Computational protocol: A Comprehensive Analysis of the Transcriptomes of Marssonina brunnea and Infected Poplar Leaves to Capture Vital Events in Host-Pathogen Interactions

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Protocol publication

[…] All five or six M. brunnea-infected leaves derived from the same condition (treatment, line and time point) were homogenized using Bertin Precellys 24 (plus beads), and then the suspension was mixed together as a sample. No biological replications were performed in subsequent sequencing part. RNA was extracted using the TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Genomic DNA was removed by DNase I (TaKaRa, Japan), and libraries were constructed using the Illumina standard kit (TruSeqTM DNA Sample Prep Kit), as described in the manufacturer’s protocol. All sequencing was performed using an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA). All the RNAseq data (fastq format) were submitted to NCBI SRA database (SRP042102, http://www.ncbi.nlm.nih.gov/sra/?term=SRP042102).The RNA-seq reads were mapped onto the genomes of M. brunnea and Populus trichocarpa separately (v2.10, http://genome.jgi-psf.org/poplar/poplar.home.html) using TopHat (v2.0.8, http://tophat.cbcb.umd.edu/, allowing 2 mismatches per read as the default). The Samtools rmdup function was used to eliminate the bias introduced by PCR amplification, and paired reads that mapped to different chromosomes were discarded. Shared mapped reads (that could be mapped to the fungus and poplar genomes) were excluded from our gene expression profile analysis. The numerical measure of the mapped fragments (a fragment means a paired read) was used to evaluate the relative expression level of a certain gene. The value for a multi-mapped read, one that mapped to multiple positions, was divided by the number of positions. For instance, each position of a read that mapped to 10 positions will count as 0.1. In an individual library, the gene expression measure was normalized using the total number of mapped fragments. The reads count normalization was performed on each organism separately. Differentially expressed genes (DEGs) between two libraries were identified using Fisher’s exact test. Principal component analysis (PCA) was used to explore the overall expression pattern (poplar genes) of these samples during infection. All perl scripts and processed data can be downloaded from the website (http://homepage.fudan.edu.cn/zhouyan/interactions/). [...] The secretome was used to predict the candidate effectors of the fungus using domain analysis. The secreted proteins were identified using the TargetP [] and TMHMM [] tools and the criteria that proteins with extracellular signals had 0 or 1 transmembrane domains but no GPI (glycosyl-phosphatidylinositol) anchor domains. The secreted proteins containing an effector-specific domain significantly enriched in known effectors [] were considered to be candidate effectors. […]

Pipeline specifications

Software tools TargetP, TMHMM
Applications Protein sequence analysis, Membrane protein analysis
Organisms Marssonina brunnea, Fungi
Diseases Infection