Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

真菌病原菌的分泌型效应蛋白是病害发生发展的关键因子。然而，已鉴定的效应蛋白间普遍缺乏序列保守性，长期以来这一问题制约了真菌效应蛋白组的预测工作。本研究针对亚麻锈菌（Melampsora lini）的无毒（avirulence, Avr）基因及候选效应蛋白的表达特征展开探究。我们针对未萌发孢子、萌发孢子、分离吸器以及植物侵染过程中接种亚麻锈菌CH5菌株的亚麻幼苗所提取的RNA，开展了转录组测序与实时荧光定量聚合酶链反应（real-time quantitative PCR, qPCR）实验。本研究还针对编码两类亚麻锈菌蛋白的基因展开了详细分析，分别为无毒蛋白与植物细胞壁降解酶（cell wall degrading enzymes, CWDEs）。对亚麻锈菌转录组中623个预测分泌蛋白编码基因的表达谱分析结果显示，6个已知无毒基因（即AvrM (avrM)、AvrM14、AvrL2、AvrL567、AvrP123 (AvrP)及AvrP4）隶属于一类共64个表达模式相似的基因：这类基因在植物体内被诱导表达，于侵染早期达到表达峰值，随后随产孢进程逐渐下调。该基因簇中还包含AvrL2的2个旁系同源基因、1个AvrL567毒性等位基因，以及多个编码推定效应蛋白的基因。与之形成鲜明对比的是，编码植物细胞壁降解酶的亚麻锈菌基因分布于不同的表达簇中，其聚类结果通常与其催化活性或底物靶标并无关联。上述结果表明，亚麻锈菌无毒蛋白的合成可能受到协同调控，而基于表达谱的分析方法在候选无毒基因的鉴定中具备显著的预测价值。