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Host Preparation |
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Materials and Methods |
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Host material
B. napus lines with EDS1-RNAi construct, B. juncea (“Cutlass”, susceptible to A. candida race 2V), B. rapa (“Torch” and “Reward”, resistant to A. candida race 2V) and B. napus (“DH12075”, resistant to A. candida race 2V) and Arabidopsis lines with a PI-RNAi construct were used in this study. All plants were planted in coco-soilless mix and grown in a growth chamber under 16 hours light at 22˚C and 8 hours dark at 16˚C.
Inoculation
Albugo candida spores were suspended in water and incubated at 12˚C for three hours to release zoospores which were observed “swimming” in the inoculum through a microscope. Brassica seedlings were inoculated with A. candida race 2V (Ac2V), by placing 10µl drops of the incubated suspension on the cotyledons. 10-14 days after inoculation, the numbers of seedlings susceptible and resistant to Ac2V were counted.
DNA Extraction
0.5 cm disks of leaf tissue were placed into 1.5 mL centrifuge tubes. DNA was extracted using the Sigma Extraction and Amplification system.
PCR Amplification
PCR was used to confirm the presence of the EDS1 RNAi construct. BAR primers were used to amplify the BAR gene associated with the EDS1 RNAi construct. PCR products were visualized by agarose gel electrophoresis.
Microscopy
Cotyledons from the infected seedlings were collected and stained with Lactophenol Trypan Blue, by heating to 100˚C for 20 minutes. The tissue was placed in chloral hydrate for two hours to clear the tissue.
Microscopy slides were prepared with the stained cotyledons. Using a Zeiss microscope, the pathogen growth was observed. |
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Inoculation |
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Centrifuge used during DNA Extraction |
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Electrophoresis apparatus used to visualize PCR products |
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Zeiss (Imager Z1) microscope used to view the pathogen growth |
Modifying Plant CharacteristicsUsing RNA Interference |