Increased herbicide resistance or tolerance in plants

Abstract

The present invention relates to the field of herbicide resistance and tolerance in plants. In particular, the present invention relates to genes involved in conferring increased herbicide resistance or tolerance to plants. In particular, the invention contemplates increased plant resistance or tolerance to herbicides that targets cellulose synthesis in plants.

Description

Increased herbicide resistance or tolerance in plantsTECHNICAL FIELD

[0001] The present invention relates to the field of herbicide resistance and tolerance in plants. In particular, the present invention relates to genes involved in conferring increased herbicide resistance or tolerance to plants. In particular, the invention contemplates increased plant resistance or tolerance to herbicides that inhibits cellulose synthesis in plants.SEQUENCE LISTING

[0002] The present application includes a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety.BACKGROUND ART

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0004] Herbicides that inhibit cellulose synthesis prevent cell division primarily in developing root tips. Such herbicides include benzamides, such as isoxaben (N-[3(1-ethyl-1-methylpropyl)- 5-isoxazolyl]) and are primarily used as a pre-emergence herbicides for controlling broadleaf weeds mainly in the horticultural sector. For example, isoxaben causes strong phytotoxicity to dicotyledonous plants, while most monocotyledonous species display tolerance. Notably, benzamides, such as isoxaben stand out as rare instances where little or no resistant weed species have emerged.

[0005] Although the phytotoxic mechanism is not completely understood, it is known that benzamides, such as isoxaben interfere with cellulose synthesis, where cellulose is the main load-bearing component of plant cell walls, consisting of a long chain of p-1 ,4-linked D-glucose units. In this regard, certain mutations in the cellulose synthase complex proteins can confer isoxaben tolerance. However, isoxaben tolerance introduced by such mutations results in compromised cellulose synthesis and diminished plant growth. As such, cellulose synthase mutations are unsuitable for generating increased resistance ortolerance to benzamides in plants.

[0006] Notwithstanding, benzamides, such as isoxaben, hold untapped potential for the broadacre cropping sector in Australia, which includes large scale crop production. For instance, wild radish (Raphanus raphanistrum) populations are becoming increasingly resistant to several commonly used herbicides from different modes of action while pre-emergent applications of benzamides, such as isoxaben can effectively control this weed.

[0007] Despite its successful use for horticultural crops, benzamides, such as isoxaben have had limited usage in cereal crops, mainly due to its prolonged residual activity and long plant- back periods. This imposes significant constraints on the cultivation of important broadleaf crops, such as canola and legumes, following cereal crops treated with benzamides, such as isoxaben due to their heightened susceptibility to these herbicides. Consequently, the application of benzamides is not feasible in broadleaf crop cultivation sites because of their high sensitivity. Moreover, the use of benzamides such as isoxaben is also very limited in cereal cultivation sites, where subsequent planting of canola or other broadleaf crops such as chickpeas is practised as a common rotation in Australia.

[0008] Previously, increased resistance and tolerance to the benzamide, isoxaben in crop plants has been explored. However, increased resistance and tolerance typically resulted in crop plant growth penalties due to reduced cellulose production. Thus, it was generally believed that it is not possible to develop a non-target site mechanism for benzamide tolerance, for example isoxaben tolerance, and therefore impossible to avoid crop growth penalty.

[0009] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.SUMMARY OF INVENTION

[0010] The present invention relates to increased herbicide resistance or tolerance in plants. Specifically, the present invention relates to increasing plant resistance or tolerance to a herbicide that inhibits cellulose synthesis. Notably, the mechanism of the present invention provides a novel approach that does not rely on a target site mechanism. This surprising approach allows for increased resistance or tolerance to herbicide that inhibits cellulose synthesis in plants without incurring growth penalties / reductions or cellulose reduction. This in turn offers a new and effective means to manage weeds at plant cultivation sites that have previously proven challenging to control. Further, there are currently no or few reported cases of weeds developing resistance to herbicides that inhibit cellulose synthesis, such as isoxaben. In contrast, other herbicides, such as glyphosate commonly face varying levels of resistance in weeds.

[0011] Accordingly, in a first aspect, the present invention provides a plant or plant part that exhibits a reduction or loss of Ferredoxin-NADP+ oxidoreductase-like (FNRL) function and thereby exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0012] In another aspect, the present invention provides a plant or plant part that exhibits a reduction or loss of FNRL function and thereby exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, wherein the plant or plant part is not Arabidopsis thaliana.

[0013] In another aspect, the present invention provides a plant or plant part that exhibits a reduction or loss of FNRL function wherein the reduction or loss of FNRL function has been shown to confer to the plant or plant part, increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0014] In certain embodiments, the plant or plant part comprises a mutated fnrl gene, wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the plant or plant part exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0015] In certain embodiments, the reduction or loss of FNRL function results from transcriptional FNRL gene silencing. In other embodiments, the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing, this includes but is not limited to gene silencing produced by RNA interference (RNAi).

[0016] In certain embodiments, the herbicide is a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0017] In certain embodiments, the herbicide is isoxaben.

[0018] In certain embodiments, the plant or plant part according to the invention is a crop plant or a crop plant part, such as a dicotyledonous plant or plant part, including for example a broadleaf crop plant or plant part.

[0019] In certain embodiments, the crop plant or crop plant part is an oil crop, a legume, sugar beet, a fruit plant, potato or a plant or plant part of the Brassica genus.

[0020] In certain embodiments, the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress.

[0021] In certain embodiments, the legume is selected from lentil, pea, chickpea, peanut or bean plants or plant parts, which for example include broad bean, soybean, green bean, lima bean, kidney bean, navy bean, cannellini bean, and adzuki bean plants or plant parts.

[0022] In certain embodiments, the plant or plant part of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

[0023] In certain embodiments, the plant part is a seed, pollen, ovule, pod, flower, leaf, root or stem tissue.

[0024] In another aspect, the present invention provides a seed, wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, or a plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, such as a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0025] In another aspect, the present invention provides a seed, wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, or a plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, such as a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben, wherein the seed is not Arabidopsis thaliana.

[0026] In another aspect, the present invention provides a seed, wherein the seed exhibits a reduction or loss of FNRL function and wherein the reduction or loss of FNRL function has been shown to confer to the seed or plant or plant part produced from the seed, increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis. In certain aspects the herbicide that inhibits cellulose synthesis includes a benzamide, for example chlorthiamid, dichlobenil, flupoxam or isoxaben.

[0027] In certain embodiments, the seed of the invention comprises a mutated fnrl gene, wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the seed, a plant or plant part produced from the seed exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0028] In certain embodiments, in the seed of the invention the reduction or loss of FNRL function results from transcriptional FNRL gene silencing. In certain embodiments, in the seed of the invention the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing, including but not limited to RNA interference (RNAi).

