山田胶锈菌和亚洲胶锈菌吸器的比较转录组分析

Comparative transcriptomic analysis of the haustoria of Gymnosporangium yamadae and G. asiaticum

摘要:

为防止锈病的传播，培育抗病品种以及减少产量损失，基于山田胶锈菌（Gymnosporangium yamadae）和亚洲胶锈菌（Gymnosporangium asiaticum）吸器阶段的转录组差异分析揭示了胶锈菌侵染寄主植物时的专化性选择机制。对山田胶锈菌和亚洲胶锈菌担孢子侵染寄主时形成的吸器进行转录组测序，分别获得了21 213条和13 015条单基因（unigenes）；从山田胶锈菌和亚洲胶锈菌中分别选择5个基因进行实时荧光定量PCR验证，显示其表达情况与转录组分析结果基本一致，表明转录组分析结果可靠；用Nr、GO、KEGG、KOG等7个数据库进行基因功能注释和富集分析，发现两种胶锈菌的基因主要富集在细胞进程、翻译、代谢相关通路；使用SignalP和TMHMM在线网站以及dbCAN、BLAST、HMMER等软件分析显示山田胶锈菌和亚洲胶锈菌吸器中的候选效应蛋白分别有343个（2.51%）和175个（2.79%），其中分别含有14个和5个蛋白酶，10个和3个脂酶；利用OrthoFinder、BLSAT和KaKs Calculator软件分析了两种胶锈菌的进化关系，在一对一同源基因中，比对率大于82%的基因对被认为处于保守选择，有12.37%的基因对处于正向选择，在亚洲胶锈菌中有5个效应子处于进化选择，其中1个为脂酶。山田胶锈菌和亚洲胶锈菌表达基因富集中没有发现显著差异，表明虽然其种间存在典型的寄主选择性，但吸器所涉及的生物学过程相对保守，而两个物种之间存在较低的蛋白相似性，说明它们受到较大的寄主选择压力而产生了明显的进化分歧，这可能与对寄主的特异性选择机制相关。在吸器阶段，胶锈菌可能可以利用植物的脂质作为能量来源，而效应子的主要目的可能是调控植物的生理进程等而不是直接攻击寄主。

关键词:锈菌;转录组;吸器;物种进化;效应子

Abstract:

To provide a theoretical basis for controlling the spread of rust disease, cultivating disease-resistant varieties and reducing yield losses, we investigated the transcriptome differences between Gymnosporangium yamadae and Gymnosporangium asiaticum at the haustorial stage and revealed a specialized selection mechanism for Gymnosporangium species to infect host plants. We sequenced the transcriptomes of the haustoria in rust-infected leaves when basidiospores of G. yamadae and G. asiaticum infected their hosts, and obtained 21 213 and 13 015 unigenes, respectively. Real-time fluorescence quantitative PCR validation of five genes selected from G. yamadae and G. asiaticum, respectively, showed that their expression profiles were generally consistent with the results of transcriptome analysis, demonstrating the reliability of the transcriptome data. We used seven databases such as Nr, GO, KEGG, and KOG to perform gene function annotation and enrichment analysis, and found that the genes from both rusts were mainly enriched in cellular processes, translation, and metabolism-related pathways. Moreover, we used SignalP, TMHMM online website and other software such as dbCAN, BLSAT, HMMER to show that there were 343 (2.51%) and 175 (2.79%) candidate effector proteins containing 14 and 5 proteases and 10 and 3 lipases in the haustoria of G. yamadae and G. asiaticum, respectively. Furthermore, we used OrthoFinder, BLAST and KaKs Calculator software to analyze the evolutionary relationship of the two fungi. Among one-to-one homologous genes, gene pairs with > 82% alignment were considered to be under conservative selection, and 12.37% under positive selection. Five effectors of G. asiaticum were under positive selection, and one of which was a lipase. No significant differences were found in the enrichment of expressed genes between G. yamadae and G. asiaticum, indicating the biological processes involved in haustoria were relatively conserved, despite the typical host selectivity between species. The low protein similarity between the two species suggested that they were under greater host selective pressure and there was significant evolutionary divergence, which might be related to the host-specific selection mechanism. In the haustorial, the main purpose of the effectors might be to regulate physiological processes in the plants rather than attacking the host directly, and G. yamadae and G. asiaticum might use plant lipids as energy sources.

Key words:rust fungi;transcriptome;haustoria;species evolution;effectors