Illumina HiSeq 1500 sequencing; The Rhizoctonia solani transcriptome during interaction with the host plant Lactuca sativa
The Rhizoctonia solani transcriptome during interaction with the host plant Lactuca sativa
For the inoculation of lettuce leaves five days old mycelia disks of approximately 5-7 mm were used. From each individual plant nine detached leafs were placed in plastic containers with a layer of sterile agarose on the bottom, this was done for three different plants with mature heads. From each set eight leafs were inoculated with a single randomly placed mycelia disk, the remaining leaves functioned as blanks. During incubation the containers were loosely covered with clear plastic to retain moisture and allow for light penetration. Leaves were incubated for three days at room temperature. Subsequently samples were taken from the three interaction zones of each of the infected leaves, these cuttings were pooled per plant of origin. Interaction zone 1 is the dark brown lesion around the inoculation site, zone 2 was sampled from the lighter brown lesion and zone 3 was sampled from the green material outside of the lesion zones. After flash freezing in liquid nitrogen the samples were stored at -80 °C prior to further processing. The L. sativa cultivar Tizian (Syngenta Seeds GmbH) was cultivated under greenhouse conditions at the Bielefeld University (Germany). As substrate a 1:1 volume ratio of quartz sand and Archut Fruhstorfer einheitserde type N (Hawita gruppe GmbH, Germany) was used in one liter pots. The plants were fertilized weekly with a 0.1% Wuxal Top N solution (Wilhelm Haug GmbH and Co. KG, Germany). The rest of the week only water was supplemented based on requirement. The greenhouse had an average temperature of 18 °C during the cultivation period, the plants were exposed to natural light supplemented with artificial light (400 watt SON-T Agro Philips) on a 16/8 day and night regime.  R. solani AG1-IB isolate 7/3/14 was obtained from the IGZ in Groβbeeren. The fungus was pre-cultivated on Difco Potato dextrose agar (Becton Dickinson, Heidelberg, Germany) at room temperature. The samples were grinded to a fine powder and per sample 0.75 ml was used for total RNA isolation with the RNeasy plant mini kit (Qiagen GmbH, Hilden, Germany) Per sample 2.25 μg of RNA was used for library preparation with the TruSeq® mRNA Sample Preparation Kit (stranded) (Illumina Inc., San Diego, U.S.A.).
Experimental Factor: control
sampling site