (b) exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In an additional embodiment of this aspect, the effective amount of the CEP peptide includes at least 0.1 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.75 μM CEP peptide, at least 1 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.75 μM CEP peptide, or at least 2 μM CEP peptide. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant or the part thereof is exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the CEP peptide. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the CEP peptide. In a further embodiment of this aspect, the phosphate level around the plant roots includes less than 1000 μM phosphate, less than 800 μM phosphate, less than 600 μM phosphate, less than 500 μM phosphate, less than 400 μM phosphate, less than 300 μM phosphate, less than 200 μM phosphate, or less than 100 μM phosphate. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is greater than 2.75 mM, greater than 3 mM, greater than 3.25 mM, greater than 3.5 mM, greater than 3.75 mM, greater than 4 mM, greater than 4.25 mM, greater than 4.5 mM, greater than 4.75 mM, greater than 5 mM, or greater than 5.5 mM. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley, maize, rice, wheat, another cereal crop, cassava, potato, soy, or a legume crop. An additional embodiment of this aspect includes the plant being barley.
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
An additional aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of NSP1 or NSP2, including: (a) transforming a plant cell, tissue, or other explant with a vector including a first nucleic acid sequence encoding a NSP1 protein or a NSP2 protein operably linked to a second nucleic acid sequence encoding a promoter; (b) selecting successful transformation events by means of a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with increased activity of the NSP1 protein or the NSP2 protein as compared to an untransformed WT plant. Yet another embodiment of this aspect further includes identifying successful introduction of the one or more genetic alterations by screening or selecting the plant cell, tissue, or other explant prior to step (b); screening or selecting plantlets between step (b) and (c); or screening or selecting plants after step (c). Still another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes transformation being done using a transformation method selected from the group of particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the NSP1 protein includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207; or the NSP2 protein includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208. In a further embodiment of this aspect, the NSP1 protein includes SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207; or the NSP2 protein includes SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the promoter is selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the first nucleic acid sequence and the second nucleic acid sequence are stably integrated into a nuclear genome of the plant.
A further aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of NSP1 or NSP2, including (a) transforming a plant cell, tissue, or other explant with one or more gene editing components that target a nuclear genome sequence operably linked to an endogenous NSP1 protein or an endogenous NSP2 protein; (b) selecting successful transformation events by means of a screening technology, an enriching technology, a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with overexpression of the NSP1 protein or the NSP2 protein as compared to an untransformed WT plant. In an additional embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex that targets the nuclear genome sequence; a vector including a TALEN protein encoding sequence, wherein the TALEN protein targets the nuclear genome sequence; a vector including a ZFN protein encoding sequence, wherein the ZFN protein targets the nuclear genome sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genome sequence; or a vector CRISPR/Cas enzyme encoding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genome sequence.
An additional aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of CEP peptide, including: (a) transforming a plant cell, tissue, or other explant with a vector including a first nucleic acid sequence encoding a CEP peptide operably linked to a second nucleic acid sequence encoding a promoter; (b) selecting successful transformation events by means of a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with increased activity of the CEP peptide as compared to an untransformed WT plant. Yet another embodiment of this aspect further includes identifying successful introduction of the one or more genetic alterations by screening or selecting the plant cell, tissue, or other explant prior to step (b); screening or selecting plantlets between step (b) and (c); or screening or selecting plants after step (c). Still another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes transformation being done using a transformation method selected from the group of particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 75% sequence identity to, at least 80% sequence identity to, at least 85% sequence identity to, at least 90% sequence identity to, at least 95% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the promoter is selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the first nucleic acid sequence and the second nucleic acid sequence are stably integrated into a nuclear genome of the plant.
A further aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of CEP peptide, including (a) transforming a plant cell, tissue, or other explant with one or more gene editing components that target a nuclear genome sequence operably linked to an endogenous CEP peptide; (b) selecting successful transformation events by means of a screening technology, an enriching technology, a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with overexpression of the CEP peptide as compared to an untransformed WT plant. In an additional embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex that targets the nuclear genome sequence; a vector including a TALEN protein encoding sequence, wherein the TALEN protein targets the nuclear genome sequence; a vector including a ZFN protein encoding sequence, wherein the ZFN protein targets the nuclear genome sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genome sequence; or a vector CRISPR/Cas enzyme encoding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genome sequence.
The following description sets forth exemplary methods, parameters, and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.
An aspect of the disclosure includes methods of cultivating a genetically altered plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a phosphate level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) providing the genetically altered plant, wherein the plant or a part thereof includes one or more genetic alterations, wherein the one or more genetic alterations reduce the phosphate level suppression of mycorrhization and/or symbiotic responses; and (b) cultivating the genetically altered plant under the phosphate level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a wild type (WT) plant grown under the same conditions. An additional embodiment of this aspect includes the one or more genetic alterations resulting in increased activity of one or more of a NODULATION SIGNALING PATHWAY 1 (NSP1) protein or a NODULATION SIGNALING PATHWAY 2 (NSP2) protein. Yet another embodiment of this aspect includes the increased activity being at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least 180% greater, at least 190% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. A further embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the increased activity being no greater than 500%, no greater than 475%, no greater than 450%, no greater than 425%, no greater than 400%, no greater than 375%, no greater than 350%, no greater than 325%, no greater than 300%, no greater than 275%, no greater than 250%, no greater than 225%, no greater than 200%, no greater than 175%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the NSP1 protein including an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207. In an additional embodiment of this aspect, the NSP1 protein includes SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207. A gene tree of NSP1 homologs is shown in
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes one or more of the NSP1 protein and the NSP2 protein being endogenous. A further embodiment of this aspect includes increased activity of the one or more endogenous NSP1 protein and the endogenous NSP2 protein being achieved using a gene editing technique to introduce the one or more genetic alterations. Still another embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter, modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein.
Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, includes the increased activity being due to heterologous expression of one or more of the NSP1 protein and the NSP2 protein. A further embodiment of this aspect includes increased activity of the one or more of the heterologous NSP1 protein and the heterologous NSP2 protein being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. An additional embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots inhibits mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. Bioavailable nitrogen may be present in soil in the form of nitrate, ammonium, or amino acids. In an additional embodiment of this aspect, the nitrogen around the plant roots is present in the form of nitrate, and wherein the nitrate level around the plant roots is less than 2.5 mM, less than 2.4 mM, less than 2.3 mM, less than 2.2 mM, less than 2.1 mM, less than 2.0 mM, less than 1.9 mM, less than 1.8 mM, less than 1.7 mM, less than 1.6 mM, less than 1.5 mM, less than 1.4 mM, less than 1.3 mM, less than 1.2 mM, less than 1.1 mM, less than 1.0 mM, less than 0.95 mM, less than 0.9 mM, less than 0.85 mM, less than 0.8 mM, less than 0.75 mM, less than 0.7 mM, less than 0.65 mM, less than 0.6 mM, less than 0.55 mM, less than 0.5 mM, less than 0.45 mM, less than 0.4 mM, less than 0.35 mM, less than 0.3 mM, less than 0.25 mM, less than 0.2 mM, less than 0.15 mM, less than 0.1 mM, or less than 0.05 mM. In yet another embodiment of this aspect, the nitrate level around the plant roots is about 0 mM. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots includes at least 100 μM phosphate, at least 125 μM phosphate, at least 150 μM phosphate, at least 175 μM phosphate, at least 200 μM phosphate, at least 225 μM phosphate, at least 250 μM phosphate, at least 275 μM phosphate, at least 300 μM phosphate, at least 325 μM phosphate, at least 350 μM phosphate, at least 375 μM phosphate, at least 400 μM phosphate, at least 425 μM phosphate, at least 450 μM phosphate, at least 475 μM phosphate, at least 500 μM phosphate, at least 525 μM phosphate, at least 550 μM phosphate, at least 575 μM phosphate, at least 600 μM phosphate, at least 625 μM phosphate, at least 650 μM phosphate, at least 675 μM phosphate, at least 700 μM phosphate, at least 725 μM phosphate, at least 750 μM phosphate, at least 800 μM phosphate, at least 850 μM phosphate, at least 900 μM phosphate, at least 950 μM phosphate, at least 1000 μM phosphate, at least 1250 μM phosphate, at least 1500 μM phosphate, at least 1750 μM phosphate, at least 2000 μM phosphate, at least 2250 μM phosphate, at least 2500 μM phosphate, at least 2750 μM phosphate, at least 3000 μM phosphate, at least 3250 μM phosphate, at least 3500 μM phosphate, at least 3750 μM phosphate, at least 4000 μM phosphate, at least 4250 μM phosphate, at least 4500 μM phosphate, at least 4750 μM phosphate, or at least 5000 μM phosphate.
Phosphorus and nitrogen are the principal elemental nutrients in the soil that limit plant productivity, and the availability of these nutrients is important in both natural and agricultural ecosystems. In particular, the pools of these two nutrients that are available to plants (e.g., plant extractable, bioavailable) determines whether mycorrhization is suppressed. High phosphate levels (e.g., replete phosphate) and/or high nitrogen levels (e.g., replete nitrate) may suppress mycorrhization. The combination of both high phosphate and high nitrogen levels (e.g., replete phosphate and replete nitrate) is particularly potent in suppressing mycorrhization.
