【作者】 陈在杰；

【导师】 王宗华； 陈松彪；

【作者基本信息】 福建农林大学 ， 植物病理学， 2019， 博士

【摘要】 硅(Si)增强植物抗病性主要包括物理与生化两个层面。物理屏障、先天免疫防御反应启动、激素稳态改变以及防御信号网络调控等,都是Si增强植物抗性反应的可能机制,但对这些机制的解析还存在着争议。系统解析Si介导植物抗病性的分子机制,对发展农作物病害绿色防控新策略具有重要意义。水稻ssb(super susceptibility to blast)是一个从籼稻恢复系闽恢3301经γ辐射诱变筛选获得,且极易被稻瘟菌侵染的突变体。苗期人工接种证实,ssb突变体对亲和菌株501-3的抗性几乎丧失,但对非亲和菌株KJ201抗性未受影响。经回交方式培育携带抗稻瘟病基因Pi-zt的ssb材料,接种转化了Avr-pizt的稻瘟菌501-3,表现出抗病表型,说明ssb突变不影响抗病基因与无毒基因互作介导的抗病反应。与野生型亲本相比,ssb突变体应答Flg22、几丁质诱导的ROS迸发水平显著降低,进一步说明ssb突变影响水稻的基础抗病反应。通过图位克隆鉴定了ssb的突变基因,其与编码NOD26蛋白/Si转运蛋白OsLsi1的Os02g0745100为同一个基因。从闽恢3301中克隆了包含上游启动子的全长6.9 kb的Lsi1基因,构建了互补表达载体p CX1300-Lsi1,通过农杆菌介导的方法转入到突变体中。获得了24个T0代阳性克隆,以亲和菌株501-3进行苗期接种鉴定,结果发现8个T1代株系恢复了野生型的基础抗性表型。ssb突变基因编码的蛋白序列存在一个A202V突变。亚细胞定位分析发现,野生型OsLsi1蛋白定位于细胞膜,而SSB/OsLsi1(A202V)则分布于整个细胞,表明A202V突变致使OsLsi1丧失膜蛋白构象。扫描电子显微镜观察结果表明,ssb突变体叶片表面硅化细胞发育严重异常。对ssb突变体叶片进行Si含量测定,发现其显著低于野生型亲本,表明A202V突变使OsLsi1功能丧失,导致ssb突变体Si缺失。以接种24 h水稻叶片为材料,进行转录组分析。结果发现,水稻材料在接种稻瘟病菌后,一些保护性酶和保护性次生代谢物的合成基因以及病程相关基因等被诱导表达。另一方面,突变体ssb接种亲和菌株后,保护性酶和次生代谢物合成基因表达水平明显低于野生型亲本中相关基因的受诱导表达水平,说明Si缺失在生理、生化层面上影响水稻对稻瘟病菌的免疫反应。转录组分析还发现,在应答亲和菌株时,ssb突变体SA、JA、ET等途径相关基因的表达也受影响;对苗期水稻材料喷施Si、SA、JA等激素或类似物,以及ET抑制剂后进行稻瘟菌接种,发现ssb突变体的基础抗性获得提升,表明SA、JA、ET等抗性途径不依赖于Si。为研究通过超量表达OsLsi1、提升水稻Si含量并增强基础抗病性,培育了组成型启动子Ubiquitin驱动OsLsi1表达的植株,但发现超量表达OsLsi1会导致水稻植株发育不正常。本研究创制了化学诱导系统驱动OsLsi1表达的水稻材料,为后续研究OsLsi1表达水平、水稻Si含量以及水稻基础抗病性的协调关系奠定了材料基础。综上,通过克隆水稻ssb/OsLsi1基因,从水稻先天免疫的主动防御与被动防御二个层面,初步解析了硅缺失对水稻稻瘟菌基础抗性的影响,物理层面的硅细胞角质层与硅在质外体的沉积提供了防御真菌侵入的物理屏障,硅通过维护植物细胞壁的完整性,阻碍稻瘟菌PAMPs与PRRs的胞内与胞外识别,使水稻先天免疫得以充分激活,进而影响了ROS迸发,植物抗毒素合成,病程相关基因的表达,并影响激素调节复杂的信号通路网络和激活防御相关基因的表达。该机制的解析将为培育广谱抗性的水稻提供技术方案。更多还原

