核盘菌致病机理研究进展

2018-03-31 17:16羊国根程家森
生物技术通报 2018年4期
关键词:盘菌寄主植物草酸

羊国根 程家森

(1. 安徽农业大学植物保护学院,合肥 230036;2. 华中农业大学农业微生物学国家重点实验室 华中农业大学湖北省作物病害监测和安全控制重点实验室,武汉 430070)

核盘菌(S. sclerotiorum(Lib.)de Bary)是一类世界性分布的重要植物病原真菌。在分类地位上,归属于真菌界(Fungi)、子囊菌门(Ascomycota)、锤舌菌纲(Leotiomycetes)、锤舌菌亚纲(Leotiomycetidae)、柔膜菌目(Helotiales)、核盘菌科(Sclerotiniaceae)、核盘菌属(Sclerotinia)[1]。核盘菌可侵染75个科450多种植物,主要侵染双子叶植物,如油菜、大豆、向日葵和番茄等;也可以侵染单子叶植物,如洋葱和郁金香等[2]。核盘菌引起的植物病害称为菌核病,也称作白腐病、茎腐病和软腐病等。核盘菌在侵染后期可形成菌核进行越夏越冬,菌核可以在土壤中存活很多年[3]。菌核病的病害循环复杂,菌核在适宜的温度和湿度条件下,可萌发形成子囊盘,释放子囊孢子侵染寄主,也可直接萌发成菌丝侵染植物[4]。核盘菌是典型的死体营养型植物病原真菌,致病机理复杂。已有报道仍主要集中研究核盘菌分泌的植物细胞壁降解酶类(Plant cell wall degrading enzymes,PCWDEs)和草酸(Oxalic acid,OA)等致病因子在侵染过程中的作用及其机制,而最近研究表明分泌蛋白也参与了核盘菌的致病过程并发挥重要功能。本文综述了有关核盘菌致病机理的最新研究进展,可为核盘菌的分子致病机制研究和抗菌核病分子育种提供重要参考。

1 水解酶类(Hydrolase)

主要包括角质酶(Cutinase)、细胞壁降解酶类和蛋白酶(Protease)类等。例如,核盘菌的角质酶编码基因SsCut在侵染叶片1 h后,表达量显著上调[5];重组蛋白SsCut可引起植物细胞坏死,诱导寄主植物产生抗性,并增强植物对核盘菌等病原菌的抗性[6]。核盘菌在侵染寄主植物时可分泌不同的植物细胞壁降解酶,包括纤维素酶(Cellulases)、半纤维素酶(Hemicellulose)、果胶酶(Pectinases)和木聚糖酶(Xylanases)等。核盘菌总共有183个植物细胞壁降解相关酶(包括木质素酶),其中果胶降解酶类有33个,果胶酶类占比在所有死体营养型真菌中是较高的[7],其研究报道也较多,尤以多聚半乳糖醛酸酶(Polygalacturonases,PGs)为主,分别有5个内切多聚半乳糖醛酸酶(endo-PGs)和5个外切多聚半乳糖醛酸酶(exo-PGs)[7-9]。endo-PGs在侵染植物时表达存在差异,次生代谢产物的积累和酸性pH环境可激活或者抑制不同endo-PGs的表达[9-10]。SsPG1参与了核盘菌早期侵染和病斑扩展,碳水化合物缺乏可以诱导Sspg1显著表达,但半乳糖醛酸可以抑制该基因的表达[5]。SsPG3和SsPG6可以在拟南芥上引起依赖于光周期的细胞坏死,而体外真核表达的BnPGIP1重组蛋白可以抑制核盘菌SsPG6酶的活性[11]。其它植物细胞壁降解酶类的研究相对较少,Yu等[12]报道了一个编码 endo-β-1,4-xylanase的基因SsXyl1,该基因与核盘菌菌丝生长、菌核形成及致病过程均密切相关。此外,植物细胞壁还含有丰富的蛋白质,核盘菌可以分泌大量的蛋白酶,如天冬氨酸蛋白酶家族等,如SsAp1在核盘菌侵染油菜和菜豆的早期表达量显著上升,推测其可能参与了核盘菌早期侵染过程[9,13]。