The total phosphorus pool includes a soluble phosphorus pool (proportionally very small) as well as a plant available pool (often <3% of the total pool). Bioavailable phosphorus is primarily available in soil in the form of phosphate (PO4−). There are a range of methods available for determining the amount of soluble phosphorus in soil (described in detail in Pierzynski (ed.), Methods for phosphorus analysis for soils, sediments, residuals, and waters. Southern Cooperative Series Bull. No. 408, June 2009, ISBN: 1-58161-408-x). Four commonly used soil phosphorus test methods are Bray and Kurtz P-1, Mehlich 1, Mehlich 3, and Olsen P (Carter, M. R., and E. G. Gregorich. 2007. Soil sampling and methods of analysis, second edition. CRC Press, Boca Raton, FL.; Frank, K., D. Beegle, and J. Denning. 1998. Phosphorus. p. 21-30. In J. R. Brown (ed.) Recommended Chemical Soil Test Procedures for the North Central Region. North Central Reg. Res. Publ. No. 221 (revised); Kuo, S. 1996. Phosphorus. p. 869-919. In D. L. Sparks. (ed.) Methods of Soil Analysis: Part 3—Chemical Methods. SSSA, Madison, WI.; SERA-IEG-6 (Southern Extension Research Activity—Information Exchange Group) 1992. Donohue, S. J. (ed.) Reference Soil and Media Diagnostic procedure for the southern region of the United States. So. Coop. Series Bulletin 374. Va. Agric. Exp. Station, Blacksburg, VA.; Sims, J. T., and A. M. Wolf. (ed.) 1995. Recommended soil testing procedures for the Northeastern United States. (2nd ed.). Bull. No. 493. Univ. Delaware, Newark, DE; SPAC (Soil and Plant Analysis Council). 1992. Handbook on reference methods for soil analysis. Georgia Univ. Stn., Athens, GA). The soil pH may be used to determine which method of these or others would be most advantageous to use (https://www.nres.usda.gov/Internet/FSE_DOCUMENTS/nresl42p2_051918.pdf). Additional commonly used soil phosphorus test methods include Morgan's and Modified Morgan's (Lunt, H. A., C. L. W. Swanson, and H. G. M. Jacobson. 1950. The Morgan Soil Testing System. Bull. No. 541, Conn. Agr. Exp. Stn., New Haven, CT; Morgan, M. F. 1941. Chemical soil diagnosis by the universal soil testing system. Conn. Agric. Exp. Stn. Bull. No. 450; SPAC (Soil and Plant Analysis Council). 1992. Handbook on reference methods for soil analysis. Georgia Univ. Stn., Athens, GA).
The total nitrogen pool is primarily composed of organic matter (about 98%) and referred to as the organic nitrogen fraction. The remaining about 2% of the total nitrogen pool is referred to as the mineral nitrogen pool, and is primarily present as nitrate (NO3−) or ammonium (NH4′). The mineral nitrogen pool is continually replenished by mineralisation processes, i.e., the conversion of organic to mineral forms, and is immediately plant available. Mineral nitrogen is used as a measure of the amount of bioavailable nitrogen in the soil. Commonly used tests to quantify immediately available mineral nitrogen are described in Maynard et al., Nitrate and Exchangeable Ammonium Nitrogen, Chapter 4, Soil Sampling and Methods of Analysis, M. R. Carter (ed.), Canadian Society of Soil Science and in https://www.udel.edu/content/dam/udelImages/canr/pdfs/extension/factsheets/soiltest-recs/CHAP4.pdf. In addition to this immediately available mineral nitrogen pool, a proportion of the organic nitrogen pool is considered to be medium-term potentially available nitrogen for plants. This potentially available pool is generally thought to be composed of organic nitrogen that is converted to mineral nitrogen by microorganisms, but plants are also able to directly absorb free amino acids from the soil (Nasholm T, Kielland K, Ganeteg U. Uptake of organic nitrogen by plants. New Phytologist. 2009; 182:31-48; Hill P W, Quilliam R S, DeLuca T H, Farrar J, Farrell M, Roberts P, Newsham K K, Hopkins D W, Bardgett R D, Jones D L. Acquisition and assimilation of nitrogen as peptide-bound and D-enantiomers of amino acids by wheat. PLoS ONE. 2011; 6: e19220; Jones D L, Clode P L, Kilburn M R, Stockdale E A, Murphy D V. Competition between plant and bacterial cells at the microscale regulates the dynamics of nitrogen acquisition in wheat (Triticum aestivum) New Phytol. 2013 November; 200(3): 796-807; Jones D L, Shannon D, Junvee-Fortune T, Farrar J F. Plant capture of free amino acids is maximized under high soil amino acid concentrations. Soil Biol Biochem 2005; 37:179-81; Kielland K. Amino acid absorption by arctic plants: implications for plant nutrition and nitrogen cycling. Ecology 1994; 75:2373-83). The measurement of potentially available nitrogen is complex, and therefore generally not used as a measure of the amount of bioavailable nitrogen in the soil (Herrmann A M, Ritz K, Nunan N., Clode P L, Pett-Ridge J, Kilburn M R, Murphy D V, O'Donnell A G, Stockdale E A. Nano-scale secondary ion mass spectrometry A new analytical tool in biogeochemistry and soil ecology: A review article. Soil Biol Biochem 2007; 39(8): 1835-1850). Immediately and potentially available nitrogen pools together are usually less than 10% of the total nitrogen in soil.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley (e.g., Hordeum vulgare), maize (e.g., corn, Zea mays), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), another cereal crop such as sorghum (e.g., Sorghum bicolor), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Setaria italica, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), teff (e.g., Eragrostis tef), oat (e.g., Avena sativa), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), rye (e.g., Secale cereale, Secale cereanum), or wild rice (e.g., Zizania spp., Porteresia spp.), cassava (e.g., manioc, yucca, Manihot esculenta), potato (e.g., russet potatoes, yellow potatoes, red potatoes, Solanum tuberosum), soy (e.g., soybean, soja, sojabean, Glycine max, Glycine soja), or a legume crop such as peanut (e.g., Arachis duranensis, Arachis hypogaea, Arachis ipaensis), pigeon pea (e.g., Cajanus cajan), chickpea (e.g., Cicer arietinum), cowpea (e.g., black-eyed pea, Vigna unguiculata), velvet bean (e.g., Mucuna pruriens), bean (e.g., Phaseolus vulgaris), pea (e.g., Pisum sativum), adzuki bean (e.g., Vigna angularis, Vigna angularis var. angularis), mung bean (e.g., Vigna radiata var. radiata), clover (e.g., Trifolium pratense, Trifolium subterraneum), or lupine (e.g., lupin, Lupinus angustifolius). Yet another embodiment of this aspect includes the plant being barley (e.g., Hordeum vulgare).
In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses are induced by a plant's perception of LCOs produced by bacteria or fungi. Symbiotic responses are associated with the interactions of plants with beneficial microorganisms, including nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (Oldroyd, G. E. D. Nature Reviews Microbiology 2013, 11), and may include symbiotic association of a plant with nitrogen-fixing bacteria (e.g., nodule formation), mycorrhizal fungi, or other beneficial commensal microorganisms. Symbiotic responses may also include the activation of the symbiosis (Sym) signaling pathway, and/or the presence of nuclear-associated calcium oscillations (also known as symbiotic calcium oscillations, or calcium spiking). In further embodiments of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses include the activation of the expression of symbiosis-associated genes, such as HA1 or Vapyrin.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein the genetically altered plant of step a) further includes one or more genetic alterations that reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses, and wherein step b) further includes cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In a further embodiment of this aspect, the one or more genetic alterations result in increased activity of a C-TERMINALLY ENCODED PEPTIDE (CEP peptide). In still another embodiment of this aspect, the increased activity is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least 180% greater, at least 190% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is no greater than 500%, no greater than 475%, no greater than 450%, no greater than 425%, no greater than 400%, no greater than 375%, no greater than 350%, no greater than 325%, no greater than 300%, no greater than 275%, no greater than 250%, no greater than 225%, no greater than 200%, no greater than 175%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide is endogenous. Yet another embodiment of this aspect includes increased activity of the endogenous CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In a further embodiment of this aspect, which may be combined with any of the preceding aspects that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter; modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is due to heterologous expression of the CEP peptide. An additional embodiment of this aspect includes increased activity of the heterologous CEP peptide being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. A further embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein step a) further includes cultivating the plant under conditions including the nitrogen level around the plant roots, and wherein step b) further includes exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In still another embodiment of this aspect, the effective amount of the CEP peptide includes at least 0.1 μM CEP peptide, at least 0.2 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.3 μM CEP peptide, at least 0.4 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.6 μM CEP peptide, at least 0.7 μM CEP peptide, at least 0.75 μM CEP peptide, at least 0.8 μM CEP peptide, at least 0.9 μM CEP peptide, at least 1 μM CEP peptide, at least 1.1 μM CEP peptide, at least 1.2 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.3 μM CEP peptide, at least 1.4 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.6 μM CEP peptide, at least 1.7 μM CEP peptide, at least 1.75 μM CEP peptide, at least 1.8 μM CEP peptide, at least 1.9 μM CEP peptide, or at least 2 μM CEP peptide. Further embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, include the plant or the part thereof being exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In additional embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23 In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). Alignments of CEP proteins are shown in
An additional aspect of the disclosure includes methods of cultivating a plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a phosphate level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) cultivating the plant under conditions including the phosphate level around the plant roots; and (b) exposing the plant or a part thereof to an effective amount of a butenolide agent, wherein the effective amount of the butenolide agent increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the butenolide agent. In yet another embodiment of this aspect, the effective amount of the butenolide agent includes at least 0.1 μM butenolide agent, at least 0.2 μM butenolide agent, at least 0.25 μM butenolide agent, at least 0.3 μM butenolide agent, at least 0.4 μM butenolide agent, at least 0.5 μM butenolide agent, at least 0.6 μM butenolide agent, at least 0.7 μM butenolide agent, at least 0.75 μM butenolide agent, at least 0.8 μM butenolide agent, at least 0.9 μM butenolide agent, at least 1 μM butenolide agent, at least 1.1 μM butenolide agent, at least 1.2 μM butenolide agent, at least 1.25 μM butenolide agent, at least 1.3 μM butenolide agent, at least 1.4 μM butenolide agent, at least 1.5 μM butenolide agent, at least 1.6 μM butenolide agent, at least 1.7 μM butenolide agent, at least 1.75 μM butenolide agent, at least 1.8 μM butenolide agent, at least 1.9 μM butenolide agent, or at least 2 μM butenolide agent. A further embodiment of this aspect includes the plant or the part thereof being exposed to the butenolide agent by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the butenolide agent being a strigolactone. Still another embodiment of this aspect includes the strigolactone being selected from the group of 5-deoxystrigol, strigol, sorgomol, sorgolactone, other strigol-like compounds, 4-deoxyorobanchol, orobanchol, fabacyl acetate, solanocol, other orobanchol-like compounds, GR24, or any combination thereof. An additional embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the butenolide agent being a karrikin. Yet another embodiment of this aspect includes the karrikin being selected from the group of karrikin1 (KAR1), karrikin2 (KAR2), karrikin3 (KAR3), karrikin4 (KAR4), karrikin5 (KAR5), karrikin6 (KAR6), a mixture of karrikin1 and karrikin2 (KAR1+KAR2), GR24, karrikin contained in liquid smoke, or any combination thereof. A further embodiment of this aspect includes the karrikin being karrikin1 (KAR1), karrikin2 (KAR2), or a mixture of karrikin1 and karrikin2 (KAR1+KAR2). GR24 is a synthetic strigolactone analog that activates both strigolactone and karrikin signaling pathways. The effect of treatment with strigolactones or karrikins on LCO-induced (i.e., symbiotic) nuclear calcium oscillations in M. truncatula is shown in
