EP4646098A1

EP4646098A1 - Nal1 in rice

Info

Publication number: EP4646098A1
Application number: EP24700672.9A
Authority: EP
Inventors: Wladimir Igor Leander TAMELING
Original assignee: Dcm Shriram Ltd; Keygene NV
Current assignee: Dcm Shriram Ltd; Keygene NV
Priority date: 2023-01-03
Filing date: 2024-01-03
Publication date: 2025-11-12
Also published as: WO2024146898A1

Abstract

The invention pertains to a mutant NAL1 protein for increasing test weight, and a nucleic acid molecule, chimeric gene, vector and host cell comprising a sequence encoding said mutant NAL1 protein. The invention further pertains to a host cell comprising said mutant protein, nucleic acid molecule, chimeric gene and/or vector, a method for producing a plant having an increased test weight, a method of screening plants having an increased test weight and a plant comprising a sequence encoding the mutant NAL1 protein.

Description

NAL1 in rice

Field of the invention

The invention is in the field of agriculture, in particular in the field of crop production, more particularly in the field of providing an increase in yield in rice.

Background of the invention

Rice consumption across the world steadily grows around 1.1 % per year and is expected to do so until 2025, when it will reach an expected volume of 570 million tonnes (mt) worldwide. This increase in rice consumption is supported by an increase in rice production, not only in the two largest rice producing countries, China and India, but also in other countries in the Indo-Gangetic plain (IGP), Nepal, Pakistan and Bangladesh. The fertile grounds of the IGP are responsible for 85% of rice cropping in south east Asia. Especially Bangladesh has shown an impressive increase in yield (tonne per hectare) since 1998, due to the use of modern technologies, such as improved irrigation, and balanced fertilizer application. However, in India the increase in yield seems stagnant and may even decline in the future, due to over- exploitation of natural resources. This problem is not limited to India, but extends to other countries in the IGP as well as China. Other factors that make rice a resource intensive crop, is the fact it needs plenty of water, labour- intensive cultivation, it degrades the quality of the soil and produces low quality of byproducts. In general rice cultivation is regarded as a water, energy and capital exhaustive practice.

To combat the hardships associated with rice cultivation, several strategies have been deployed to increase rice productivity, decrease (post-harvest) loss of grains and improve overall yield. Crop rotation practices which intercrops rice with legumes are effective in places with low water supplies and will increase the soil fertility, while the use of advanced irrigation systems and fertilizers increases yield as well. Another way to improve rice yield, is the use of modern rice varieties, which are better adaptable to changing climate conditions, have increased resistances and improved nutrient uptake efficiency. The use of new breeding techniques enable the development of not only new rice varieties with increased beneficial properties, but also new varieties of other cereal crops. As the increase in demand for rice and cereals increases, farmers will more and more rely on high quality seeds to meet the demand for rice production.

Introgression of the favourable NAL1 allele from the new plant type (NPT) cultivar YP9 into high- yielding modern rice varieties resulted in an increase in grain yield, largely due to an increase in the number of grains per panicle and an improved source-sink situation. However, as a trade-off the test weight (thousand grain weight) was decreased in these lines, wherein the favourable NAL1 allele is of the “HVI-type”, i.e. having three SNPs (having a R233H, A475V and V484I) as compared to the “RAV-type” allele present in the high-yielding rice varieties (Fujita et al., Proc Natl Acad Sci U S A. 2013, 110(51): 20431-20436)., In 2018 another study, performed by Huang et al. in 2018, showed that an increase expression of the RAV-type NAL1 in rice resulted in an increase of the number of grains per panicle, but a decrease in grain length. Both these results show that increased expression of NAL1 in rice has a positive effect on particular yield subcomponent traits, while it has a negative (trade-off) effect on others. As such there remains a need in the art to uncover new molecular targets and their related proteins (e.g. nucleic acids and/or amino acids) that can be altered or manipulated to increase the yield of field grown rice without negative pleiotropic effects.

Summary of the invention

Described is the discovery of a novel mutant NAL1 protein resulting in an increase in the test weight (TW). Test weight for cereals is a measure for grain weight, a yield subcomponent trait that is an important contributor to grain yield and therefore a highly relevant to keep up with the growing demand for food with a reduced input in agriculture.

Therefore, provided is this mutant NAL1 protein having an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto, wherein the mutant NAL1 protein, when expressed in a plant, results in an increased test weight as compared to a control plant, wherein the control plant is a plant that has the same genetic background and does not express the mutant NAL1 protein.

Preferably, the mutant NAL1 protein comprises a sequence analogous to SEQ ID NO: 1 and has an amino acid substitution at position 180, or at a position analogous to position 180 of SEQ ID NO: 1 .

Preferably, the mutant NAL1 protein further results in at least one of an increased number of grains per panicle and increased yield as compared to the control plant.

Preferably, the amino acid substitution on position 180, or a position analogous thereto, is a proline to serine conversion.

Preferably, the mutant NAL1 protein has the sequence of SEQ ID NO: 2.

Also provided is a nucleic acid molecule comprising a sequence encoding the mutant NAL1 protein provided herein.

Preferably, the nucleic acid molecule comprises the coding sequence of SEQ ID NO: 4.

Further provided is a chimeric gene comprising a promotor operably linked to the mutant nucleic acid molecule sequence as provided herein.

Further provided is a vector comprising the nucleic acid molecule provided herein.

Further provided is a host cell comprising the mutant NAL1 protein, nucleic acid, chimeric gene, or vector provided herein.

Further provided is a method for producing a plant having an increased test weight as compared to the control plant, wherein said method comprises at least the step of introducing a nucleic sequence provided herein.

Preferably, the introduction of the nucleic acid sequence provided herein is performed by mutating an endogenous NAL1 coding sequence by random or targeted mutagenesis.

Preferably, the introduction of the nucleic acid sequence provided herein is performed by introgressing into said plant a gene encoding the mutant NAL1 protein provided herein. Preferably, the introduction is performed by:

(a) inserting a nucleic acid molecule encoding the mutant NAL1 protein into a plant cell, wherein said nucleic acid molecule further comprises (i) a promoter functional in a plant cell and (ii) a terminator, wherein said promoter and terminator is operably linked to a sequence encoding the mutant NAL1 protein;

(b) obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises said sequence encoding the mutant NAL1 protein; and

(c) generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises said sequence encoding the mutant NAL1 protein.

Further provided is a method of screening for plants having an increased test weight, wherein said screening comprises the steps of: providing a heterogenic population of plants; performing a molecular marker assay to identify the presence or absence of: o a mutant NAL1 protein having an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto; and/or o a gene encoding a mutant Nall protein having an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto; and selecting one or more plants comprising the mutation.

Further provided is a plant comprising a sequence encoding the mutant NAL1 protein provided wherein said plant has an increased yield as compared to a control plant.

