背景:几种植物病原体产生约18-30个核苷酸(nt)的小的非编码RNA,其转录后调节基因表达。通常称为小RNA(sRNA),据报道,这些小分子也存在于坏死性病原体菌核病中。菌核病菌在400多种植物中引起疾病,包括重要的油料作物甘蓝型油菜。sRNA可进一步分类为微小RNA(miRNA)和短干扰RNA(siRNA)。某些miRNA可以激活产生更多sRNA的基因座;这些次级sRNA产生基因座被称为“定相siRNA”(PHAS)基因座,仅在植物中进行了描述。迄今为止,很少有研究在硬核链球菌中表征sRNAs及其内源性靶标。
结果:我们使用Illumina测序来表征来自遍布欧洲油菜叶片的菌丝菌丝菌的真菌菌丝垫的sRNA。总的来说,从体外制备了八个sRNA文库,接种后12小时(HPI),和24个HPI菌丝垫样品。聚类分析鉴定了354个丰富的sRNA簇,其读数超过每百万100个读数(RPM)。差异表达分析显示,在12和24HPI时,34和57个基因座上调,分别,与体外样品相比。其中,25个基因座通常被上调。总之,从25个基因座的主要RNA中鉴定出343个内源性靶标。几乎88%的这些目标被注释为重复元件基因,而其余靶标是非重复元件基因。真菌降解体读数证实了一种上调的sRNA对两种转座因子的切割。总之,用成熟和milRNA*(星形)序列预测了24个milRNA基因座;这些都是先前与实验验证的miRNA相关的标准。Degradome测序数据证实了14个靶标的裂解。这些目标与重复元件基因有关,磷酸乙酰转移酶,RNA结合因子,和交换因素。PHAS基因预测工具鉴定了26个可能的分阶段干扰基因座,其中147个phasiRNAs来自硬化链球菌基因组,这表明这种病原体可能产生的sRNAs在高等真核生物中的功能与miRNAs相似。
结论:我们的结果为sRNA群体提供了新的见解,并为菌核链球菌中sRNA的研究增加了新的资源。
BACKGROUND: Several phytopathogens produce small non-coding RNAs of approximately 18-30 nucleotides (nt) which post-transcriptionally regulate gene expression. Commonly called small RNAs (sRNAs), these small molecules were also reported to be present in the necrotrophic pathogen Sclerotinia sclerotiorum. S. sclerotiorum causes diseases in more than 400 plant species, including the important oilseed crop Brassica napus. sRNAs can further be classified as microRNAs (miRNAs) and short interfering RNAs (siRNAs). Certain miRNAs can activate loci that produce further sRNAs; these secondary sRNA-producing loci are called \'phased siRNA\' (PHAS) loci and have only been described in plants. To date, very few studies have characterized sRNAs and their endogenous targets in S. sclerotiorum.
RESULTS: We used Illumina sequencing to characterize sRNAs from fungal mycelial mats of S. sclerotiorum spread over B. napus leaves. In total, eight sRNA libraries were prepared from in vitro, 12 h post-inoculation (HPI), and 24 HPI mycelial mat samples. Cluster analysis identified 354 abundant sRNA clusters with reads of more than 100 Reads Per Million (RPM). Differential expression analysis revealed upregulation of 34 and 57 loci at 12 and 24 HPI, respectively, in comparison to in vitro samples. Among these, 25 loci were commonly upregulated. Altogether, 343 endogenous targets were identified from the major RNAs of 25 loci. Almost 88% of these targets were annotated as repeat element genes, while the remaining targets were non-repeat element genes. Fungal degradome reads confirmed cleavage of two transposable elements by one upregulated sRNA. Altogether, 24 milRNA loci were predicted with both mature and milRNA* (star) sequences; these are both criteria associated previously with experimentally verified miRNAs. Degradome sequencing data confirmed the cleavage of 14 targets. These targets were related to repeat element genes, phosphate acetyltransferases, RNA-binding factor, and exchange factor. A PHAS gene prediction tool identified 26 possible phased interfering loci with 147 phasiRNAs from the S. sclerotiorum genome, suggesting this pathogen might produce sRNAs that function similarly to miRNAs in higher eukaryotes.
CONCLUSIONS: Our results provide new insights into sRNA populations and add a new resource for the study of sRNAs in S. sclerotiorum.