[0029] In a further aspect, the present invention provides a method of controlling undesired vegetation at a plant cultivation site, the method comprising the step of applying to the site an effective amount of a herbicide that inhibits cellulose synthesis, and wherein the plant cultivation site is used for cultivating a plant according to the invention. In certain embodiments, the plant cultivation site is used for cultivating a plant that is resistant to the herbicide that inhibits cellulose synthesis followed by cultivating a plant according to the invention. In certain embodiments, the plant that is resistant to the herbicide that inhibits cellulose synthesis is a cereal crop. In certain embodiments, in the method of the invention, the herbicide that inhibits cellulose synthesis is a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0030] In yet a further aspect, the present invention provides a method of producing a plant or plant part that exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis the method comprising the step of reducing or abrogating FNRL function in the plant or plant part, wherein reduction or loss of FNRL function increases resistance or tolerance to a herbicide that targets cellulose synthesis.

[0031] In a further aspect, the present invention provides a method of producing a plant or plant part that exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis the method comprising the step of reducing or abrogating FNRL function in the plant or plant part, wherein reduction or loss of FNRL function increases resistance or tolerance to a herbicide that targets cellulose synthesis, wherein the plant or plant part is not Arabidopsis thaliana.

[0032] In a further aspect, the present invention provides a plant or plant part when produced by the method of the invention.

[0033] In certain embodiments of the method of the invention, the herbicide that inhibits cellulose synthesis is a benzamide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben. In certain embodiments of the method of the invention, the plant or plant part comprises a mutated fnrl gene, wherein the mutated fnrl gene results in a reduction or loss of FNRL function. In certain embodiments of the method of the invention, the reduction or loss of FNRL function results from transcriptional FNRL gene silencing. In certain embodiments of the invention, the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing, including but not limited to RNA interference (RNAi).

[0034] In certain embodiments of the method of the invention, the herbicide is isoxaben.

[0035] In certain embodiments of the method of the invention, the plant is a crop plant or a crop plant part, including but not limited to a broadleaf crop. In certain embodiments thebroadleaf crop includes but is not limited to an oil crop, a legume, sugar beet, a fruit plant, potato or a plant of the Brassica genus. In certain embodiments, the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress. In certain embodiments, the legume is selected from lentil, pea, chickpea, peanut or bean plants. In certain embodiments, the bean plant is selected from broad bean, soybean, green bean, lima bean, kidney bean, navy bean, cannellini bean, and adzuki bean plants. In certain embodiments, the plant of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

[0036] The unexpected advancement of the present invention facilitates the use of a herbicides that inhibit cellulose synthesis in plants and plant parts, such as but not limited to crop plants and crop plant parts. In particular, the present invention has surprisingly developed a non-target site approach for increasing resistance or tolerance to a herbicide that targets cellulose synthesis in a plant or plant part, such as a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, furoxan and isoxaben, that avoids disadvantages, such as plant growth penalty.

[0037] The present invention addresses a crucial market need by offering an alternative choice for herbicides. As weeds increasingly develop resistance to commonly used herbicides, including glyphosate, the availability of a new herbicide tolerance trait becomes essential. Farmers and the agricultural industry require effective tools to combat these resistant weeds and maintain crop productivity. Thus, this innovation provides an economically valuable solution by expanding the range of herbicides available and helping address the growing challenge of herbicide-resistant weeds in agriculture.

[0038] Thus, the present invention, in certain embodiments, relates to effective weed control, particularly for herbicide resilient weeds such as but not limited to wild radish, that have developed resistance to known herbicides.BRIEF DESCRIPTION OF THE FIGURES

[0039] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying Figures in which:

[0040] Figure 1 : fnrl mutants show resistance to isoxaben. A) Representative images of ten-day-old Arabidopsis thaliana seedlings (wild-type Col-0, or fnrl mutants 1 or 2) grown on half MS medium supplemented with 1 % sucrose, 0.8% agar, and either 2.5nM isoxaben or Mock treatment. B) Quantification of primary root length in ten-day-old Arabidopsis thaliana seedlings grown under 2.5nM isoxaben or Mock conditions. C) Cellulose quantification in the roots of seedlings in B. Col-0 roots show reduced cellulose in response to isoxaben while fnrl-1 and fnrl- 2 mutants do not show any decrease in cellulose. AIR: Alcohol insoluble residue. D) Representative images of four-week-old Arabidopsis thaliana rosettes from wild-type and fnrl mutant plants grown in soil. E) Root length phenotype of indicated genotypes grown on Mock and 2.5nM isoxaben media for 10 days. pFNRL:FNRL:GFP plants complement the isoxaben tolerance phenotype of fnrl-1 mutants. This shows that by putting functional FNRL gene back in the fnrl mutants the sensitivity to isoxaben is restored. Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01 ; ***, P < 0.001 and ; ****, P < 0.0001) according to Student’s t test. The graphics and statistical analysis were generated using the ggpubR package in R.

[0041] Figure 2: FNRL mutations show resistance / tolerance to isoxaben at molecular level A) Representative images demonstrating Reactive Oxygen Species (ROS) activity through H2DCFDA signal in the root tips of ten-days-old Arabidopsis thaliana seedlings exposed to 2.5nM isoxaben are presented. The genotypes are labelled. The data shows that the fnrl mutants exhibit a diminished signal as compared to wild-type plants. B) Quantification of the results presented in panel A. C) Phloroglucinol signal intensity indicating lignin accumulation in roots of ten-days-old Arabidopsis thaliana seedlings exposed to 600nM isoxaben. Genotypes are indicated, fnrl mutants show a reduced response to isoxaben treatment as compared to wild type. D) Quantification of the results presented in panel C. E) Gene expression levels of the isoxaben marker gene TCH4 determined by qPCR in ten-days-old seedlings exposed to 2.5nM isoxaben. The results from three biological replicates are shown. Mean ± SD is presented. Statistical significance is denoted by asterisks (*, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 0.0001) according to Student’s t-test. The graphics and statistical analysis were performed using the ggpubR package in R.

[0042] Figure. 3 Transcriptomic Profiling Reveals Impaired Response of fnrl Mutants to Isoxaben Treatment A) Principal component analysis (PCA) plot showing the separation of wildtype plants treated with isoxaben from mock-treated plants. Genotypes are indicated. B) Number of differentially expressed genes (DEGs) identified in response to isoxaben treatment in wild-type plants and fnrl-1 and fnrl-2 mutants using stringent criteria (log2FoldChange >1 or <-1 and padj<0.05). Wild-type plants exhibited regulation of 444 genes, while fnrl-1 and fnrl-2 mutants showed regulation of only 4 and 5 genes, respectively.

[0043] Figure 4 Mitochondria Retrograde Signalling Response is activated in fnrl MutantsA). Expression levels of genes belonging to the mitochondrial dysfunction stimulon, which share a common cis-element for the transcriptional response to mitochondrial stress, in fnrl mutant lines and wild type. Gene expression is presented as Iog2 fold change. B). Gene expression levels of mitochondria-nucleus retrograde signalling marker genes measured by qPCR in ten- days-old seedlings treated with 2.5nM isoxaben or mock. The results are shown for three biological replicates. Marker genes and genotypes are indicated. Mean ± SD is presented.Statistical significance is indicated by asterisks (*, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 0.0001) based on Student’s t-test.