Preferably said plant is not exclusively obtained by an essential biological method.

Legend to the Figure

Figure 1 : the results of two independent field trials (M4 and M5) of the NAL1 P180S mutant. A: the increase of test weight, B: the increase in yield (average yield per plant) and C: the increase of grains per panicle. A star denotes a significant difference (p<0.05) using a two-sided t-test, the error bars represent the standard deviation.

Definitions

Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. It is clear for the skilled person that any methods and materials similar or equivalent to those described herein can be used for practicing the present invention.

Methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2012; Ausubel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; the series Methods in Enzymology, Academic Press, San Diego and JM Walker, the series Methods in Molecular Biology, Springer Protocols.

The singular terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The indefinite article "a" or "an" thus usually means "at least one".

“Analogous to” in respect of a domain, sequence or position of a protein, in relation to an indicated domain, sequence or position of a reference protein, is to be understood herein as a domain, sequence or position that aligns to the indicated domain, sequence or position of the reference protein upon alignment of the protein to the reference nucleic acid using alignment algorithms as described herein, such as Needleman Wunsch, and has the same or similar function. “Analogous to” in respect of a domain, sequence or position of a nucleic acid, in relation to an indicated domain, sequence or position of a reference nucleic acid, is to be understood herein as a domain, sequence or position that aligns to the indicated domain, sequence or position of the reference nucleic acid upon alignment of the nucleic acid to the reference nucleic acid using alignment algorithms as described herein, such as Needleman Wunsch, and wherein the domain, sequence or position of the nucleic acid and/or the encoded amino acid sequence has the same or similar function.

The term “and/or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, the term “about” is used to describe and account for small variations. For example, the term can refer to less than or equal to ± (+ or -) 10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1 %, less than or equal to ±0.5%, less than or equal to ±0.1%, or less than or equal to ±0.05%. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

The term “comprising” is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 dimensional structure or origin. A “fragment” or “portion” of a protein may thus still be referred to as a “protein”. An “isolated protein” is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant cell. The protein of the invention may be at least one of a recombinant, synthetic or artificial protein.

"Plant" refers to either the whole plant or to parts of a plant, such as cells, protoplasts, calli, tissue, organs (e.g. embryos pollen, ovules, seeds, gametes, roots, leaves, flowers, flower buds, anthers, fruit, etc.) obtainable from the plant, as well as derivatives of any of these and progeny derived from such a plant by selfing or crossing. Non-limiting examples of plants include crop plants and cultivated plants, such as African eggplant, alliums, artichoke, asparagus, barley, beet, bell pepper, bitter gourd, bladder cherry, bottle gourd, cabbage, canola, carrot, cassava, cauliflower, celery, chicory, common bean, corn salad, cotton, cucumber, eggplant, endive, fennel, gherkin, grape, hot pepper, lettuce, maize, melon, oilseed rape, okra, parsley, parsnip, pepino, pepper, potato, pumpkin, radish, rice, ridge gourd, rocket, rye, snake gourd, sorghum, spinach, sponge gourd, squash, sugar beet, sugar cane, sunflower, tomatillo, tomato, tomato scion, vegetable Brassica, watermelon, wax gourd, wheat and zucchini.

"Plant cell(s)" include protoplasts, gametes, suspension cultures, microspores, pollen grains, etc., either in isolation or within a tissue, organ or organism. The plant cell can e.g. be part of a multicellular structure, such as a callus, meristem, plant organ or an explant.

“Similar conditions” for culturing the plant / plant cells means among other things the use of a similar temperature, humidity, nutrition and light conditions, and similar irrigation and day/night rhythm.

“Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleotide (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods. The percentage sequence identity / similarity can be determined over the full length of the sequence.

“Sequence identity” and “sequence similarity” can be determined by alignment of two amino acid or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BI_ASTn and BI_ASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, word length = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, word length = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

A “nucleic acid” or “polynucleotide” according to the present invention may include any polymer or oligomer of pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982) which is herein incorporated by reference in its entirety for all purposes). The present invention contemplates any deoxyribonucleotide, ribonucleotide or nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogeneous or homogenous in composition, and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA (optionally cDNA) or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double-stranded form, including homoduplex, heteroduplex, and hybrid states. An “isolated nucleic acid” is used to refer to a nucleic acid which is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant cell. The nucleic acid of the invention may be at least one of a recombinant, synthetic or artificial nucleic acid.

The terms “nucleic acid construct”, “nucleic acid vector”, “vector” and “expression construct” are used interchangeably herein and is herein defined as a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The terms “nucleic acid construct” and “nucleic acid vector” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The vector backbone may for example be a binary or superbinary vector (see e.g. U.S. Pat. No. 5,591 ,616, US 2002138879 and WO 95/06722), a co-integrate vector or a T-DNA vector, as known in the art, into which a chimeric gene is integrated or, if a suitable transcription regulatory sequence is already present, only a desired nucleic acid (e.g. comprising a coding sequence, an antisense or an inverted repeat sequence) is integrated downstream of the transcription regulatory sequence. Vectors can comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like.

The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site.

“Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, e.g. a regulatory non-coding RNA or an RNA which is capable of being translated into a biologically active protein or peptide. Expression in relation to a protein or peptide is to be understood herein as the process of gene expression resulting in production of said protein or peptide.

The term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid region is “operably linked” when it is placed into a functional relationship with another nucleic acid region. For instance, a promoter, or rather a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked may mean that the DNA sequences being linked are contiguous.

“Promoter” refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acids. A promoter fragment is preferably located upstream (5’) with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation site(s) and can further comprise any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

Optionally, the term “promoter” may also include the 5’ UTR region (5’ Untranslated Region) (e.g. the promoter may herein include one or more parts upstream of the translation initiation codon of transcribed region, as this region may have a role in regulating transcription and/or translation). A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically (e.g. by external application of certain compounds) or developmentally regulated. A “tissue specific” promoter is only active in specific types of tissues or cells.

The term “wild type” as used in the context of the present invention in combination with a protein or nucleic acid means that said protein or nucleic acid consists of an amino acid or nucleotide sequence, respectively, that occurs as a whole in nature and can be isolated from organisms in nature as such, e.g. is not the result of modification techniques such as targeted or random mutagenesis or the like. A wild type protein is expressed in at least a particular developmental stage under particular environmental conditions, e.g. as it occurs in nature.

The term “endogenous” as used in the context of the present invention in combination with a protein or nucleic acid (e.g. gene) means that said protein or nucleic acid originates from the plant. Often an endogenous protein or nucleic acid will be present in its normal genetic context in the plant. In the present invention, an endogenous protein or nucleic acid may be modified in situ (in the plant or plant cell) using standard molecular biology methods, e.g. gene silencing, random mutagenesis or targeted mutagenesis.

