Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (5): 1445-1454.DOI: 10.1016/j.jia.2022.08.127

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mgr-mir-9通过靶向效应子MgPDI调控拟禾本科根结线虫侵染水稻

  

  • 收稿日期:2022-05-23 接受日期:2022-06-22 出版日期:2023-05-20 发布日期:2022-06-22

mgr-mir-9 implicates Meloidogyne graminicola infection in rice by targeting the effector MgPDI

TIAN Zhong-ling1, ZHOU Jia-yan1, ZHENG Jing-wu2, HAN Shao-jie2, 3#   

  1. 1 Key Laboratory of Pollution Exposure and Health Intervention of Zhejiang Province, Interdisciplinary Research Academy (IRA), Zhejiang Shuren University, Hangzhou 310015, P.R.China

    2 Laboratory of Plant Nematology, Institute of Biotechnology, College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, P.R.China

    3 Ministry of Agriculture Key Lab of Molecular Biology of Crop Pathogens and Insect Pests, Zhejiang University, Hangzhou 310058, P.R.China

  • Received:2022-05-23 Accepted:2022-06-22 Online:2023-05-20 Published:2022-06-22
  • About author:TIAN Zhong-ling, E-mail: zltian@zjsru.edu.cn; #Correspondence HAN Shao-jie, E-mail: hanshaojie@zju.edu.cn
  • Supported by:

    This work was financially supported by the National Natural Science Foundation of China (32001877).

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摘要：

MicroRNAs (miRNAs)是一类重要的非编码RNA，在包括植物寄生线虫在内的一系列动物中均是重要的内源性基因调控因子。拟禾本科根结线虫系一种定居性植物内寄生虫，主要侵染危害水稻，严重影响水稻生产。目前关于拟禾本科根结线虫的研究主要集中在效应子在线虫寄生过程中的功能研究方面。而拟禾本科根结线虫miRNA如何对其自身效应子表达进行调控而影响线虫侵染的机制鲜有报道。本研究对拟禾本科根结线虫二龄幼虫（J2）的3个文库进行小RNA测序，共获得49,767,105条clean reads。通过进一步的注释，共鉴定出233个已知miRNAs和21个新miRNAs。在已知的miRNAs中，mgr-lin-4、mgr-mir-1、mgr-mir-100、mgr-mir-86、mgr-mir-279、mgr-mir-87、mgr-mir-71、mgr-mir-9、mgr-mir-50、mgr-mir-72和mgr-mir-34的丰度最高。基于实时荧光定量PCR对随机选取的5个miRNA表达水平进行了测定，定量检测结果与小RNA测序结果相符。由于miRNA是动物体内重要的调控因子，我们推测这些miRNAs可能在拟禾本科根结线虫的J2龄期调控效应子的表达，而促进其侵染。利用双荧光素酶报告基因检测验证mgr-mir-9能够靶向拟禾本科根结线虫侵染相关的重要效应子基因MgPDI；此外，用mgr-mir-9 mimics浸泡后的二龄幼虫体内MgPDI基因表达下调，线虫繁殖能力下降，证实了mgr-mir-9参与调控拟禾本科根结线虫的早期侵染。本研究首次报道了拟禾本科根结线虫miRNA在早期侵染过程中通过调控自身效应子的表达而行使重要功能，为应用相关miRNA及其靶向的效应因子作为拟禾本科根结线虫防治的新靶标提供了理论支持。

Abstract:

MicroRNAs (miRNAs), a class of small non-coding RNAs, are crucial endogenous gene regulators in a range of animals, including plant-parasitic nematodes.  Meloidogyne graminicola is an obligate sedentary endoparasite of rice and causes significant yield losses.  A number of studies focused on the roles of M. graminicola effectors during the parasitic process; however, how nematode miRNAs regulate its effectors needs elucidating.  In this research, we analyzed a cluster of M. graminicola miRNAs obtained at the second-stage juveniles (J2s) stage that are closely linked to the regulation of M. graminicola effectors.  There are 49 767 105 total clean reads obtained from three libraries.  A total of 233 known miRNAs and 21 novel miRNAs were identified.  Among the known miRNAs, mgr-lin-4, mgr-mir-1, mgr-mir-100, mgr-mir-86, mgr-mir-279, mgr-mir-87, mgr-mir-71, mgr-mir-9, mgr-mir-50, mgr-mir-72, and mgr-mir-34 are the most abundant 11 miRNAs families.  Moreover, the expression levels of selected miRNAs were validated by real-time quantitative PCR.  We hypothesized that these miRNAs might regulate the expression of secreted effectors during the J2s stage to facilitate its infection.  Consistent with this, we found that mgr-mir-9 targets MgPDI, an important M. graminicola effector mRNA.  In addition to that, J2s treated with mgr-mir-9 mimics showed down-regulation of MgPDI expression and reduced reproductive ability, alluding mgr-mir-9 is involved in nematode infection.  These results provide novel insight into the regulatory functions of M. graminicola miRNAs during the infection and identify miRNAs and their effector targets as potential key management targets to limit parasite survival during the early stages of infection.

Key words: microRNA function ,  Meloidogyne graminicola , deep sequencing ,  Dual-Luciferase Reporter Assay System ,  protein disulfide isomerase