In situ hybridization was performed on 5-μm sections using an acetylcholine receptor in situ hybridization Kit (BioChain Institute, Hayward, CA) with some modifications to the instructions provided by the manufacturers.
For this purpose we normally do 10 reactions per RNA using the MEGAscript T7 kit (Ambion) with the following modification to the manufacturer's instructions: instead of the recommended incubation time for transcription at 37°C, we usually get better yield using longer incubation times of at least 6 h.
Genomic DNA was extracted from adult and larval D. ponderosae specimens using QIAGEN (Toronto, ON, Canada) DNeasy 96 Blood & Tissue kits as per the manufacturer's instructions, with modification to the lysis step.
Libraries were prepared according to the manufacturer's instructions without any modification to the manufacturer's protocol.
This proved to be very helpful and led to minor modifications to the questionnaire instruction.
Immunoprecipitation (IP) was carried out using either Invitrogen M-280 Tosylactivated Dynabeads (Carlsbad, CA, USA) (for HNE DJ-1) or Santa Cruz ImmunoCruz IP/WB Optima F System (Santa Cruz, CA, USA) (for phosphorylated DJ-1) with slight modifications to the manufacturer's instructions.
Genomic DNA was extracted using the Qiagen DNeasy tissue kit (Qiagen, Valencia, CA) with slight modifications to the manufacturer's instructions.
Mature leaf tissue was ground in liquid nitrogen, and total RNA extracted using TRI reagent (Molecular Research Center) with minor modifications to the manufacturer's instructions.
Mature leaf tissue was ground in liquid nitrogen, and total RNA extracted using TRI reagent (Molecular Research Center, Cincinnati, OH) with minor modifications to the manufacturer's instructions.
Separation of the amplified bacterial community DNA via DGGE was performed using the Bio-Rad D GENE system (Bio-Rad, Hercules, CA) with slight modifications to the manufacturer's instructions.
