Liberibacter crescens is a cultured surrogate for functional genomics of uncultured pathogenic ' Candidatus Liberibacter' spp. and is naturally competent for transformation

' Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. . L. asiaticus, causal agent of Huanglongbing (HLB) or citrus "greening", and . L. solanacearum, causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, (Lcr) has been axenically cultured. Lcr strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step and highly reproducible protocols for axenic culture, transformation and targeted gene knockouts of Lcr are described. In the course of developing these protocols, we found that Lcr is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA as compared to linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of ∼900 transformants per μg plasmid, while electroporation using the same plasmid resulted in 6×10 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of non-replicative plasmids yielded 10-12 transformation events per μg DNA, while similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per μg DNA.