【作者】 张鑫；

【导师】 于峰；

【作者基本信息】 湖南大学 ， 化学， 2020， 博士

【摘要】 植物寄生线虫是全球性的粮食作物病虫害之一,我国每年遭受线虫病害的作物种植面积高达上亿亩,造成数百亿元的经济损失。植物与寄生线虫始终处于持续的“军备竞赛”之中。植物寄生线虫通过口针分泌大量效应蛋白,干预宿主植物的免疫系统,促进线虫的寄生。然而,目前线虫与宿主植物相互作用的机理尚不清楚。位于细胞膜的信号感受蛋白FERONIA(FER)是目前植物学领域研究最为深入的受体蛋白激酶之一。FER在植物中控制生长发育、逆境响应等多个环节,同时影响作物的产量与品质。快速碱化因子(Rapid ALkalinization Factor,简称RALF),作为受体蛋白FER的一类配体分子,会激活下游信号通路,调节质膜氢泵(H~+-ATPase,AHA2)活性和钙离子流传递,同时也可影响免疫复合物的稳定性以及免疫信号通路关键蛋白的稳定性,进而调节植物的生长发育和免疫应答。根结线虫(如南方根结线虫)是目前植物寄生线虫领域研究最为深入的对象之一,与其它寄生线虫(如孢囊线虫)具有相似的生活方式,但在形成取食位点方面具有明显区别。相对于孢囊线虫,根结线虫可通过调控取食位置附近的细胞大小(主要是调控细胞的液泡大小),形成取食位点并获取营养。由于受体蛋白激酶FER介导的RALF信号通路控制植物细胞大小,且调控液泡发育,因此RALF-FER信号通路可能与根结线虫的寄生发育密切相关。在植物中,RALF23多肽通过受体蛋白FER调控免疫复合物FLS2-BAK1的形成,影响flg22诱导的活性氧(ROS)激活,进而影响植物免疫反应。同时,RALF23多肽还可抑制受体蛋白FER对茉莉酸(Jasmonic Acid,简称JA)信号通路关键转录因子MYC2的磷酸化,并维持MYC2的蛋白稳定性,最终影响JA信号通路组成的植物免疫应答。不仅如此,尖孢镰刀菌(Fusarium oxysporum)分泌的F-RALF多肽能够被植物中的受体蛋白FER识别,抑制AHA2氢泵活性,碱化质外体,促进真菌体内的丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinase,简称MAPK)磷酸化,从而促进真菌侵染。说明可调控植物免疫的RALF多肽具有跨界信号转导的潜能。基于此,本课题组将利用生物化学和分子生物学手段探究:(1)植物寄生线虫的寄生是否与受体蛋白FER有关;(2)线虫寄生过程中分泌的效应蛋白中是否存在RALF多肽等可与植物受体蛋白结合的配体分子;(3)植物寄生线虫如何参与并调控植物受体蛋白FER介导的RALF信号通路。本论文依据此研究思路开展了一系列的分子、生化与遗传学等实验,论文的具体研究结果如下:(1)缺失受体蛋白FER的拟南芥和水稻突变体,将会降低其对南方根结线虫的敏感性,具体表现为:降低植物对南方根结线虫的吸引能力,抑制南方根结线虫对植物的侵染效率,延缓南方根结线虫在植物体内的发育,抑制南方根结线虫在植物体内的生殖。说明受体蛋白FER作为植物信号通路交叉会话的关键节点,在南方根结线虫的寄生过程十分重要。(2)在探究上述现象的潜在机制时,我们从已有的数据库中鉴定出18个新的RALF类似物(RALF-like)。该类RALF-like与植物RALF具有较高的序列相似性和结构保守性,且均来自于根结线虫,而非孢囊线虫等寄生线虫。从进化关系来看,18个RALF-like散落在进化树的不同分支,而非聚类成簇。说明线虫RALF-like的进化起源,并非垂直遗传,可能是水平转移或协同进化。以南方根结线虫Mi RALF1和Mi RALF3为代表,研究线虫RALF-like的时空特异性表达。发现Mi RALF1和Mi RALF3在食管腺细胞表达,并在线虫发育的寄生阶段高表达,符合植物寄生线虫典型的效应蛋白特征。(3)线虫RALF-like具有植物RALF典型的生物活性,即抑制植物根伸长,提高植物胞外碱化,同时具有与植物RALF一致的转录表达谱。我们通过GST pull-down、竞争性结合、免疫共沉淀(Co-IP)等生化手段,证明了线虫RALF-like可与受体蛋白FER胞外结构域结合。同时,利用分子对接(docking)和微量热涌动(MST)实验手段,从结构模拟和分子热力学方面进一步证明了线虫RALF-like可与FER胞外结构域结合,且测定了不同种RALF-like与FER胞外结构域的亲和力。(4)实验证明线虫RALF-like可引起植物MAPK磷酸化,并抑制JA信号通路关键转录因子MYC2的蛋白稳定性,说明线虫RALF-like对植物免疫应答具有一定的影响。通过对不同种RALF处理的ROS信号检测,发现线虫RALF-like和植物RALF类似,也可抑制flg22诱导的ROS信号通路。利用寄主诱导的基因沉默(Host Induced Gene Silencing,简称HIGS)技术,敲低线虫RALF-like表达,会延缓南方根结线虫在植物体内的发育和繁殖。同时,在植物中过表达RALF-like将会提高植物对南方根结线虫的敏感性。这项研究表明,线虫编码的RALFs通过植物编码的受体蛋白激酶FER促进寄生,并为研究寄主-病原相互作用提供了一个新的范式。更多还原

