title: Attachment of Agrobacterium tumefaciens to leaf tissue in response to infiltration conditions.
abstract: Transient expression of recombinant proteins in plant tissues following Agrobacterium-mediated gene transfer is a promising technique for rapid protein production. However, transformation rates and transient expression levels can be sub-optimal depending on process conditions. Attachment of Agrobacterium tumefaciens to plant cells is an early, critical step in the gene transfer pathway. Bacterial attachment levels and patterns may influence transformation and, by extension, transient expression. In this study, attachment of A. tumefaciens to lettuce leaf tissue was investigated in response to varying infiltration conditions, including bacterial density, surfactant concentration, and applied vacuum level. Bacterial density was found to most influence attachment levels for the levels tested (10(8) , 10(9) , and 10(10) CFU/mL), with the relationship between bacterial density and attachment levels following a saturation trend. Surfactant levels tested (Break-Thru S240: 1, 10, 100, and 1,000 µL/L) also had a significant positive effect on bacterial attachment while vacuum level (5, 25, and 45 kPa) did not significantly affect attachment in areas exposed to bacteria. In planta transgene transient expression levels were measured following infiltration with 10(8) , 10(9) , and 10(10) CFU/mL bacterial suspension. Notably, the highest attachment level tested led to a decrease in transient expression, suggesting a potential link between bacterial attachment levels and downstream phenomena that may induce gene silencing. These results illustrate that attachment can be controlled by adjusting infiltration conditions and that attachment levels can impact transgene transient expression in leaf tissue.
authors.affiliation: Dept. of Biological and Agricultural Engineering, University of California, Davis, One Shields Avenue, Davis, CA; Dept. of Food Science and Technology, University of California, Davis, One Shields Avenue, Davis, CA.
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Simmons
authors.forename: Jean S
authors.initials: JS
authors.lastname: VanderGheynst
authors.forename: N
authors.initials: N
authors.lastname: Nitin
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001422:Bacterial Adhesion / Q000187:drug effects / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D018545:Lactuca / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology*; D013501:Surface-Active Agents / Q000494:pharmacology; D014162:Transfection; D019076:Transgenes / Q000235:genetics*; D014618:Vacuum
chemical_list: D001426:Bacterial Proteins; D013501:Surface-Active Agents; D005966:Glucuronidase
keywords: Agrobacterium tumefaciens; agroinfiltration; bacterial attachment; confocal microscopy; plant-microbe interaction