Still another embodiment of this aspect, which may be combined with any of the preceding embodiments, includes the phosphate level around the plant roots completely suppressing mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the butenolide agent. In an additional embodiment of this aspect, includes the phosphate level around the plant roots inhibits mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the butenolide agent. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the butenolide agent. In yet another embodiment of this aspect, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is less than 2.5 mM, less than 2.4 mM, less than 2.3 mM, less than 2.2 mM, less than 2.1 mM, less than 2.0 mM, less than 1.9 mM, less than 1.8 mM, less than 1.7 mM, less than 1.6 mM, less than 1.5 mM, less than 1.4 mM, less than 1.3 mM, less than 1.2 mM, less than 1.1 mM, less than 1.0 mM, less than 0.95 mM, less than 0.9 mM, less than 0.85 mM, less than 0.8 mM, less than 0.75 mM, less than 0.7 mM, less than 0.65 mM, less than 0.6 mM, less than 0.55 mM, less than 0.5 mM, less than 0.45 mM, less than 0.4 mM, less than 0.35 mM, less than 0.3 mM, less than 0.25 mM, less than 0.2 mM, less than 0.15 mM, less than 0.1 mM, or less than 0.05 mM. In yet another embodiment of this aspect, the nitrate level around the plant roots is about 0 mM. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots includes at least 100 μM phosphate, at least 125 μM phosphate, at least 150 μM phosphate, at least 175 μM phosphate, at least 200 μM phosphate, at least 225 μM phosphate, at least 250 μM phosphate, at least 275 μM phosphate, at least 300 μM phosphate, at least 325 μM phosphate, at least 350 μM phosphate, at least 375 μM phosphate, at least 400 μM phosphate, at least 425 μM phosphate, at least 450 μM phosphate, at least 475 μM phosphate, at least 500 μM phosphate, at least 525 μM phosphate, at least 550 μM phosphate, at least 575 μM phosphate, at least 600 μM phosphate, at least 625 μM phosphate, at least 650 μM phosphate, at least 675 μM phosphate, at least 700 μM phosphate, at least 725 μM phosphate, at least 750 μM phosphate, at least 800 μM phosphate, at least 850 μM phosphate, at least 900 μM phosphate, at least 950 μM phosphate, at least 1000 μM phosphate, at least 1250 μM phosphate, at least 1500 μM phosphate, at least 1750 μM phosphate, at least 2000 μM phosphate, at least 2250 μM phosphate, at least 2500 μM phosphate, at least 2750 μM phosphate, at least 3000 μM phosphate, at least 3250 μM phosphate, at least 3500 μM phosphate, at least 3750 μM phosphate, at least 4000 μM phosphate, at least 4250 μM phosphate, at least 4500 μM phosphate, at least 4750 μM phosphate, or at least 5000 μM phosphate. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley (e.g., Hordeum vulgare), maize (e.g., corn, Zea mays), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), another cereal crop such as sorghum (e.g., Sorghum bicolor), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Setaria italica, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), teff (e.g., Eragrostis tef), oat (e.g., Avena sativa), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), rye (e.g., Secale cereale, Secale cereanum), or wild rice (e.g., Zizania spp., Porteresia spp.), cassava (e.g., manioc, yucca, Manihot esculenta), potato (e.g., russet potatoes, yellow potatoes, red potatoes, Solanum tuberosum), soy (e.g., soybean, soja, sojabean, Glycine max, Glycine soja), or a legume crop such as peanut (e.g., Arachis duranensis, Arachis hypogaea, Arachis ipaensis), pigeon pea (e.g., Cajanus cajan), chickpea (e.g., Cicer arietinum), cowpea (e.g., black-eyed pea, Vigna unguiculata), velvet bean (e.g., Mucuna pruriens), bean (e.g., Phaseolus vulgaris), pea (e.g., Pisum sativum), adzuki bean (e.g., Vigna angularis, Vigna angularis var. angularis), mung bean (e.g., Vigna radiata var. radiata), clover (e.g., Trifolium pratense, Trifolium subterraneum), or lupine (e.g., lupin, Lupinus angustifolius). Yet another embodiment of this aspect includes the plant being barley (e.g., Hordeum vulgare).
In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses are induced by a plant's perception of LCOs produced by bacteria or fungi. Symbiotic responses are associated with the interactions of plants with beneficial microorganisms, including nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (Oldroyd, G. E. D. Nature Reviews Microbiology 2013, 11), and may include symbiotic association of a plant with nitrogen-fixing bacteria (e.g., nodule formation), mycorrhizal fungi, or other beneficial commensal microorganisms. Symbiotic responses may also include the activation of the symbiosis (Sym) signaling pathway, and/or the presence of nuclear-associated calcium oscillations (also known as symbiotic calcium oscillations, or calcium spiking). In further embodiments of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses include the activation of the expression of symbiosis-associated genes, such as HA1 or Vapyrin.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein the genetically altered plant of step a) further includes one or more genetic alterations that reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses, and wherein step b) further includes cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In a further embodiment of this aspect, the one or more genetic alterations result in increased activity of a CEP peptide. In still another embodiment of this aspect, the increased activity is at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least 180% greater, at least 190% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is no greater than 500%, no greater than 475%, no greater than 450%, no greater than 425%, no greater than 400%, no greater than 375%, no greater than 350%, no greater than 325%, no greater than 300%, no greater than 275%, no greater than 250%, no greater than 225%, no greater than 200%, no greater than 175%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the CEP peptide is endogenous. Yet another embodiment of this aspect includes increased activity of the endogenous CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In a further embodiment of this aspect, which may be combined with any of the preceding aspects that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter; modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity of a CEP peptide, the increased activity is due to heterologous expression of the CEP peptide. An additional embodiment of this aspect includes increased activity of the heterologous CEP peptide being achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. A further embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
Yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, further includes cultivating the genetically altered plant under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, wherein step a) further includes cultivating the plant under conditions including the nitrogen level around the plant roots, and wherein step b) further includes exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In still another embodiment of this aspect, the effective amount of the CEP peptide includes at least 0.1 μM CEP peptide, at least 0.2 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.3 μM CEP peptide, at least 0.4 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.6 μM CEP peptide, at least 0.7 μM CEP peptide, at least 0.75 μM CEP peptide, at least 0.8 μM CEP peptide, at least 0.9 μM CEP peptide, at least 1 μM CEP peptide, at least 1.1 μM CEP peptide, at least 1.2 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.3 μM CEP peptide, at least 1.4 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.6 μM CEP peptide, at least 1.7 μM CEP peptide, at least 1.75 μM CEP peptide, at least 1.8 μM CEP peptide, at least 1.9 μM CEP peptide, or at least 2 μM CEP peptide. Further embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, include the plant or the part thereof being exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In additional embodiments of this aspect, which may be combined with any of the preceding embodiments that have the plant or a part thereof being exposed to a CEP peptide, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that does not have the nitrogen level around the plant roots being permissive of mycorrhization and/or symbiotic responses, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In a further embodiment of this aspect, the nitrogen level around the plant roots inhibits mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. An additional embodiment of this aspect includes the nitrogen around the plant roots being present in the form of nitrate, and the nitrate level around the plant roots being greater than 2.75 mM, greater than 2.8 mM, greater than 2.9 mM, greater than 3 mM, greater than 3.1 mM, greater than 3.2 mM, greater than 3.25 mM, greater than 3.3 mM, greater than 3.4 mM, greater than 3.5 mM, greater than 3.6 mM, greater than 3.7 mM, greater than 3.75 mM, greater than 3.8 mM, greater than 3.9 mM, greater than 4 mM, greater than 4.1 mM, greater than 4.2 mM, greater than 4.25 mM, greater than 4.3 mM, greater than 4.4 mM, greater than 4.5 mM, greater than 4.6 mM, greater than 4.7 mM, greater than 4.75 mM, greater than 4.8 mM, greater than 4.9 mM, greater than 5 mM, greater than 5.25 mM, or greater than 5.5 mM.
A further aspect of the disclosure includes methods of cultivating a genetically altered plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) providing the genetically altered plant, wherein the plant or a part thereof includes one or more genetic alterations, wherein the one or more genetic alterations reduce the nitrogen level suppression of mycorrhization and/or symbiotic responses; and (b) cultivating the genetically altered plant under the nitrogen level around the plant roots, wherein the genetically altered plant has increased mycorrhization and/or promoted symbiotic responses as compared to a WT plant grown under the same conditions. In an additional embodiment of this aspect, the one or more genetic alterations result in increased activity of a CEP peptide. Yet another embodiment of this aspect includes the increased activity being at least 10% greater, at least 15% greater, at least 20% greater, at least 25% greater, at least 30% greater, at least 35% greater, at least 40% greater, at least 45% greater, at least 50% greater, at least 55% greater, at least 60% greater, at least 65% greater, at least 70% greater, at least 75% greater, at least 80% greater, at least 85% greater, at least 90% greater, at least 95% greater, at least 100% greater, at least 110% greater, at least 120% greater, at least 130% greater, at least 140% greater, at least 150% greater, at least 160% greater, at least 170% greater, at least 180% greater, at least 190% greater, or at least 200% greater than the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. Still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the increased activity is no greater than 500%, no greater than 475%, no greater than 450%, no greater than 425%, no greater than 400%, no greater than 375%, no greater than 350%, no greater than 325%, no greater than 300%, no greater than 275%, no greater than 250%, no greater than 225%, no greater than 200%, no greater than 175%, no greater than 150%, or no greater than 125% of the activity of the corresponding one or more proteins in the WT plant grown under the same conditions. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In yet another embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23.