[0044] Figure 5 ANAC017 transcription factor plays a key role in fr) / 7-mediated isoxaben tolerance. A) Representative images of ten-day-old seedlings grown on half MS medium supplemented with 1% sucrose, 0.8% agar, and either 2.5nM isoxaben or Mock treatment.B) Quantification of primary root length in ten-day-old seedlings grown under 2.5nM isoxaben or Mock conditions. Mean ± SD is presented. Statistical significance is indicated by asterisks (*, P < 0.05; **, P < 0.01 ; ***, P < 0.001 ; ****, P < 0.0001) based on Student’s t-test.DESCRIPTION OF EMBODIMENTS

[0045] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0046] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising" and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

[0047] Other definitions for selected terms used herein may be found within the detailed description of the invention and apply throughout. Unless otherwise defined, all other scientific and technical terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the invention belongs.

[0048] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0049] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0050] The term “crop” or “crop plant” or the like refers to any plant that can be grown and harvested for profit or subsistence. This includes, for example, food crops, feed crops, fiber crops, oil crops, ornamental crops, and industrial crops. Specific examples of crop plants include, but are not limited to, wheat, corn, feed grains, rice, soybean and oil crops, such as canola, vegetables and pulses, potato, sugar cane, cotton and fruit, nut trees, including but not limited to almond, Brazil nut, cashew nut, hazelnut, macadamia, pecan, pine nut, pistachio, walnut, and peanut. The term “crop” or “crop plant” or the like does not include Arabidopsis thaliana.

[0051] Broadleaf crops include but are not limited to soybean, canola, sunflower, field pea, dry bean, sugar beet, potatoes, flax, lentil, chickpea, fruit trees or vines such as but not limited to, grape, orange and tangerine, pineapple, melon, berry, apricot, mango, plum, lychee and rambutan, durian, banana, tomato, apple, pear, coffee bean, peach, and cherry.

[0052] Plants of the invention may pass on the nucleotide of the invention to progeny. As used herein, “progeny” includes any plant, seed, plant cell, and / or plant part, including regenerable plant part comprising the fnrl mutant resulting in a reduction or loss of FNRL function derived from an ancestor plant and / or a nucleotide having at least one of the nucleotide sequences of the invention.

[0053] The term “plant part” includes any part of a plant that may be used to regenerate a plant and includes, but is not limited to, seed, pollen, ovule, pod, flower, leaf, root or stem tissue.

[0054] In the context of the present invention, the term “a reduction or loss of FNRL function” means that the fnrl gene has been altered by any means, for example known mutation or gene editing methodologies known to the skilled person. These well-known methods include using Agrobacterium tumefaciens to introduce genetic modifications into plants and plant cells, via Agrobacterium tumefaciens- mediated transformation or AtMT. For example, the movement of transfer DNA (T-DNA) from a plasmid to the genome of a plant genome. Agrobacterium tumefaciens-meditated transformation techniques are well described in the scientific literature. See, for example Horsch et al, Science, 233:496-498, 1984, and Fraley et al, Proc. Natl. Acad.Sci. USA, 90:4803, 1983. Other gene delivery systems can include a “gene gun” which for example fires gold particles carrying the foreign DNA into plant cells. Microinjection can also be used to introduce foreign DNA into plant cells.

[0055] Methods for the modification of plant genes are also well known and include for example, CRISPR, transcription activator-like effector nucleases (TALEN) gene editing, Zinc finger gene editing, meganucleases, and homology recombination methods, that results in a reduction or loss of function from the FNRL protein. For example, delivering gene modification agents, such as CRISPR, TALEN or ZINC finger agents to a plant or plant cell can be carried out by known methods, such as, but not limited to agroinfiltration.

[0056] A reduction or loss of FNRL function includes an fnrl gene that has been modified so that a functional fnrl RNA transcript is not produced. This means that either an fnrl transcript is not produced or, alternatively, the fnrl transcript is incapable of expressing a functional FNRL protein. Such modifications to the fnrl gene can, for example, include frameshift insertion(s) or deletion(s), nucleotide substitutions, insertion of gene fragments or complete or partial removal of the fnrl gene. Loss of FNRL function also includes, for example, the expression of a protein from an fnrl RNA transcript, where the protein is incapable of performing the function associated with wild-type FNRL. A reduction or loss of function can also include transcriptional gene silencing and in the context of the present invention, transcriptional FNRL gene silencing. This can include reduction or loss of FNRL function from post-transcriptional FNRL gene silencing, including but not limited to gene silencing produced by RNA interference (RNAi), which would be known to the skilled person.

[0057] In the context of the present invention, the term “undesired vegetation” includes a plant having little or no value that grows on cultivated land to the exclusion or detriment of a desired crop. The skilled person would understand that the term “undesired vegetation” includes for example, weeds, which is a well understood term in the relevant art. Undesired vageation may include, but are not limited to, Polygonum (Polygonum) species as Rhizoma fagopyri cymosi (Polygonum convolvolus), Amaranthus species are as kitchen garden (Amaranthus retroflexus), Chenopodium species are as common lamb's-quarters (Chenopodium album L.), chrysanthemum harvest spp (Sida) species are as bur (Sida spinosa L.), Ambrosia (Ambrosia) species are as common ragweed (Ambrosia artemisiifolia), sting luxuriant Chrysanthemum (Aca nth os perm urn) species, Anthemis (Anthemis) species, Atriplex (Atriplex) species, Cirsium (Cirsium) species, Japanese bearbind belongs to (Convolvulus) species, Conyza (Conyza) species, Cassia (Cassia) species, Commelina communis belongs to (Commelina) species, Datura (Datura) species, Euphorbia (Euphorbia) species, Geranium (Geranium) species, ox achyranthes chrysanthemum spp (Galinsoga) species, morning glory (Ipomoea (Ipomoea)species), lamium (Lamium) species, mallow (Malva) species, Matricaria (Matricaria) species, Sistrurus (Sysimbrium) species, Solarium species, Xanthium (Xanthium) species, Veronica species, Viola (Viola) species, common chickweed (Stellaria media), piemarker (Abutilon theophrasti), large fruit sesbania (Hempsesbania (Sesbania exaltata Cory)), have apart from single tree mallow (Anoda cristata), beggar-ticks (Bidens pilosa), field mustard (Brassica kaber), shepherd's purse (Capsella bursa-pastoris), cornflower (Centaurea cyanus), yellow weasel hemp nettle (Galeopsis tetrahit), clearvers, sunflower (Helianthus annuus), desmodium (Desmodium tortuosum), kochia scoparia (Kochiascoparia), mountain wood blue (Mercurialis annua), true forget menot (Myosotis arvensis), corn poppy (Papaver rhoeas), corn radish (Raphanus raphanistrum), saltwort (Salsolakali), wild mustard (Sinapis arvensis), corn sow thistle (Sonchus arvensis), penny cress (Thlaspiarvense), Aztec marigold (Tagetes minuta), ink clover (Richardia brasiliensis), Arabidopsis thaliana. etc.