【Abstract】 Plant-parasitic nematodes（PPNs）are devastating pathogens that cause crop diseases and subsequently large area of infections leading to severe national economic losses（up to tens of billions of RMB）.Plants and PPNs are always competing in an arms race.PPNs can secret a number of effectors via stylet to benefit its parasitism and simultaneously aid evade the attack of their host immune system.However,in this field,our understanding of how plant-parasitic nematodes interact with its host is limited and warrants further studies.As a receptor kinase on plasma membrane,FERONIA（FER）is one of well-studied signaling networks in plants so far.FER modulates a multitude of essential biological processes in plants（e.g.,growth,reproduction,and stress responses）and influences the yield and quality of crops.RALFs,one of the peptide ligands perceived by FER,trigger the casecade downstream,modulate the activity of AHA2 and Ca²⁺transport,and influence the stability of immune-related proteins and immune complex,further regulating plant growth and development and immune responses.Root-knot nematode（e.g.,Meloidogyne incognita）is most well-studied in PPNs so far.It has a similarity for the lifestyle of root-knot nematode,but a significant difference in the feeding site,with other species.Compared with cyst nematode,root-knot nematode modulates the cell size（e.g.,modulating the vacuole size）,further forming the feeding site.As previously reported,RALF-FER signaling controls plant cell size and regulates its development of vacuole,both of which benefit the parasitism of nematode.In plants,RALF23 peptide regulates the immune response via modulating the formation of immune complex（FLS2-BAK1）through FER and influencing the flg22-induced ROS burst.Meanwhile,RALF23 peptide inhibits FER-dependent phosphorylation of MYC2,a key transcription factor in Jasmonic Acid（JA）signaling,further affecting the JA signaling and immune responses.It has been shown that Fusarium oxysporum encodes RALF-likes that can modulate plant immune response via FER,consequentially inhibits the activity of AHA2,alkalinizes the apoplast and phosphorylates MAPK in Fusarium oxysporum.This indicates that RALF peptides have the potency of cross-kingdom signal transduction.Based on this,we aim to（1）confirm the interaction of nematode parasitism with receptor kinase FER;（2）explore whether there are ligands such as RAFL peptides that can bind to plant receptor protein in the secreted effectors during nematode parasitism;（3）determine how PPN invovle in and regulate the RALF signaling pathway mediated by plant receptor protein FER.A series of molecular,biochemical and genetic experiments were conducted,and the results of this study are as follows:（1）Loss or mutation of FERONIA in Arabidopsis or rice lost susceptibility to root-knot nematodes parasitism.These mutants reduced the capacity to attract Meloidogyne incognita,inhibited the efficiency of infection,delayed the development of Meloidogyne incognita and suppressed the reproduction of Meloidogyne incognita in plants.These results indicated that the key node of FER in the plant signaling pathway also plays an important role in the parasitism of Meloidogyne incognita.（2）To confirm the underlying mechanisms associated with above-mentioned findings,we have identified 18 novel RALF-likes from existing databases.These RALF-likes had high similarity with plant RALFs and conserved domain with plant RALFs,and nematode RALF-likes all originated from root-knot nematodes,not cyst nematodes.From the evolutionary relationship,18 RALF-like scattered,rather than clustered,in different branches of the evolutionary tree,which indicates nematode RALF-likes may originate from horizontal transfer or coevolution,instead of vertical genetics.Taking Mi RALF1 and Mi RALF3 as representatives,the temporal and spatial specific expression of RALF-likes in Meloidogyne incognita was studied.It showed that Mi RALF1 and Mi RALF3 were expressed in esophageal gland cells and especially higher during parasitism.（3）RALF-likes had the typical biological activity of plant RALF peptides:root elongation inhibition,extracellular alkalization.They also had a simialr transcriptional expression profile with plant RALF.RALF-likes can bind to the extracellular domain of receptor protein FER in GST pull-down,competitive binding and Co-IP assays.Meanwhile,docking assay and MST assay also confirm this from the aspects of structure simulation and molecular thermodynamics.The affinities between RALF/RALF-likes and FER extracellular domain were measured as well.（4）Our results showed that RALF-likes could induce MAPK phosphorylation in plants and inhibit the stability of MYC2 protein,a key transcription factor in JA signaling pathway.It was found that RALF-like of nematodes could inhibit the flg22induced ROS signal pathway similar to plant RALF.Knock-down of RALF-like expression by host induced gene silencing（HIGS）delayed the development and reproduction of Meloidogyne incognita in plants.Meanwhile,overexpression of RALF-like in plants increased the sensitivity of plants to Meloidogyne incognita.In summary,this study indicated that nematode-encoded RALFs facilitate parasitism via plant-encoded FERONIA and provides a novel paradigm for studying host-pathogen interactions.更多还原