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the CEP peptide is endogenous. A further embodiment of this aspect includes increased activity of the CEP peptide being achieved using a gene editing technique to introduce the one or more genetic alterations. An additional embodiment of this aspect includes the gene editing technique being selected from the group of transcription activator-like effector nuclease (TALEN) gene editing techniques, clustered Regularly Interspaced Short Palindromic Repeat (CRISPR/Cas) gene editing techniques, or zinc-finger nuclease (ZFN) gene editing techniques. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments that has a gene editing technique to introduce the one or more genetic alterations, the one or more genetic alterations that increase the activity of the endogenous protein are selected from the group of inactivating a repressor element that represses expression of the endogenous protein, removing a repressor element that represses expression of the endogenous protein, modulating the methylation state of a repressor element that represses expression of the endogenous protein, activating an enhancer element that increases expression of the endogenous protein, adding an enhancer element that increases expression of the endogenous protein, modulating the methylation state of an enhancer element that increases expression of the endogenous protein, adding a transcriptional activator recruiting or binding element that activates expression of the endogenous protein, replacing the endogenous promoter with an overexpression promoter that directs expression of the endogenous protein, modulating the methylation state of the endogenous promoter, modulating the methylation state of the endogenous coding sequence, adding elements that stabilize an endogenous mRNA encoding the endogenous protein, removing elements that destabilize the endogenous mRNA encoding the endogenous protein, modifying a coding sequence to increase stability of the endogenous protein, or modifying a coding sequence for the endogenous protein to increase activity of the endogenous protein.
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments that has the one or more genetic alterations resulting in increased activity, the increased activity is due to heterologous expression of the CEP peptide. In a further embodiment of this aspect, increased activity of the heterologous CEP peptide is achieved using a vector including a first nucleic acid encoding the heterologous protein operably linked to a second nucleic acid encoding a promoter. An additional embodiment of this aspect includes the promoter being selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In a further embodiment of this aspect, the nitrogen level around the plant roots inhibits mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the WT plant grown under the same conditions. In yet another embodiment of this aspect, the phosphate level around the plant roots includes less than 1000 μM phosphate, less than 950 μM phosphate, less than 900 μM phosphate, less than 850 μM phosphate, less than 800 μM phosphate, less than 750 μM phosphate, less than 725 μM phosphate, less than 700 μM phosphate, less than 675 μM phosphate, less than 650 μM phosphate, less than 625 μM phosphate, less than 600 μM phosphate, less than 575 μM phosphate, less than 550 μM phosphate, less than 525 μM phosphate, less than 500 μM phosphate, less than 475 μM phosphate, less than 450 μM phosphate, less than 425 μM phosphate, less than 400 μM phosphate, less than 375 μM phosphate, less than 350 μM phosphate, less than 325 μM phosphate, less than 300 μM phosphate, less than 275 μM phosphate, less than 250 μM phosphate, less than 225 μM phosphate, less than 200 μM phosphate, less than 175 μM phosphate, less than 150 μM phosphate, less than 125 μM phosphate, less than 100 μM phosphate, or less than 50 μM phosphate. In yet another embodiment of this aspect, the phosphate level around the plant roots is about 0 μM. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is greater than 2.75 mM, greater than 2.8 mM, greater than 2.9 mM, greater than 3 mM, greater than 3.1 mM, greater than 3.2 mM, greater than 3.25 mM, greater than 3.3 mM, greater than 3.4 mM, greater than 3.5 mM, greater than 3.6 mM, greater than 3.7 mM, greater than 3.75 mM, greater than 3.8 mM, greater than 3.9 mM, greater than 4 mM, greater than 4.1 mM, greater than 4.2 mM, greater than 4.25 mM, greater than 4.3 mM, greater than 4.4 mM, greater than 4.5 mM, greater than 4.6 mM, greater than 4.7 mM, greater than 4.75 mM, greater than 4.8 mM, greater than 4.9 mM, greater than 5 mM, greater than 5.25 mM, or greater than 5.5 mM. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley (e.g., Hordeum vulgare), maize (e.g., corn, Zea mays), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), another cereal crop such as sorghum (e.g., Sorghum bicolor), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Setaria italica, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), teff (e.g., Eragrostis tef), oat (e.g., Avena sativa), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), rye (e.g., Secale cereale, Secale cereanum), or wild rice (e.g., Zizania spp., Porteresia spp.), cassava (e.g., manioc, yucca, Manihot esculenta), potato (e.g., russet potatoes, yellow potatoes, red potatoes, Solanum tuberosum), soy (e.g., soybean, soja, sojabean, Glycine max, Glycine soja), or a legume crop such as peanut (e.g., Arachis duranensis, Arachis hypogaea, Arachis ipaensis), pigeon pea (e.g., Cajanus cajan), chickpea (e.g., Cicer arietinum), cowpea (e.g., black-eyed pea, Vigna unguiculata), velvet bean (e.g., Mucuna pruriens), bean (e.g., Phaseolus vulgaris), pea (e.g., Pisum sativum), adzuki bean (e.g., Vigna angularis, Vigna angularis var. angularis), mung bean (e.g., Vigna radiata var. radiata), clover (e.g., Trifolium pratense, Trifolium subterraneum), or lupine (e.g., lupin, Lupinus angustifolius). Yet another embodiment of this aspect includes the plant being barley (e.g., Hordeum vulgare).
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses are induced by a plant's perception of LCOs produced by bacteria or fungi. Symbiotic responses are associated with the interactions of plants with beneficial microorganisms, including nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (Oldroyd, G. E. D. Nature Reviews Microbiology 2013, 11), and may include symbiotic association of a plant with nitrogen-fixing bacteria (e.g., nodule formation), mycorrhizal fungi, or other beneficial commensal microorganisms. Symbiotic responses may also include the activation of the symbiosis (Sym) signaling pathway, and/or the presence of nuclear-associated calcium oscillations (also known as symbiotic calcium oscillations, or calcium spiking). In further embodiments of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses include the activation of the expression of symbiosis-associated genes, such as HA1 or Vapyrin.
A further aspect of this disclosure includes methods of cultivating a plant with increased mycorrhization and/or promoted symbiotic responses under conditions including a nitrogen level around the plant roots that suppresses mycorrhization and/or symbiotic responses, including: (a) cultivating the plant under conditions including the nitrogen level around the plant roots; and (b) exposing the plant or a part thereof to an effective amount of a CEP peptide, wherein the effective amount of the CEP peptide increases mycorrhization and/or promotes symbiotic responses in the plant or plant part as compared to the plant grown under the same conditions without the CEP peptide. In an additional embodiment of this aspect, the effective amount of the CEP peptide at least 0.1 μM CEP peptide, at least 0.2 μM CEP peptide, at least 0.25 μM CEP peptide, at least 0.3 μM CEP peptide, at least 0.4 μM CEP peptide, at least 0.5 μM CEP peptide, at least 0.6 μM CEP peptide, at least 0.7 μM CEP peptide, at least 0.75 μM CEP peptide, at least 0.8 μM CEP peptide, at least 0.9 μM CEP peptide, at least 1 μM CEP peptide, at least 1.1 μM CEP peptide, at least 1.2 μM CEP peptide, at least 1.25 μM CEP peptide, at least 1.3 μM CEP peptide, at least 1.4 μM CEP peptide, at least 1.5 μM CEP peptide, at least 1.6 μM CEP peptide, at least 1.7 μM CEP peptide, at least 1.75 μM CEP peptide, at least 1.8 μM CEP peptide, at least 1.9 μM CEP peptide, or at least 2 μM CEP peptide. In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant or the part thereof is exposed to the CEP peptide by direct application, application through irrigation or spraying, application in a seed coating, application in a seed coating with a mycorrhizal inoculum, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19).
In yet another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen level around the plant roots completely suppresses mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the CEP peptide. In a further embodiment of this aspect, the nitrogen level around the plant roots inhibits mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the CEP peptide. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the phosphate level around the plant roots is permissive of mycorrhization and/or symbiotic responses in the plant grown under the same conditions without the CEP peptide. In a further embodiment of this aspect, the phosphate level around the plant roots includes less than 1000 μM phosphate, less than 950 μM phosphate, less than 900 μM phosphate, less than 850 μM phosphate, less than 800 μM phosphate, less than 750 μM phosphate, less than 725 μM phosphate, less than 700 μM phosphate, less than 675 μM phosphate, less than 650 μM phosphate, less than 625 μM phosphate, less than 600 μM phosphate, less than 575 μM phosphate, less than 550 μM phosphate, less than 525 μM phosphate, less than 500 μM phosphate, less than 475 μM phosphate, less than 450 μM phosphate, less than 425 μM phosphate, less than 400 μM phosphate, less than 375 μM phosphate, less than 350 μM phosphate, less than 325 μM phosphate, less than 300 μM phosphate, less than 275 μM phosphate, less than 250 μM phosphate, less than 225 μM phosphate, less than 200 μM phosphate, less than 175 μM phosphate, less than 150 μM phosphate, less than 125 μM phosphate, less than 100 μM phosphate, or less than 50 μM phosphate. In yet another embodiment of this aspect, the phosphate level around the plant roots is about 0 μM. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the nitrogen around the plant roots is present in the form of nitrate, and the nitrate level around the plant roots is greater than 2.75 mM, greater than 2.8 mM, greater than 2.9 mM, greater than 3 mM, greater than 3.1 mM, greater than 3.2 mM, greater than 3.25 mM, greater than 3.3 mM, greater than 3.4 mM, greater than 3.5 mM, greater than 3.6 mM, greater than 3.7 mM, greater than 3.75 mM, greater than 3.8 mM, greater than 3.9 mM, greater than 4 mM, greater than 4.1 mM, greater than 4.2 mM, greater than 4.25 mM, greater than 4.3 mM, greater than 4.4 mM, greater than 4.5 mM, greater than 4.6 mM, greater than 4.7 mM, greater than 4.75 mM, greater than 4.8 mM, greater than 4.9 mM, greater than 5 mM, greater than 5.25 mM, or greater than 5.5 mM. In a further embodiment of this aspect, which may be combined with any of the preceding embodiments, the plant is barley (e.g., Hordeum vulgare), maize (e.g., corn, Zea mays), rice (e.g., indica rice, japonica rice, aromatic rice, glutinous rice, Oryza sativa, Oryza glaberrima), wheat (e.g., common wheat, spelt, durum, einkorn, emmer, kamut, Triticum aestivum, Triticum spelta, Triticum durum, Triticum urartu, Triticum monococcum, Triticum turanicum, Triticum spp.), another cereal crop such as sorghum (e.g., Sorghum bicolor), millet (e.g., finger millet, fonio millet, foxtail millet, pearl millet, barnyard millets, Eleusine coracana, Panicum sumatrense, Panicum milaceum, Setaria italica, Pennisetum glaucum, Digitaria spp., Echinocloa spp.), teff (e.g., Eragrostis tef), oat (e.g., Avena sativa), triticale (e.g., X Triticosecale Wittmack, Triticosecale schlanstedtense Wittm., Triticosecale neoblaringhemii A. Camus, Triticosecale neoblaringhemii A. Camus), rye (e.g., Secale cereale, Secale cereanum), or wild rice (e.g., Zizania spp., Porteresia spp.), cassava (e.g., manioc, yucca, Manihot esculenta), potato (e.g., russet potatoes, yellow potatoes, red potatoes, Solanum tuberosum), soy (e.g., soybean, soja, sojabean, Glycine max, Glycine soja), or a legume crop such as peanut (e.g., Arachis duranensis, Arachis hypogaea, Arachis ipaensis), pigeon pea (e.g., Cajanus cajan), chickpea (e.g., Cicer arietinum), cowpea (e.g., black-eyed pea, Vigna unguiculata), velvet bean (e.g., Mucuna pruriens), bean (e.g., Phaseolus vulgaris), pea (e.g., Pisum sativum), adzuki bean (e.g., Vigna angularis, Vigna angularis var. angularis), mung bean (e.g., Vigna radiata var. radiata), clover (e.g., Trifolium pratense, Trifolium subterraneum), or lupine (e.g., lupin, Lupinus angustifolius). An additional embodiment of this aspect includes the plant being (e.g., Hordeum vulgare).
