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Open Access Publications from the University of California

CRAGE-Duet Facilitates Modular Assembly of Biological Systems for Studying Plant-Microbe Interactions.

  • Author(s): Wang, Bing
  • Zhao, Zhiying
  • Jabusch, Lauren K
  • Chiniquy, Dawn M
  • Ono, Koyo
  • Conway, Jonathan M
  • Zhang, Zheyun
  • Wang, Gaoyan
  • Robinson, David
  • Cheng, Jan-Fang
  • Dangl, Jeffery L
  • Northen, Trent R
  • Yoshikuni, Yasuo
  • et al.

Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.

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