title: A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants.
abstract: Ablation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve sufficient specificity of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter specificity to be used to enhance targeting specificity. Using a transient system, we first show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.
authors.affiliation: DNA Plant Technologies, 6701 San Pablo Avenue, Oakland, CA 94608, USA. diburgess2@attbi.com
authors.forename: Diane G
authors.initials: DG
authors.lastname: Burgess
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Ralston
authors.forename: William G
authors.initials: WG
authors.lastname: Hanson
authors.forename: Matthew
authors.initials: M
authors.lastname: Heckert
authors.forename: Minh
authors.initials: M
authors.lastname: Ho
authors.forename: Tina
authors.initials: T
authors.lastname: Jenq
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Palys
authors.forename: Keliang
authors.initials: K
authors.lastname: Tang
authors.forename: Neal
authors.initials: N
authors.lastname: Gutterson
mesh_terms: D001426:Bacterial Proteins; D001483:Base Sequence; D016923:Cell Death; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D018551:Solanum lycopersicum / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D059828:Plant Cells; D010944:Plants / Q000201:enzymology* / Q000235:genetics*; D030821:Plants, Genetically Modified; D012098:Reproduction / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D018744:DNA, Plant; D010446:Peptide Fragments; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease