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Potential complications when developing gene deletion clones in Xylella fastidiosa

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 eventua...

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Autores principales: Johnson, Kameka L, Cursino, Luciana, Athinuwat, Dusit, Burr, Thomas J, Mowery, Patricia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403849/
https://www.ncbi.nlm.nih.gov/pubmed/25880211
http://dx.doi.org/10.1186/s13104-015-1117-9
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author Johnson, Kameka L
Cursino, Luciana
Athinuwat, Dusit
Burr, Thomas J
Mowery, Patricia
author_facet Johnson, Kameka L
Cursino, Luciana
Athinuwat, Dusit
Burr, Thomas J
Mowery, Patricia
author_sort Johnson, Kameka L
collection PubMed
description 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.
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spelling pubmed-44038492015-04-21 Potential complications when developing gene deletion clones in Xylella fastidiosa Johnson, Kameka L Cursino, Luciana Athinuwat, Dusit Burr, Thomas J Mowery, Patricia BMC Res Notes Research Article 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. BioMed Central 2015-04-16 /pmc/articles/PMC4403849/ /pubmed/25880211 http://dx.doi.org/10.1186/s13104-015-1117-9 Text en © Johnson et al.; licensee BioMed Central. 2015 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Johnson, Kameka L
Cursino, Luciana
Athinuwat, Dusit
Burr, Thomas J
Mowery, Patricia
Potential complications when developing gene deletion clones in Xylella fastidiosa
title Potential complications when developing gene deletion clones in Xylella fastidiosa
title_full Potential complications when developing gene deletion clones in Xylella fastidiosa
title_fullStr Potential complications when developing gene deletion clones in Xylella fastidiosa
title_full_unstemmed Potential complications when developing gene deletion clones in Xylella fastidiosa
title_short Potential complications when developing gene deletion clones in Xylella fastidiosa
title_sort potential complications when developing gene deletion clones in xylella fastidiosa
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403849/
https://www.ncbi.nlm.nih.gov/pubmed/25880211
http://dx.doi.org/10.1186/s13104-015-1117-9
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