2 草酸

Godoy等[14]在1990年报道核盘菌产生的草酸是其致病的决定因子,他们发现紫外诱变获得的突变体A2不能产草酸,同时丧失了致病力;而在加入琥珀酸钠后,突变体A2恢复了产草酸能力并可在大豆叶片上形成病斑。随后更多研究表明草酸在核盘菌致病过程中的作用主要体现在以下几个方面:(1)草酸可以螯合植物细胞中游离的Ca2+,形成草酸钙结晶。植物细胞壁被降解后会产生游离的Ca2+,草酸可螯合这些Ca2+,保护侵染点区域的菌丝免受高浓度Ca2+的伤害[15]。核盘菌侵染油菜6 h和72 h时,侵染点的茎秆分别有46%和100%可见草酸钙结晶[16];(2)草酸使寄主植物保卫细胞功能失调,阻止气孔正常关闭。寄主植物被核盘菌侵染后,气孔在夜间仍处于打开状态,导致水分蒸发较快,从而引起植物叶片的萎蔫;草酸也可抑制脱落酸引起的气孔关闭[17];(3)草酸可抑制寄主植物的活性氧(Reactive oxygen species,ROS)爆发。侵染早期,核盘菌分泌草酸抑制活性氧爆发和胼胝质的积累,促进核盘菌菌丝的定殖;侵染后期,草酸又可刺激寄主植物产生大量的ROS,诱发植物组织的程序性细胞死亡(Programmed cell death,PCD),促进核盘菌侵染和扩展[18];(4)草酸可抑制寄主植物的细胞自噬(Autophagy)[19];(5)草酸可以降低周围环境的pH值,从而有利于核盘菌的侵染。有研究表明是草酸导致的低pH环境,而不是草酸根本身,在核盘菌致病中发挥重要作用。核盘菌体内存在着一个pH感应转录因子pacC/RIM1同源蛋白pac1,伴随着环境pH值升高而积累量升高,激活pac1介导的下游信号转导,有利于草酸的生物合成[20]。草酸缺失的核盘菌突变体可直接侵染叶片表面pH值低的豆科植物,而利用缓冲液降低叶片表面的pH值后,草酸缺失的核盘菌突变体也可成功侵染这些豆科植物[21],进一步证实了草酸营造的低pH环境在核盘菌致病过程中发挥重要作用。

3 分泌蛋白

效应子(Effector)在活体营养型病原菌和半活体营养型病原菌与寄主植物互作中发挥着重要作用[22-23];死体营养型病原真菌也可分泌效应子促进其侵染[24-26]。有研究者认为,核盘菌可能也存在短暂的活体营养阶段,菌丝在侵染初期在植物细胞的质外体空间生长而不穿透植物的细胞壁,通过分泌草酸和效应蛋白来抑制植物的免疫反应,促进核盘菌的侵染[27]。早期研究发现,核盘菌中存在一个类似整联蛋白(Ss-Integrin-like,SSITL)的分泌蛋白,该蛋白有典型的整联蛋白的FG-GAP重复结构域。SSITL基因在侵染早期表达急剧上升,该基因沉默后引起核盘菌致病力下降,而超表达SSITL的寄主植株也更加感病,进一步研究表明SSITL蛋白参与了核盘菌抑制JA/ET信号途径介导的局部和系统性抗病反应,因此SSITL在核盘菌致病过程中发挥类似效应子的功能[28]。而核盘菌的分泌蛋白质组分析结果表明,有486个植物诱导表达的小分泌蛋白参与了核盘菌与寄主植物的互作,其中78个被认为是候选的效应蛋白[29]。Derbyshire等[8]利用单分子实时测序和RNA-seq手段也鉴定到了70个候选效应蛋白,但与Guyon等[29]预测的有所不同。上述结果提示核盘菌中也存在大量的候选效应子并可能在其致病过程中发挥重要作用。

