Thionin-D4E1 chimeric protein protects plants against bacterial infections
Inventors
Stover, Eddie W • Gupta, Goutam • Hao, Guixia
Assignees
US Department of Agriculture USDA • Triad National Security LLC
Publication Number
US-9725734-B2
Publication Date
2017-08-08
Expiration Date
2035-04-21
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Abstract
The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.
Core Innovation
This invention describes the generation of a chimeric protein consisting of three domains: a first domain encoding either a pro-thionin or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker separating the first and second domains. The chimeric protein can have either the thionin or D4E1 domain at the amino terminus, and the other domain at the carboxyl terminus. This chimeric protein exhibits antibacterial activity.
The problem being addressed is the need to protect plants, particularly citrus, against devastating bacterial diseases such as citrus canker caused by Xanthomonas citri ssp. citri, and Huanglongbing (HLB or citrus greening) caused by Candidatus Liberibacter species. Current methods lack effective treatment, and these diseases severely impact agricultural production. The invention proposes genetically altered plants capable of expressing the chimeric protein to resist these bacterial diseases by killing the infecting bacteria.
The chimeric protein is designed so that each domain (thionin or pro-thionin and D4E1 or pro-D4E1) can fold into its proper three-dimensional shape and retain its antibacterial activity, helped by the peptide linker ranging from approximately three to forty-four amino acids. Optimized versions of the thionin domain have been developed to increase antibacterial activity and reduce plant toxicity. Genetically altered plants expressing these chimeric proteins show enhanced resistance to gram-negative bacterial diseases, including citrus canker and HLB, compared to plants expressing either domain alone or wild-type plants.
Claims Coverage
The patent includes several independent claims directed to genetically altered plants expressing chimeric proteins and methods for generating such plants with increased resistance to Xanthomonas citri ssp. citri.
Genetically altered plant producing a chimeric protein with enhanced disease resistance
A genetically altered plant, part, or progeny comprising a promoter operably linked to a polynucleotide encoding a chimeric protein of SEQ ID NO: 8 or SEQ ID NO: 18 that produces the chimeric protein and exhibits increased resistance to Xanthomonas citri ssp. citri compared to wild-type plants.
Method of constructing genetically altered plants expressing chimeric proteins
Introducing a polynucleotide encoding a chimeric protein comprising optimized thionin or optimized pro-thionin as the first domain, D4E1 or pro-D4E1 as the second domain, and a peptide linker of three to approximately forty-four amino acids as the third domain, ensuring the domains fold properly and retain antibacterial activity, selecting plants that express the protein and have increased resistance to Xanthomonas citri ssp. citri.
Location of domains within the chimeric protein
Configurations where the optimized thionin or its pro-form is at the amino terminus and D4E1 at the carboxyl terminus, or D4E1 (or pro-D4E1) is at the amino terminus and optimized thionin at the carboxyl terminus of the chimeric protein.
Method of enhancing resistance by transformation
Transforming wild-type plant cells with polynucleotides encoding the chimeric protein with described domains and linkers to generate genetically altered plants producing the chimeric protein that kills Xanthomonas citri ssp. citri and exhibit greater resistance than wild-type plants.
Use of specific peptide linkers
Employing peptide linkers with amino acid sequences selected from SEQ ID NOs: 10, 39, 40, 41, and 42 to separate the thionin and D4E1 domains in the chimeric protein.
The claims cover genetically altered plants producing specific chimeric proteins comprising thionin and D4E1 domains connected by designated peptide linkers, methods of constructing such plants, and configurations of domain locations, all providing increased resistance to the bacterial pathogen Xanthomonas citri ssp. citri.
Stated Advantages
The chimeric protein expressed in genetically altered plants provides greater antibacterial activity than plants expressing either the optimized thionin or D4E1 alone, resulting in enhanced resistance to bacterial diseases such as citrus canker and Huanglongbing.
The peptide linker facilitates correct three-dimensional folding of each domain, preserving individual and synergistic antibacterial activities.
Optimized thionin exhibits increased antibacterial activity with reduced toxicity to the plant compared to wild-type thionin.
The genetically altered plants exhibit significantly reduced disease symptoms and better overall health when exposed to bacterial pathogens compared to non-genetically altered plants.
Documented Applications
Protection of citrus plants against citrus canker caused by Xanthomonas citri ssp. citri.
Protection of citrus plants against Huanglongbing (HLB or citrus greening) caused by Candidatus Liberibacter species.
General use in genetically altered plants to enhance resistance against gram-negative bacterial diseases affecting various plant species.
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