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Abstract：
Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici (P. capsici), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25-26 nt in HY, SP, and ZO but distributed at 18-34 nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici.
摘要：
小RNA(sRNAs)是重要的非编码RNA调节因子,在植物病原菌的发育和致病过程中起着关键作用。以及其他生物过程。然而,这些丰富和变化的sRNAs是否参与疫霉的发育或感染仍然是一个谜。在这项研究中,辣椒疫霉4个无性阶段的sRNA测序(P.capsici),即,作为菌丝体（HY），孢子囊（SP），游动孢子（ZO），囊肿（CY），和辣椒感染P.capsici（IN），被执行，然后进行sRNA分析,microRNA(miRNA)鉴定,和miRNA靶标预测。sRNAs主要分布在HY的25-26nt,SP,和ZO，但在CY和IN中以18-34nt分布。在HY中鉴定了92、42、176、39和148个已知的miRNA和15、19、54、13和1个新的miRNA，SP,ZO,CY,在,分别。发现已知miRNA的表达谱在不同阶段差异很大，可以分为4类。新的miRNAs大多属于I部分。对已知的miRNA靶向基因的基因本体论（GO）分析表明，它们参与了催化活性途径，绑定函数，和其他生物过程。新的miRNA靶向基因的京都基因和基因组百科全书（KEGG）分析表明，它们参与赖氨酸降解途径。使用定量逆转录-聚合酶链反应(qRT-PCR)验证候选miRNA的表达,和miRNA在PcDCL1或PcAGO1突变体中下调。为了进一步探索检测到的miRNA的功能，一种新的miRNA的前体，miR91被CRISPR-Cas9敲除，突变体显示菌丝生长减少，孢子囊生产,和游动孢子生产。发现503142（肌醇多磷酸5-磷酸酶和相关蛋白）可以预测为miR91的靶标，并使用烟草瞬时表达系统验证了miR91与503142之间的相互作用。总的来说,我们的结果表明，不同和差异表达的sRNAs参与了辣椒假单胞菌的发育和发病机制。
