Anti-fungal polypeptides

Abstract

The present invention relates to a composition comprising at least one polypeptide, which polypeptide comprises the amino acid sequence set out in any one of SEQ ID NOs: 1 to 51 or 101 to 111 or an amino acid sequence having at least about 80% sequence identify thereto and which polypeptide is capable of binding to a fungus. The invention further relates to a composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in any one of SEQ ID NOs: 52 to 67 or 112 to 122, a CDR2 region having the amino acid sequence set out in any one of SEQ ID NOs: 68 to 83 or 123 to 133, and a CDR3 region having the amino acid sequence set out in any one of SEQ ID NOs: 84 to 100 or 134 to 144 and which polypeptide is capable of binding to a fungus. The compositions may be used as anti-fungal compositions.

1 . A composition comprising at least one polypeptide, which polypeptide is capable of binding to a fungus, thereby causing retardation of growth of a spore of the said fungus and/or lysis of a spore of the fungus. 
     
     
         2 . A composition according to  claim 1 , wherein said at least one polypeptide specifically binds to at least one plasma membrane component of said fungus. 
     
     
         3 . A composition comprising at least one polypeptide, wherein said at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of  Botrytis cinerea , said lipid-containing fraction being obtainable by a method comprising:
 fractionating hyphae of  Botrytis cinerea  by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.   
     
     
         4 . A composition comprising at least one polypeptide, which polypeptide comprises the amino acid sequence set out in any one of SEQ ID NOs: 1 to 51 or 101 to 111 or an amino acid sequence having at least about 80% sequence identify to any thereto and which polypeptide is capable of binding to a fungus. 
     
     
         5 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region, optionally wherein the CDR3 region has as sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144, and which polypeptide is capable of binding to a fungus. 
     
     
         6 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 52, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 84 and which polypeptide is capable of binding to a fungus. 
     
     
         7 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 53, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 69, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 85 and which polypeptide is capable of binding to a fungus. 
     
     
         8 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 54, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 70, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 86 and which polypeptide is capable of binding to a fungus. 
     
     
         9 . A composition comprising at least one polypeptide, which polypeptide comprises the amino acid sequence set out in any one of SEQ ID NOs: 1 to 51 to 101 to 111, or an amino acid sequence having at least about 80% sequence identify to any thereto and which polypeptide is capable of binding to a fungus, wherein the polypeptide comprises one or more substitutions resulting in increased positive charge in comparison to any one of SEQ ID NOs: 1 to 51 to 101 to 111. 
     
     
         10 . A composition according to any one of the preceding claims, wherein the said at least one polypeptide is an antibody or a functional fragment thereof. 
     
     
         11 . A composition according to any one of the preceding claims, wherein the said at least one polypeptide is a heavy chain variable domain of an antibody (V H  or V HH ) or a functional fragment thereof. 
     
     
         12 . A composition according to any one of  claims 4  to  11 , wherein said at least one polypeptide specifically binds to at least one plasma membrane component of said fungus. 
     
     
         13 . A composition according to  claim 12 , wherein said at least one polypeptide is capable of binding to a lipid-containing fraction of the plasma membrane of  Botrytis cinerea , said lipid-containing fraction being obtainable by a method comprising:
 fractionating hyphae of  Botrytis cinerea  by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.   
     
     
         14 . A composition according to any one of the preceding claims, wherein the concentration of said at least one polypeptide in said composition ranges from 0.0001% to 50% by weight. 
     
     
         15 . A composition according to any one of the preceding claims, which is an agrochemical composition. 
     
     
         16 . A composition according to  claim 15 , which further comprises an agrochemically suitable carrier and/or one or more suitable adjuvants. 
     
     
         17 . A composition according to any one of the preceding claims, wherein said fungus is a plant pathogenic fungus. 
     
     
         18 . A composition according to  claim 17 , wherein the genus of said plant pathogenic fungus is chosen from the group comprising  Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum, Diplodia, Erysiphe, Fusarium, Leptosphaeria, Gaeumanomyces, Helminthosporium, Macrophomina, Nectria, Penicillium, Peronospora, Phoma, Phymatotrichum, Phytophthora, Plasmopara, Podosphaera, Puccinia, Pyrenophora, Pyricularia, Pythium, Rhizoctonia, Scerotium, Sclerotinia, Septoria, Thielaviopsis, Uncinula, Venturia, Verticillium, Magnaporthe, Blumeria, Mycosphaerella, Ustilago, Melampsora, Phakospora, Monilinia, Mucor, Rhizopus , and  Aspergillus.    
     
