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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.


Cq value comparisons using 96.96 dynamic array and Stratagene Mx3005P. cDNAs were synthesized using polydT(15) primers and 10 μg of total RNA from leaves untreated (a) or treated with BTH (b), Fosetyl-Al (c). Bars represent the means of Ct values from three biological replicates. Open bars: Stratagene MX3005P system and closed bars: 96.96 Fluidigm dynamic array
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Fig2: Cq value comparisons using 96.96 dynamic array and Stratagene Mx3005P. cDNAs were synthesized using polydT(15) primers and 10 μg of total RNA from leaves untreated (a) or treated with BTH (b), Fosetyl-Al (c). Bars represent the means of Ct values from three biological replicates. Open bars: Stratagene MX3005P system and closed bars: 96.96 Fluidigm dynamic array

Mentions: The Cq values obtained on a subset of 23 genes were compared for the same samples in two real-time PCR systems: the Stratagene Mx3005P and the Biomark HD, a Fluidigm® integrated fluidic circuits (IFCs) by automating PCR reactions in nanoliter volumes [46]. Twenty-two out of 23 mRNAs exhibited lower Cq values in the Fluidigm dynamic array than those obtained with the Stratagene MX3005P (15.60 ± 0.42 for the 96.96 dynamic array and 19.54 ± 0.42 for the Stratagene, mean difference, 3.96 ± 0.17), suggesting that the microfluidic technology exhibited a greater sensitivity than the Stratagene while the amounts of cDNA used in this technique were 70–150 times lower (Fig. 2a, b and c).Fig. 2


High-throughput gene-expression quantification of grapevine defense responses in the field using microfluidic dynamic arrays
Cq value comparisons using 96.96 dynamic array and Stratagene Mx3005P. cDNAs were synthesized using polydT(15) primers and 10 μg of total RNA from leaves untreated (a) or treated with BTH (b), Fosetyl-Al (c). Bars represent the means of Ct values from three biological replicates. Open bars: Stratagene MX3005P system and closed bars: 96.96 Fluidigm dynamic array
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Related In: Results  -  Collection

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

Fig2: Cq value comparisons using 96.96 dynamic array and Stratagene Mx3005P. cDNAs were synthesized using polydT(15) primers and 10 μg of total RNA from leaves untreated (a) or treated with BTH (b), Fosetyl-Al (c). Bars represent the means of Ct values from three biological replicates. Open bars: Stratagene MX3005P system and closed bars: 96.96 Fluidigm dynamic array
Mentions: The Cq values obtained on a subset of 23 genes were compared for the same samples in two real-time PCR systems: the Stratagene Mx3005P and the Biomark HD, a Fluidigm® integrated fluidic circuits (IFCs) by automating PCR reactions in nanoliter volumes [46]. Twenty-two out of 23 mRNAs exhibited lower Cq values in the Fluidigm dynamic array than those obtained with the Stratagene MX3005P (15.60 ± 0.42 for the 96.96 dynamic array and 19.54 ± 0.42 for the Stratagene, mean difference, 3.96 ± 0.17), suggesting that the microfluidic technology exhibited a greater sensitivity than the Stratagene while the amounts of cDNA used in this technique were 70–150 times lower (Fig. 2a, b and c).Fig. 2

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.