US2021340556A1PendingUtilityA1

Transgenic plant comprising a polynucleotide encoding a variable domain of heavy-chain antibody

Individually held — no corporate assignee on recordPriority: Nov 5, 2014Filed: Dec 7, 2020Published: Nov 4, 2021

Est. expiryNov 5, 2034(~8.3 yrs left)· nominal 20-yr term from priority

Inventors:Peter Verheesen Jan Geerinck João Filipe Veloso Vieira Marnix Peferoen Inge Elodie Van Daele

C12N 15/8279C07K 16/14C07K 2317/569C07K 2317/30C07K 2317/22C07K 2317/567C07K 2317/565C12N 15/8282C07K 2317/52

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References

Claims

Abstract

This disclosure relates to a transgenic plant or plant tissue. In particular, this disclosure relates to a transgenic plant or plant tissue or plant cell comprising at least one polynucleotide comprising at least one sequence encoding a variable domain of a heavy-chain antibody (VHH) specifically binding to a sphingolipid of a fungus. Advantageously, the expression of the polynucleotide in at least part of the transgenic plant or plant tissue or plant cell (i) protects at least part of the transgenic plant or plant tissue or plant cell from an infection with a plant pathogenic fungus, (ii) inhibits the growth of a plant pathogenic fungus on at least part of the transgenic plant or plant tissue or plant cell, or (iii) increases the resistance of at least part of the transgenic plant or plant tissue or plant cell against a plant pathogenic fungus. This disclosure also relates to a method for protecting at least part of a plant or plant tissue or plant cell from an infection with a plant pathogen, for inhibiting the growth of a plant pathogen on at least part of a plant or plant tissue or plant cell, or for increasing pathogen resistance of at least part of a plant or plant tissue or plant cell, comprising expressing in at least part of the plant or plant tissue or plant cell at least one polynucleotide encoding a VHH specifically binding to a pathogen.

Claims

exact text as granted — not AI-modified

1 - 18 . (canceled) 
     