随着研究的深入,更多分泌蛋白在核盘菌中的作用及其作用机制被阐述,为进一步理解核盘菌的致病机制提供了新的思路和视角。Lyu等[30]在核盘菌上分离鉴定了一个富含半胱氨酸的小分泌蛋白SsSSVP1,该蛋白不含任何已知保守结构域,编码有163个氨基酸,其中有8个半胱氨酸残基,半胱氨酸含量超过4%。SsSSVP1仅在核盘菌属和灰葡萄孢属中存在同源蛋白。SsSSVP1在核盘菌侵染早期(3 hpi)表达即明显升高,该基因沉默后引起核盘菌的致病力下降。SsSSVP1瞬时表达可以引起烟草叶片的坏死,荧光定位及突变试验证明从菌丝分泌后,SsSSVP1可以自主转运至寄主植物的细胞质中,进而劫持寄主植物的线粒体蛋白QCR8,干扰QCR8正常的亚细胞定位和功能,促进核盘菌的侵染,因此SsSSVP1在核盘菌侵染过程中发挥类似效应子的功能。核盘菌SsCP1是cerato-platanin(CP)蛋白家族的典型成员,被证实是一个可以被植物识别的PAMP,可引起依赖于水杨酸途径的植物免疫反应,增加寄主植物对病原菌的抗性。但另一方面,SsCP1可在寄主植物的质外体与PR1互作。菌丝分泌的SsCP1与PR1互作降低PR1对核盘菌菌丝的抑制作用,从而有利于核盘菌的侵染。与此同时,随着侵染过程的发展和SsCP1的累积,高浓度的SsCP1可引起寄主植物细胞坏死,从而有利于死体营养型的核盘菌获取营养物质[31]。有趣的是,SsSSVP1和SSCP1两个效应分子的互作蛋白QCR8和PR1均为植物中非常保守并且功能重要的蛋白,这与核盘菌的寄主范围广泛这一特性是相吻合的。

诱导寄主植物细胞死亡的效应子有利于死体营养型病原菌的侵染[32]。前述分泌蛋白SsSSVP1和SSCP1均可促进寄主植物细胞的死亡,与核盘菌死体营养型的特性是符合的。此外报道显示核盘菌中其它一些分泌蛋白也可导致寄主植物细胞死亡,如核盘菌中有2个编码坏死和乙烯诱导多肽(NEPs)的基因SsNep1和SsNep2,在本氏烟中瞬时表达均可诱导植物细胞坏死,同时SsNep2在坏死区域和侵染顶端的菌丝都能表达,并依赖于Ca2+和环磷酸腺苷的信号转导[33]。灰葡萄孢大量分泌的一个类似IgE结合蛋白BcIEB1,BcIEB1可以引起植物的细胞死亡和抑制幼苗生长[34];BcIEB1可以诱导植物产生PTI,同时可以与PR5(Osmotin)结合抑制PR5的抗真菌活性[35]。我们发现核盘菌的基因组中也存在着2个编码IEB1的保守蛋白,并且其氨基酸序列基本一致,可能存在类似的功能[8]。子囊菌中特有的分泌蛋白SsCDI1,可以诱导本氏烟等茄科植物的细胞坏死,但不能在拟南芥、大豆等双子叶植物以及单子叶植物上引起细胞坏死[36]。

