5 protocols
Solanum lycopersicum ‘Moneymaker’ plants were grown in controlled growth chambers at 22C, a photoperiod of 16 h, supplemented by artificial light and 70% humidity. The third leaf from the top of every plant was detached and placed upside down in humid transparent plastic trays and a controlled incubator with the same settings as in the growth chamber. Phytophthora capsici wild type strain LT1534 was grown in Petri dishes on V8 agar medium in a dark climate chamber at 25C for three days and for two days under bright light at 25C. To induce zoospore release, plates were flooded with ice-cold distilled water and spores were harvested from sporulating mycelia by dislodging the sporangia with a sterile glass spreader. Sporangial suspensions were collected and incubated at room temperature under bright light conditions. Release of zoospores was followed and the amounts counted under the microscope in a haemocytometer and adjusted to 1x10^5 ml-1. The detached leaves were inoculated with four droplets (each 20ul) of the zoospore solution.
Target RNA (up to 100 ng) was labeled as recommended (version 6.5).
RNA was isolated from frozen leaf tissue following the protocol RNeasy Plant Cells Mini Kit (QIAGEN) and treated afterwards with DNAse (Ambion) to remove genomic DNA contamination according to the manufacturers instructions.
Sporangia and germinating cysts were collected from a 10 ml zoospore solution after an incubation time of 1 h and 24 h at room temperature, respectively, by centrifugation for 1 min at 3500 rpm. The pellets were collected, frozen in liquid nitrogen and stored at -80C. Mycelium was prepared by inoculating Pea broth with zoospores, suspension used or tomato infection. Mycelium was grown for 48 hours before harvesting. Harvested mycelium was rinsed with sterile water before flash freezing in liquid nitrogen and storage at -80C.