| Autor: |
Jain M; Department of Plant Pathology, University of Florida, Gainesville, FL 32611., Cai L; Department of Plant Pathology, University of Florida, Gainesville, FL 32611., Fleites LA; Department of Plant Pathology, University of Florida, Gainesville, FL 32611., Munoz-Bodnar A; Department of Plant Pathology, University of Florida, Gainesville, FL 32611., Davis MJ; Department of Plant Pathology, Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850., Gabriel DW; Department of Plant Pathology, University of Florida, Gainesville, FL 32611. |
| Jazyk: |
angličtina |
| Zdroj: |
Phytopathology [Phytopathology] 2019 Oct; Vol. 109 (10), pp. 1811-1819. Date of Electronic Publication: 2019 Aug 13. |
| DOI: |
10.1094/PHYTO-04-19-0129-R |
| Abstrakt: |
' 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 × 10 4 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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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