In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, the mycorrhization includes a symbiotic association of one or more plant parts selected from the group of a root system, a root, a root primordia, a root tip, a lateral root, a root meristem, or a root cell, with mycorrhizal fungi. An additional embodiment of this aspect includes the mycorrhizal fungi being selected from the group of Acaulosporaceae spp., Diversisporaceae spp., Gigasporaceae spp., Pacisporaceae spp., Funneliformis spp., Glomus spp., Rhizophagus spp., Sclerocystis spp., Septoglomus spp., Claroideoglomus spp., Ambispora spp., Archaeospora spp., Geosiphon pyriformis, Paraglomus spp., other species in the division Glomeromycota, or any combination thereof. In still another embodiment of this aspect, which may be combined with any of the preceding embodiments, increased mycorrhization enhances plant uptake of nutrients surround the plant roots selected from the group of phosphate, nitrate, or potassium, and increased mycorrhization optionally enhances plant uptake of water.
In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses are induced by a plant's perception of LCOs produced by bacteria or fungi. Symbiotic responses are associated with the interactions of plants with beneficial microorganisms, including nitrogen-fixing bacteria and arbuscular mycorrhizal fungi (Oldroyd, G. E. D. Nature Reviews Microbiology 2013, 11), and may include symbiotic association of a plant with nitrogen-fixing bacteria (e.g., nodule formation), mycorrhizal fungi, or other beneficial commensal microorganisms. Symbiotic responses may also include the activation of the symbiosis (Sym) signaling pathway, and/or the presence of nuclear-associated calcium oscillations (also known as symbiotic calcium oscillations, or calcium spiking). In further embodiments of this aspect, which may be combined with any of the preceding embodiments, symbiotic responses include the activation of the expression of symbiosis-associated genes, such as HA1 or Vapyrin.
An additional aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of NSP1 or NSP2, including: (a) transforming a plant cell, tissue, or other explant with a vector including a first nucleic acid sequence encoding a NSP1 protein or a NSP2 protein operably linked to a second nucleic acid sequence encoding a promoter; (b) selecting successful transformation events by means of a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with increased activity of the NSP1 protein or the NSP2 protein as compared to an untransformed WT plant. Yet another embodiment of this aspect further includes identifying successful introduction of the one or more genetic alterations by screening or selecting the plant cell, tissue, or other explant prior to step (b); screening or selecting plantlets between step (b) and (c); or screening or selecting plants after step (c). Still another embodiment of this aspect, which may be combined with any preceding embodiments, includes transformation being done using a transformation method selected from the group of particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the NSP1 protein includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207; or the NSP2 protein includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208. In a further embodiment of this aspect, the NSP1 protein includes SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, or SEQ ID NO: 207; or the NSP2 protein includes SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, or SEQ ID NO: 208. In yet another embodiment of this aspect, which may be combined with any preceding embodiments, the promoter is selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof. In still another embodiment of this aspect, which may be combined with any preceding embodiments, the first nucleic acid sequence and the second nucleic acid sequence are stably integrated into a nuclear genome of the plant.
A further aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of NSP1 or NSP2, including (a) transforming a plant cell, tissue, or other explant with one or more gene editing components that target a nuclear genome sequence operably linked to an endogenous NSP1 protein or an endogenous NSP2 protein; (b) selecting successful transformation events by means of a screening technology, an enriching technology, a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with overexpression of the NSP1 protein or the NSP2 protein as compared to an untransformed WT plant. In an additional embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex that targets the nuclear genome sequence; a vector including a TALEN protein encoding sequence, wherein the TALEN protein targets the nuclear genome sequence; a vector including a ZFN protein encoding sequence, wherein the ZFN protein targets the nuclear genome sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genome sequence; or a vector CRISPR/Cas enzyme encoding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genome sequence.
An additional aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of CEP peptide, including: (a) transforming a plant cell, tissue, or other explant with a vector including a first nucleic acid sequence encoding a CEP peptide operably linked to a second nucleic acid sequence encoding a promoter; (b) selecting successful transformation events by means of a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with increased activity of the CEP peptide as compared to an untransformed WT plant. Yet another embodiment of this aspect further includes identifying successful introduction of the one or more genetic alterations by screening or selecting the plant cell, tissue, or other explant prior to step (b); screening or selecting plantlets between step (b) and (c); or screening or selecting plants after step (c). Still another embodiment of this aspect, which may be combined with any preceding embodiments, includes transformation being done using a transformation method selected from the group of particle bombardment (i.e., biolistics, gene gun), Agrobacterium-mediated transformation, Rhizobium-mediated transformation, or protoplast transfection or transformation. In an additional embodiment of this aspect, which may be combined with any of the preceding embodiments, the CEP peptide includes an amino acid sequence with at least 70% sequence identity to, at least 71% sequence identity to, at least 72% sequence identity to, at least 73% sequence identity to, at least 74% sequence identity to, at least 75% sequence identity to, at least 76% sequence identity to, at least 77% sequence identity to, at least 78% sequence identity to, at least 79% sequence identity to, at least 80% sequence identity to, at least 81% sequence identity to, at least 82% sequence identity to, at least 83% sequence identity to, at least 84% sequence identity to, at least 85% sequence identity to, at least 86% sequence identity to, at least 87% sequence identity to, at least 88% sequence identity to, at least 89% sequence identity to, at least 90% sequence identity to, at least 91% sequence identity to, at least 92% sequence identity to, at least 93% sequence identity to, at least 94% sequence identity to, at least 95% sequence identity to, at least 96% sequence identity to, at least 97% sequence identity to, at least 98% sequence identity to, or at least 99% sequence identity to SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide includes SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In a further embodiment of this aspect, the CEP peptide is selected from the group of CEP1 (e.g., SEQ ID NO: 17), CEP2 (e.g., SEQ ID NO: 18), CEP3 (e.g., SEQ ID NO: 19), CEP4 (e.g., SEQ ID NO 20), CEP5 (e.g., SEQ ID NO: 21), CEP6 (e.g., SEQ ID NO: 22), or CEP7 (e.g., SEQ ID NO: 23). In an additional embodiment of this aspect, the CEP peptide is CEP3 (e.g., SEQ ID NO: 19). In yet another embodiment of this aspect, which may be combined with any preceding embodiments, the promoter is selected from the group of a CaMV35S promoter, a ubiquitin promoter, a pBdUBI10 promoter, a pPvUBI2 promoter, a pPvUBI1 promoter, a pZmUBI promoter, a pOsPGD1 promoter, a p35s promoter, a pOsUBI3 promoter, a pBdEF1α promoter, a pAtUBI10 promoter, a pOsAct1 promoter, a pOsRS2 promoter, a pZmEF1α promoter, a pZmTUB1α promoter, a pHvIDS2 promoter, a ZmRsyn7 promoter, a pSiCCaMK promoter, or any combination thereof. In still another embodiment of this aspect, which may be combined with any preceding embodiments, the first nucleic acid sequence and the second nucleic acid sequence are stably integrated into a nuclear genome of the plant.
A further aspect of this disclosure includes methods of producing the genetically altered plant of any of the preceding embodiments that has increased activity of CEP peptide, including (a) transforming a plant cell, tissue, or other explant with one or more gene editing components that target a nuclear genome sequence operably linked to an endogenous CEP peptide; (b) selecting successful transformation events by means of a screening technology, an enriching technology, a selection agent, marker-assisted selection, or selective media; (c) regenerating the transformed cell, tissue, or other explant into a genetically altered plantlet; and (d) growing the genetically altered plantlet into a genetically altered plant with overexpression of the CEP peptide as compared to an untransformed WT plant. In an additional embodiment of this aspect, the one or more gene editing components include a ribonucleoprotein complex that targets the nuclear genome sequence; a vector including a TALEN protein encoding sequence, wherein the TALEN protein targets the nuclear genome sequence; a vector including a ZFN protein encoding sequence, wherein the ZFN protein targets the nuclear genome sequence; an oligonucleotide donor (OND), wherein the OND targets the nuclear genome sequence; or a vector CRISPR/Cas enzyme encoding sequence and a targeting sequence, wherein the targeting sequence targets the nuclear genome sequence.
One embodiment of the present invention provides genetically altered plants or plant cells containing one or more genetic alterations, which increase activity of one or more of a NODULATION SIGNALING PATHWAY 1 (NSP1) protein or a NODULATION SIGNALING PATHWAY 2 (NSP2) protein. In addition, the present disclosure provides genetically altered plants or plant cells containing one or more genetic alterations that increase activity of CEP peptides.
Transformation and generation of genetically altered monocotyledonous and dicotyledonous plant cells is well known in the art. See, e.g., Weising, et al., Ann. Rev. Genet. 22:421-477 (1988); U.S. Pat. No. 5,679,558; Agrobacterium Protocols, ed: Gartland, Humana Press Inc. (1995); and Wang, et al. Acta Hort. 461:401-408 (1998). The choice of method varies with the type of plant to be transformed, the particular application and/or the desired result. The appropriate transformation technique is readily chosen by the skilled practitioner.