     
         19 . A composition comprising at least one polypeptide, which polypeptide comprises the amino acid sequence set out in any one of SEQ ID NOs: 1 to 51 or 101 to 111, or an amino acid sequence having at least about 80% sequence identify to either thereto and which polypeptide is capable of binding to a fungus for use as an anti-fungal agent. 
     
     
         20 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 to 67 and 112 to 122, a CDR2 region having an amino acid sequence selected from the group consisting of SEQ ID NOs: 68 to 83 and 123 to 133, and a CDR3 region, optionally wherein the CDR3 region has as sequence selected from the group consisting of SEQ ID NOs: 84 to 100 and 134 to 144, and which polypeptide is capable of binding to a fungus for use as an anti-fungal agent. 
     
     
         21 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 52, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 68, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 84 and which polypeptide is capable of binding to a fungus for use as an anti-fungal agent. 
     
     
         22 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 53, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 69, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 85 and which polypeptide is capable of binding to a fungus for use as an anti-fungal agent. 
     
     
         23 . A composition comprising at least one polypeptide, which polypeptide comprises a CDR1 region having the amino acid sequence set out in SEQ ID NO: 54, a CDR2 region having the amino acid sequence set out in SEQ ID NO: 70, and a CDR3 region having the amino acid sequence set out in SEQ ID NO: 86 and which polypeptide is capable of binding to a fungus for use as an anti-fungal agent. 
     
     
         24 . Use of a composition according to any of one of the preceding claims as an anti-fungal agent. 
     
     
         25 . Use according to  claim 24  as an anti-fungal agent on plants. 
     
     
         26 . A method for protecting or treating a plant or a part of said plant from an infection with a plant pathogenic fungus, at least comprising the step of applying directly or indirectly to said plant or to a part of said plant, a composition according to any one of  claims 1  to  23 , under conditions effective to protect or treat said plant or a part of said plant against said infection with said plant pathogenic fungus. 
     
     
         27 . A post-harvest treatment method for protecting or treating a harvested plant or a harvested part of said plant from an infection with a plant pathogenic fungus, at least comprising the step of applying directly or indirectly to said harvested plant or to a harvested part of said plant, a composition according to any one of  claims 1  to  23 , under conditions effective to protect or treat said harvested plant or a harvested part of said plant against said infection with said plant pathogenic fungus. 
     
     
         28 . A method of inhibiting or killing the growth of a plant pathogenic fungus, comprising at least the step of applying directly or indirectly to a plant or to a part of said plant, a composition according to any of one of  claims 1  to  23 . 
     
     
         29 . A polypeptide as defined in any one of  claims 1  to  23 . 
     
     
         30 . A method for the preparation of a polypeptide which specifically binds to and/or has affinity to a fungus, which method comprises:
 immunizing an animal with a fungal target, or with a suitable antigenic determinant based thereon or derived therefrom, such as an antigenic part thereof, a fragment thereof, a region thereof, a domain thereof, a loop thereof or other epitope thereof;   obtaining from the immunized animal a collection or sample of cells expressing polypeptide sequences;   screening the collection or sample of cells for cells that express an amino acid sequence that binds to and/or has affinity for the fungal target;   either (i) isolating the amino acid sequence, or (ii) isolating from the collection or sample of cells a nucleic acid sequence that encodes the amino acid sequence; and   expressing said amino acid sequence,   thereby to prepare a polypeptide which specifically binds to and/or has affinity to a fungus.   
     
     
         31 . A method according to  claim 30 , wherein the fungal target is a lipid-containing fraction of the plasma membrane of  Botrytis cinerea , said lipid-containing fraction being obtainable by a method comprising:
 fractionating hyphae of  Botrytis cinerea  by total lipid extract thin-layer chromatography and selecting the fraction with a Retention Factor (Rf) higher than the ceramide fraction and lower than the non-polar phospholipids fraction.   
     
     
         32 . A method for the preparation of an anti-fungal composition, which method comprises:
 preparing an anti-fungal polypeptide according to the method of  claim 30  or  31 ; and   combining the anti-fungal polypeptide with one or more suitable carriers and/or one or more suitable adjuvants.   
     
     
         33 . A transgenic plant, plant part, seed, or plant cell comprising a nucleic acid sequence encoding a polypeptide as defined in any one of  claims 1  to  23 .