【作者】 李鹏；

【导师】 姜子德；

【作者基本信息】 华南农业大学 ， 植物病理学， 2023， 博士

【摘要】 荔枝霜疫霉(Peronophythora litchii Chen ex KO et al.)是荔枝生产上的重要病原菌,由其引发的荔枝霜疫病严重威胁着荔枝产业。由于缺乏抗病品种,目前防治荔枝霜疫病仍主要依赖对环境有影响的化学农药。研究病原菌与荔枝的互作机制有助于培育抗病品种,故本研究基于实验室前期对荔枝霜疫霉菌株SHS3的全基因测序与转录组测序数据,选择48个侵染前期高表达的RXLR效应分子并分析了它们对ICD(INF1-triggered cell death)的抑制能力,重点研究了PlAvh202和PlAvh222的功能以及在寄主中的靶标,为明确病原菌的致病机理提供了重要的材料和依据。主要研究结果如下:1.从荔枝霜疫霉菌株SHS3中成功克隆了48个RXLR效应分子,利用农杆菌介导的烟草瞬时转化,发现PlAvh42、PlAvh202、PlAvh208和PlAvh222能抑制致病疫霉激发子INF1引起的烟草细胞坏死,而PlAvh202、PlAvh208和PlAvh222还能抑制无毒蛋白受体对Avr3a/R3a引起的细胞坏死;将PlAvh202和PlAvh222的氨基酸序列分别与不同地区的荔枝霜疫霉氨基酸序列进行比对,发现不同地区荔枝霜疫霉中的PlAvh202和PlAvh222氨基酸序列完全一致,说明PlAvh202和PlAvh222序列具有高度的保守性,可能是病原菌侵染过程中的重要致病因子。2.利用CRISPR/Cas9技术分别对PlAvh202与PlAvh222进行基因敲除,各得到3个敲除突变体。将PlAvh202和PlAvh222的敲除突变体、野生型(WT)及对照(CK)分别接种荔枝嫩叶,48 h后统计病斑直径。结果表明在分别敲除PlAvh202和PlAvh222后,荔枝霜疫霉突变体造成的荔枝叶片病斑明显小于野生型和对照,说明PlAvh202与PlAvh222对荔枝霜疫霉毒力有重要贡献;同时,所有敲除突变体在胡萝卜营养培养基上的生长速率与野生型和对照相比没有明显差异,说明PlAvh202与PlAvh222的敲除对霜疫霉的生长没有影响。3.通过烟草的瞬时表达技术探究了PlAvh202和PlAvh222对INF1诱导的PTI反应的影响。结果表明在INF1诱导后的第36 h和48 h,PlAvh202和PlAvh222均能显著抑制植物免疫maker基因NbPR1、NbPR2、NbLOX、NbPDF1.2、NbRboh A和NbRboh B的表达,同时也能抑制INF1诱导的活性氧迸发;此外,PlAvh202与PlAvh222能促进辣椒疫霉侵染本氏烟。以上结果说明PlAvh202与PlAvh222可以抑制植物的免疫反应并提高植物对病原菌的敏感度。为了探究PlAvh202的功能域,本研究构建了4个缺失突变体,烟草的瞬时表达实验表明PlAvh202促进辣椒疫霉侵染和抑制ICD均依赖于其C端的重复序列IR2,说明PlAvh202的毒力和其抑制细胞死亡存在一定联系。4.利用免疫共沉淀和液相色谱串联质谱(LC-MS/MS)技术鉴定到了PlAvh202的烟草靶标,即乙烯合成相关蛋白NbSAMS2-like;接着通过萤火素酶互补、免疫共沉淀和Pull-down实验进一步证明NbSAMS2-like的4个荔枝同源蛋白(LcSAMS2、LcSAMS3、LcSAMS4和LcSAMS5)在植物体内、体外都能与PlAvh202互作。本研究还以PlAvh222为诱饵蛋白,通过酵母双杂交系统筛选出了6个候选互作蛋白,鉴于其中的钙离子结合蛋白LcCa M出现次数最多,故对其进行免疫共沉淀验证,结果表明两者在植物体内存在互作关系。5.烟草中过表达LcSAMS会增加植物对辣椒疫霉的抗性,然而通过病毒诱导的基因沉默(virus induced gene silencing,VIGS)技术沉默了本氏烟草中的NbSAMSs基因后发现烟草更加感病且PlAvh202失去促进辣椒疫霉侵染能力;同时,NbSAMSs的沉默也在一定程度上削弱了ICD。这些结果说明SAMS正调控植物免疫,PlAvh202通过负调控SAMS来抑制植物免疫。进一步的研究表明PlAvh202能通过26S蛋白酶体来降解荔枝中的SAMS蛋白,进而抑制植物乙烯的生物合成,导致荔枝更加感病。以上这些结果证明PlAvh202通过破坏SAMS蛋白的稳定性来削弱乙烯介导的荔枝抗性。综上所述,本研究明确了PlAvh202和PlAvh222对植物免疫系统的干扰能力,是病原菌侵染过程中的致病因子;阐明了PlAvh202靶向并促进植物SAMS降解进而抑制乙烯所介导的植物免疫机制,为荔枝病害防控提供了新的靶标,为人们深入了解和利用植物免疫系统提供了有价值的理论依据。更多还原