Any methodology known in the art to delete, insert or otherwise modify the cellular DNA (e.g., genomic DNA and organelle DNA) can be used in practicing the inventions disclosed herein. For example, a disarmed Ti plasmid, containing a genetic construct for deletion or insertion of a target gene, in Agrobacterium tumefaciens can be used to transform a plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using procedures described in the art, for example, in EP 0116718, EP 0270822, PCT publication WO 84/02913 and published European Patent application (“EP”) 0242246. Ti-plasmid vectors each contain the gene between the border sequences, or at least located to the left of the right border sequence, of the T-DNA of the Ti-plasmid. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0233247), pollen mediated transformation (as described, for example in EP 0270356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611), plant RNA virus-mediated transformation (as described, for example in EP 0 067 553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation (as described, for example in U.S. Pat. No. 4,536,475), and other methods such as the methods for transforming certain lines of corn (e.g., U.S. Pat. No. 6,140,553; Fromm et al., Bio/Technology (1990) 8, 833-839); Gordon-Kamm et al., The Plant Cell, (1990) 2, 603-618) and rice (Shimamoto et al., Nature, (1989) 338, 274-276; Datta et al., Bio/Technology, (1990) 8, 736-740) and the method for transforming monocots generally (PCT publication WO 92/09696). For cotton transformation, the method described in PCT patent publication WO 00/71733 can be used. For soybean transformation, reference is made to methods known in the art, e.g., Hinchee et al. (Bio/Technology, (1988) 6, 915) and Christou et al. (Trends Biotech, (1990) 8, 145) or the method of WO 00/42207.
Genetically altered plants of the present invention can be used in a conventional plant breeding scheme to produce more genetically altered plants with the same characteristics, or to introduce the genetic alteration(s) in other varieties of the same or related plant species. Seeds, which are obtained from the altered plants, preferably contain the genetic alteration(s) as a stable insert in nuclear DNA or as modifications to an endogenous gene or promoter. Plants comprising the genetic alteration(s) in accordance with the invention include plants comprising, or derived from, root stocks of plants comprising the genetic alteration(s) of the invention, e.g., fruit trees or ornamental plants. Hence, any non-transgenic grafted plant parts inserted on a transformed plant or plant part are included in the invention.
Introduced genetic elements, whether in an expression vector or expression cassette, which result in the expression of an introduced gene, will typically utilize a plant-expressible promoter. A ‘plant-expressible promoter’ as used herein refers to a promoter that ensures expression of the genetic alteration(s) of the invention in a plant cell. Examples of promoters directing constitutive expression in plants are known in the art and include: the strong constitutive 35S promoters (the “35S promoters”) of the cauliflower mosaic virus (CaMV), e.g., of isolates CM 1841 (Gardner et al., Nucleic Acids Res, (1981) 9, 2871-2887), CabbB S (Franck et al., Cell (1980) 21, 285-294; Kay et al., Science, (1987) 236, 4805) and CabbB JI (Hull and Howell, Virology, (1987) 86, 482-493); promoters from the ubiquitin family (e.g., the maize ubiquitin promoter of Christensen et al., Plant Mol Biol, (1992) 18, 675-689, or the Arabidopsis UBQ10 promoter of Norris et al. Plant Mol. Biol. (1993) 21, 895-906), the gos2 promoter (de Pater et al., The Plant J (1992) 2, 834-844), the emu promoter (Last et al., Theor Appl Genet, (1990) 81, 581-588), actin promoters such as the promoter described by An et al. (The Plant J, (1996) 10, 107), the rice actin promoter described by Zhang et al. (The Plant Cell, (1991) 3, 1155-1165); promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. (Plant Mol Biol, (1998) 37, 1055-1067), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S4 or S7 promoter), an alcohol dehydrogenase promoter, e.g., pAdh1S (GenBank accession numbers X04049, X00581), and the TR1′ promoter and the TR2′ promoter (the “TR1′ promoter” and “TR2′ promoter”, respectively) which drive the expression of the 1′ and 2′ genes, respectively, of the T DNA (Velten et al., EMBO J, (1984) 3, 2723 2730).
In preferred embodiments, plant-expressible promoters for achieving high levels of expression in cereal roots are used (described in Feike et al., Plant Biotechnology Journal (2019), 1-12, doi: 10.1111/pbi.13135). Non-limiting examples include pBdUBI10, pPvUBI2, pPvUBI1, pZmUBI, pOsPGD1, p35s, pOsUBI3, pBdEF1α, pAtUBI10, pOsAct1, pOsRS2, pZmEF1a, pZmTUB1a, pHvIDS2, ZmRsyn7, or pSiCCaMK (Feike et al., Plant Biotechnology Journal (2019), 1-12, doi: 10.1111/pbi.13135).
Alternatively, a plant-expressible promoter can be a tissue-specific promoter, i.e., a promoter directing a higher level of expression in some cells or tissues of the plant, e.g., in root cells. These plant promoters can be combined with enhancer elements, they can be combined with minimal promoter elements, or they can comprise repeated elements to ensure the expression profile desired.
In some embodiments, genetic elements to increase expression in plant cells can be utilized. For example, an intron at the 5′ end or 3′ end of an introduced gene, or in the coding sequence of the introduced gene, e.g., the hsp70 intron. Other such genetic elements can include, but are not limited to, promoter enhancer elements, duplicated or triplicated promoter regions, 5′ leader sequences different from another transgene or different from an endogenous (plant host) gene leader sequence, 3′ trailer sequences different from another transgene used in the same plant or different from an endogenous (plant host) trailer sequence.
An introduced gene of the present invention can be inserted in host cell DNA so that the inserted gene part is upstream (i.e., 5′) of suitable 3′ end transcription regulation signals (e.g., transcript formation and polyadenylation signals). This is preferably accomplished by inserting the gene in the plant cell genome (nuclear or chloroplast). Preferred polyadenylation and transcript formation signals include those of the nopaline synthase gene (Depicker et al., J. Molec Appl Gen, (1982) 1, 561-573), the octopine synthase gene (Gielen et al., EMBO J, (1984) 3:835 845), the SCSV or the Malic enzyme terminators (Schunmann et al., Plant Funct Biol, (2003) 30:453-460), and the T DNA gene 7 (Velten and Schell, Nucleic Acids Res, (1985) 13, 6981 6998), which act as 3′ untranslated DNA sequences in transformed plant cells. In some embodiments, one or more of the introduced genes are stably integrated into the nuclear genome. Stable integration is present when the nucleic acid sequence remains integrated into the nuclear genome and continues to be expressed (e.g., detectable mRNA transcript or protein is produced) throughout subsequent plant generations. Stable integration into and/or editing of the nuclear genome can be accomplished by any known method in the art (e.g., microparticle bombardment, Agrobacterium-mediated transformation, CRISPR/Cas9, electroporation of protoplasts, microinjection, etc.).
The term recombinant or modified nucleic acids refers to polynucleotides which are made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of polynucleotides by genetic engineering techniques or by chemical synthesis. In so doing one may join together polynucleotide segments of desired functions to generate a desired combination of functions.
As used herein, the terms “overexpression” and “upregulation” refer to increased expression (e.g., of mRNA, polypeptides, etc.) relative to expression in a wild type organism (e.g., plant) as a result of genetic modification. In some embodiments, the increase in expression is a slight increase of about 10% more than expression in wild type. In some embodiments, the increase in expression is an increase of 50% or more (e.g., 60%, 70%, 80%, 100%, etc.) relative to expression in wild type. In some embodiments, an endogenous gene is overexpressed. In some embodiments, an exogenous gene is overexpressed by virtue of being expressed. Overexpression of a gene in plants can be achieved through any known method in the art, including but not limited to, the use of constitutive promoters, inducible promoters, high expression promoters, enhancers, transcriptional and/or translational regulatory sequences, codon optimization, modified transcription factors, and/or mutant or modified genes that control expression of the gene to be overexpressed.
Where a recombinant nucleic acid is intended for expression, cloning, or replication of a particular sequence, DNA constructs prepared for introduction into a host cell will typically comprise a replication system (e.g. vector) recognized by the host, including the intended DNA fragment encoding a desired polypeptide, and can also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Additionally, such constructs can include cellular localization signals (e.g., plasma membrane localization signals). In preferred embodiments, such DNA constructs are introduced into a host cell's genomic DNA, chloroplast DNA or mitochondrial DNA.
In some embodiments, a non-integrated expression system can be used to induce expression of one or more introduced genes. Expression systems (expression vectors) can include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides can also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, cell wall, or be secreted from the cell.
Selectable markers useful in practicing the methodologies of the invention disclosed herein can be positive selectable markers. Typically, positive selection refers to the case in which a genetically altered cell can survive in the presence of a toxic substance only if the recombinant polynucleotide of interest is present within the cell. Negative selectable markers and screenable markers are also well known in the art and are contemplated by the present invention. One of skill in the art will recognize that any relevant markers available can be utilized in practicing the inventions disclosed herein.
Screening and molecular analysis of recombinant strains of the present invention can be performed utilizing nucleic acid hybridization techniques. Hybridization procedures are useful for identifying polynucleotides, such as those modified using the techniques described herein, with sufficient homology to the subject regulatory sequences to be useful as taught herein. The particular hybridization techniques are not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied by one of skill in the art. Hybridization probes can be labeled with any appropriate label known to those of skill in the art. Hybridization conditions and washing conditions, for example temperature and salt concentration, can be altered to change the stringency of the detection threshold. See, e.g., Sambrook et al. (1989) vide infra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y., for further guidance on hybridization conditions.
Additionally, screening and molecular analysis of genetically altered strains, as well as creation of desired isolated nucleic acids can be performed using Polymerase Chain Reaction (PCR). PCR is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al. (1985) Science 230:1350-1354). PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands of the target sequence. The primers are oriented with the 3′ ends pointing towards each other. Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary sequences, and extension of the annealed primers with a DNA polymerase result in the amplification of the segment defined by the 5′ ends of the PCR primers. Because the extension product of each primer can serve as a template for the other primer, each cycle essentially doubles the amount of DNA template produced in the previous cycle. This results in the exponential accumulation of the specific target fragment, up to several million-fold in a few hours. By using a thermostable DNA polymerase such as the Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated. Other enzymes which can be used are known to those skilled in the art.