     
         19 . A transgenic plant or plant tissue or plant cell comprising at least one polynucleotide comprising at least one sequence encoding a variable domain of a heavy-chain antibody (VHH) that specifically binds to a glucosylceramide of a fungus; wherein the at least one polynucleotide comprises a sequence encoding a VHH comprising:
 (i) any one or more of SEQ ID NOS:1 to 84, and/or   (ii) a CDR1 region having SEQ ID NO: 85, a CDR2 region having SEQ ID NO: 169, and a CDR3 region having SEQ ID NO: 253, or   a CDR1 region having SEQ ID NO: 86, a CDR2 region having SEQ ID NO: 170, and a CDR3 region having SEQ ID NO: 254, or   a CDR1 region having SEQ ID NO: 87, a CDR2 region having SEQ ID NO: 171, and a CDR3 region having SEQ ID NO: 255, or   a CDR1 region having SEQ ID NO: 88, a CDR2 region having SEQ ID NO: 172, and a CDR3 region having SEQ ID NO: 256, or   a CDR1 region having SEQ ID NO: 89, a CDR2 region having SEQ ID NO: 173, and a CDR3 region having SEQ ID NO: 257, or   a CDR1 region having SEQ ID NO: 90, a CDR2 region having SEQ ID NO: 174, and a CDR3 region having SEQ ID NO: 258, or   a CDR1 region having SEQ ID NO: 91, a CDR2 region having SEQ ID NO: 175, and a CDR3 region having SEQ ID NO: 259, or   a CDR1 region having SEQ ID NO: 92, a CDR2 region having SEQ ID NO: 176, and a CDR3 region having SEQ ID NO: 260, or   a CDR1 region having SEQ ID NO: 93, a CDR2 region having SEQ ID NO: 177, and a CDR3 region having SEQ ID NO: 261, or   a CDR1 region having SEQ ID NO: 94, a CDR2 region having SEQ ID NO: 178, and a CDR3 region having SEQ ID NO: 262, or   a CDR1 region having SEQ ID NO: 95, a CDR2 region having SEQ ID NO: 179, and a CDR3 region having SEQ ID NO: 263, or   a CDR1 region having SEQ ID NO: 96, a CDR2 region having SEQ ID NO: 180, and a CDR3 region having SEQ ID NO: 264, or   a CDR1 region having SEQ ID NO: 97, a CDR2 region having SEQ ID NO: 181, and a CDR3 region having SEQ ID NO: 265, or   a CDR1 region having SEQ ID NO: 98, a CDR2 region having SEQ ID NO: 182, and a CDR3 region having SEQ ID NO: 266, or   a CDR1 region having SEQ ID NO: 99, a CDR2 region having SEQ ID NO: 183, and a CDR3 region having SEQ ID NO: 267, or   a CDR1 region having SEQ ID NO: 100, a CDR2 region having SEQ ID NO: 184, and a CDR3 region having SEQ ID NO: 268, or   a CDR1 region having SEQ ID NO: 101, a CDR2 region having SEQ ID NO: 185, and a CDR3 region having SEQ ID NO: 269, or   a CDR1 region having SEQ ID NO: 102, a CDR2 region having SEQ ID NO: 186, and a CDR3 region having SEQ ID NO: 270, or   a CDR1 region having SEQ ID NO: 103, a CDR2 region having SEQ ID NO: 187, and a CDR3 region having SEQ ID NO: 271, or   a CDR1 region having SEQ ID NO: 104, a CDR2 region having SEQ ID NO: 188, and a CDR3 region having SEQ ID NO: 272, or   a CDR1 region having SEQ ID NO: 105, a CDR2 region having SEQ ID NO: 189, and a CDR3 region having SEQ ID NO: 273, or   a CDR1 region having SEQ ID NO: 106, a CDR2 region having SEQ ID NO: 190, and a CDR3 region having SEQ ID NO: 274, or   a CDR1 region having SEQ ID NO: 107, a CDR2 region having SEQ ID NO: 191, and a CDR3 region having SEQ ID NO: 275, or   a CDR1 region having SEQ ID NO: 108, a CDR2 region having SEQ ID NO: 192, and a CDR3 region having SEQ ID NO: 276, or   a CDR1 region having SEQ ID NO: 109, a CDR2 region having SEQ ID NO: 193, and a CDR3 region having SEQ ID NO: 277, or   a CDR1 region having SEQ ID NO: 110, a CDR2 region having SEQ ID NO: 194, and a CDR3 region having SEQ ID