The term “NAL1 protein” refers to a protein belonging to the family of NARROW LEAF proteins, which preferably is, or is a homologue or orthologue of the protein comprising or consisting of the amino acid sequence of SEQ ID NO 1. A NAL1 gene is to be understood herein as a gene comprising a sequence encoding a NAL1 protein. The NAL1 gene may be NAL1 of SEQ ID NO: 7.

“Mutagenesis” and/or “modification of a gene or nucleic acid” may be random mutagenesis or targeted mutagenesis resulting in an altered or mutated nucleic acid. Random mutagenesis may be, but is not limited to, chemical mutagenesis and gamma radiation. Non-limiting examples of chemical mutagenesis include, but are not limited to, EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), NaN3 (sodium azide) D), ENU (N-ethyl-N-nitrosourea), AzaC (azacytidine) and NQO (4-nitroquinoline 1-oxide). Optionally, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference) may be used to generate plant lines with a modified gene as defined herein. TILLING uses traditional chemical mutagenesis (e.g. EMS mutagenesis) followed by high-throughput screening for mutations. Thus, plants, seeds and tissues comprising a gene having one or more of the desired mutations may be obtained using TILLING. Targeted mutagenesis is mutagenesis that can be designed to alter a specific nucleotide or nucleic acid sequence, such as but not limited to, oligo-directed mutagenesis, RNA-guided endonucleases (e.g. CRISPR-technology), TALENs or Zinc finger technology.

A “control plant” as referred to herein is a plant of the same species and preferably same genetic background as the plant that is, or is a progeny of, a plant (or “putative test plant” or “test plant”) that has been subjected to a method as taught herein, i.e. a method for test weight, grains per panicle and/or yield. In case the test plant is a plant having a modified (endogenous) NAL1 gene, the control plant preferably comprises a wild type, preferably an endogenous or unmodified, NAL1 gene. Preferably, the control plant only differs from the putative test plant in that the control plant lacks the protein, nucleic acid and/or vector or construct of the invention.

Preferably the control plant is grown under the same conditions as the test plant comprising the protein and/or nucleic acid of the invention

The term “differential expression of a gene” as used herein, refers to a situation wherein the level of RNA and/or protein derived (wherein “derived” in this respect is to be understood as transcribed and/or translated, respectively) from said gene in a modified plant is reduced or increased as compared to the level of said RNA and/or protein that is produced in a suitable control plant (e.g., a wild type plant) under similar conditions. Preferably, expression of a gene is reduced or increased when the level of RNA and/or protein derived from said gene in a plant is at least 1 %, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% lower or higher, respectively, than the level of RNA and/or protein derived from said gene in the control plant. A decrease of 100% is understood herein that the RNA and/or protein is absent in the modified plant. Alternatively or in addition, expression of a gene is reduced or increased when the level of RNA or protein encoded by said gene in a plant is statistically significantly lower or higher, respectively, than the level of RNA or protein that is produced in the control plant.

The term “differential expression of a protein” as used herein, refers to a situation wherein the level of said protein in a modified plant is reduced or increased as compared to the level of said protein produced in a suitable control plant (e.g., a wild type plant) under similar conditions. Preferably, expression of a protein is reduced or increased when the level of said protein produced in a plant is at least 1 %, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% lower or higher, respectively, than the level of said protein that is produced in the control plant. Alternatively or in addition, expression of a protein is reduced or increased when the level of said protein produced in a plant is statistically significantly lower or higher, respectively, than the level of protein that is produced in the control plant. It is understood herein that “differential expression” may be due both e.g. to a modification of one or more regulatory elements and/or a modification of the coding sequence. As a non-limiting example, modification of a regulatory element may result in lower transcript levels, while a modification of the coding sequence may result in the complete absence of any transcripts encoding the wild type protein. The term “differential activity of a protein” as used herein refers to a situation wherein activity of a protein, preferably the natural (or “wild type”) activity, such as for example its ability to bind to a promoter element, to bind to a receptor, to catalyze an enzymatic reaction, to regulate gene expression, etcetera, is altered, reduced, blocked, inhibited, increased or induced for instance due to a modification in structure, as compared to the activity of the same protein albeit without said modification, preferably in a plant maintained under similar conditions. Preferably, the activity of a modified protein may be considered to be differentiated when the activity of said modified protein produced in a plant is at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 50%, 70%, 80%, 90%, or even 100% lower or higher than the activity of the same protein without said modification as produced in a control plant. The skilled person will readily be capable of establishing whether or not activity of a protein is differentiated.

“Test weight”, or TW, is to be understood herein as the weight of a defined number (preferably 100, even more preferably 1000) of, preferably randomly selected, filled grains per plant. In comparing the effect of the mutant NAL1 gene of the invention on test weight, preferably the average test weight of multiple plants comprising the mutant NAL1 gene (test plants) is compared to the average test weight of multiple control plants. Preferably test weight is assessed on grains that were harvested and threshed from fully matured plants. Prior to weighing a predefined number of filled grains on a precision balance, the grains have to be dried to a preset moisture content. For rice, preferably the defined number of grains is 100 and the preset moisture content is 13% moisture content according to the Standard Evaluation System (SES) of the International Rice Research Institute (IRRI).

“Number of grains per panicle” is to be understood herein as the number of filled grains per main panicle. The main panicle is to be understood as the panicle formed on the main culm of the plant. In comparing the effect of the mutant NAL1 gene of the invention on test weight, preferably the average number of grains per (main) panicle of multiple (preferably 20) plants comprising the mutant NAL1 gene (test plants) is compared to the average number of grains per (main) panicle of multiple (preferably 20) control plants. Preferably grains per panicle is assessed at the mature stage of the crop. After threshing and cleaning, grains can be counted in an automatic seed counter.

“Yield” is to be understood herein as the average weight of all grains per plant. For rice, preferably, filled grains are weight drying to a 13% moisture content. In comparing the effect of the mutant NAL1 gene of the invention on yield, preferably the average weight of all grains of multiple (preferably 20) plants comprising the mutant NAL1 gene (test plants) is compared to the average weight of all grains of multiple (preferably 20) control plants. Preferably yield is assessed at the mature stage of the crop.

Detailed description of the invention

In a first aspect provided is a modified NAL1 protein, wherein said protein comprises amino acid substitution or conversion on position 180 of SEQ ID NO: 1 , or a position analogous thereto. The modified NAL1 protein is also indicated herein as “the modified protein”, “the modified protein of the invention” or “the protein of the invention”. Preferably the modified protein is derived from a wild type, preferably endogenous NAL1 protein, by said one or more modifications. Preferably, said wild type or endogenous protein comprises a Proline (P) on position 180, or a position analogous thereto. Preferably, said wild type or endogenous NAL1 protein is of the RAV-type, i.e. having an Arginine (R) on position 233, or a position analogous thereto, an Alanine (A) on position 475, or a position analogous thereto, and a Valine (V) on position 484, or a position analogous thereto. Alternatively, said wild type or endogenous NAL1 protein is of the HVI-type, i.e. having an Histidine (H) on position 233, or a position analogous thereto, a Valine (V) on position 475, or a position analogous thereto, and a Isoleucine (I) on position 484, or a position analogous thereto.

