Optimized thionin 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-10378024-B2
Publication Date
2019-08-13
Expiration Date
2035-04-21
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Abstract
Two small proteins with anti-bacterial activity are generated and called optimized thionin and optimized pro-thionin. Optimized pro-thionin contains a signal sequence for intracellular trafficking of the protein and is cleaved off to yield optimized thionin. Genetically altered plants and their progeny expressing a polynucleotide encoding optimized pro-thionin or optimized thionin resist diseases caused by bacteria.
Core Innovation
This invention concerns a chimeric protein comprising a first domain of thionin or pro-thionin, a second domain of D4E1 or pro-D4E1, and a third domain which is a peptide linker separating the first and second domains to enable proper folding and retention of bacterial activity. The chimeric protein can have either thionin or optimized thionin at the amino terminus and D4E1 at the carboxyl terminus, or vice versa. The peptide linker ranges from about three to approximately forty-four amino acids to reduce steric hindrance and may enhance solubility and synergistic antibacterial effects. Optimized thionin includes specific amino acid substitutions and extensions enhancing antibacterial activity and reducing plant toxicity compared to wild-type thionin.
The invention also encompasses genetically altered plants expressing the chimeric proteins, or expressing either optimized pro-thionin, optimized thionin, or D4E1 alone, conferring resistance to bacterial diseases caused by gram-negative and gram-positive bacteria. The plants expressing the chimeric protein demonstrate superior antibacterial activity compared to plants expressing only one of the constituent proteins, attributed to a synergistic effect. The chimeric protein is produced in plants via polynucleotides encoding the protein under the control of promoters, introduced through transformation or introgression methods, generating genetically altered plants and their progeny with enhanced bacterial resistance.
The problem addressed relates to devastating bacterial diseases in plants, particularly citrus greening (HLB) caused by Candidatus Liberibacter species and citrus canker caused by Xanthomonas citri ssp. citri, which currently lack effective treatments. There is an unmet need for methods to prevent and treat these bacterial diseases in citrus and other plants. Natural antimicrobial peptides offer some protection, but synthetic peptides like D4E1 show enhanced biocontrol activity, and combining such peptides with thionins in chimeric proteins promises improved protection. The invention provides such chimeric proteins and genetically altered plants expressing them, providing broad-spectrum antibacterial activity to combat these plant diseases.
Claims Coverage
The patent presents fourteen claims, including compositions of optimized thionin and pro-thionin, polynucleotides and expression vectors encoding these proteins, genetically altered plants producing these proteins, and methods for constructing genetically altered citrus plants with enhanced resistance to bacterial diseases.
Optimized thionin protein composition
An optimized thionin comprising the amino acid sequence of SEQ ID NO: 14.
Optimized pro-thionin protein composition
An optimized pro-thionin comprising the amino acid sequence of SEQ ID NO: 6.
Polynucleotide encoding optimized pro-thionin
A polynucleotide comprising a DNA sequence encoding the optimized pro-thionin of claim 2.
Expression vector with optimized pro-thionin encoding sequence
An expression vector comprising a promoter operably linked to a polynucleotide encoding optimized pro-thionin.
Genetically altered plants producing optimized thionin
A genetically altered plant, part or progeny thereof comprising a promoter operably linked to a polynucleotide encoding optimized thionin, producing the optimized thionin protein.
Polynucleotide encoding optimized thionin
A polynucleotide comprising a DNA sequence encoding optimized thionin.
Expression vector with optimized thionin encoding sequence
An expression vector comprising a promoter operably linked to a polynucleotide encoding optimized thionin.
Citrus plants genetically altered to produce optimized thionin or pro-thionin
Genetically altered citrus plants, parts or progeny thereof comprising polynucleotides encoding and expressing optimized thionin or pro-thionin.
Method to construct genetically altered citrus plants with resistance to citrus greening and canker
A method comprising introducing an expression vector encoding optimized thionin or optimized pro-thionin into wild-type plant cells, selecting altered cells that produce optimized thionin with antibacterial activity against Candidatus Liberibacter species or X. citri ssp. citri, and growing genetically altered citrus plants with increased disease resistance compared to non-altered plants.
Introgression or transformation as method of introducing expression vector
Introduction of the expression vector into plants is conducted via introgression or genetic transformation.
The claims comprehensively cover compositions of optimized thionin and optimized pro-thionin proteins, their encoding polynucleotides and expression vectors, genetically altered plants expressing these proteins particularly citrus plants, and methods for generating such genetically altered plants with enhanced resistance to citrus greening disease and canker. The inventive features focus on optimized thionin variants and their use in genetic engineering for disease resistance.
Stated Advantages
Enhanced antibacterial activity of the chimeric protein compared to individual constituent peptides due to a synergistic effect.
Reduced toxicity to plants from optimized thionin compared to wild-type thionins.
Broad-spectrum antibacterial activity effective against gram-negative and gram-positive bacteria.
Genetically altered plants expressing the chimeric proteins show increased resistance to devastating plant diseases such as citrus greening and citrus canker.
The chimeric protein allows for protection of economically important crops by killing bacteria that infect the plants.
Documented Applications
Protection of citrus plants against citrus greening disease (HLB) caused by Candidatus Liberibacter species.
Protection of citrus plants against citrus canker caused by Xanthomonas citri ssp. citri.
General prevention and treatment of bacterial diseases in plants by genetically altering plants to express chimeric proteins possessing antibacterial activity.
Generation of genetically altered plants expressing chimeric proteins for enhanced resistance to gram-negative and gram-positive bacterial infections.
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