NO: 278, or   a CDR1 region having SEQ ID NO: 111, a CDR2 region having SEQ ID NO: 195, and a CDR3 region having SEQ ID NO: 279, or   a CDR1 region having SEQ ID NO: 112, a CDR2 region having SEQ ID NO: 196, and a CDR3 region having SEQ ID NO: 280, or   a CDR1 region having SEQ ID NO: 113, a CDR2 region having SEQ ID NO: 197, and a CDR3 region having SEQ ID NO: 281, or   a CDR1 region having SEQ ID NO: 114, a CDR2 region having SEQ ID NO: 198, and a CDR3 region having SEQ ID NO: 282, or   a CDR1 region having SEQ ID NO: 115, a CDR2 region having SEQ ID NO: 199, and a CDR3 region having SEQ ID NO: 283, or   a CDR1 region having SEQ ID NO: 116, a CDR2 region having SEQ ID NO: 200, and a CDR3 region having SEQ ID NO: 284, or   a CDR1 region having SEQ ID NO: 117, a CDR2 region having SEQ ID NO: 201, and a CDR3 region having SEQ ID NO: 285, or   a CDR1 region having SEQ ID NO: 118, a CDR2 region having SEQ ID NO: 202, and a CDR3 region having SEQ ID NO: 286, or   a CDR1 region having SEQ ID NO: 119, a CDR2 region having SEQ ID NO: 203, and a CDR3 region having SEQ ID NO: 287, or   a CDR1 region having SEQ ID NO: 120, a CDR2 region having SEQ ID NO: 204, and a CDR3 region having SEQ ID NO: 288, or   a CDR1 region having SEQ ID NO: 121, a CDR2 region having SEQ ID NO: 205, and a CDR3 region having SEQ ID NO: 289, or   a CDR1 region having SEQ ID NO: 122, a CDR2 region having SEQ ID NO: 206, and a CDR3 region having SEQ ID NO: 290, or   a CDR1 region having SEQ ID NO: 123, a CDR2 region having SEQ ID NO: 207, and a CDR3 region having SEQ ID NO: 291, or   a CDR1 region having SEQ ID NO: 124, a CDR2 region having SEQ ID NO: 208, and a CDR3 region having SEQ ID NO: 292, or   a CDR1 region having SEQ ID NO: 125, a CDR2 region having SEQ ID NO: 209, and a CDR3 region having SEQ ID NO: 293, or   a CDR1 region having SEQ ID NO: 126, a CDR2 region having SEQ ID NO: 210, and a CDR3 region having SEQ ID NO: 294, or   a CDR1 region having SEQ ID NO: 127, a CDR2 region having SEQ ID NO: 211, and a CDR3 region having SEQ ID NO: 295, or   a CDR1 region having SEQ ID NO: 128, a CDR2 region having SEQ ID NO: 212, and a CDR3 region having SEQ ID NO: 296, or   a CDR1 region having SEQ ID NO: 129, a CDR2 region having SEQ ID NO: 213, and a CDR3 region having SEQ ID NO: 297, or   a CDR1 region having SEQ ID NO: 130, a CDR2 region having SEQ ID NO: 214, and a CDR3 region having SEQ ID NO: 298, or   a CDR1 region having SEQ ID NO: 131, a CDR2 region having SEQ ID NO: 215, and a CDR3 region having SEQ ID NO: 299, or   a CDR1 region having SEQ ID NO: 132, a CDR2 region having SEQ ID NO: 216, and a CDR3 region having SEQ ID NO: 300, or   a CDR1 region having SEQ ID NO: 133, a CDR2 region having SEQ ID NO: 217, and a CDR3 region having SEQ ID NO: 301, or   a CDR1 region having SEQ ID NO: 134, a CDR2 region having SEQ ID NO: 218, and a CDR3 region having SEQ ID NO: 302, or   a CDR1 region having SEQ ID NO: 135, a CDR2 region having SEQ ID NO: 219, and a CDR3 region having SEQ ID NO: 303, or   a CDR1 region having SEQ ID NO: 136, a CDR2 region having SEQ ID NO: 220, and a CDR3 region having SEQ ID NO: 304, or   a CDR1 region having SEQ ID NO: 137, a CDR2 region having SEQ ID NO: 221, and a CDR3 region having SEQ ID NO: 305, or   a CDR1 region having SEQ ID NO: 138, a CDR2 region having SEQ ID NO: 222, and a CDR3 region having the amino acid sequence NRY, or   a CDR1 region having SEQ ID NO: 139, a CDR2 region having SEQ ID NO: 223, and a CDR3 region having SEQ ID NO: 