Preferably, expression of the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, results in an increase in test weight. Preferably, the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, increases the number of grains per panicle. Preferably, the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, increases the grain yield. Preferably, in addition to increase in test wait, the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, increases the number of grains per panicle. Preferably, in addition to increase in test wait, the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, increases the yield. Preferably, in addition to increase in test wait, the modified protein when present in a plant, preferably in the absence of its unmodified counterpart, increases both the number of grains per panicle and the yield. The unmodified counterpart preferably is the protein encoded by the unmodified endogenous gene. Preferably, said unmodified gene encodes a NAL1 protein that is of the RAV-type, i.e. having an Arginine (R) on position 233, or a position analogous thereto, an Alanine (A) on position 475, or a position analogous thereto, and a Valine (V) on position 484, or a position analogous thereto. Alternatively, said unmodified gene encodes a NAL1 protein that is of the HVI-type, i.e. having an Histidine (H) on position 233, or a position analogous thereto, a Valine (V) on position 475, or a position analogous thereto, and a Isoleucine (I) on position 484, or a position analogous thereto. Preferably said gene is, or is a homologue or orthologue of the NAL1 gene having the sequence of SEQ ID NO: 7. In other words, the invention encompasses a modified protein having a modification that increases test weight when expressed and/or is present in a plant. Preferably the modified protein is a modified endogenous protein of said plant, which is encoded by a modified endogenous gene. Preferably the modified protein is a modified endogenous protein of said plant. Preferably, the test weight of a plant comprising the modified endogenous gene encoding the modified protein of the invention is increased as compared to a control plant not comprising said modified protein. The modification may be one or more amino acid insertions, deletions and/or substitutions.

Preferably said the modified protein is encoded by a nucleic-acid derived from a wild type or endogenous nucleic-acid by having one or more modifications in the coding sequence of the gene encompassed in the wild type or endogenous nucleic acid. The modified protein may be a mutant protein. The mutant protein may be a naturally occurring mutant protein or a man-made mutant protein, e.g. obtainable by a technical process, such as but not limited to, targeted and/or random mutagenesis of the nucleic-acid sequence encoding the protein.

Preferably the modified protein may be produced synthetically, or in vivo (in cell or in planta) for instance by transcription and translation of a gene or construct as defined herein, optionally comprising a transgene encoding such protein, e.g. a wild type gene modified to encode said protein, or by transcription and translation of an endogenous sequence modified to encode such protein. Preferably, the protein of the invention is derived from a wild type and/or endogenous protein. The expression of the protein of the invention may be controlled by an endogenous promoter, such as, but not limited to, the promoter naturally controlling the expression of the wild type or endogenous protein from which the modified protein of the invention is derived.

In a further aspect, the invention encompasses a nucleic acid encoding the modified NAL1 protein of the invention. Preferably the nucleic acid comprises a gene or a modified gene, wherein said gene encompasses one or more modifications resulting in expression of the modified protein of the invention. Preferably the modified gene is derived from a wild type, preferably endogenous gene, by said one or more modification. Preferably, the modified gene is derived from the gene having the sequence of SEQ ID NO: 7, or a homologue or orthologue thereof. Preferably the modified gene is derived from a gene having SEQ ID NO: 7, or a sequence that is at least 60%, 65% 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, wherein preferably the modified gene encodes a modified NAL1 protein of the invention. Optionally, said modified gene is derived from a gene, preferably endogenous gene, having the sequence of SEQ ID NO: 7, or a gene that is a homologue or orthologue thereof, by the mutation of a nucleotide (a SNP or Single Nucleotide Polymorphism) on a position analogous to position Chr4:31 ,211 ,923 of said gene. Optionally, said modified gene is derived from a gene, preferably endogenous gene, having the sequence of SEQ ID NO: 7, or a gene that is a homologue or orthologue thereof, by the mutation of a nucleotide (a SNP or Single Nucleotide Polymorphism) on a position analogous to position 1961 of SEQ ID NO: 7. Preferably said SNP is a Cytosine to Thymine (indicated herein as “C to T”) conversion. Preferably, the modified gene is derived from the gene of SEQ ID NO: 7 by having a mutation in exon 2 resulting in an amino acid substitution, i.e. a non-silent mutation. Preferably, said amino acid substitution is a non-conservative amino acid change. A non-conservative amino acid change is to be understood herein as the replacement of an amino acid by an amino acid with different (non-comparable) properties, wherein these properties may be chemical properties, for example polarity, charge, solubility, hydrophobicity, hydrophilicity or amphipathic nature of the residues. Preferably, said amino acid substitution is the substitution of a neutral-nonpolar amino acid to a neutral-polar amino acid. Preferably said amino acid substitution is on position 180 of SEQ ID NO: 1 , or a position analogous thereof. Preferably, said amino acid substitution is a Proline to an Serine or Threonine mutation, preferably a Proline to Serine mutation. Optionally, the protein of the invention further comprises at least one, optionally all, of the amino acid substitutions selected from the group consisting of: an Arginine (R) to Histidine (H) conversion on position 233, or a position analogous thereto, an Alanine (A) to Valine (V) conversion on position 475, or a position analogous thereto, and a Valine (V) to Isoleucine (I) on position 484on position 484, or a position analogous thereto. Preferably, the modified gene is, or is a homologue of SEQ ID NO: 8. Preferably, said modified gene has the sequence of SEQ ID NO: 8, or a gene that is a homologue or orthologue thereof, and has a T on a position analogous to position 1961 of SEQ ID NO: 8.

Hence, the nucleic acid provided herein may also be considered a nucleic acid comprising a modified wild type gene or a modified endogenous gene. Said modified gene may be a mutant gene. The mutant gene may be a naturally occurring mutant gene or a man-made mutant gene, e.g. obtainable by a technical process, such as, but not limited to, targeted and/or random mutagenesis. When present in the plant cell, preferably in the absence of expression of the unmodified gene, the nucleic acid of the invention results in an increase in test weight as compared to a control plant, wherein said control plant preferably the unmodified (wild type and/or endogenous) gene. Alternatively or in addition, when present in the plant cell, preferably in the absence of expression of the unmodified gene, the nucleic acid of the invention results in an increase in number of grains per panicle as compared to a control plant, wherein said control plant preferably the unmodified (wild type and/or endogenous) gene. Alternatively or in addition, when present in the plant cell, preferably in the absence of expression of the unmodified gene, the nucleic acid of the invention results in an increase in yield as compared to a control plant, wherein said control plant preferably the unmodified (wild type and/or endogenous) gene.

