Transcriptome analysis of the plant pathogen Sclerotinia sclerotiorum interaction with resistant and susceptible canola (Brassica napus) lines

Sclerotinia stem rot is an economically important disease of canola (Brassica napus) and is caused by the fungal pathogen Sclerotinia sclerotiorum. This study evaluated the differential gene expression patterns of S. sclerotiorum during disease development on two canola lines differing in susceptibility to this pathogen. Sequencing of the mRNA libraries derived from inoculated petioles and mycelium grown on liquid medium generated approximately 164 million Illumina reads, including 95 million 75-bp-single reads, and 69 million 50-bp-paired end reads. Overall, 36% of the quality filter-passed reads were mapped to the S. sclerotiorum reference genome. On the susceptible line, 1301 and 1214 S. sclerotiorum genes were differentially expressed at early (8–16 hours post inoculation (hpi)) and late (24–48 hpi) infection stages, respectively, while on the resistant line, 1311 and 1335 genes were differentially expressed at these stages, respectively. Gene ontology (GO) categories associated with cell wall degradation, detoxification of host metabolites, peroxisome related activities like fatty acid ß-oxidation, glyoxylate cycle, oxidoreductase activity were significantly enriched in the up-regulated gene sets on both susceptible and resistant lines. Quantitative RT-PCR of six selected DEGs further validated the RNA-seq differential gene expression analysis. The regulation of effector genes involved in host defense suppression or evasion during the early infection stage, and the expression of effectors involved in host cell death in the late stage of infection provide supporting evidence for a two-phase infection model involving a brief biotrophic phase during early stages of infection. The findings from this study emphasize the role of peroxisome related pathways along with cell wall degradation and detoxification of host metabolites as the key mechanisms underlying pathogenesis of S. sclerotiorum on B. napus.菌核茎腐病（Sclerotinia stem rot）是甘蓝型油菜（Brassica napus）具有重要经济价值的病害，其病原菌为真菌核盘菌（Sclerotinia sclerotiorum）。本研究针对两种对该病原菌抗性存在差异的油菜品系，解析了核盘菌在病害侵染过程中的差异基因表达模式。对接种叶柄以及液体培养基上生长的菌丝体构建的mRNA文库进行测序，共获得约1.64亿条Illumina测序读段（Illumina reads），其中包括9500万条75 bp单端读段以及6900万条50 bp双端读段。经质量质控的读段中，共计36%可比对至核盘菌参考基因组。在感病品系上，核盘菌分别在侵染早期（接种后8~16小时，hpi）和侵染后期（接种后24~48小时，hpi）检测到1301和1214个差异表达基因；而在抗病品系上，两个阶段的差异表达基因数分别为1311和1335个。基因本体（Gene Ontology，GO）功能分类分析显示，在感病与抗病品系的上调基因集中，与细胞壁降解、宿主代谢物解毒、过氧化物酶体相关功能（如脂肪酸β-氧化、乙醛酸循环、氧化还原酶活性）相关的条目均显著富集。针对6个筛选得到的差异表达基因（Differentially Expressed Genes，DEGs）的实时定量逆转录PCR（quantitative RT-PCR）实验，进一步验证了RNA测序（RNA-seq）差异基因表达分析结果的可靠性。侵染早期参与宿主防御抑制或逃逸的效应基因调控，以及侵染后期介导宿主细胞死亡的效应基因表达，为“早期侵染存在短暂活体营养阶段”的双相感染模型提供了支持证据。本研究结果表明，过氧化物酶体相关通路、细胞壁降解以及宿主代谢物解毒，是核盘菌侵染甘蓝型油菜的关键致病机制。