306, or   a CDR1 region having SEQ ID NO: 140, a CDR2 region having SEQ ID NO: 224, and a CDR3 region having SEQ ID NO: 307, or   a CDR1 region having SEQ ID NO: 141, a CDR2 region having SEQ ID NO: 225, and a CDR3 region having SEQ ID NO: 308, or   a CDR1 region having SEQ ID NO: 142, a CDR2 region having SEQ ID NO: 226, and a CDR3 region having SEQ ID NO: 309, or   a CDR1 region having SEQ ID NO: 143, a CDR2 region having SEQ ID NO: 227, and a CDR3 region having SEQ ID NO: 310, or   a CDR1 region having SEQ ID NO: 144, a CDR2 region having SEQ ID NO: 228, and a CDR3 region having SEQ ID NO: 311, or   a CDR1 region having SEQ ID NO: 145, a CDR2 region having SEQ ID NO: 229, and a CDR3 region having SEQ ID NO: 312, or   a CDR1 region having SEQ ID NO: 146, a CDR2 region having SEQ ID NO: 230, and a CDR3 region having SEQ ID NO: 313, or   a CDR1 region having SEQ ID NO: 147, a CDR2 region having SEQ ID NO: 231, and a CDR3 region having SEQ ID NO: 314, or   a CDR1 region having SEQ ID NO: 148, a CDR2 region having SEQ ID NO: 232, and a CDR3 region having SEQ ID NO: 315, or   a CDR1 region having SEQ ID NO: 149, a CDR2 region having SEQ ID NO: 233, and a CDR3 region having SEQ ID NO: 316, or   a CDR1 region having SEQ ID NO: 150, a CDR2 region having SEQ ID NO: 234, and a CDR3 region having SEQ ID NO: 317, or   a CDR1 region having SEQ ID NO: 151, a CDR2 region having SEQ ID NO: 235, and a CDR3 region having SEQ ID NO: 318, or   a CDR1 region having SEQ ID NO: 152, a CDR2 region having SEQ ID NO: 236, and a CDR3 region having SEQ ID NO: 319, or   a CDR1 region having SEQ ID NO: 153, a CDR2 region having SEQ ID NO: 237, and a CDR3 region having SEQ ID NO: 320, or   a CDR1 region having SEQ ID NO: 154, a CDR2 region having SEQ ID NO: 238, and a CDR3 region having SEQ ID NO: 321, or   a CDR1 region having SEQ ID NO: 155, a CDR2 region having SEQ ID NO: 239, and a CDR3 region having SEQ ID NO: 322, or   a CDR1 region having SEQ ID NO: 156, a CDR2 region having SEQ ID NO: 240, and a CDR3 region having SEQ ID NO: 323, or   a CDR1 region having SEQ ID NO: 157, a CDR2 region having SEQ ID NO: 241, and a CDR3 region having SEQ ID NO: 324, or   a CDR1 region having SEQ ID NO: 158, a CDR2 region having SEQ ID NO: 242, and a CDR3 region having SEQ ID NO: 325, or   a CDR1 region having SEQ ID NO: 159, a CDR2 region having SEQ ID NO: 243, and a CDR3 region having SEQ ID NO: 326, or   a CDR1 region having SEQ ID NO: 160, a CDR2 region having SEQ ID NO: 244, and a CDR3 region having SEQ ID NO: 327, or   a CDR1 region having SEQ ID NO: 161, a CDR2 region having SEQ ID NO: 245, and a CDR3 region having SEQ ID NO: 328, or   a CDR1 region having SEQ ID NO: 162, a CDR2 region having SEQ ID NO: 246, and a CDR3 region having SEQ ID NO: 329, or   a CDR1 region having SEQ ID NO: 163, a CDR2 region having SEQ ID NO: 247, and a CDR3 region having SEQ ID NO: 330, or   a CDR1 region having SEQ ID NO: 164, a CDR2 region having SEQ ID NO: 248, and a CDR3 region having SEQ ID NO: 331, or   a CDR1 region having SEQ ID NO: 165, a CDR2 region having SEQ ID NO: 249, and a CDR3 region having SEQ ID NO: 332, or   a CDR1 region having SEQ ID NO: 166, a CDR2 region having SEQ ID NO: 250, and a CDR3 region having SEQ ID NO: 333, or   a CDR1 region having SEQ ID NO: 167, a CDR2 region having SEQ ID NO: 251, and a CDR3 region having SEQ ID NO: 334, or   a CDR1 region having SEQ ID NO: 168, a CDR2 region having SEQ ID NO: 252, and a CDR3 region having SEQ ID NO: 335.   
     
