P. sojae reference strain P6497 (race 2) was used in this research. Mycelia (MY) were cultivated in 10% V8 liquid medium at 25ºC in darkness for 48h, then blotted dry with absorbent paper and preserved in liquid nitrogen for RNA isolation. Zoosporangia (SP) were induced by repeatedly washing 48- to 72-hour-old mycelial mats with sterile distilled water at 25ºC in darkness until sporangia formed abundantly. Zoospores (ZO) were released by placing the zoosporangial mycelial mat into 10 ml sterile distilled water at 5-10ºC for 10-15 min, then at 25ºC for 10-30 min. The zoospores were then concentrated by centrifugation at 2,000 rpm at 0ºC to a concentration of >150 zoospores /µl then preserved in liquid nitrogen for RNA isolation. The zoospores were counted under microscope based on 2µl sucked zoospore suspension sample and estimated from the average of three repeats. Cysts (CY) were produced by vortexing the zoospores suspension at room temperature for 30 seconds, then collected by centrifugation at 2,000 rpm at 0ºC and preserved in liquid nitrogen for RNA isolation. Germinating cysts (GC) were obtained by cultivating cysts with 5% V8 liquid medium at 25ºC, 150 rpm for 1h, then harvesting them by centrifugation at 2,000 rpm at 0ºC. For mycelia infection (IF1.5h- IF24h), the soybean cultivar Williams, which is susceptible to P6497, was grown in a greenhouse at 22-28ºC and used at the second leaf stage. Soybean leaves were treated with 0.05% v/v solution of tween 20 to improve wetting. A mycelial mat was washed with sterile distilled water and then laid on and sandwiched between upper surfaces of two leaves at 25ºC respectively for 1.5h, 3h, 6h, 12h, 24h after infection. For the 1.5 h and 3 h time points, the mycelial mat was carefully peeled from the leaves and preserved in liquid nitrogen. For the later time points, the regions of the leaves in contact with the mycelia were excised together with the mycelia and preserved in liquid nitrogen.
Extracted molecule
total RNA
Extraction protocol
Tag library preparation for the samples was performed in parallel using Illumina gene expression sample preparation kit. Briefly, total RNA from the three samples was used for mRNA capture with magnetic oligo(dT) beads. First and second strand cDNA were synthesized and bead-bound cDNA was subsequently digested with NlaIII. The cDNA fragments with 3' ends were then purified with magnetic beads and Illumina adapter 1 was added to their 5' ends. The junction of Illumina adapter 1 and CATG site is the recognition site of MmeI, which cuts 17 bp downstream of the CATG site, producing tags with adapter 1. After removing 3' fragments with magnetic beads precipitation, Illumina adapter 2 was introduced at 3' ends of tags, acquiring tags with different adapters at both ends to form a tag library. After 15 cycles of linear PCR amplification, 85 base strips were purified by PAGE gel electrophoresis. These strips were then digested, and the single-chain molecules were fixed onto the Illumina sequencing chip for sequencing.
Library strategy
RNA-Seq
Library source
transcriptomic
Library selection
cDNA
Instrument model
Illumina Genome Analyzer
Description
Digital gene expression tag profiling
Data processing
Illumina Pipeline Software version 1.0 was used for data processing. Raw sequences have 3' adaptor fragments as well as a few low-quality sequences and several types of impurities. Raw sequences were transformed into clean 21 bp (CATG+17bp) tags by the following steps: (1) 3' adaptor sequence was trimmed, resulting in 21 bp tags from 35 bp, of raw sequence (2) empty reads were removed (reads with only 3' adaptor sequences but no tags); (3) low quality tags were removed (tags with ambiguous base calls); (4) tags of unusual length were removed, leaving only tags of 21 bp; (5) non-redundant tags were removed (each tag needs to be detected at least twice to be considered reliable).
Submission date
Jun 01, 2011
Last update date
May 15, 2019
Contact name
Wenwu Ye
Organization name
Nanjing Agricultral University, China
Street address
Department of Plant Pathology, Nanjing Agricultural University
