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Potential complications when developing gene deletion clones in Xylella fastidiosa.

Johnson KL, Cursino L, Athinuwat D, Burr TJ, Mowery P - BMC Res Notes (2015)

Bottom Line: 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.As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. johnson3@yahoo.com.

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.

No MeSH data available.


Related in: MedlinePlus

Confirmation of Xylella fastidiosa pilJ gene deletion. The pilJA/pilJD (AD) primers amplify a 3082 bp fragment from wild-type control cells (wt) or a 2200 bp fragment form the XfΔpilJ mutant (J) and deletion plasmid (P). The pilJE/pilJF (EF) primers amplify a 2030 bp band for the wild-type control strain and no band for the mutant cells or deletion plasmid. RST31/33 (RST) primers confirm that the bacteria were X. fastidiosa.
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Fig2: Confirmation of Xylella fastidiosa pilJ gene deletion. The pilJA/pilJD (AD) primers amplify a 3082 bp fragment from wild-type control cells (wt) or a 2200 bp fragment form the XfΔpilJ mutant (J) and deletion plasmid (P). The pilJE/pilJF (EF) primers amplify a 2030 bp band for the wild-type control strain and no band for the mutant cells or deletion plasmid. RST31/33 (RST) primers confirm that the bacteria were X. fastidiosa.

Mentions: The pilJ gene encodes a putative chemotaxis receptor of interest [16]. The gene was deleted from X. fastidiosa Temecula 1 using site directed replacement with a kanamycin resistant marker [10]. Transformants were selected on antibiotic plates at 10 μg/mL since the minimum inhibitory concentration of kanamycin for X. fastidiosa Temecula 1 is 4 μg/mL [11]. All subsequent work with the transformed cells (Xf∆pilJ mutants) were performed with 50 μg/mL kanamycin. Deletion of the pilJ gene was confirmed by PCR using multiple primer sets (Figure 1). The pilJA/pilJD (AD) primers amplified a 3082 bp band from wild-type control bacteria and a 2200 bp band from the deletion plasmid pUC19-pilJ and the Xf∆pilJ strain (Figure 2). The pilJE/pilJF (EF) primers are complementary to sequences within the pilJ gene producing a 2030 bp band for wild-type control cells and no fragments for the Xf∆pilJ bacteria or plasmid control. The X. fastidiosa-specific RST31/33 primers [17] confirmed that the bacteria were X. fastidiosa. As expected, these primers failed to amplify a band from the pUC19-pilJ plasmid. The Xf∆pilJ strain was subsequently tested in a number of behavioral assays to explore the role of the PilJ protein (data not shown). The Xf∆pilJ strain was placed in storage at -80°C in PD2 with 7% DMSO.Figure 1


Potential complications when developing gene deletion clones in Xylella fastidiosa.

Johnson KL, Cursino L, Athinuwat D, Burr TJ, Mowery P - BMC Res Notes (2015)

Confirmation of Xylella fastidiosa pilJ gene deletion. The pilJA/pilJD (AD) primers amplify a 3082 bp fragment from wild-type control cells (wt) or a 2200 bp fragment form the XfΔpilJ mutant (J) and deletion plasmid (P). The pilJE/pilJF (EF) primers amplify a 2030 bp band for the wild-type control strain and no band for the mutant cells or deletion plasmid. RST31/33 (RST) primers confirm that the bacteria were X. fastidiosa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4403849&req=5

Fig2: Confirmation of Xylella fastidiosa pilJ gene deletion. The pilJA/pilJD (AD) primers amplify a 3082 bp fragment from wild-type control cells (wt) or a 2200 bp fragment form the XfΔpilJ mutant (J) and deletion plasmid (P). The pilJE/pilJF (EF) primers amplify a 2030 bp band for the wild-type control strain and no band for the mutant cells or deletion plasmid. RST31/33 (RST) primers confirm that the bacteria were X. fastidiosa.
Mentions: The pilJ gene encodes a putative chemotaxis receptor of interest [16]. The gene was deleted from X. fastidiosa Temecula 1 using site directed replacement with a kanamycin resistant marker [10]. Transformants were selected on antibiotic plates at 10 μg/mL since the minimum inhibitory concentration of kanamycin for X. fastidiosa Temecula 1 is 4 μg/mL [11]. All subsequent work with the transformed cells (Xf∆pilJ mutants) were performed with 50 μg/mL kanamycin. Deletion of the pilJ gene was confirmed by PCR using multiple primer sets (Figure 1). The pilJA/pilJD (AD) primers amplified a 3082 bp band from wild-type control bacteria and a 2200 bp band from the deletion plasmid pUC19-pilJ and the Xf∆pilJ strain (Figure 2). The pilJE/pilJF (EF) primers are complementary to sequences within the pilJ gene producing a 2030 bp band for wild-type control cells and no fragments for the Xf∆pilJ bacteria or plasmid control. The X. fastidiosa-specific RST31/33 primers [17] confirmed that the bacteria were X. fastidiosa. As expected, these primers failed to amplify a band from the pUC19-pilJ plasmid. The Xf∆pilJ strain was subsequently tested in a number of behavioral assays to explore the role of the PilJ protein (data not shown). The Xf∆pilJ strain was placed in storage at -80°C in PD2 with 7% DMSO.Figure 1

Bottom Line: 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.As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Plant-Microbe Biology, Cornell University New York State Agricultural Experiment Station, Geneva, NY, 14456, USA. johnson3@yahoo.com.

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.

No MeSH data available.


Related in: MedlinePlus