title: Potential complications when developing gene deletion clones in Xylella fastidiosa.
abstract: BACKGROUND
  The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers.
  
  
  METHODS
  Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations.
  
  
  RESULTS
  We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present.
  
  
  CONCLUSIONS
  We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. johnson3@yahoo.com.
authors.forename: Kameka L
authors.initials: KL
authors.lastname: Johnson
authors.affiliation: Department of Biology, Hobart and William Smith Colleges, Geneva, NY, 14456, USA. cursino@hws.edu.
authors.forename: Luciana
authors.initials: L
authors.lastname: Cursino
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. athinova6@hotmail.com.|Current address: Major of Organic Farming Management, Faculty of Science and Technology, Thammasat University, Pathum Thani, Thailand. athinova6@hotmail.com.
authors.forename: Dusit
authors.initials: D
authors.lastname: Athinuwat
authors.affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. tjb1@cornell.edu.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Burr
authors.affiliation: Department of Biology, Hobart and William Smith Colleges, Geneva, NY, 14456, USA. mowery@hws.edu.
authors.forename: Patricia
authors.initials: P
authors.lastname: Mowery
mesh_terms: D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010935:Plant Diseases / Q000382:microbiology*; D027843:Vitis / Q000382:microbiology*; D044167:Xylella / Q000235:genetics* / Q000472:pathogenicity