title: Engineering of Bacteriophages Y2::dpoL1-Cand Y2::luxABfor Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora.
abstract: Erwinia amylovorais the causative agent of fire blight, a devastating plant disease affecting members of theRosaceaeAlternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-rangeE. amylovoravirus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene ofE. amylovoravirus L1 (dpoL1-C) or a bacterialluxABfusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid proteinorf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-Cdemonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability ofE. amylovorato colonize the surface of detached flowers. The reporter phage Y2::luxABtransduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, bothin vitroand in plant material.IMPORTANCEFire blight, caused byErwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduceE. amylovoraon flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection ofE. amylovoracells.
authors.affiliation: Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland.|Agroscope, Research Division Plant Protection, Wädenswil, Switzerland.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Born
authors.affiliation: Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland.
authors.forename: Lars
authors.initials: L
authors.lastname: Fieseler
authors.affiliation: Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland.
authors.forename: Valentin
authors.initials: V
authors.lastname: Thöny
authors.affiliation: Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland.
authors.forename: Nadja
authors.initials: N
authors.lastname: Leimer
authors.affiliation: Agroscope, Research Division Plant Protection, Wädenswil, Switzerland.
authors.forename: Brion
authors.initials: B
authors.lastname: Duffy
authors.affiliation: Institute of Food, Nutrition, and Health, ETH Zurich, Zürich, Switzerland martin.loessner@ethz.ch.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Loessner
mesh_terms: D001435:Bacteriophages / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D044084:Erwinia amylovora / Q000502:physiology / Q000821:virology*; D015967:Gene Expression Regulation, Viral; D005818:Genetic Engineering; D027845:Malus / Q000382:microbiology; D010935:Plant Diseases / Q000382:microbiology / Q000517:prevention & control*; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014774:Virulence
chemical_list: D014764:Viral Proteins
keywords: bacteriophage; depolymerase; fire blight; luciferase; recombinant phage; reporter; reporter phage