Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens 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 L. crescens are described. In the course of developing these protocols, we found that L. crescens 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 compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Identification and Characterization of Menadione and Benzethonium Chloride as Potential Treatments of Pierce's Disease of Grapevines.

Xylella fastidiosa infects a wide range of plant hosts and causes Pierce's disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naphthoquinone, vitamin K) from the phytochemical library and benzethonium chloride (a topical disinfectant) from the chemical library both showed significant bactericidal activity against X. fastidiosa. Both menadione and benzethonium chloride foliar spray (15 and 5 mM, respectively) and soil drench (5 and 25 mM, respectively) treatments were equally effective in reducing PD symptoms by 54 to 59% and revealed that the effects of both chemical treatments became systemic. However, menadione was phytotoxic when applied as a foliar spray at effective concentrations, causing significant loss of photosynthetic capacity.