Preferably the modified gene of the nucleic acid of the invention is derived from a wild type and/or endogenous gene by a step of genetic modification. Said wild type and/or endogenous gene is preferably a plant gene. The modified gene of the nucleic acid of the invention preferably is a modified endogenous gene that shows at least one of a differential expression and differential activity of the encoded protein when present in a plant, as compared to the endogenous gene in a control plant, preferably under similar conditions.

Optionally, the modified gene is obtained from said wild type and/or endogenous gene by deletion, insertion and/or substitution of at least one nucleotide, wherein said deletion, insertion and/or substitution results in a gene with differential expression, differential activity and/or differential function of the encoded protein. Said modified gene may be obtained via random or targeted mutagenesis. Such modification may be within the coding sequence of said gene, resulting in a modified protein which may be less functional as compared to the protein encoded by the unmodified gene or which is a dysfunctional protein, wherein a dysfunctional protein is to be understood as a protein not being capable of fulfilling the function of the protein encoded by the unmodified gene. Alternatively, such modification may result in a modified protein which may show higher activity and/or an increase or induced function as compared to the protein encoded by the unmodified gene.

Preferably, the unmodified, wild type and/or endogenous gene encodes for a protein that is, or is a homologue or orthologue of, a protein having an amino acid sequence of SEQ ID NO: 1 , and/or comprises the coding sequence of SEQ ID NO: 3 respectively. Preferably, the modified gene shows differential expression and/or activity of the encoded protein as compared to the corresponding (unmodified) endogenous gene under similar and suitable conditions.

The modified gene of the nucleic acid of the invention may be derived by genetic modification from a NAL1 protein coding gene that comprises any one of:

(a) a sequence encoding a protein of SEQ ID NO: 1 ;

(b) a CDS of SEQ ID NO: 3; and,

(c) a homologue or orthologue of any one of (a) and (b), wherein preferably, said modified gene shows differential expression and/or activity of the encoded protein as compared to the (unmodified) endogenous gene encoding the protein of SEQ ID NO: 1 under similar and suitable conditions, preferably when present in a plant cell, wherein preferably said plant cell is of a plant of the family of Poaceae, more preferably of the genus Oryza. Preferably, said modified gene encodes for the modified protein having amino acid sequence of SEQ ID NO: 2. Preferably, said modified gene comprises the CDS that has the nucleotide sequence of SEQ ID NO: 4.

A modified protein derived from wild type, preferably an endogenous, protein as indicated herein preferably means that said modified protein differs from said wild type, preferably endogenous, protein in that it has one or more amino acid substitutions, insertions and/or deletions, optionally a truncation, resulting in a protein having a differential function, a dysfunctional protein, and/or a truncated protein.

A modified gene derived from an endogenous gene as indicated herein preferably means that said gene differs from said endogenous gene in that it has one or more modifications, preferably one or more nucleotide substitutions, insertions and/or deletions. Apart from the modification resulting in an amino-acid substitution on position 180 of SEQ ID NO: 1 , or a position analogous thereof, the modified gene may comprise one or more further The further modification of the endogenous gene may be a modification in the coding region and/or in a non-coding region of the gene, e.g. by modifying the coding sequence, altering a regulatory sequence, and/or introducing or removing a splice (donor / acceptor) site. Optionally, the further modification may be a synonymous or a non-synonymous alteration of a codon. Optionally, the further modification may result in a decrease or increase in expression of the encoded gene, for instance by modification of a regulatory sequence such as the promoter sequence of said gene.

Further comprises is a construct or expression vector comprising the nucleic acid of the invention, comprising the modified gene of the invention and/or encoding the modified gene protein of the invention.

In a further aspect provided is a plant or plant cell comprising the protein, nucleic acid and/or construct of the invention, and/or a plant or plant cell obtainable from a method as defined herein. The plant or plant cell may comprise a mutation in an endogenous NAL1 gene as defined herein. The plant or plant cell may comprise a mutation in the coding sequence of an endogenous NAL1 gene, wherein the mutation results in the differential expression and/or activity as compared to an the protein encoded by the unmodified endogenous NAL1 gene. Preferably, all homologous genes within the genome of said plant or plant cell are modified to result in a nucleic acid of the invention. The modification of the homologous genes does not have to be the same or identical modification. Preferably, all NAL1 alleles of the plant or plant cell are modified to result in the nucleic acid of the invention. Preferably, the plant or plant cell is homozygously expressing the modified protein of the invention. Put differently, said plant or plant cell comprises the nucleic acid of the invention in its genome homozygously. The plant or plant cell of the invention may be characterized by a modified protein, which shows a differential, increased, decreased or lost function and/or activity. Further, the plant or plant cell of the invention may be characterized by a induced, reduced or abolished expression of the endogenous protein as defined herein. Preferably, the plant, or plant cell is of plant that, is desired to have at least one of an increased test weight, increased number of grains per panicle and an increased yield. Preferably, the plant has at least one of, an increased test weight, increased number of grains per panicle and increased yield, as compared to a control plant, which can be tested for and/or screened for as indicated herein. As defined herein, the plant may be a plant part or plant tissue. Optionally the plant tissue or plant part of the invention is a leaf, fruit, panicle, and/or scion. Said the plant of the invention may be from any plant species. Preferably the plant of the invention is a seed plant, even more preferred said plant is a flowering plant e.g. an angiosperm. Preferably the plant of the invention is a monocot, preferably a cereal. Preferably, the plant of the invention belongs to the family Poaceae. The plant of the invention may be any species belonging to the genus: Aegilops, Arabidopsis, Beta, Brassica, Cannabis, Capsicum, Cicer, Citrullus, Citrus, Coffea, Cucumis, Cucurbita, Daucus, Eucalyptus, Glycine, Helianthus, Hordeum, Lactuca, Lagenaria, Lepidium, Manihot, Musa, Nicothiana, Oryza, Petunia, Pisum, Populos, Prunus, Raphanus, Setaria, Solanum, Sorgum, Theobroma, Trifolium, Triticum, Vitis or Zea, preferably the species, Aegilops tauschii, Arabidopsis thaliana, Beta vulgaris, Brassica napus, Brassica rapa, Brassica oleracea, Cannabis sativa, Capsicum annuum, Cicer arietinum, Citrullus lanatus, Citrus Clementina, Coffea canephora, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Eucalyptus grandis, Glycine max, Helianthus annuus, Hordeum vulgare, Lactuca sativa, Musa acuminata, Nicotiana tabacum, Oryza indica, Oryza sativa, Petunia axillaris, Petunia inflata, Pisum sativum, Populus trichocarpa, Prunus persica, Raphanus sativus, Solanum lycopersicum, Solanum melongena, Solanum pennellii, Solanum tuberosum, Sorghum bicolor, Trifolium pratense, Triticum aestivum, Triticum urartu, Vitis vinifera or Zea mais.