Nucleic acids and proteins of the present invention can also encompass homologues of the specifically disclosed sequences. Homology (e.g., sequence identity) can be 50%-100%. In some instances, such homology is greater than 80%, greater than 85%, greater than 90%, or greater than 95%. The degree of homology or identity needed for any intended use of the sequence(s) is readily identified by one of skill in the art. As used herein percent sequence identity of two nucleic acids is determined using an algorithm known in the art, such as that disclosed by Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN, BLASTP, and BLASTX, programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (BLASTN and BLASTX) are used. See www.ncbi.nih.gov. One of skill in the art can readily determine in a sequence of interest where a position corresponding to amino acid or nucleic acid in a reference sequence occurs by aligning the sequence of interest with the reference sequence using the suitable BLAST program with the default settings (e.g., for BLASTP: Gap opening penalty: 11, Gap extension penalty: 1, Expectation value: 10, Word size: 3, Max scores: 25, Max alignments: 15, and Matrix: blosum62; and for BLASTN: Gap opening penalty: 5, Gap extension penalty:2, Nucleic match: 1, Nucleic mismatch −3, Expectation value: 10, Word size: 11, Max scores: 25, and Max alignments: 15).
Preferred host cells are plant cells. Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated nucleic molecule, contain one or more deleted or otherwise non-functional genes normally present and functional in the host cell, or contain one or more genes to produce at least one recombinant protein. The nucleic acid(s) encoding the protein(s) of the present invention can be introduced by any means known to the art which is appropriate for the particular type of cell, including without limitation, transformation, lipofection, electroporation or any other methodology known by those skilled in the art.
“Isolated”, “isolated DNA molecule” or an equivalent term or phrase is intended to mean that the DNA molecule or other moiety is one that is present alone or in combination with other compositions, but altered from or not within its natural environment. For example, nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” from its natural setting within the scope of this disclosure so long as the element is not within the genome of the organism in which it is naturally found, the element is altered from its natural form, or the element is not at the location within the genome in which it is naturally found. Similarly, a nucleotide sequence encoding a protein or any naturally occurring variant of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence was not within the DNA of the organism from which the sequence encoding the protein is naturally found in its natural location or if that nucleotide sequence was altered from its natural form. A synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring protein would be considered to be isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., the nucleotide sequence of the DNA inserted into the genome of the cells of a plant, alga, fungus, or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium.
Having generally described this invention, the same will be better understood by reference to certain specific examples, which are included herein to further illustrate the invention and are not intended to limit the scope of the invention as defined by the claims.
The present disclosure is described in further detail in the following examples which are not in any way intended to limit the scope of the disclosure as claimed. The attached figures are meant to be considered as integral parts of the specification and description of the disclosure. The following examples are offered to illustrate, but not to limit the claimed disclosure.
The following example describes experiments measuring activation of symbiosis signaling and immunity signaling in Medicago truncatula in response to oligosaccharide perception and nutrient levels.
The wild type M. truncatula cv. Jemalong A17 background was used. Seedlings were grown on modified Buffered Nodulation Media (BNM) with 100 nM Aminoethoxyvinylglycine (AVG; Sigma-Aldrich) for five days under different nutrient conditions. Nitrogen and phosphorus levels were manipulated by altering the concentrations of potassium nitrate (KNO3) and potassium dihydrogen phosphate (KH2PO4) in the media. For the replete nitrate and replete phosphate (+N+P) condition, BNM was modified with 5 mM KNO3 and 3.75 mM KH2PO4. For the limiting nitrate and replete phosphate (i.e., no nitrate and replete phosphate, −N+P) condition, BNM was modified with 3.75 mM KH2PO4. For the replete nitrate and limiting phosphate (+N−P) condition, BNM was modified with 0.0075 mM KH2PO4 and 5 mM KNO3. For the limiting nitrate and limiting phosphate (i.e., no nitrate and low phosphorus, −N−P) condition, BNM was modified with 0.0075 mM KH2PO4. The high phosphate concentration used for replete phosphate conditions was based on concentrations previously shown to suppress mycorrhization in M. truncatula (Balzergue, C. et al. Frontiers in plant science 4: article 426).
For calcium analyses M. truncatula seedlings were grown on Buffered Nodulation Media (BNM) agar with 100 nM Aminoethoxyvinylglycine (AVG; Sigma-Aldrich) until lateral roots emerged (
Where Sf is the flattened signal, So is the original signal, and MA is the moving average of the value. For each element i, MA is calculated as:
where m is the arbitrary number of points to calculate the average, w=[m/2] and xj is the signal at point j.
M. truncatula Gene Expression
Gene expression was measured by qRT-PCR (
Phytophthora
palmivora EF1a
M. truncatula primary roots growing on different nutrient conditions for 5 days were cut into 0.5 cm strips and incubated in 200 μL liquid medium containing different nutrient conditions in a 96-well plate (Greiner Bio-one) overnight. After incubation, the water was removed from each well and exchanged with 200 μL reaction buffer containing 0.5 mM L-012 (Wako Chemicals, USA) according to experiments performed. The fungal germinated spores exudates (GSE, 10 times concentrated) were used to detect ROS production in M. truncatula roots. Luminescence was recorded with a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific) (
M. truncatula wild type seedlings were grown on either high nitrate and high phosphate medium (+N+P, BNM modified with 5 mM KNO3 and 3.75 mM KH2PO4) or no nitrogen and low phosphorus medium (−N−P, BNM modified with 0.0075 mM KH2PO4). Plants for nodulation were transferred to a 1:1 mix of sand: terragreen (Oil-Dri Company, Wisbech, UK) and S. meliloti 1021 was inoculated by watering on plant roots with OD600=0.02. Nodules were counted at 1 week, 2 weeks, and 3 weeks post inoculation using a Leica M205FA stereo microscope.
M. truncatula plants were grown in pots (4×4×4.5 cm3) containing aluminum silicate/sand and inoculated with 200 spores of Rhizophagus irregularis produced by Premier Tech (Quebec, Canada). Mycorrhizal colonized roots were collected and incubated in 10% KOH (Sigma-Aldrich) at 95° C. for 10 minute and then stained with 5% ink (Waterman) in acetic acid (Sigma-Aldrich) at 5 weeks (Giovanetti M., et al. New Phytol. 84: 489-500). The grid line intersect method (Giovanetti M., et al. New Phytol. 84: 489-500) was used to quantify mycorrhizal colonization: roots were cut into 1 cm segments and spread randomly in plastic petri dishes in which a grid with 1 cm×1 cm squares was affixed to the base. 120 intersections for each root sample were counted to measure roots with or without mycorrhizal infection on a Leica DM6000 light microscope.
Phytophthora palmivora Infection Assays
Phytophthora palmivora was grown on V8 juice agar medium for 7 days until mycelium were fully expanded over the whole plate. The plates were then kept in a fume hood for 24 h, to dry the medium. 10 ml sterilized cool water was poured on each plate and kept for 1 h to release zoospores. The concentration of spores was quantified on a hemacytometer. M. truncatula seedlings were grown on +N+P (5 mM KNO3 and 3.75 mM KH2PO4) and −N−P (0.0075 mM KH2PO4) plates for 1-3 days and root tip regions were inoculated with 1×105/ml P. palmivora spores. To quantify P. palmivora growth, 30 seedlings for each ecotype 48 h post-inoculation were used to measure the lesion size and this was normalized to the individual root length (
The effects of oligosaccharides on M. truncatula colonization by symbiotic mycorrhizal fungi was examined. Oscillations in nuclear-associated calcium levels in root epidermal cells have been shown to be a component of M. truncatula symbiotic signaling. The COs CO4 and CO8, as well as the LCOs NS-LCO and SmLCO, were tested for their ability to promote nuclear calcium oscillations in M. truncatula root epidermal cells under different nutrient conditions. As shown in
As shown in
Lipochitooligosaccharides (LCO) and chitooligosaccharide (CO) perception has been shown to be essential for mycorrhizal colonization in M. truncatula (Feng et al. Nature Comms 2019 10: 5047). The CO receptor complex has been shown to either promote or restrict fungal colonization (Bozsoki et al. Proc. Natl. Acad. Sci. 2017 10.1073/pnas.1706795114; Feng et al. Nature Comms 2019 10:5047), highlighting the need for additional decision points that dictate the outcome of fungal recognition through the perception of COs. Taken together, these results indicate that this additional decision was defined by the prior nutrient status of the plant: symbiosis signaling was enhanced by nutrient starvation, whereas immunity signaling was suppressed (
The following example describes the symbiotic relationship between monocots and mycorrhizal fungi. In particular, the following example describes the contributions of oligosaccharide perception and nutrient levels to Hordeum vulgare (barley) and Zea mays (maize) symbioses with mycorrhizal fungi, and/or immunity-related signaling.
The wild type Z. mays W22 background was used.
Hordeum vulgare cv. Golden Promise was transformed as previously described (Bartlett et al. Plant Biotechnol. J. 2008 7:856-866). Leaf tissue (1-2 cm leaf material) from individual hygromycin-resistant transgenic barley plants was frozen in liquid nitrogen.
H. vulgare plants tested in
H. vulgare plants were grown with high nitrate (HN; 3 mM NO3−) concentration in combination with a range of phosphate concentrations, including 10 μM phosphate, 500 μM phosphate, 1 mM phosphate, or 2.5 mM (
Mycorrhizal colonization of H. vulgare with R. irregularis was measured 5 weeks or 7 weeks post inoculation. Mycorrhizal colonization of Z. mays with R. irregularis was measured 7 weeks post inoculation. Fungal colonization was quantified by tryptan blue staining. Samples of root pieces of approximately 1 cm in size were incubated in 10% KOH for 30 min at 96° C. followed by three washes with distilled water. Afterwards, the samples were incubated in 0.3 M HCl for 30-120 minutes at room temperature. The samples were boiled at 96° C. for 8 minutes in a 0.1% w/v tryptan blue staining solution in a 2:1:1 mixture of lactic acid:glycerol:distilled water before they were de-stained with a 1:1 solution of glycerol and 0.3 M HCl. 10 root pieces per sample were mounted on a cover slide, and total fungal colonization as well as the presence of specific fungal structures was quantified at 10 representative random points per root piece microscopically. All fungal structures present at one random point were recorded.
For H. vulgare calcium analyses, plants were grown on the relevant nutrient medium (see Plant materials and growth conditions above) for 5 or 16 days (
H. vulgare roots were grown on different nutrient conditions for 5 days, and then cut into 0.5 cm strips and incubated in 200 μL liquid medium containing different nutrients in a 96-well plate (Greiner Bio-one) overnight. After incubation, the medium was removed from each well and exchanged with 200 μL reaction buffer containing 0.5 mM L-012 (Wako Chemicals, USA). Luminescence was recorded with a Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific).
