RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, β-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include β-1,4-glucosidases, β-1,4-glucanases, β-1,4-galactanases, a β-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade β-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of β-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.RNA测序（RNA-Seq）分析表明，寄生疫霉（Phytophthora parasitica）基因组中预测的转录本有超过60%（12962个）在羽扇豆根系侵染的前60小时内得以表达。该侵染转录组涵盖了该菌431个细胞壁降解酶编码基因中的278个。本转录组数据为编码靶向植物细胞壁主要组分的蛋白的基因存在全局转录级联反应提供了有力证据。一类核心的果胶酶家族在侵染早期呈现高表达特征；随着侵染进程推进，转录组的表达谱逐渐转向以编码纤维素酶、半纤维素酶、β-1,3-葡聚糖酶（β-1,3-glucanases）和糖蛋白的转录本为主。表达量最高的寄生疫霉碳水化合物活性酶基因包含两个CBM1纤维素结合结构域（CBM1 cellulose binding module），且不具备催化结构域。排名前200的差异表达基因包含β-1,4-葡萄糖苷酶（β-1,4-glucosidases）、β-1,4-葡聚糖酶（β-1,4-glucanases）、β-1,4-半乳糖苷酶（β-1,4-galactanases）、一种β-1,3-葡聚糖酶、一种α-1,4-多聚半乳糖醛酸酶、一种果胶脱乙酰酶以及一种果胶甲酯酶。对基因表达谱的细致解析揭示了复合碳水化合物内部不同糖苷键的降解时序：（i）果胶同聚半乳糖醛酸的去甲基化先于其脱乙酰化过程；（ii）果胶鼠李糖半乳糖醛酸聚糖I的主链裂解早于其侧链的降解；（iii）侵染早期，非催化型纤维素结合蛋白与携带辅助活性模块的酶对纤维素微纤丝的初始攻击，可辅助后续糖基水解酶及携带CBM1纤维素结合结构域的酶发挥降解作用；（iv）在对半纤维素主链进行广泛的内部裂解后，才会靶向其末端残基；（v）糖蛋白表面的碳水化合物链在侵染后期才被降解。寄生疫霉侵染转录组的一个显著特征是，在侵染中晚期，编码β-1,3-葡聚糖酶的基因转录水平显著升高。本研究结果提示，高表达的β-1,3-葡聚糖酶可有效降解植物在防御反应中合成的胼胝质。