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High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays

View Article: PubMed Central - PubMed

ABSTRACT

Background: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.

Results: We have developed a pioneering tool (“NeoViGen96” chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4 h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the “NeoViGen96” chip as well as their efficacy against downy mildew.

Results: BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway.

Results: FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes.

Conclusions: The NeoViGen96” chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4 h and 1,500 €), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270 h and 9,000 €) thus a throughput 60–70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3304-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Correlation scatter plots of fold expression of “BioMolChem” chip genes (n = 24) using Stratagene or Biomark HD systems obtained with three replicates of leaves treated with BTH (red), with FOS (green) or untreated (black). Correlation of fold expression obtained by Stratagene MX3005P system (x axis) and microfluidic dynamic array (y axis). R2 = coefficient of correlation of the simple linear regression, PPMCC = Pearson product–moment correlation coefficient (Pearson’s correlation). Significant correlation was determined at a level of p-value < 0.05
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Fig4: Correlation scatter plots of fold expression of “BioMolChem” chip genes (n = 24) using Stratagene or Biomark HD systems obtained with three replicates of leaves treated with BTH (red), with FOS (green) or untreated (black). Correlation of fold expression obtained by Stratagene MX3005P system (x axis) and microfluidic dynamic array (y axis). R2 = coefficient of correlation of the simple linear regression, PPMCC = Pearson product–moment correlation coefficient (Pearson’s correlation). Significant correlation was determined at a level of p-value < 0.05

Mentions: We compared fold-change expression of defense-related gene in the same samples measured by the 96.96 dynamic array with those obtained from the “BioMolChem” chip with the Stratagene Mx3005P (Fig. 4). Fold change comparisons were similar between the two platforms, which indicated a perfect significant correlation between the two technologies (R2 = 0.737 and Pearson’s correlation (PPMCC) =0.86; p-value < 0.05). The maximum fold change detected by the Stratagene was 2.59 compared to 2.28 by the 96.96 dynamic array (Fig. 4).Fig. 4


High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays
Correlation scatter plots of fold expression of “BioMolChem” chip genes (n = 24) using Stratagene or Biomark HD systems obtained with three replicates of leaves treated with BTH (red), with FOS (green) or untreated (black). Correlation of fold expression obtained by Stratagene MX3005P system (x axis) and microfluidic dynamic array (y axis). R2 = coefficient of correlation of the simple linear regression, PPMCC = Pearson product–moment correlation coefficient (Pearson’s correlation). Significant correlation was determined at a level of p-value < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120521&req=5

Fig4: Correlation scatter plots of fold expression of “BioMolChem” chip genes (n = 24) using Stratagene or Biomark HD systems obtained with three replicates of leaves treated with BTH (red), with FOS (green) or untreated (black). Correlation of fold expression obtained by Stratagene MX3005P system (x axis) and microfluidic dynamic array (y axis). R2 = coefficient of correlation of the simple linear regression, PPMCC = Pearson product–moment correlation coefficient (Pearson’s correlation). Significant correlation was determined at a level of p-value < 0.05
Mentions: We compared fold-change expression of defense-related gene in the same samples measured by the 96.96 dynamic array with those obtained from the “BioMolChem” chip with the Stratagene Mx3005P (Fig. 4). Fold change comparisons were similar between the two platforms, which indicated a perfect significant correlation between the two technologies (R2 = 0.737 and Pearson’s correlation (PPMCC) =0.86; p-value < 0.05). The maximum fold change detected by the Stratagene was 2.59 compared to 2.28 by the 96.96 dynamic array (Fig. 4).Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

Background: The fight against grapevine diseases due to biotrophic pathogens usually requires the massive use of chemical fungicides with harmful environmental effects. An alternative strategy could be the use of compounds able to stimulate plant immune responses which significantly limit the development of pathogens in laboratory conditions. However, the efficiency of this strategy in natura is still insufficient to be included in pest management programs. To understand and to improve the mode of action of plant defense stimulators in the field, it is essential to develop reliable tools that describe the resistance status of the plant upon treatment.

Results: We have developed a pioneering tool (&ldquo;NeoViGen96&rdquo; chip) based on a microfluidic dynamic array platform allowing the expression profiling of 85 defense-related grapevine genes in 90 cDNA preparations in a 4&nbsp;h single run. Two defense inducers, benzothiadiazole (BTH) and fosetyl-aluminum (FOS), have been tested in natura using the &ldquo;NeoViGen96&rdquo; chip as well as their efficacy against downy mildew.

Results: BTH-induced grapevine resistance is accompanied by the induction of PR protein genes (PR1, PR2 and PR3), genes coding key enzymes in the phenylpropanoid pathway (PAL and STS), a GST gene coding an enzyme involved in the redox status and an ACC gene involved in the ethylene pathway.

Results: FOS, a phosphonate known to possess a toxic activity against pathogens and an inducing effect on defense genes provided a better grapevine protection than BTH. Its mode of action was probably strictly due to its fungicide effect at high concentrations because treatment did not induce significant change in the expression level of selected defense-related genes.

Conclusions: The NeoViGen96&rdquo; chip assesses the effectiveness of plant defense inducers on grapevine in vineyard with an excellent reproducibility. A single run with this system (4&nbsp;h and 1,500 &euro;), corresponds to 180 qPCR plates with conventional Q-PCR assays (Stragene system, 270&nbsp;h and 9,000 &euro;) thus a throughput 60&ndash;70 times higher and 6 times cheaper. Grapevine responses after BTH elicitation in the vineyard were similar to those obtained in laboratory conditions, whereas our results suggest that the protective effect of FOS against downy mildew in the vineyard was only due to its fungicide activity since no activity on plant defense genes was observed. This tool provides better understanding of how the grapevine replies to elicitation in its natural environment and how the elicitor potential can be used to reduce chemical fungicide inputs.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3304-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus