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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

Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔpilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A. The pilJE/pilJF (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔpilJ mutant strains or the deletion plasmid (P). B. The pilJA/pilJD (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔpilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H2O, was used as negative controls for each PCR reaction.
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Fig4: Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔpilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A. The pilJE/pilJF (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔpilJ mutant strains or the deletion plasmid (P). B. The pilJA/pilJD (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔpilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H2O, was used as negative controls for each PCR reaction.

Mentions: The Xf∆pilJ strain was streaked onto PW agar plates amended with kanamycin after -80°C storage. The Xf∆pilJ mutant was observed to have behavioral phenotypes different from that previously observed for the mutant before storage but similar to wild-type X. fastidiosa (data not shown). The genotype of the Xf∆pilJ mutant was therefore assessed by PCR. Xf∆pilJ mutant directly from the -80°C stock was streaked onto PW agar plates with kanamycin to obtain single colonies and assessed by PCR. Of the twelve single colonies analyzed with the EF primers, 11 gave bands suggestive of non-transformed cells (Figure 3). Colony 12, which lacked a fragment with the EF primers, suggesting it was the Xf∆pilJ mutant strain, was streaked onto PW-kanamycin for a second round of single colony isolation. Subsequently, 32 colonies from round two were transferred to new PW plates with kanamycin and used for PCR with the AD and EF primer sets (Figure 4). Five colonies (colonies 1, 2, 4, 15, 17) appeared to be the Xf∆pilJ mutant strain, as they did not give a band with the EF primers and had a 2200 bp band with the AD primers, while 11 colonies exhibited results typical of non-transformed cells, as they gave bands with the EF primers and a 3082 bp band with the AD primers. Mixed colonies of non-transformed and transformed cells were also observed (colony 22); where the EF primers amplified a band indicating the non-transformed strain was present, and the AD primers amplified a mutant size band indicating that the Xf∆pilJ mutant was also present. Samples that appeared to be non-transformed or failed to amplify a band with AD primers, whether they amplified a band with EF primers or not, were not further analyzed. Two of the five transformed colonies were streaked onto PW agar plates with kanamycin to obtain single colonies for a third round of isolation. Of the 16 colonies examined in round three, 13 gave the mutant phenotype with the EF and AD primers (Figure 5). Again, those colonies appearing to be non-transformed, failing to amplify a band with AD primers, or giving a very small band with the AD primers were not further examined. Four of the Xf∆pilJ mutants were restreaked onto PW with kanamycin and assessed by real-time PCR for the presence of the pilJ gene (data not shown). None of these colonies were positive for the pilJ gene, therefore the samples were stored at -80°C in PD2 with 7% DMSO. This phenomenon of contamination by non-transformed cells was not limited to the Xf∆pilJ mutant strain, but observed with a number of our X. fastidiosa deletion mutants (data not shown).Figure 3


Potential complications when developing gene deletion clones in Xylella fastidiosa.

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

Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔpilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A. The pilJE/pilJF (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔpilJ mutant strains or the deletion plasmid (P). B. The pilJA/pilJD (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔpilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H2O, was used as negative controls for each PCR reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Mixture of wild-type and mutant Xylella fastidiosa strains after second isolation. The XfΔpilJ mutant confirmed in Figure 3 (isolate 12) was streaked onto periwinkle agar plates amended with kanamycin and the genotype assessed for 32 single colonies. Each number denotes a single colony. A. The pilJE/pilJF (EF) primers amplified a 2030 bp band for non-transformed bacteria and no equivalent bands for the transformed XfΔpilJ mutant strains or the deletion plasmid (P). B. The pilJA/pilJD (AD) primers amplified a 3082 bp band for non-transformed cells and a 2200 bp fragment from the XfΔpilJ strain and the deletion plasmid (P). Wild-type X. fastidiosa DNA (wt) was used as a positive control for the PCR reactions, while primer reaction without template DNA represented by H2O, was used as negative controls for each PCR reaction.
Mentions: The Xf∆pilJ strain was streaked onto PW agar plates amended with kanamycin after -80°C storage. The Xf∆pilJ mutant was observed to have behavioral phenotypes different from that previously observed for the mutant before storage but similar to wild-type X. fastidiosa (data not shown). The genotype of the Xf∆pilJ mutant was therefore assessed by PCR. Xf∆pilJ mutant directly from the -80°C stock was streaked onto PW agar plates with kanamycin to obtain single colonies and assessed by PCR. Of the twelve single colonies analyzed with the EF primers, 11 gave bands suggestive of non-transformed cells (Figure 3). Colony 12, which lacked a fragment with the EF primers, suggesting it was the Xf∆pilJ mutant strain, was streaked onto PW-kanamycin for a second round of single colony isolation. Subsequently, 32 colonies from round two were transferred to new PW plates with kanamycin and used for PCR with the AD and EF primer sets (Figure 4). Five colonies (colonies 1, 2, 4, 15, 17) appeared to be the Xf∆pilJ mutant strain, as they did not give a band with the EF primers and had a 2200 bp band with the AD primers, while 11 colonies exhibited results typical of non-transformed cells, as they gave bands with the EF primers and a 3082 bp band with the AD primers. Mixed colonies of non-transformed and transformed cells were also observed (colony 22); where the EF primers amplified a band indicating the non-transformed strain was present, and the AD primers amplified a mutant size band indicating that the Xf∆pilJ mutant was also present. Samples that appeared to be non-transformed or failed to amplify a band with AD primers, whether they amplified a band with EF primers or not, were not further analyzed. Two of the five transformed colonies were streaked onto PW agar plates with kanamycin to obtain single colonies for a third round of isolation. Of the 16 colonies examined in round three, 13 gave the mutant phenotype with the EF and AD primers (Figure 5). Again, those colonies appearing to be non-transformed, failing to amplify a band with AD primers, or giving a very small band with the AD primers were not further examined. Four of the Xf∆pilJ mutants were restreaked onto PW with kanamycin and assessed by real-time PCR for the presence of the pilJ gene (data not shown). None of these colonies were positive for the pilJ gene, therefore the samples were stored at -80°C in PD2 with 7% DMSO. This phenomenon of contamination by non-transformed cells was not limited to the Xf∆pilJ mutant strain, but observed with a number of our X. fastidiosa deletion mutants (data not shown).Figure 3

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