【Abstract】 Peronophythora litchii,the most destructive pathogen in litchi,can cause litchi downy blight,which is a great threat in litchi production.Because the control of P.litchii mainly depends on chemical fungicides with the environment damage,exploring the pathogenic mechanism of P.litchii is valuable to cultivate disease-resistant varieties of litchi.In this study,48 highly-expressed RXLR effectors of P.litchii,based on previous genome and transcriptome sequencing,were tested to see if they could suppress INF1-triggered cell death(ICD).We focused on the virulence function and host targets of PlAvh202 and PlAvh222 to reveal the pathogenic mechanism.The main research results are as follows:1.The genes encoding 48 RXLR effectors were successfully cloned from c DNA library of P.litchii,and then were transiently expressed in Nicotiana benthamiana,in which INF1or Avr3a/R3a was expressed at 24 h before effector expression.PlAvh42,PlAvh202,PlAvh208,and PlAvh222 were found able to suppress ICD,and PlAvh202,PlAvh208,and PlAvh222 could also suppress Avr3a/R3a-triggered cell death.The amino acid sequence of PlAvh202 or PlAvh222 in P.litchii from different provinces were aligned and compared,and the result showed that these two effectors are highly conserved.2.PlAvh202 or PlAvh222 was deleted using CRISPR/Cas9 system.Three PlAvh202 or PlAvh222-deleted mutants,WT,and CK(random-insertion)of P.litchii were inoculated on litchi leaves,lesion diameters were measured at 48 h post inoculation(hpi).The results showed that PlAvh202 or PlAvh222 deletion led to smaller lesion on the infected leaves,compared those infected by WT or CK,suggesting that PlAvh202 and PlAvh222 contributed to virulence of P.litchii.However,PlAvh202 or PlAvh222 deletion did not affect the growth of P.litchii.3.PlAvh202 or PlAvh222 was transiently expressed in N.benthamiana to examine their effect on INF-triggered PTI.The results showed that PlAvh202 and PlAvh222 could suppress the expression of immune maker genes(NbPR1,NbPR2,NbLOX,NbPDF1.2,NbRboh A,and NbRboh B)and ROS burst.In addition,PlAvh202 and PlAvh222 also promoted Phytophthora capsici infection.Four truncated versions of PlAvh202 were constructed to explore the functional region.IR2 is required for PlAvh202 to suppress ICD and promote plant susceptibility,suggesting that there is a link between ICD suppression and virulence of PlAvh202.4.By co-immunoprecipitation(Co-IP)and LC-MS/MS,we identified NbSAMS2-like,a key enzyme in ethylene(ET)biosynthesis,as the target of PlAvh202.Four homologs of NbSAMS2-like in litchi were then shown to interact with PlAvh202 in vivo and in vitro by Split luciferase complementation(SLC),Co-IP and Pull-down.In addition,we found 6candidate targets of PlAvh222 using yeast two-hybrid(Y2H)screening.LcCa M was selected for subsequent experiments because it appears most frequently among all 6 targets.Co-IP showed that PlAvh222 interacted with LcCa M in vivo.5.Overexpression of LcSAMS promotes N.benthamiana resistance to P.capsici,whereas silencing NbSAMS by VIGS(virus induced gene silencing)enhances plant susceptibility to P.capsici and disables the ability of PlAvh202 to promote P.capsici infection.Moreover,diminished ICD was observed in NbSAMS-silenced plants.These results suggests that LcSAMS positively regulates plant immunity,and PlAvh202 suppresses plant immunity by negatively regulating LcSAMS.Further research revealed that PlAvh202 could suppress ethylene-mediated litchi resistance by targeting and destabilizing plant SAMS in a26S proteasome-dependent manner.In summary,our study demonstrates that PlAvh202 and PlAvh222 can interfere with plant immune system.Moreover,we reveal a mechanism that PlAvh202 targets and destabilizes host SAMS by 26S proteasome degradation system to suppress ET-mediated litchi resistance.This study identifies a new target of RXLR effectors,and provide a theoretical basis for understanding and utilization of plant immune system.更多还原