De novo transcriptome of Phakopsora pachyrhizi by Illumina short-read sequencing

<i>Phakopsora pachyrhizi</i> is an obligate biotrophic fungal pathogen of soybean that causes Asian soybean rust (ASR), a devastating disease that can cause yield losses of 80% or greater. <i>P. pachyrhizi</i> secretes an arsenal of effector proteins to manipulate host immunity and promote disease. Current knowledge of the <i>P. pachyrhizi </i>genome is limited and only a small number of the total <i>P. pachyrhizi </i>effectors have been identified. We therefore sequenced the transcriptome of <i>P. pachyrhizi</i> during infection to identify <i>P. pachyrhizi </i>Candidate Secreted Effector Proteins (CSEPs). Total RNA was extracted from <i>P. pachyrhizi</i>-infected soybean leaf tissue collected at 3, 7, 10, and 14 days after inoculation (dai). Strand-specific cDNA libraries were prepared from ribosomal depleted RNA using NEBNext Ultra II RNA library prep kit according to the manufacturer's instructions. Paired-end short-read Illumina sequencing was performed on dual-indexed cDNA libraries using HiSeq3000 (150bp from each end; https://www.illumina.com/) and MiSeq (300bp from each end; https://www.illumina.com/). The raw reads obtained from Illumina short-read sequencing were quality assessed using FastQC v.0.11.2. Paired-end read trimming was conducted by Trimmomatic 0.36 using sliding window 4:15 and excluding read below a minimal length of 36. Trimmed paired-end RNA-Seq reads from 3, 7, 10, and 14 dai were mapped against the soybean genome v2.1 (https://plants.ensembl.org/Glycine_max) using STAR 2.5.3a aligner to remove soybean reads. The non-soybean short-reads were <i>de novo</i> assembled using Trinity v2.6.6. BLASTN (Basic Local Alignment Search Tool) search was performed on non-soybean transcripts using Blastplus v2.6.0 (NCBI: National Center for Biotechnology Information) to remove any plant transcripts with a query coverage greater than 80% and identity greater than 95%. The final <i>de novo </i>transcriptome containing non-plant, non-soybean transcripts for each time point was used for prediction of candidate effectors.<br><b>Citation:</b>M.G.Elmore, S.Banerjee, K.F.Pedley, A.Ruck, S.A.Whitham. <i>De novo</i> transcriptome of <i>Phakopsora</i> <i>pachyrhizi</i> uncovers putative effector repertoire during infection. Physiological and Molecular Plant Pathology.,<br>110 (2020),101464, 10.1016/j.pmpp.2020.101464

<i>Phakopsora pachyrhizi</i>是大豆的专性活体营养型真菌病原体，可引发亚洲大豆锈病（Asian soybean rust，ASR）——一种毁灭性病害，产量损失可达80%或更高。<i>P. pachyrhizi</i>分泌大量效应蛋白以调控宿主免疫反应、促进病害发生。目前对<i>P. pachyrhizi</i>基因组的认知有限，仅鉴定出其全部效应蛋白中的一小部分。因此，我们对感染期间的<i>P. pachyrhizi</i>转录组进行测序，以鉴定其候选分泌效应蛋白（Candidate Secreted Effector Proteins，CSEPs）。从接种后3、7、10和14天（days after inoculation，dai）采集的<i>P. pachyrhizi</i>感染大豆叶片组织中提取总RNA，按制造商说明使用NEBNext Ultra II RNA文库制备试剂盒从去核糖体RNA构建链特异性cDNA文库。使用HiSeq3000（每端150bp；https://www.illumina.com/）和MiSeq（每端300bp；https://www.illumina.com/）对双索引cDNA文库进行Illumina双端短读长测序。通过FastQC v.0.11.2评估原始读数质量，用Trimmomatic 0.36以滑动窗口4:15修剪双端读数并排除长度小于36的序列。使用STAR 2.5.3a比对工具将各时间点修剪后的双端RNA-Seq读数与大豆基因组v2.1（https://plants.ensembl.org/Glycine_max）比对以去除大豆来源序列，再用Trinity v2.6.6对非大豆短读长进行从头组装。通过Blastplus v2.6.0（NCBI：美国国家生物技术信息中心）对非大豆转录本进行BLASTN（基本局部比对搜索工具）分析，去除查询覆盖率＞80%且一致性＞95%的植物转录本。最终包含各时间点非植物、非大豆转录本的从头组装转录组用于候选效应蛋白预测。<br><b>引用：</b>M.G.Elmore、S.Banerjee、K.F.Pedley、A.Ruck、S.A.Whitham. <i>Phakopsora pachyrhizi</i>的从头转录组揭示感染期间的推定效应蛋白库. 《Physiological and Molecular Plant Pathology》, <br>110 (2020), 101464, 10.1016/j.pmpp.2020.101464