     
         20 . A method for the production of a transgenic plant or plant tissue or plant cell, the method comprising the introduction of at least one polynucleotide comprising at least one sequence encoding a variable domain of a heavy-chain antibody (VHH) that specifically binds to a glucosylceramide of a fungus, into the genome of a plant or plant tissue or plant cell. 
     
     
         21 . The method of  claim 20 , wherein the expression of the polynucleotide in at least part of the transgenic plant or plant tissue or plant cell (i) protects at least part of the transgenic plant or plant tissue or plant cell from an infection with a plant pathogenic fungus, (ii) inhibits the growth of a plant pathogenic fungus on at least part of the transgenic plant or plant tissue or plant cells, and/or (iii) increases the resistance of at least part of the transgenic plant or plant tissue or plant cell against a plant pathogenic fungus. 
     
     
         22 . The method of  claim 20 , wherein the introduction of the at least one polynucleotide into the genome of a plant or plant tissue or plant cell comprises transformation using liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen or microprojection, or any combination thereof. 
     
     
         23 . The method of  claim 20 , wherein the introduction of the at least one polynucleotide into the genome of a plant or plant tissue or plant cell comprises  Agrobacterium -mediated transformation. 
     
     
         24 . The method of  claim 20 , wherein the introduction of the at least one polynucleotide comprises transformation in planta. 
     
     
         25 . The method of  claim 20 , wherein the introduction of the at least one polynucleotide comprises vacuum infiltration. 
     
     
         26 . The method of  claim 20 , further comprising selecting plants, plant tissue or plant cells for the presence of one or more markers that are encoded by plant-expressible genes co-transfected with the at least one sequence encoding a variable domain of a heavy chain antibody (VHH). 
     
     
         27 . The method of  claim 20 , comprising subjecting the plant, plant tissue or plant cell to selective conditions to distinguish transformed plants, plant tissue or plant cells from untransformed plants, plant tissue or plant cells. 
     
     
         28 . The method of  claim 20 , further comprising propagating the transformed plant, plant tissue or plant cell. 
     
     
         29 . The method of  claim 20 , wherein the transformed plant is propagated by clonal propagation, classical breeding techniques, or any combination thereof. 
     
     
         30 . The method of  claim 20 , wherein the introduction of the at least one polynucleotide into the genome of a plant or plant tissue or plant cell is stable. 
     
     
         31 . The method of  claim 20 , wherein the at least one polynucleotide is integrated into the plant genome. 
     
     
         32 . The method of  claim 20 , wherein the at least one polynucleotide comprises a promoter suitable for expression in plants, a plant tissue or plant cell. 
     
     
         33 . The method of  claim 20 , wherein the promoter is the 35S Cauliflower Mosaic Virus (CaMV) promoter, a plant tissue or plant cell specific promoter, or an inducible promoter. 
     
     
         34 . The method of  claim 20 , wherein the at least one polynucleotide further comprises at least one sequence encoding a target signal for secretion, for location to the cytoplasm, or for location to cellular compartments or organelles, such as the ER lumen, the apoplast, the vacuole, or the intra- and/or exterior membranes. 
     
     
         35 . The method of  claim 20 , wherein the signal for location to the cytoplasm is an ATG start codon. 
     
     
         36 . The method of  claim 20 , wherein the polynucleotide is in the form of a vector, such as a plasmid, cosmid, yeast artificial chromosome (YAC), a viral vector or transposon. 
     
     
         37 . The method of  claim 20 , wherein the plant pathogenic fungus for which the VHH is specific is selected from the group consisting of  Alternaria, Ascochyta, Botrytis, Cerospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scenrotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakopsora, Monilinia, Mucor, Rhizopus  and  Aspergillus.    
     
     
         38 . The method of  claim 20 , wherein the at least one polynucleotide is introduced to a tissue target selected from the group consisting of leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue, such as apical meristem, axillary buds and root meristems, and induced meristem tissue, such as cotyledon meristem and hypocotyl meristem. 
     
     
         39 . The method of  claim 20 , wherein the transgenic plant or plant tissue or plant cell is selected from the group consisting of corn, rice, wheat, barley, sorghum, millet oats, rye, triticale, cereals, soybean, alfalfa, leguminous crops, sugar beet, fodder beet, papaya, banana, plantains, fruits, grapevines, nuts, oilseed rape, sunflower, oil crops, squash cucumber, melons, cucurbits, cotton, fiber plants, sugarcane, palm, jatropha, fuel crops, cabbages, tomato, pepper, vegetables, ornamentals, shrubs, poplar, eucalyptus, trees, evergreens, grasses, coffee plants, tea plants, tobacco plants, hop plants, rubber plants, and latex plants. 
     
     
         40 . The method of  claim 20 , wherein the at least one polynucleotide further comprises at least one sequence encoding a tag, preferably a His6, c-myc, FLAG, C-tag, 3×FLAG, His5, His10, HA, T7, strep, HSV, and/or an E-tag. 
     
     
         41 . The transgenic plant or plant tissue or plant cell produced by the method of  claim 20 . 
     
     
         42 . Use of at least one polynucleotide encoding a variable domain of a heavy-chain antibody (VHH) that specifically binds to a glucosylceramide of a fungus, for the production of a transgenic plant or plant tissue or plant cell. 
     
     
         43 . Harvestable parts and propagation materials from the transgenic plant, plant tissue or plant cell produced by the method of  claim 20 .

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