The level of mycorrhizal colonization of monocot plants of different genotypes was tested (
Next, it was tested whether LysM receptor-like kinase homologs were required for mycorrhizal colonization of H. vulgare. 10 LysM receptor-like kinase genes were found in H. vulgare, in particular three (including RLK4 and RLK5) that showed very close homology to CERK1, the M. truncatula CO receptor, and one (RLK10) that showed closed homology to NFP, the M. truncatula LCO receptor. Of the LysM receptor-like kinase mutants tested, rlk4-1 mutant barley were found to have a defect in mycorrhizal colonization, with almost no colonization occurring (
H. vulgare epidermal root cells were treated with COs, LCOs, or peptidoglycan, and tested for their ability to generate nuclear-associated calcium oscillations associated with symbiosis signaling (symbiotic calcium oscillations) (
In addition, the role of nutrient status in mycorrhizal colonization of H. vulgare was examined. Symbiotic calcium oscillations upon treatment with SmLCO occurred in H. vulgare at a higher level under limiting nitrate and limiting phosphate conditions than they did under replete nitrate and replete phosphate conditions (
The following example describes experiments to determine the roles of strigolactones, karrikins, and CEP peptides in the H. vulgare symbiotic response under nutrient limiting conditions.
Wild type H. vulgare (H vulgare cv. Golden Promise) was grown in sand, watered with modified liquid BNM containing either replete nitrate and replete phosphate (+N+P, 5 mM KNO3 and 0.5 mM KH2PO4), replete nitrate and limiting phosphate (+N−P, 5 mM KNO3, no phosphate), limiting nitrate and replete phosphate (−N+P, no nitrate, 3.75 mM KH2PO4), or limiting nitrate and limiting phosphate (−N−P, no nitrate or phosphate).
Wild type H. vulgare tested in
Treatment with Strigolactones and Karrikins
Wild type H. vulgare plants tested in
H. vulgare RT-qPCR primer sequences
In
For nuclear calcium imaging, roots were removed and transferred to liquid media and treated for 12 hours with either buffer alone, 1 μM 5-deoyxstrigol (
H. vulgare was treated with the synthetic strigolactone analog GR24 (
In order to determine the effect of strigolactones and karrikins on symbiotic signaling in M. truncatula (
In addition, the effect of different nutrient levels and/or strigolactone and karrikin treatment on expression levels of H. vulgare LysM receptor-like kinase homologs was examined. As shown in
The impacts of strigolactone/karrikin treatments in H. vulgare were shown to be entirely dependent on the nitrogen status of the plant: if nitrate levels were high, there was only limited LCO induction of calcium oscillations following strigolactone/karrikin treatment (“1 μM SL” trace in
CEP peptides are recognized by receptors present in the shoot, which in turn generate mobile signals to control nodule number in the root (Kereszt et al. Frontiers in plant science 2018 10:3389). It was tested whether CEP peptides also regulated symbiotic processes in cereals, in particular the regulation of mycorrhization, and whether CEP peptides were the missing second signal that coordinated the response to nitrogen levels. H. vulgare plants grown under replete nitrate and phosphate showed no nuclear calcium signaling (
Across a time course of nutrient deprivation in H. vulgare, a similar phenomenon was observed, whereby a single root became responsive to LCOs at early stages of nutrient deprivation, but, as nutrient deprivation continued, a majority of the root system became LCO responsive (
Transcriptional expression of some CEP genes is induced in H. vulgare under nitrogen (i.e., nitrate) starvation, as shown in
Effect of Exogenous Administration of Strigolactone and/or CEP Peptides on H. vulgare Mycorrhizal Colonization
Mycorrhizal colonization of H. vulgare when treated with the synthetic strigolactone analog GR24 was examined under different nutrient conditions. GR24 promoted mycorrhizal colonization when under high phosphate and low nitrate conditions when measured at 6 weeks post inoculation (
The following example describes the role of the transcription factors NSP1 and NSP2 in regulating mycorrhizal colonization, as well as the engineering of NSP transcription factor expression levels in H. vulgare.
M. truncatula Gene Expression Analyses
To determine the expression levels of the NSP genes as shown in
M. truncatula qRT-PCR primer sequences
NSP Gene Expression in H. vulgare
To determine the expression levels of the NSP genes as shown in
LysM Receptor-Like Kinase and Strigolactone Biosynthesis Gene Expression in H. vulgare
Wild type H. vulgare tested in
Engineering of NSP Genes in H. vulgare
Mutation of NSP2: Hordeum vulgare cv. Golden Promise was transformed as previously described (Bartlett et al. Plant Biotechnol. J. 2008 7:856-866). Leaf tissue (1-2 cm leaf material) from individual hygromycin-resistant transgenic barley plants was frozen in liquid nitrogen. NSP2 was mutated via CRISPR/Cas9 activity using the target sequences of guide 2A (gacggcggccacgacctccacgg, SEQ ID NO: 188) and guide 2B (gtgaccatggaggacgtggtggg, SEQ ID NO: 189). The nsp2-2 line was generated by a 314 basepair deletion between guides 2A and 2B, and the nsp2-4 line was generated by a 3 basepair deletion at guide 2A and a 1 bp insertion at guide 2B. The nsp2-1 line was found to have the same deletion as the nsp2-4 line. A summary of the H. vulgare lines with NSP2 mutations that were generated is provided below in Table 4.
Overexpression of NSP1 and NSP2: M. truncatula NSP1 and/or NSP2 were overexpressed in H. vulgare using the maize ubiquitin promoter pZmUBI1 (Lee, L. Y. et al, Plant Physiol. 2007 145:1294-1300), as described in Feike et al (Feike, D. et al, Plant Biotechnology Journal 2019 12:2234-2245) (see
M. truncatula NSP1 and/or NSP2
Western blotting: Overexpression of M. truncatula NSP homologs in H. vulgare was assessed by Western blot (
Mycorrhizal colonization assays were performed as described in Example 2.
Two transcription factors, NSP1 and NSP2, have been previously shown to be required for both nodulation and mycorrhization in M. truncatula (Kalo et al. Science 2005 308: 1786-1798; Smit et al. Science 2005 308: 1789-1791; Delaux et al. New Phytologist 2013 199: 59-65). Further, both NSP1 and NSP2 regulate strigolactone biosynthesis in M. truncatula (
The function of the NSP transcription factors was examined in H. vulgare. Multiple NSP homologs were found in the H. vulgare genome (
Furthermore, the expression levels of the H. vulgare LysM receptor-like kinase homologs that showed increased expression under limiting nitrate (see
In addition, the expression levels of H. vulgare strigolactone biosynthesis genes were analyzed. As shown in
The effects of nutrient starvation and strigolactone and/or karrikin treatment on the expression of M. truncatula LysM receptor-like kinase (LysM RLK) genes were tested. As shown in
As discussed above, NSP1 and NSP2 regulate strigolactone biosynthesis in M. truncatula. It was therefore tested whether overexpression of these transcription factors alone was sufficient to override phosphate suppression. Lines of H. vulgare overexpressing M. truncatula NSP1, NSP2, or NSP1 and NSP2 were generated (
Furthermore, overexpression of NSP1 and NSP2 overrode the inhibitory effects of replete nutrient conditions on gene expression (
Furthermore, NSP1 and NSP2 were also codon-optimized for expression in H. vulgare and overexpressed (
The following example describes additional characterization of the H. vulgare mutant lines and H. vulgare overexpression lines described in Example 4. Further H. vulgare lines will also be generated. In particular, the genes RLK10 and NSP1 will be further characterized.
Engineering of H. vulgare
Transgenic H. vulgare lines will be generated using the procedures described in Example 4. Independent transgenic H. vulgare lines mutant in NSP1, mutant in RLK10, and overexpressing RLK10 will be generated.
Materials and Methods for Characterizing H. vulgare Lines
Transgenic H. vulgare lines from Example 4 and newly engineered transgenic H. vulgare lines will be characterized in order to determine whether RLK10 is an LCO receptor and whether NSP1 is also necessary for strigolactone biosynthetic gene expression and symbiotic colonization.
The expression of strigolactone biosynthetic genes in H. vulgare lines mutant in NSP1 will be determined using the materials and methods described in Example 4. Symbiotic colonization of H. vulgare lines mutant in NSP1 will be determined using the materials and methods described in Example 2.
Symbiotic colonization of H. vulgare lines mutant in RLK10 will be determined using the materials and methods described in Example 2. H. vulgare lines mutant in RLK10 will be tested for transcriptional responses to treatment with COs and LCOs.
H. vulgare overexpression lines with overexpressed NSP1 and/or NSP2 will be characterized in order to determine whether they are colonized by associative bacteria, including rhizobia.
NSP1 is necessary for symbiotic colonization of H. vulgare.
RLK10 is an LCO receptor. RLK10 is necessary for H. vulgare mycorrhizal colonization and transcriptional responses to COs and LCOs.
The following example describes engineering of M. truncatula and M polymorpha overexpression lines. In particular, the transcription factors NSP1 and NSP2 will be overexpressed in, and their effect on mycorrhizal colonization and gene expression will be tested.
M. truncatula lines will be generated to overexpress NSP1 and/or NSP2. M. truncatula lines will be generated as described in Example 2. NSP1 and/or NSP2 will be overexpressed.
M. polymorpha lines will be generated to overexpress NSP1 and/or NSP2.
M. truncatula and M. polymorpha overexpression lines with overexpressed NSP1 and/or NSP2 will be characterized in order to determine whether they have elevated levels of mycorrhizal colonization. Mycorrhizal colonization levels for M. truncatula and M polymorpha will be determined using the materials and methods described in Example 2.
Gene expression levels will be measured in M. truncatula roots by qPCR. For example, the expression levels of the strigolactone biosynthetic enzyme genes GGPS, D27, CCD7, CCD8, and MAX1 will be measured using the qPCR primers in Table 6. M. polymorpha gene expression levels will also be measured.
M. truncatula qPCR primer sequences
Overexpression of NSP1 and/or NSP2 results in an increase in strigolactone biosynthetic enzyme gene expression in M. truncatula and M polymorpha.
Overexpression of NSP1 and/or NSP2 results in increased mycorrhizal colonization in M. truncatula
This application is a national stage application under 35 U.S.C. § 371 of International Application No. PCT/EP2021/054816, filed internationally on Feb. 26, 2021 which claims the benefit of U.S. Provisional Application No. 62/983,433, filed Feb. 28, 2020, each of which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/054816 | 2/26/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62983433 | Feb 2020 | US |
