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Selection of reference genes from two leafhopper species challenged by phytoplasma infection, for gene expression studies by RT-qPCR.

Galetto L, Bosco D, Marzachì C - BMC Res Notes (2013)

Bottom Line: This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity.Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens.Phylogenetic indications were also drawn from sequence analysis of these genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy. l.galetto@ivv.cnr.it.

ABSTRACT

Background: Phytoplasmas are phloem-limited phytopathogenic wall-less bacteria and represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. For gene expression studies based on mRNA quantification by RT-qPCR, stability of housekeeping genes is crucial. The aim of this study was the identification of reference genes to study the effect of phytoplasma infection on gene expression of two leafhopper vector species. The identified reference genes will be useful tools to investigate differential gene expression of leafhopper vectors upon phytoplasma infection.

Results: The expression profiles of ribosomal 18S, actin, ATP synthase β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tropomyosin were determined in two leafhopper vector species (Hemiptera: Cicadellidae), both healthy and infected by "Candidatus Phytoplasma asteris" (chrysanthemum yellows phytoplasma strain, CYP). Insects were analyzed at three different times post acquisition, and expression stabilities of the selected genes were evaluated with BestKeeper, geNorm and Normfinder algorithms. In Euscelidius variegatus, all genes under all treatments were stable and could serve as reference genes. In Macrosteles quadripunctulatus, BestKeeper and Normfinder analysis indicated ATP synthase β, tropomyosin and GAPDH as the most stable, whereas geNorm identified reliable genes only for early stages of infection.

Conclusions: In this study a validation of five candidate reference genes was performed with three algorithms, and housekeeping genes were identified for over time transcript profiling of two leafhopper vector species infected by CYP. This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity. Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens. Phylogenetic indications were also drawn from sequence analysis of these genes.

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Amplification profiles of candidate reference genes. Box plot of qPCR cycle threshold values (Cq) for candidate reference genes in the two phytoplasma vector species. The median is depicted as the line across the box; the box indicates the 25th and 75th percentiles; whiskers represent the 90th and 10th percentiles.
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Figure 1: Amplification profiles of candidate reference genes. Box plot of qPCR cycle threshold values (Cq) for candidate reference genes in the two phytoplasma vector species. The median is depicted as the line across the box; the box indicates the 25th and 75th percentiles; whiskers represent the 90th and 10th percentiles.

Mentions: Cycle threshold (Cq) values obtained amplifying the five putative housekeeping genes from the two vector species were plotted (Figure 1). Cq values for the five genes ranged from 12.26 to 38.52 in E. variegatus and from 10.25 to 34.03 in M. quadripunctulatus. Ribosomal 18S showed the lowest Cq values in both species (13.52 ± 0.74, mean Cq ± std. dev. for E. variegatus, and 12.65 ± 1.82 for M. quadripunctulatus), being expressed at a very high level compared to other protein encoding mRNAs. A similar amplification profile is reported for the 18S gene of the vector Delphacodes kuscheli challenged by Mal de Rio Cuarto fijivirus[28]. Amplification of actin, ATP synthase β, GAPDH and tropomyosin of E. variegatus showed mean Cqs of 32.89 ± 1.84, 28.05 ± 1.45, 21.19 ± 1.04, 27.62 ± 1.42, respectively. Mean Cqs of M. quadripunctulatus actin, ATP synthase β, GAPDH and tropomyosin were 25.66 ± 3.25, 30.57 ± 1.45, 30.30 ± 1.04, 22.23 ± 1.77, respectively.


Selection of reference genes from two leafhopper species challenged by phytoplasma infection, for gene expression studies by RT-qPCR.

Galetto L, Bosco D, Marzachì C - BMC Res Notes (2013)

Amplification profiles of candidate reference genes. Box plot of qPCR cycle threshold values (Cq) for candidate reference genes in the two phytoplasma vector species. The median is depicted as the line across the box; the box indicates the 25th and 75th percentiles; whiskers represent the 90th and 10th percentiles.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852609&req=5

Figure 1: Amplification profiles of candidate reference genes. Box plot of qPCR cycle threshold values (Cq) for candidate reference genes in the two phytoplasma vector species. The median is depicted as the line across the box; the box indicates the 25th and 75th percentiles; whiskers represent the 90th and 10th percentiles.
Mentions: Cycle threshold (Cq) values obtained amplifying the five putative housekeeping genes from the two vector species were plotted (Figure 1). Cq values for the five genes ranged from 12.26 to 38.52 in E. variegatus and from 10.25 to 34.03 in M. quadripunctulatus. Ribosomal 18S showed the lowest Cq values in both species (13.52 ± 0.74, mean Cq ± std. dev. for E. variegatus, and 12.65 ± 1.82 for M. quadripunctulatus), being expressed at a very high level compared to other protein encoding mRNAs. A similar amplification profile is reported for the 18S gene of the vector Delphacodes kuscheli challenged by Mal de Rio Cuarto fijivirus[28]. Amplification of actin, ATP synthase β, GAPDH and tropomyosin of E. variegatus showed mean Cqs of 32.89 ± 1.84, 28.05 ± 1.45, 21.19 ± 1.04, 27.62 ± 1.42, respectively. Mean Cqs of M. quadripunctulatus actin, ATP synthase β, GAPDH and tropomyosin were 25.66 ± 3.25, 30.57 ± 1.45, 30.30 ± 1.04, 22.23 ± 1.77, respectively.

Bottom Line: This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity.Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens.Phylogenetic indications were also drawn from sequence analysis of these genes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy. l.galetto@ivv.cnr.it.

ABSTRACT

Background: Phytoplasmas are phloem-limited phytopathogenic wall-less bacteria and represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. For gene expression studies based on mRNA quantification by RT-qPCR, stability of housekeeping genes is crucial. The aim of this study was the identification of reference genes to study the effect of phytoplasma infection on gene expression of two leafhopper vector species. The identified reference genes will be useful tools to investigate differential gene expression of leafhopper vectors upon phytoplasma infection.

Results: The expression profiles of ribosomal 18S, actin, ATP synthase β, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and tropomyosin were determined in two leafhopper vector species (Hemiptera: Cicadellidae), both healthy and infected by "Candidatus Phytoplasma asteris" (chrysanthemum yellows phytoplasma strain, CYP). Insects were analyzed at three different times post acquisition, and expression stabilities of the selected genes were evaluated with BestKeeper, geNorm and Normfinder algorithms. In Euscelidius variegatus, all genes under all treatments were stable and could serve as reference genes. In Macrosteles quadripunctulatus, BestKeeper and Normfinder analysis indicated ATP synthase β, tropomyosin and GAPDH as the most stable, whereas geNorm identified reliable genes only for early stages of infection.

Conclusions: In this study a validation of five candidate reference genes was performed with three algorithms, and housekeeping genes were identified for over time transcript profiling of two leafhopper vector species infected by CYP. This work set up an experimental system to study the molecular basis of phytoplasma multiplication in the insect body, in order to elucidate mechanisms of vector specificity. Most of the sequences provided in this study are new for leafhoppers, which are vectors of economically important plant pathogens. Phylogenetic indications were also drawn from sequence analysis of these genes.

Show MeSH
Related in: MedlinePlus