[0058] Sequences relevant to the present disclosure, including sequences included in the constructs referred to in the Examples are listed below.

[0059] Arabidopsis thaliana Ferredoxin-NADP+ oxidoreductase-like (FNRL) gene. At1G15140 protein sequence.MSTLPFAPSVTHAHFSHSLS PMFILRHLPLTRHLRLSRNNRVASWSAAAVRQDASLWTPAPLSLIESAA ESLFHI SIDI SNAPDLVASYTRPGQYLQIRVPDVEKPSFMAIASPPSLASSRGAFEFLVKSIAGSTAEIL CGLKKGETVELS SVMGNGFNIDLIDPPEEYPTVLI FATGSGI SPI RSLIESGFGADRRSDVRLYYGARNL NRMAYQEKFKEWESAGVKWPVLSQPDDGWKGETGYVQAAFARAKQLSAPKATGAVLCGQKQMAEEITSM LVADGVSNDKLLKNF (SEQ ID NO: 2)

[0060] Arabidopsis thaliana Ferredoxin-NADP+ oxidoreductase-like (FNRL) gene. At1G15140 nucleotide sequence ATGTCCACTCTTCCTTTCGCGCCTTCTGTCACCCATGCGCACTTTAGCCACTCCCTCTCCCCCATGTTTATCCTGCGCCATCTCCCTCTTACACGCCATCTCCGTTTATCCCGAAATAACCGCGTCGCTTCCGTTGTATC CGCCGCCGCCGTTCGCCAGGACGCTTCCCTCTGGACTCCAGCTCCTCTCTCTCTAATCGAATCAGCTGCA GAATCGCTTTTCCACATCTCAATCGACATTTCAAACGCTCCGGATCTCGTAGCTTCGTACACGAGACCCG GTCAGTATCTCCAGATCCGTGTTCCTGATGTTGAGAAGCCTTCTTTTATGGCAATCGCTTCTCCTCCTTC CTTGGCGTCTTCTCGTGGTGCTTTCGAATTCTTGGTCAAGAGCATCGCTGGTTCTACCGCGGAGATTCTC TGCGGGTTGAAGAAAGGAGAGACCGTTGAGCTTAGCTCTGTGATGGGTAATGGTTTCAATATCGATCTGA TTGATCCTCCTGAGGAATATCCCACCGTTTTGATTTTCGCCACTGGATCTGGAATTAGCCCCATCCGCTC ACTAATCGAATCAGGATTTGGCGCTGATAGAAGATCTGACGTAAGACTCTATTACGGGGCTAGGAACCTG AATAGGATGGCTTACCAGGAAAAGTTTAAAGAGTGGGAATCAGCGGGTGTCAAAGTTGTGCCGGTATTATCACAGCCAGATGATGGGTGGAAAGGGGAAACTGGATATGTGCAGGCTGCTTTTGCAAGGGCTAAACAGCTATCAGCTCCCAAGGCCACGGGAGCGGTGCTGTGCGGACAGAAACAAATGGCTGAGGAGATAACTTCAATG CTTGTAGCTGATGGAGTCTCAAATGACAAGCTGCTCAAAAACTTTTGA (SEQ ID NO: 1)

[0061] There is a high degree of conservation of the fnrl gene across the plant kingdom, see Table 1 below. As such, there is a strong expectation that a reduction or loss of FNRL function in any plant that is sensitive to herbicide that inhibits cellulose synthesis will result in increased resistance, or tolerance, to the herbicide that inhibits cellulose synthesis. For example, benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben, and in particular isoxaben. Moreover, the skilled person would readily be able to identify and modify the fnrl gene of any plant using known molecular biology methodologies and to obtain a plant that exhibits a reduction or loss of FNRL function and thereby exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, for example a benzamide, such as isoxaben.

[0062] Homologs of the fnrl gene were identified in various monocot species, including Oryza sativa (rice) and Zea mays (corn), as well as dicot species such as Solanum lycopersicum (tomato), Populus trichocarpa (Black cottonwood), Brassica rapa, and capsicum annum.

[0063] The person skilled in the art would be familiar with a variety of known molecular biology methodologies that could be used to produce a plant or plant part exhibiting a reduction or loss of FNRL function. These methodologies include, but are not limited to, plant gene editing methodologies such as involving Agrobacterium tumefaciens and CRISPR / Cas gene editing, transcription activator-like effector nucleases (TALEN) gene editing, Zinc Finger gene editing, meganucleases, and homology recombination methods. The skilled person would understand that known gene editing methods could be used to create a reduction or loss of FNRL function by, for example, the introduction or deletion of nucleotide mutations, such as frame shift mutations, nucleotide substitutions, the introduction of gene segments into the fnrl gene or complete or partial removal of the fnrl gene. A reduction or loss of function can also include transcriptional gene silencing and in the context of the present invention, transcriptional FNRL gene silencing. This can include reduction or loss of FNRL function from post-transcriptional FNRL gene silencing, including but not limited to gene silencing produced by RNA interference (RNAi), which would be known to the skilled person. The skilled person would also understand that a reduction or loss of fnrl gene function can also include transcriptional gene silencing and in the context of the present invention, transcriptional FNRL gene silencing. This can include reduction or loss of FNRL function from post-transcriptional FNRL gene silencing, including but not limited to gene silencing produced by RNA interference (RNAi), which would be known to the skilled person.

[0064] It would be clear to the skilled person that given the high degree of conservation of the fnrl gene across the plant kingdom, producing any plant exhibiting a reduction or loss of FNRL function and thereby an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, would be readily practical and realizable using known methodologies and the disclosure of the present invention. For example, based on the present disclosure, the skilled person could readily produce a crop plant or plant part that exhibits a reduction or loss of FNRL function and thereby an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, for example isoxaben.

[0065] The generation of plants, such as crop plants, that exhibit increased resistance or tolerance to herbicide that inhibits cellulose synthesis has the potential to significantly expand the market for known and hitherto underutilized herbicides, such as benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam and isoxaben. This represents a substantial economic opportunity and could contribute to more effective weed control strategies in agriculture.

[0066] The inventors, using a genetic screening process involving Arabidopsis thaliana mutants, have surprisingly identified a gene, Ferredoxin-NADP(+) oxidoreductase-like (FNRL, At1g15140), that, when mutated, imparts increased tolerance to herbicide that inhibits cellulose synthesis such as benzamide herbicide, for example isoxaben. This enhanced resistance or tolerance can be characterized by a reduction in primary root stunting, lower accumulation of reactive oxygen species, and a decreased occurrence of ectopic lignification.

[0067] Notably, when exposed to the benzamide herbicide, isoxaben, the roots of fnrl mutants did not exhibit the typical reduction in cellulose levels, suggesting that FNRL functions upstream of isoxaben-induced cellulose inhibition.

[0068] The inventors have also revealed that the isoxaben detoxification process mediated by fnrl is dependent on the transcription factor ANAC017, which appears to serve as a regulator of the mitochondria-retrograde signalling pathway associated with a plant’s ability to cope with isoxaben-related stress.