Preferably, the nucleic acid and/or protein of the invention is present in the plant as defined herein. Preferably, the nucleic acid and/or protein of the invention is derived from a wild type, preferably an endogenous, gene and/or protein of said plant.

In a further aspect, provided is a host cell comprising the nucleic acid and/or protein of the invention. Preferably the nucleic acid of the invention is comprised within the genome of said host cell. Optionally, said host cell is a bacterium, cynobacterium, virus, fungi, insect, yeast, or a plant cell. Optionally, said host cell is a bacterium, preferably an Agrobacterium tumefaciens or Escherichia coli.

Preferably, said host cell is a plant cell. Even more preferably, said host cell is a plant cell from a plant as of the invention as defined herein. In an aspect, the host cell of the invention is produced by at least one of mutagenesis or transformation of a nucleic acid as defined herein. In an aspect, the host cell can be a mutagenized or transgenic host cell.

In a further aspect, provided is a method for producing a plant having at least one of an increased test weight, increased number of grains per panicle and increased yield as compared to a control plant, wherein said method comprises at least the step of introducing the nucleic acid provided herein.

Said method comprises the step of introducing the modified NAL1 gene and/or modified NAL1 protein of the invention, optionally by modifying the endogenous NAL1 gene as defined herein. Preferably, said method further comprises a step of regenerating the plant cell or plant tissue into a plant. The method for producing a plant having at least one of an increased test weight, increased number of grains per panicle and increased yield can also be regarded as a method of conferring the increased test weight trait, increased number of grains per panicle trait and increased yield trait, respectively, to said plant. Optionally, the method of the invention comprises the step of introducing expression of the modified protein of the invention. Optionally, the method for producing a plant having increased test weight as compared to the control plant may comprise the steps of:

(a) introducing into said plant a nucleic acid molecule encoding the mutant NAL1 protein as defined herein and further comprising (i) a promoter functional in a plant cell and (ii) a terminator, wherein said promoter and terminator are operably linked to the sequence encoding the mutant NAL1 protein;

Introducing expression of said protein may be achieved by transfection (transient or stable) of the plant by a nucleic acid and/or a construct encoding said protein. Such method may comprise an additional or simultaneous step of reducing or abolishing the expression of its endogenous counterpart. The step of introducing the expression of the protein of the invention may be performed using any conventional means known to the skilled person. Direct transformation of a nucleic acid encoding the protein of the invention into a plant can occur by one of many techniques known to one skilled in the art and the manner selected is not critical to the practice of the invention. Methods for introducing nucleic acids, constructs and expression vectors into plant tissue available to one skilled in the art are varied and will depend on the plant selected. Procedures for transforming a wide variety of plant species are well known and described throughout the literature. A nucleic acid encoding the protein of the invention may be introduced into a plant to introduce expression of the protein. The nucleic acid expressing the protein of the invention can be introduced into a plant using any conventional method known in the art. As a nonlimiting example, this can occur by direct transformation methods, such as Agrobacterium transformation of plant tissue, microprojectile bombardment, electroporation, transfection or any one of many methods known to one skilled in the art. Alternatively or in addition, introducing expression of the modified protein of the invention may be achieved by mutating an endogenous gene in a plant, resulting in decreased expression of the endogenously encoded protein. The endogenous coding sequence may be modified by mutagenesis to result in a sequence encoding the modified protein of the invention. Optionally, the modification results in a non-naturally occurring gene, i.e. a gene that does not occur in nature, and optionally the modification results in expression of a non-natural protein, i.e. a protein not occurring in nature.

The expression of the protein of the invention may be controlled by an endogenous promoter, such as, but not limited to the promoter controlling the expression of the endogenous protein in a control plant. Preferably, expression of the modified NAL1 protein of the invention is controlled by promoter of the unmodified (endogenous) NAL1 gene. Alternatively or in addition, expression of the protein of the invention may be controlled by a promoter that is not a native promoter, i.e. the promoter sequence is introduced in the plant. Optionally, the method of the invention comprises a step of modifying a regulatory sequence of the gene, such as the promoter sequence resulting in reduced or increased expression of the encoded protein. In such case, expression of the protein of the invention may be controlled by a modified promoter, preferably a modified endogenous promoter, wherein said modification results in reduced expression as compared to the expression level of said protein that is under the control of an unmodified (native) promoter, preferably an unmodified endogenous promoter.

The plant or plant part (e.g. a scion) of the method of the invention may be a monocot or dicot, and may be from a plant of any species belonging to any species belonging to the genus: Aegilops, Arabidopsis, Beta, Brassica, Cannabis, Capsicum, Cicer, Citrullus, Citrus, Coffea, Cucumis, Cucurbita, Daucus, Eucalyptus, Glycine, Helianthus, Hordeum, Lactuca, Lagenaria, Lepidium, Manihot, Musa, Nicothiana, Oryza, Petunia, Pisum, Populos, Prunus, Raphanus, Setaria, Solanum, Sorgum, Theobroma, Trifolium, Triticum, Vitis or Zea, preferably the species, Aegilops tauschii, Arabidopsis thaliana, Beta vulgaris, Brassica napus, Brassica rapa, Brassica oleracea, Cannabis sativa, Capsicum annuum, Cicer arietinum, Citrullus lanatus, Citrus Clementina, Coffea canephora, Cucumis melo, Cucumis sativus, Cucurbita maxima, Cucurbita moschata, Eucalyptus grandis, Glycine max, Helianthus annuus, Hordeum vulgare, Lactuca sativa, Musa acuminata, Nicotiana tabacum, Oryza indica, Oryza sativa, Petunia axillaris, Petunia inflata, Pisum sativum, Populus trichocarpa, Prunus persica, Raphanus sativus, Solanum lycopersicum, Solanum melongena, Solanum pennellii, Solanum tuberosum, Sorghum bicolor, Trifolium pratense, Triticum aestivum, Triticum urartu, Vitis vinifera or Zea mais.

The plant may be, or may be obtainable from, the family of Poaceae. The plant may be of the genus Oryza, more preferably said plant is a Oryza sativa or Oryza indica plant. The plant produced by the method of the invention preferably has a modification in the NAL1 gene.

In addition or alternatively, the plant may be of the genus Oyza, more preferably of the species Oryza sativa, even more preferably Oryza sativa subsp. indica, and preferably the modified protein of the invention is derived from the N AL 1 protein. Optionally, the method of the invention further comprises a step for transferring the nucleic acid of the invention to offspring of the plant produced by the method of the invention, which may be performed by introgression. Breeding techniques for introgression are well known to one skilled in the art.