此外,有些分泌蛋白参与了核盘菌侵染垫的形成。例如,分泌蛋白Ss-Caf1含有EF-hand结构,在核盘菌侵染过程中发挥重要作用,Ss-Caf1的T-DNA插入突变体其草酸产量是野生型菌株的4倍,但突变体不能在健康叶片上致病,可以在有伤口的叶片上致病。电镜观察发现,突变体不能形成正常的侵染垫,表明侵染垫在核盘菌致病中有着重要作用[37]。具有Rhs重复结构的分泌蛋白Ss-Rhs1参与了复合侵染垫的形成,基因沉默突变体在拟南芥和油菜叶片上形成较小的病斑[38]。有研究表明核盘菌在SA类似物苯并噻二唑(BTH)预处理后的油菜上形成的病斑减少约40%,表达降解水杨酸的NahG拟南芥对核盘菌更加敏感,表明SA在植物抵抗核盘菌侵染中具有积极作用[39-40]。Kabbage等[27]也报道了在核盘菌中存在一个类似效应子的分泌型分支变位酶SsCm1,与玉米黑粉病菌的Cmu1高度同源,将分支酸转化为预苯酸阻断水杨酸的合成,从而抑制植物的免疫反应促进核盘菌的侵染[19,40-41]。还有一些分泌蛋白被证实与核盘菌的致病密切相关,但具体功能及作用机制需要进一步探讨,如核盘菌的一个小分泌蛋白SsCVNH(Cyanovirin-N homology)在核盘菌致病和菌核发育同样发挥着重要作用[42];核盘菌发酵液中的蛋白激发子SCFE1,可以诱导植物产生依赖于受体蛋白RLP30的PTI,RLP30突变体对核盘菌更加感病,证实SCFE1有利于核盘菌的侵染[43];编码假定蛋白的ssv263缺失后,突变体的致病力显著下降[44]。

4 其它致病相关蛋白

除分泌蛋白外,其它致病相关蛋白在核盘菌致病过程中也发挥重要作用,如核盘菌NADPH氧化酶(SsNOX1和SsNOX2)与ROS产生相关,Ssnox1沉默突变体中ROS水平降低,草酸产量下降,表明清除ROS或提高氧化激发的耐受力,在核盘菌侵染过程中也发挥作用[45]。编码γ-谷氨酰转肽酶的Ss-Ggt1基因影响核盘菌侵染垫的形成,在没有伤口的叶片上形成病斑的时间推迟,而在有伤口的叶片上没有区别,Ss-Ggt1与核盘菌的早期侵染相关[46]。SsSOD1编码一个Cu/Zn超氧化物歧化酶,基因破坏后不会影响核盘菌的菌丝生长,而突变体致病力受到影响,进一步研究表明SsSOD1是耐受ROS和氧化应激所必须的[47-48]。Ss-Bi1编码一个凋亡相关的Bax抑制子,与核盘菌响应各种环境压力相关,基因沉默后引起致病力下降,表明细胞凋亡的精细调控与致病力相关[49]。转录因子SsFKH1与核盘菌的致病力也密切相关,其基因沉默突变体在番茄叶片上的致病力显著下降[50]。

5 展望

核盘菌寄主范围广泛,其引起的菌核病导致作物产量下降和品质降低。由于缺乏有效的抗病品种,目前菌核病的防治主要依赖杀菌剂,但田间已出现了抗药性菌株,导致化学防治效果不佳。因此,深入解析核盘菌的致病机理,开发和利用植物自身的抗病相关基因,将有助于发展菌核病绿色防控新策略。例如,草酸是核盘菌的重要致病因子,降解草酸是提高作物抗性的途径之一。在大豆、油菜和烟草中表达来自外源的草酸氧化酶,可以大大提高作物对核盘菌的抗性[51-53];表达草酸脱羧酶的大豆和番茄可降解草酸,也增强了对核盘菌的抗性[54-55]。过量表达病程相关蛋白也可提高寄主植物对核盘菌的抗性,如PR1具有结合甾醇和抑制病原菌生长的作用,过量表达PR1可提高寄主植物的抗性[31,56]。PR3(几丁质酶)和PGIP共同表达的油菜也增强了对核盘菌的抗性[57]。此外,利用植物自身的PTI也可提高植物对核盘菌的抗性纳入受体蛋白RLP23特异性识别nlp20(NLPs的保守的20 aa),介导依赖于SOBIR1-BAK1的免疫反应,异源表达RLP23的番茄对核盘菌的抗性水平显著提高[58]。

深入解析核盘菌的分子致病机理,有助于在植物中发现更多的抗病相关蛋白。利用基因组编辑技术进行基因定向改造,或精细调控抗病相关基因的表达,有望获得具有一定抗性的品种应用于菌核病的安全防控。

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