[0069] Transcriptomic analysis of fnrl mutant roots revealed a significantly diminished response to isoxaben treatment. This supports a situation in which fnrl mutants enable increased isoxaben resistance or tolerance without detrimental effects on plant cellulose levels and growth. This unexpected discovery has promising implications for herbicide that inhibits cellulose synthesis including benzamide herbicide-tolerant plants including, but not limited to, crops and weed management strategies.EXAMPLES

[0070] The present invention is not to be limited in scope by any of the specific embodiments described herein. Particular embodiments of the invention are further described by the following non-limiting examples with reference to the accompanying figures. Although the examples described for these limitations are predominantly in the context of specific plants, the relevance relates to a novel strategy that can be broadly and readily applied in range of crop plants.

[0071] Any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.

[0072] The inventors have identified fnrl as a new gene involved in the response to herbicide that inhibits cellulose synthesis, for example a benzamide, such as isoxaben. Reduced function or loss-of-function FNRL mutants displayed increased resistance or tolerance to herbicide that inhibits cellulose synthesis, a benzamide, isoxaben, suppressing the typical root stunting and swelling response (Figure. 1). Furthermore, FNRL influenced lignin regulation and the transcriptional regulation of isoxaben-responsive marker genes (Fig. 2B, 2E). These findings indicate that the function of FNRL is essential for orchestrating the well-established responses associated with the Ceil Wall Damage (CWD) response, triggered by Cellulose Biosynthesis Inhibitors (CBIs) or genetically induced cellulose deficiency.

[0073] RNA sequencing analysis showed that, in contrast to wild type, fnrl mutants were largely unresponsive to isoxaben. Comparing gene expression data between wild-type and fnrl mutants under mock conditions revealed significant differential regulation of numerous genes.

[0074] In conclusion, the identification of FNRL as a key player in conferring isoxaben increased resistance or tolerance provides valuable insights and permits the generation of isoxaben resistant plants.Example 1 : FERREDOXIN-NADP(+) OXIDOREDUCTASE-LIKE (fnrl) mutants confer increased resistance, or tolerance to isoxaben.

[0075] As a strategy to identify genes potentially associated with isoxaben tolerance, the inventors screened a selection of Transfer DNA (T-DNA ) mutants of Arabidopsis thaliana in agar media containing isoxaben. Isolated mutant plants showed tolerance to isoxaben. Mutant FNRL Arabidopsis thaliana plants were grown vertically on half Murashige and Skoog (MS)media supplemented 2.5 nM isoxaben. From this screen, it was found that a T-DNA line affecting FNRL (SALK_039715, fnrl-1. The fnrl-1 mutant carries a T-DNA insertion in the first exon) conferred isoxaben compared to the wild type (Col-0) (Fig. 1A and B). This was shown as normal plant and root growth in fnrl mutants grown with isoxaben treatment. The increased resistance or tolerance phenotype was confirmed using a second mutant allele of fnrl (SALK_048627, fnrl-2 The fnrl-2 mutant carries an insertion in the first intron.) and fnrl-1 complementation with FNRL:GFP, which restored FNRL function, and which also restored isoxaben sensitivity, (Figure 1 E). This confirms FNRL is the causal gene responsible for the increased resistance or tolerance to isoxaben (Fig. 1A and B).

[0076] The rosette growth of the fnrl mutants was similar to that of the wild type, suggesting that FNRL does not affect overall plant growth (Fig. 1 D). In other words, fnrl mutation surprisingly does not result in a growth penalty.Example 2: FNRL plays a crucial role in isoxaben-induced cell wall damage response.

[0077] Typically, isoxaben treatment leads to the accumulation of Reactive Oxygen Species (ROS) and lignin in roots. Therefore, the inventors examined these processes in fnrl mutants in response to isoxaben treatment. We cultivated plants on half-strength MS plates supplemented with 2.5 nM isoxaben or mock for ten days. To quantify ROS levels, we utilised the cellpermeant indicator 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma D6883). The fnrl mutants showed reduced ROS presence in the roots compared to the wild type under normal conditions. Additionally, when wild-type plants were treated with isoxaben, there was a significant increase in H2DCFDA signal intensity compared to the mock treatment, indicating higher ROS accumulation (Fig. 2A and B). However, this isoxaben-induced elevation of H2DCFDA signal was noticeably reduced in both fnrl mutant alleles (Fig. 2A and B). These results indicate that FNRL plays a role in regulating ROS accumulation both under normal conditions and during isoxaben treatment.

[0078] Isoxaben-induced CWD leads to alterations in cell wall biochemistry, characterised by ectopic lignin and callose deposition. We examined lignin accumulation in roots using phloroglucinol staining in Arabidopsis thaliana ten-day-old seedlings grown on half-MS liquid media. In comparison to the wild-type, we observed reduced phloroglucinol staining in the root elongation zone of fnrl mutant seedlings, indicating a decrease in ectopic lignification in response to isoxaben (Fig. 2C and D). Thus, the wild type plants show lignification, which is part of isoxaben-induced cell wall damage, upon treatment of isoxaben, whereas the fnrl mutants did not. This indicates that isoxaben is unable to act on the cellulose synthesis pathway in the fnrl mutants.

[0079] To investigate whether FNRL activity is required for the transcriptional regulation of specific marker genes in response to isoxaben-induced CWD, we next performed quantitative real-time polymerase chain reaction (qPCR) experiments using RNA isolated from ten-day-old roots. We focused on TOUCH4 (TCH4), which is a marker gene that responds to isoxaben- induced CWD. We found that the isoxaben-induced upregulation of TCH4 was notably attenuated in the fnrl mutants when compared to the wild type (Fig. 2E). Taken together, FNRL appears to promote CWD-responses when seedling roots are exposed to isoxaben.Example 3: Isoxaben-induced transcriptomic changes are attenuated in fnrl mutants.

[0080] To investigate whether other aspects of isoxaben-induced changes are evident in Arabidopsis thaliana fnrl mutants, the inventors performed RNA sequencing analyses on root tips 48 hours after isoxaben treatment. Ten-day-old plants were grown on half MS medium and then transferred to plates containing 2.5 nM isoxaben or mock for 48 hours. RNA was extracted from 2mm sections of root tips, followed by RNA sequencing, which revealed distinct gene regulation patterns in response to isoxaben treatment between wild-type and fnrl seedlings.

[0081] Principal component analysis (PCA) plots demonstrated a clear separation of wild-type seedlings treated with isoxaben compared to mock (Fig. 3A). By contrast, fnrl mutants clustered together regardless of the treatment (Fig. 3A). Differentially expressed gene (DEG) analysis showed 444 differentially regulated genes in wild-type plants upon isoxaben treatment, whereas fnrl-1 and fnrl-2 mutants showed only five and four genes, respectively (Fig. 3B). These results indicate that fnrl mutants do not respond to isoxaben at the transcriptome level.