Preferably, the method of the invention results in a plant of the invention as defined herein. Preferably said plant has at least one of an increased test weight, an increased number of grains per panicle and an increased yield compared to a control plant as defined herein.

The method of the invention may further comprise a step of screening or testing the plant for at least one of an increased test weight, an increased number of grains per panicle and an increased yield. Any screening or testing method known in the art can be used for screening the plant, such as, but not limited to, the methods described herein. Said screening or testing can be any phenotyping method to assess the test weight, increased number of grains per panicle and/or yield, known to the skilled person. Alternatively or in addition, expression levels of modified and/or unmodified protein at a molecular level (protein or mRNA) may be determined. In addition or alternatively, the presence of a nucleic acid or construct comprising the modified gene of the nucleic acid of the invention and/or encoding the modified protein of the invention may be determined. Preferably, the method comprises the screening for a nucleic- acid sequence encoding the modified NAL1 protein of the invention. Preferably, the method comprises the screening for a modification in a NAL1 gene, wherein said modification results in a sequence encoding a modified NAL1 protein, wherein said modified NAL1 protein preferably has an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto, as compared to the unmodified NAL1 gene. Preferably said screening is a SNP resulting in a Proline to Serine substitution in a NAL1 protein, preferably on position 180 of SEQ ID NO: 1 or a position analogous thereto. The person skilled in the art is aware of techniques to determine protein expression levels and/or the presence or absence of a nucleic acid sequence within a plant. Well know molecular techniques to identify such sequences are based on, e.g. Sequence Based Genotyping (Truong, et al. PLoS One. 2012; 7(5): e37565); oligo-ligation (SNPSelect; Hogers et al. PLoS One. 2018; 13(10): e0205577), AFLP (W01993/006239; Vos et al. . Nucl. Acids Res., 1995, 21 , 4407-4414). Optionally, the assay is a KASP (Kompetitive allele specific PCR) assay. In such assay, the following respective target specific sequences may be used in allele-specific primers of such KASP assay: SEQ ID NO: 5 (NAL1 wild type allele) in combination with SEQ ID NO: 6 (NAL1 mutant type allele). The method may further comprise a step of selecting the plant comprising the nucleic-acid sequence encoding the modified NAL1 protein of the invention. Optionally, the method may further comprise a step of phenotyping as further detailed herein. Optionally, multiple plants are screened.

The method for producing a plant of the invention having increased test weight as defined herein may further comprise a step of assessing expression of the protein of the invention and/or detecting the presence of the nucleic acid of the invention in said plant and optionally subsequently selecting said plant.

Expression levels of the protein of the invention can be determined using any conventional method known to the skilled person. Such methods include detecting the transcript (e.g. mRNA) or detecting the protein of the invention. Non-limiting examples for detecting the transcript include e.g. PCR, q-PCR and northern blotting. Non-limiting examples for detecting the presence of the protein of the invention includes e.g. western blotting and mass spectrometry on full polypeptides and peptide digests. The person skilled in the art is also aware of using methods for screening for the presence of the nucleic acid of the invention.

As also indicated herein above, the method may further comprise a step of producing progeny of the plant comprising the nucleic acid of the invention and/or expressing the protein of the invention. The method can comprise a further step of producing seeds from the plant expressing the protein of the invention and/or comprising the nucleic acid of the invention. The method may further comprise growing the seeds into plants that have increased test weight, optionally in combination with an increased number of grains per panicle and/or an increased yield.

In a further aspect, the invention also provides for a method of screening a plant for the presence of the nucleic acid of the invention and/or for expression of the protein of the invention. Said method comprises a step of assessing the presence of the nucleic acid of the invention in said plant and/or assessing expression of the protein of the invention in said plant. Preferably, the method of screening is for screening of a NAL1 gene encoding the mutant NAL1 protein of the invention. The screening may be for screening of a C to T conversion on a position analogous to position Chr4:31 , 211 ,923 of the gene encoding the NAL1 protein. The person skilled in the art is aware of methods to screen for mutants, such as, but not limited to a KASP assay as exemplified herein.

Optionally the method further comprises a step of selecting said plant, plant cell, plant tissue or plant part, essentially as described herein above. In case multiple plants or plant cells are screened, said method may further comprise a stop identifying, detecting and/or selecting a plant or plant cell comprising the nucleic acid of the invention and/or expressing the protein of the invention. Therefore, the invention also provides for a method of identifying, detecting and/or selecting a plant or plant cell comprising the nucleic acid of the invention and/or expressing the protein of the invention. Optionally, said method further comprises a step of phenotyping said plant, or said plant grown from said plant cell by assessing at least one of test weight, number of grains per panicle and/or yield.

In a further aspect, the invention provides for a plant obtainable from a method as defined herein, wherein said plant comprises the protein, nucleic acid and/or construct of the invention. The plant may comprise a mutation in an endogenous sequence encoding a NAL1 protein as defined to result in a nucleic acid of the invention. Preferably, all homologous genes within the genome of said plant are modified to result in a nucleic acid of the invention. Preferably, the modification results in a modified NAL1 protein. Preferably, said plant is a plant of the invention as defined herein.

The modification of the homologous genes does not have to be the same or identical modification. Preferably, said plant comprises the nucleic acid of the invention in its genome homozygously. The plant of the invention may be characterized by a modified protein. The plant comprising the modified gene of the nucleic acid of the invention and/or the modified protein of the invention has, or has at least one of an increased test weight, increased number of grains per panicle and increased yield as compared to a control plant, which can be tested for and/or screened for as indicated herein.

The skilled person is well aware how to select appropriate conditions to test for increased test weight, increased number of grains per panicle and/or increased yield.

In an aspect, the plant of the invention is not, or is not exclusively, obtained by an essentially biological process. Preferably, the plant of the invention is obtained by a method comprising a technical step. Preferably, the plant of the invention is man-made.

Preferably, the plant of the invention and/or of the method of the invention may be a crop plant or a cultivated plant, i.e. plant species which is cultivated and bred by humans. A crop plant may be cultivated for food or feed purposes (e.g. field crops), or for ornamental purposes (e.g. production of flowers for cutting, grasses for lawns, etc.). A crop plant as defined herein also includes plants from which non-food products are harvested, such as oil for fuel, plastic polymers, pharmaceutical products, cork, fibers (such as cotton) and the like. Preferably, the plant part, leaf, fruit, plant cell, seed, and/or scion as taught herein are from a crop plant.

The plant may be, or may be obtainable from, the family of Poaceae. The plant may be of the genus Oryza, more preferably said plant is a Oryza sativa or Oryza indica plant, comprising a modified gene of the invention that is derives from a NAL1 gene.

In addition or alternatively, the plant may be, or may be obtainable from the family of Poaceae, preferably of the genus Oryza, more preferably said plant is a Oryza sativa or Oryza indica plant, comprising a modified protein of the invention that is derived from a NAL1 protein.

