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A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs.

Ihlow A, Schweizer P, Seiffert U - BMC Plant Biol. (2008)

Bottom Line: In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects.Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images.In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pattern Recognition Group, IPK Gatersleben, Corrensstr, 3, D-06466 Gatersleben, Germany. ihlow@ipk-gatersleben.de

ABSTRACT

Background: To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps.

Results: We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects.

Conclusion: Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.

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Screening pipeline. After inserting the slides onto the xy-stage, the screening is started in the control program. The final output are the susceptibility indices for each slide in terms of the number of infected transformed cells over the total number of transformed cells according to Equation (1).
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Figure 3: Screening pipeline. After inserting the slides onto the xy-stage, the screening is started in the control program. The final output are the susceptibility indices for each slide in terms of the number of infected transformed cells over the total number of transformed cells according to Equation (1).

Mentions: To realize a fully automated specimen analysis, interesting regions on the slide must first be identified, before detailed images of these regions can be generated. A naive complete high-resolution scanning is out of the question due to the tight time constraints of a high-throughput system. Therefore, the screening is divided into two main temporal passes, namely a preview scan and a subsequent detailed scan. During the preview scan, an overview image of each slide is generated by assembling together coarse resolution images (432 × 342 pixels) of low magnification (5× objective). To cover the entire slide, 15 horizontal times 38 vertical steps are necessary, leading to 570 subimages per slide. After the positions of transformed cells are identified, the detailed scan starts. Subsequently, the microscope changes to the 10× objective and acquires high-resolution images (1300 × 1030 pixels) of each requested position. To overcome the limited depth of field, images of several foci are necessary. Five focal layers of 7 μm distance proved to be sufficient to provide a sharp reproduction of all details. The described pipeline is illustrated in Figure 3 as a flow chart.


A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs.

Ihlow A, Schweizer P, Seiffert U - BMC Plant Biol. (2008)

Screening pipeline. After inserting the slides onto the xy-stage, the screening is started in the control program. The final output are the susceptibility indices for each slide in terms of the number of infected transformed cells over the total number of transformed cells according to Equation (1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2262080&req=5

Figure 3: Screening pipeline. After inserting the slides onto the xy-stage, the screening is started in the control program. The final output are the susceptibility indices for each slide in terms of the number of infected transformed cells over the total number of transformed cells according to Equation (1).
Mentions: To realize a fully automated specimen analysis, interesting regions on the slide must first be identified, before detailed images of these regions can be generated. A naive complete high-resolution scanning is out of the question due to the tight time constraints of a high-throughput system. Therefore, the screening is divided into two main temporal passes, namely a preview scan and a subsequent detailed scan. During the preview scan, an overview image of each slide is generated by assembling together coarse resolution images (432 × 342 pixels) of low magnification (5× objective). To cover the entire slide, 15 horizontal times 38 vertical steps are necessary, leading to 570 subimages per slide. After the positions of transformed cells are identified, the detailed scan starts. Subsequently, the microscope changes to the 10× objective and acquires high-resolution images (1300 × 1030 pixels) of each requested position. To overcome the limited depth of field, images of several foci are necessary. Five focal layers of 7 μm distance proved to be sufficient to provide a sharp reproduction of all details. The described pipeline is illustrated in Figure 3 as a flow chart.

Bottom Line: In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects.Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images.In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pattern Recognition Group, IPK Gatersleben, Corrensstr, 3, D-06466 Gatersleben, Germany. ihlow@ipk-gatersleben.de

ABSTRACT

Background: To find candidate genes that potentially influence the susceptibility or resistance of crop plants to powdery mildew fungi, an assay system based on transient-induced gene silencing (TIGS) as well as transient over-expression in single epidermal cells of barley has been developed. However, this system relies on quantitative microscopic analysis of the barley/powdery mildew interaction and will only become a high-throughput tool of phenomics upon automation of the most time-consuming steps.

Results: We have developed a high-throughput screening system based on a motorized microscope which evaluates the specimens fully automatically. A large-scale double-blind verification of the system showed an excellent agreement of manual and automated analysis and proved the system to work dependably. Furthermore, in a series of bombardment experiments an RNAi construct targeting the Mlo gene was included, which is expected to phenocopy resistance mediated by recessive loss-of-function alleles such as mlo5. In most cases, the automated analysis system recorded a shift towards resistance upon RNAi of Mlo, thus providing proof of concept for its usefulness in detecting gene-target effects.

Conclusion: Besides saving labor and enabling a screening of thousands of candidate genes, this system offers continuous operation of expensive laboratory equipment and provides a less subjective analysis as well as a complete and enduring documentation of the experimental raw data in terms of digital images. In general, it proves the concept of enabling available microscope hardware to handle challenging screening tasks fully automatically.

Show MeSH
Related in: MedlinePlus