[0082] FNRL is highly conserved across plant kingdom. Phylogenetic analysis of the FNRL protein across major Viridiplantae lineages reveals its high conservation, typically existing as a single copy gene in dicotyledons except for recent gene duplication events in species like Populus trichocarpa and Malus domestica. In dicotyledonous plants, the sequence similarity exceeds 80%, while monocotyledonous plants exhibit at least one homologue with a remarkably high similarity approaching 80%. The FNRL gene maintains high conservation in mosses and liverworts, with a similarity above 70%. Chiorophytes, however, exhibit the most distant homologues of FNRL, with a similarity between 55 and 62%. These findings show that the FNRL sequence is remarkably conserved throughout the Viridiplantae, suggesting functional conservation across diverse plant species. This is set out in Table 1 below.Table 1 FNRL homologues and their protein sequence similarity to FNRL protein.Example 4 Materials and methodsPlant material and growth conditions

[0083] The fnrl T-DNA insertion mutant Arabidopsis thaliana lines fnrl-1 (SALK039715) and fnrl-2 (SALK048627), were obtained from the Arabidopsis Biological Resource Center (ABRC, https: / / abrc.osu.edu / ). To conduct the experiments involving isoxaben (ISX) and mock treatments (equal volume of ethanol), a vertical agar plate method was employed. The standard media composition consisted of a half-strength MS Basal Salt mixture, 0.5% (w / v) MES (Sigma- Aldrich), 0.5% (w / v) Sue (Sigma-Aldrich), and 0.8% (w / v) Difco granulated agar. For isoxaben treatment, the standard media was supplemented with 2.5 nM isoxaben (Sigma, 36138). Arabidopsis seeds were subjected to surface sterilisation using chlorine gas for three hours. Subsequently, they were resuspended in 0.1% (w / v) agarose, stratified at 4°C for two days, and then sown in a single row on 10-cm-square plates sealed with 3M Micropore tape. The plates were vertically arranged in racks within a growth chamber, which maintained a 16-hour light / 8- hour dark cycle, a light intensity of 120 E mA2 sA1 , and temperature conditions of 23 °C (day) / 19°C (night). The relative humidity inside the chamber was set to 60%.Quantitative RT-PCR

[0084] Upon harvesting, the roots were rapidly frozen in liquid nitrogen and subsequently ground using the Invitrogen tissue lyser. Approximately 100 to 150 mg of the resulting powder was employed to extract total RNA, utilising an RNeasy Plant Mini Kit (QIAGEN). To eliminate any potential DNA contamination, on-column DNase treatment was conducted using an RNase-free DNase kit (QIAGEN). For reverse transcription, 500 ng of RNA per sample was utilised in conjunction with Superscript III reverse transcriptase from Invitrogen. Subsequently, quantitative PCR was performed using the SYBR select master mix from Invitrogen. The primers employed for the PCR analysis are described in the supplemental data.ROS and lignin staining

[0085] To assess intracellular ROS accumulation in root meristems, H2DCFDA fluorescent stain (Sigma, D6883) was employed following established protocols (Juarez et al., 2015). Arabidopsis seeds were cultivated on square plates containing half-strength MS medium supplemented with 1 % sucrose, 0.8% agar and 2.5 nM isoxaben or mock treatment. After stratification at 4°C for two days, the seedlings were grown for ten days at 22°C under long-day conditions with a 10-degree incline. For imaging the seedlings were treated with 100 pM H2DCFDA staining solution for 5 minutes and then rinsed with water. Images were taken on a Leica M205 FCA stereo fluorescence microscope with a GFP ET filter. Staining for lignin (using phloroglucinol) was conducted following the previously established protocol (Gigli-Bisceglia et al., 2018). Images of seedlings exhibiting phloroglucinol staining were taken on a Leica M205 FCA stereo fluorescence microscope. Micrographs of ROS and lignin staining were quantified using Imaged software (Rueden et al., 2017). For quantification, a defined region of interest (ROI) positioned 500pm above the root tip (excluding the root cap) was selected for all samples.RNA sequencing and data analysis

[0086] Seedlings were grown for eight days on half strength MS media and then transferred to mock- or 2.5 nM isoxaben-containing plates. Two mm root tips were harvested after 48 hours of treatment. Three biological replicates were used for each genotype and treatment. The replicates consisted of 2 mm root tip sections obtained from more than one hundred pooled seedlings from at least two independent plates. Total RNA was extracted from these replicates. The TruSeq Stranded mRNA Library Prep Kit was employed to generate RNA-seq libraries, which were subsequently sequenced on an Illumina NextSeq500 instrument. The resulting reads were 70 base pairs in length. To ensure the quality and reliability of the datavFastQC software (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) was used for quality control assessment of the data to verify its integrity. Prior to alignment, the RNA-Seq reads were trimmed using trimmomatic (Bolger et al., 2014). The trimmed reads were aligned to the Arabidopsis TAIR10 genome employing STAR (v2.5.3) (Dobin et al., 2013). For each gene, the tool htseq-count was used to count the mapped reads (Anders et al., 2015). The HTseq count output served as the input for DESeq2 analysis to identify differentially expressed genes (Love et al., 2014). Gene clustering was performed with clusterprofiler package in R (Wu et al., 2021).Heatmaps were drawn using a complex-heatmap package (Gu, 2022) and the GO enrichment was performed on geneontology.org.Gene conservation analysis

[0087] Gene conservation analyses was conducted by utilizing the FNRL amino acid sequence to search for closest homologues in Phytozome (https: / / phytozome-next.jgi.doe.gov / ) from different species spread across major lineages of Viridiplantae. The similarity percentage and score for these sequences were retrieved from Phytozome.SPECIFIC EMBODIMENTS

[0088] 1. A plant or plant part that exhibits a reduction or loss of FNRL function and thereby exhibits an increased resistance or tolerance to a herbicide that inhibits cellulose synthesis.

[0089] 2. The plant or plant part according to embodiment 1 wherein the plant or plant part comprises a mutated fnrl gene, wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the plant or plant part exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0090] 3. A plant or plant part that exhibits a reduction or loss of FNRL function and thereby exhibits an increased resistance or tolerance to a herbicide that inhibits cellulose synthesis, wherein the plant or plant part is not Arabidopsis thaliana.

[0091] 4. A plant or plant part that exhibits a reduction or loss of FNRL function wherein the reduction or loss of FNRL function has been shown to confer to the plant or plant part, increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0092] 5. The plant or plant part according to embodiment 1 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

[0093] 6. The plant or plant part according to embodiment 1 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

[0094] 7. The plant or plant part according to embodiment 6 wherein the post- transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

[0095] 8. The plant or plant part according to any one of embodiments 1 to 6 wherein the herbicide is a benzamide herbicide.

[0096] 9. The plant or plant part according to any one of embodiments 1 to 6 wherein the herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0097] 10. The plant or plant part according to any one of embodiments 1 to 9 wherein the herbicide is isoxaben.

[0098] 11 . The plant or plant part according to any one of embodiments 1 to 10 wherein the plant is a crop plant or a crop plant part.

[0099] 12. The plant or plant part according to any one of embodiment 11 wherein the crop plant or crop plant part is a dicotyledonous plant or plant part.