An additional aspect of the invention pertains to plants grown from the seeds or regenerated from the plant cell, comprising the nucleic acid and/or protein of the invention as defined herein.

An additional aspect of the invention described herein pertains to progeny of the plant of the invention, wherein the progeny has increased test weight as specified herein and wherein the progeny comprises the nucleic acid and/or protein of the invention. The progeny may be obtained by selfing or breeding and selection, wherein the selected progeny retains the increased test weight of the parent plant and/or retain the expression of the protein of the invention.

In an aspect, the invention further concerns the use of a nucleic acid, protein, construct, or host cell of the invention for increasing the test weight in a plant, preferably for increasing test weight in combination with increasing number of grains per panicle and/or increased yield.

In an aspect, the invention pertains to plant parts and plant products derived from the plant of the invention and/or plant obtained or obtainable by the method of the invention, wherein the plant part and/or plant product comprise the modified gene, modified protein or parts thereof. Such plant parts and/or plant products may be seed or fruit and/or products derived therefrom. Such plant parts, plant products may also be non-propagating material. Optionally, such plant product is mill or flour, or any product derived therefrom. Examples

Example 1

In order to find new genes to increase yield and related traits in rice, an EMS mutation screen was performed on an elite hybrid indica rice parental line (Bioseed Research India). Out of all the candidates a mutation from Proline to Serine on position 180 in the NAL1 protein (NAL1 P180S) was picked up as a candidate and selected for testing. In order to phenotype this mutant multiple traits were measured, including yield, test weight, and number of grains per panicle. These traits were measured in both the M4 and M5 generation (homozygous for mutant NAL1 allele) and compared to the wild type control (C4) in a field trial set-up.

Field trials were carried out in Sathupally, Telangana, India (17.210951 , 80.831453) and Mokila, Telangana, India (17.4298237, 78,1901975) using 2 or 1 plots in the rainy season (period of June-Oct) in 2019 and 2020, respectively. The non-mutagenized wild type and the NAL1 P180S mutant with the same genetic background were sown in a field nursery. Thirty days after sowing seedlings were transplanted in the field in 3-row plots at a 20 x 20 cm distance containing 36 plants. The M4 and M5 generations of the NAL1 P180S mutant were used for the field trials in 2019 and 2020, respectively. Presence of the mutation in homozygous state was checked by the specifically designed KASP-assay. In this assay, the following respective target specific sequences were used in allele-specific primers: SEQ ID NO: 5 (NAL1 wild type allele) in combination with SEQ ID NO: 6 (NAL1 mutant type allele). Agronomic traits were assessed at crop maturity and yield-related data were collected after harvest. Phenotyping was performed on 20 plants per trial for all traits. Border plants were excluded from phenotyping. Test weight (TW) was measured by weighing 100 filled randomly selected grains per plant and an average was calculated. These grains were dried to a 13% moisture level prior to weighing. For yield, the average weight of all grains per plant was assessed by weighing the grains after drying to a 13% moisture content. The number of grains per panicle was determined by counting the number of filled grains on the main panicle (on main culm) per plant.

These field trials showed that both in the M4 and M5 generation the test weight, yield and number of grains per panicle was higher for the P180S mutant, compared to the wild type (figure 1A-1 C). These increases correspond with a 14% (M4) and 5% (M5) increase in test weight, a 69% (M4) and 29% (M5) increase in yield and a 57% (M4) and 29% (M5) increase in number of grains per panicle

Claims

1. A mutant NAL1 protein having an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto, wherein the mutant NAL1 protein, when expressed in a plant, results in an increased test weight as compared to a control plant, wherein the control plant is a plant that has the same genetic background and does not express the mutant NAL1 protein.

2. A mutant NAL1 protein according to claim 1 , wherein the mutant NAL1 protein results in increased number of grains per panicle as compared to the control plant according to claim 1 .

3. A mutant NAL1 protein according to claim 1 , wherein the mutant NAL1 protein results in increased yield as compared to the control plant according to claim 1 .

4. The mutant NAL1 protein according to claim 1-3, wherein said amino acid substitution is a proline to serine conversion.

5. The mutant NAL1 protein according to claim 1-4, wherein said mutant NAL1 protein has the sequence of SEQ ID NO: 2.

6. A nucleic acid molecule comprising a sequence encoding the mutant NAL1 protein according to any one of claims 1-5.

7. The nucleic acid molecule according to claim 6, wherein said mutant nucleic acid molecule comprises a coding sequence of SEQ ID NO: 4.

8. A chimeric gene comprising a promotor operably linked to the mutant nucleic acid molecule sequence according to claim 6 or 7.

9. A vector comprising the nucleic acid according to claims 6 or 7, or the chimeric gene according to claim 8.

10. A host cell comprising the mutant NAL1 protein according to any one of claims 1-3, the nucleic acid according to claim 6 or 7, the chimeric gene according to claim 8 and/or the vector according to claim 9, wherein preferably the host cell is a bacterium, cynobacterium, virus, fungi, insect, yeast, or a plant cell, preferably a bacterium wherein said bacterium is Agrobacterium tumefaciens or Escherichia coli.

11 . A method for producing a plant having increased test weight as compared to the control plant of claim 1-3, wherein said method comprises at least the step of introducing a nucleic sequence of claim 6 or 7.

12. The method according claim 11 , wherein said introducing is performed by at least one of: i) mutating an endogenous NAL1 coding sequence by random or targeted mutagenesis; and ii) introgressing into said plant a gene encoding the mutant NAL1 protein according to any one of claims 1-5.

13. The method according to claim 11 , wherein said introducing is performed by inserting a nucleic acid molecule of claim 6 or 7 into a plant cell, wherein said nucleic acid molecule further comprises (i) a promoter functional in a plant cell and (ii) a terminator, wherein said promoter and terminator is operably linked to a sequence encoding the mutant NAL1 protein according to any one of claims 1-5; obtaining a transformed plant cell from the plant cell of step (a), wherein said transformed plant cell comprises said sequence encoding the mutant NAL1 protein; and generating a transgenic plant from said transformed plant cell of step (b), wherein said transgenic plant comprises said sequence encoding the mutant NAL1 protein.

14. A method of screening for plants having an increased test weight, wherein said screening comprises the steps of: providing a heterogenic population of plants; performing a molecular marker assay to identify the presence or absence of: a mutant NAL1 protein having an amino acid substitution on position 180 of SEQ ID NO:

1 or a position analogous thereto; and/or a gene encoding a mutant NAL1 protein having an amino acid substitution on position 180 of SEQ ID NO: 1 or a position analogous thereto; and selecting one or more plants comprising the mutation.

15. A plant comprising a sequence encoding a mutant NAL1 protein according to any one of claims 1-5, wherein preferably said plant is not exclusively obtained by an essential biological method.