[0100] 13. The plant or plant part according to embodiment 11 or 12 wherein the crop plant or crop plant part is a broadleaf crop plant or plant part.

[0101] 14. The plant or plant part according to any one of embodiments 11 to 13 wherein the crop plant or crop plant part is an oil crop, a legume, sugar beet, a fruit plant, potato or a plant or plant part of the Brassica genus.

[0102] 15. The plant or plant part according to embodiment 14 wherein the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress.

[0103] 16. The plant or plant part according to embodiment 14 wherein the legume is selected from lentil, pea, chickpea, peanut or bean plants or plant parts.

[0104] 17. The plant or plant part according to embodiment 16 wherein bean plant or plant part is selected from broad bean, soybean, green bean, lima bean, kidney bean, navy bean, can nellini bean, and adzuki bean plants or plant parts.

[0105] 18. The plant or plant part according to embodiment 14 wherein the plant or plant part of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

[0106] 19. The plant or plant part according to any one of embodiments 1 to 18 wherein the plant part is a seed, pollen, ovule, pod, flower, leaf, root or stem tissue.

[0107] 20. A seed, wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, or a plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0108] 21. A seed, wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, or a plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis, such as a benzamide herbicide, including but not limited to chlorthiamid, dichlobenil, flupoxam or isoxaben, wherein the seed is not Arabidopsis thaliana.

[0109] 22 A seed, wherein the seed exhibits a reduction or loss of FNRL function and wherein the reduction or loss of FNRL function has been shown to confer to the seed or plant or plant part produced from the seed, increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis. In certain aspects the herbicide that inhibits cellulose synthesis includes a benzamide, for example chlorthiamid, dichlobenil, flupoxam or isoxaben

[0110] 23. The seed according to any one of embodiments 20 to 22 wherein the seed comprises a mutated fnrl gene, and wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the seed, a plant or plant part produced from the seed exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0111] 24. The seed according to any one of embodiments 20 to 23 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

[0112] 25. The seed according to any one of embodiments 20 to 23 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

[0113] 26. The seed according to embodiment 25 wherein the post-transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

[0114] 27. A seed produced by the plant or plant part of any one of embodiments 1 to 19 wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

[0115] 28. The seed according to any one of embodiments 20 to 27 wherein the herbicide is a benzamide herbicide.

[0116] 29. The seed according to any one of embodiments 20 to 28 wherein the herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0117] 30. The seed according to any one of embodiment 20 to 28 wherein the herbicide is isoxaben.

[0118] 31. A method of controlling undesired vegetation at a plant cultivation site, said method comprising the step of applying to said site an effective amount of a herbicide that inhibits cellulose synthesis, and wherein the plant cultivation site is used for cultivating a plant according to any one of embodiments 1 to 19.

[0119] 32. The method of embodiment 31 wherein the plant cultivation site is used for cultivating a plant that is resistant to the herbicide that inhibits cellulose synthesis followed by cultivating a plant according to embodiments 1 to 19.

[0120] 33. The method of embodiment 32 wherein the plant that is resistant to the herbicide that inhibits cellulose synthesis is a cereal crop.

[0121] 34. The method according any one of embodiments 31 to 33 wherein the herbicide that inhibits cellulose synthesis is a benzamide herbicide.

[0122] 35. The method according to embodiment 34 wherein the benzamide herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0123] 36. The method according to embodiment 35 wherein the benzamide herbicide is isoxaben.

[0124] 37. A method of producing a plant or plant part that exhibits increased resistance, or tolerance to a herbicide that inhibits cellulose synthesis said method comprising the step of reducing or abrogating FNRL function in said plant or plant part, wherein reduction or loss of FNRL function increases resistance or tolerance to a herbicide that targets cellulose synthesis.

[0125] 38 A method of producing a plant or plant part that exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis the method comprising the step of reducing or abrogating FNRL function in the plant or plant part, wherein reduction or loss of FNRL function increases resistance or tolerance to a herbicide that targets cellulose synthesis, wherein the plant or plant part is not Arabidopsis thaliana.

[0126] 39. The method according to embodiment 37 or 38 wherein the plant or plant part comprises a mutated fnrl gene, and wherein the mutated fnrl gene results in a reduction or loss of FNRL function.

[0127] 40. The method according to embodiment 37 or 38 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

[0128] 41. The method according to embodiment 37 or 38 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

[0129] 42. The method according to embodiment 41 wherein the post-transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

[0130] 43. The method according to any one of embodiments 37 to 42 wherein the herbicide is a benzamide herbicide.

[0131] 44. The method according to any one of embodiments 37 to 43 wherein the benzamide herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

[0132] 45. The method according to any one of embodiments 37 to 44 wherein the benzamide herbicide is isoxaben.

[0133] 46. The method according to any one of embodiment 37 to 45 wherein the plant is a crop plant or a crop plant part.

[0134] 47. The method according to embodiment 46 wherein the crop plant or crop plant part is a dicotyledonous plant or plant part.

[0135] 48. The method according to embodiment 46 or 47 wherein the crop plant or crop plant part is a broadleaf crop plant or plant part.

[0136] 49. The method according to any one of embodiment 46 to 48 wherein the crop plant or crop plant part is an oil crop, a legume, sugar beet, a fruit plant, potato or a plant or plant part of the Brassica genus.

[0137] 50. The method according to embodiment 49 wherein the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress.

[0138] 51. The method according to embodiment 49 wherein the legume is selected from lentil, pea, chickpea, peanut or bean plants or plant parts.

[0139] 52. The method according to embodiment 51 wherein bean plant or plant part is selected from broad bean, soybean, green bean, lima bean, kidney bean, navy bean, cannellini bean, and adzuki bean plants or plant parts.

[0140] 53. The method according to embodiment 49 wherein the plant or plant part of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

[0141] 54. The method according to any one of embodiments 37 to 53 wherein the plant part is a seed, pollen, ovule, pod, flower, leaf, root or stem tissue

[0142] 55. A plant or plant part when produced by the method of any one of embodiments37 to 54.REFERENCESAnders, S., Pyl, P. T. and Huber, W. (2015) HTSeq--a Python framework to work with high- throughput sequencing data. Bioinformatics, 31, 166-169.Bolger, A. M., Lohse, M. and Usadel, B. (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30, 2114-2120.Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., et al. (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics, 29, 15-21.Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R., Flick, J. S., Adams, S. P, et al. (1983) Expression of bacterial genes in plant cells. Proc Natl Acad Sci U S A, 80, 4803- 4807.Gigli-Bisceglia, N., Engelsdorf, T., Strnad, M., Vaahtera, L., Khan, G. A., Yamoune, A., et al. (2018) Cell wall integrity modulates Arabidopsis thaliana cell cycle gene expression in a cytokinin- and nitrate reductase-dependent manner. Development, 145.Gu, Z. (2022) Complex heatmap visualization. Imeta, 1, e43.Horsch, R. B., Fraley, R. T., Rogers, S. G., Sanders, P. R., Lloyd, A. and Hoffmann, N. (1984) Inheritance of functional foreign genes in plants. Science, 223, 496-498.Juarez, S. P. D., Mangano, S. and Estevez, J. M. (2015) Improved ROS measurement in root hair cells. Plant Cell Expansion: Methods and Protocols, 67-71 .Love, M. I., Huber, W. and Anders, S. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15, 550.Rueden, C. T., Schindelin, J., Hiner, M. C., DeZonia, B. E., Walter, A. E., Arena, E. T., et al. (2017) lmageJ2: Imaged for the next generation of scientific image data. BMC bioinformatics, 18, 1-26.Wu, T., Hu, E., Xu, S., Chen, M., Guo, P, Dai, Z., et al. (2021) clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. The Innovation, 2, 100141 .

Claims

CLAIMS1 . A plant or plant part that exhibits a reduction or loss of Ferredoxin-NADP+ oxidoreductase-like (FNRL) function and thereby exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

2. The plant or plant part according to claim 1 wherein the plant or plant part comprises a mutated fnrl gene, wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the plant or plant part exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

3. The plant or plant part according to claim 1 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

4. The plant or plant part according to claim 1 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

5. The plant or plant part according to claim 4 wherein the post-transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

6. The plant or plant part according to any one of claims 1 to 5 wherein the herbicide is a benzamide herbicide.

7. The plant or plant part according to any one of claims 1 to 6 wherein the herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

8. The plant or plant part according to any one of claims 1 to 7 wherein the herbicide is isoxaben.

9. The plant or plant part according to any one of claims 1 to 8 wherein the plant is a crop plant or a crop plant part.

10. The plant or plant part according to any one of claim 9 wherein the crop plant or crop plant part is a dicotyledonous plant or plant part.

11. The plant or plant part according to claim 9 or claim 10 wherein the crop plant or crop plant part is a broadleaf crop plant or plant part.

12. The plant or plant part according to any one of claims 9 to 11 wherein the crop plant or crop plant part is an oil crop, a legume, sugar beet, a fruit plant, potato or a plant or plant part of the Brassica genus.

13. The plant or plant part according to claim 12 wherein the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress.

14. The plant or plant part according to claim 12 wherein the legume is selected from lentil, pea, chickpea, peanut or bean plants or plant parts.

15. The plant or plant part according to claim 14 wherein bean plant or plant part is selected from broad bean, soybean, green bean, lima bean, kidney bean, navy bean, cannellini bean, and adzuki bean plants or plant parts.

16. The plant or plant part according to claim 12 wherein the plant or plant part of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

17. The plant or plant part according to any one of claims 1 to 16 wherein the plant part is a seed, pollen, ovule, pod, flower, leaf, root or stem tissue.

18. A seed, wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, or a plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

19. The seed according to claim 18 wherein the seed comprises a mutated fnrl gene, and wherein the mutated fnrl gene results in a reduction or loss of FNRL function and wherein the seed, a plant or plant part produced from the seed exhibits increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

20. The seed according to claim 18 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

21. The seed according to claim 18 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

22. The seed according to claim 21 wherein the post-transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

23. A seed produced by the plant or plant part of any one of claims 1 to 17 wherein the seed exhibits a reduction or loss of FNRL function and thereby the seed, plant or plant part produced from the seed, exhibits an increased resistance, or tolerance, to a herbicide that inhibits cellulose synthesis.

24. The seed according to any one of claims 18 to 23 wherein the herbicide is a benzamide herbicide.

25. The seed according to any one of claims 18 to 24 wherein the herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

26. The seed according to any one of claims 18 to 25 wherein the herbicide is isoxaben.

27. A method of controlling undesired vegetation at a plant cultivation site, said method comprising the step of applying to said site an effective amount of a herbicide that inhibits cellulose synthesis, and wherein the plant cultivation site is used for cultivating a plant according to any one of claims 1 to 17.

28. The method of claim 27 wherein the plant cultivation site is used for cultivating a plant that is resistant to the herbicide that inhibits cellulose synthesis followed by cultivating a plant according to any one of claims 1 to 17.

29. The method of claim 28 wherein the plant that is resistant to the herbicide that inhibits cellulose synthesis is a cereal crop.

30. The method according any one of claims 27 to 29 wherein the herbicide that inhibits cellulose synthesis is a benzamide herbicide.

31. The method according claim 30 wherein the benzamide herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

32. The method according claim 31 wherein the benzamide herbicide is isoxaben.

33. A method of producing a plant or plant part that exhibits increased resistance, or tolerance to a herbicide that inhibits cellulose synthesis said method comprising the step of reducingor abrogating FNRL function in said plant or plant part, wherein reduction or loss of FNRL function increases resistance or tolerance to a herbicide that targets cellulose synthesis.

34. The method according to claim 33 wherein the plant or plant part comprises a mutated fnrl gene, and wherein the mutated fnrl gene results in a reduction or loss of FNRL function.

35. The method according to claim 33 wherein the reduction or loss of FNRL function results from transcriptional FNRL gene silencing.

36. The method according to claim 33 wherein the reduction or loss of FNRL function results from post-transcriptional FNRL gene silencing.

37. The method according to claim 36 wherein the post-transcriptional FNRL gene silencing is produced by RNA interference (RNAi).

38. The method according to any one of claims 33 to 37 wherein the herbicide is a benzamide herbicide.

39. The method according to any one of claims 33 to 38 wherein the benzamide herbicide is selected from chlorthiamid, dichlobenil, flupoxam and isoxaben.

40. The method according to any one of claims 33 to 39 wherein the benzamide herbicide is isoxaben.

41. The method according to any one of claims 33 to 40 wherein the plant is a crop plant or a crop plant part.

42. The method according to claim 41 wherein the crop plant or crop plant part is a dicotyledonous plant or plant part.

43. The method according to claim 41 or claim 42 wherein the crop plant or crop plant part is a broadleaf crop plant or plant part.

44. The method according to any one of claims 41 to 43 wherein the crop plant or crop plant part is an oil crop, a legume, sugar beet, a fruit plant, potato or a plant or plant part of the Brassica genus.

45. The method according to claim 44 wherein the oil crop is selected from canola, palm, soybean, rapeseed, sunflower, mustard, flax, jatropha, coconut, hemp, olive, ground nut, castor, and pennycress.

46. The method according to claim 44 wherein the legume is selected from lentil, pea, chickpea, peanut or bean plants or plant parts.

47. The method according to claim 46 wherein bean plant or plant part is selected from broad bean, soybean, green bean, lima bean, kidney bean, navy bean, cannellini bean, and adzuki bean plants or plant parts.

48. The method according to claim 44 wherein the plant or plant part of the Brassica genus is selected from bok choy, cabbage, spinach, cauliflower, collards, broccoli, Brussel sprouts, kale, kohlrabi, swede and turnip plants.

49. The method according to any one of claims 33 to 48 wherein the plant part is a seed, pollen, ovule, pod, flower, leaf, root or stem tissue.

50. A plant or plant part when produced by the method of any one of claims 33 to 49.

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