【Abstract】 Silicon（Si）enhances plant disease resistance mainly including physical and biochemical levels.Physical barrier,innate immune defense response activation,hormone homeostasis change and defense signal network regulation are all possible mechanisms of Si enhancing plant resistance response,but the analysis of these mechanisms is still controversial.A systematic analysis of the molecular mechanism of plant disease resistance mediated by Si is of great significance for the development of new strategies for green control of crop diseases.Rice ssb（super susceptibility to blast）is a mutant easily infected by Magnaporthe oryzae,which was obtained from indica rice Minhui 3301 byγ-irradiation mutagenesis.Artificial inoculation at seedling stage confirmed that the resistance of ssb mutant to compatible strain 501-3 was almost lost,but the resistance to incompatible strain KJ201 was not affected.The ssb material carrying blast resistance gene Pi-zt was cultivated by backcross and inoculated with Avr-pizt transformed blast strain 501-3,which showed resistance phenotype,indicating that ssb mutation did not affect the resistance response mediated by interaction between resistance gene and avirulent gene.Compared with wild-type,the level of ROS burst induced by ssb mutants in response to Flg22 and chitin was significantly lower than that of wild-type,which further indicated that ssb mutations affected the basal resistance of rice.The mutant gene of ssb was identified by the map cloning,which was the same gene as the Os02g0745100 encoding the NOD26 protein/Si transporter OsLsi1.The full length Lsi1 gene（6.9 kb）containing upstream promoter was cloned from Muhui 3301,and the complementary expression vector p CX1300-Lsi1 was constructed and transferred into the mutant by Agrobacterium-mediated method.Twenty-four T0 generation positive clones were obtained,and inoculated with 501-3 strain at the seedling stage.The results showed that the eight T₁ generation lines restored the wild-type basal resistance phenotype.The protein sequence encoded by the ssb mutant gene has an A202V mutation.The subcellular localization analysis found that wild-type OsLsi1 was localized in the cell membrane and SSB/OsLsi1（A202V）was distributed throughout the cell,indicating that the A202V mutation resulted in the loss of the membrane protein conformation of OsLsi1.The results of the scanning electron microscope（SEM）show that the development of silicified cells on the leaf surface of ssb mutant was seriously abnormal.The content of Si in the leaves of ssb mutant was significantly lower than that of wild type,which indicated that A202V mutation resulted in loss of OsLsi1 function and Si deletion in ssb mutant.The rice leaves were inoculated for 24 hours,and the transcript sequence was determined.The results showed that the synthetic genes of some protective enzymes and protective secondary metabolites as well as the genes related to the course of disease were induced to express after inoculation with Magnaporthe oryzae.On the other hand,the expression level of protective enzyme and secondary metabolite synthesis gene in mutant ssb was significantly lower than that in wild type after inoculation with compatible strain.These results suggest that Si deletion affects the immune response of the rice to blast fungus on physiological and biochemical levels.The RNA-seq also showed that the expression of genes related to the pathway such as SA,JA,ET was also affected in response to the compatible strain.After spraying with hormones or analogues such as Si,SA,JA and ET inhibitors at seedling stage,the rice blast was inoculated with ssb mutants.It was found that the basic resistance of ssb mutants was enhanced,indicating that the resistance pathways of SA,JA,ET did not depend on Si.In order to study the effect of over-expression of OsLsi1,on Si content and basic disease resistance of rice,the constitutive promoter Ubiquitin-driven OsLsi1 expression plants were cultivated,but it was found that over-expression of OsLsi1 could lead to abnormal development of rice plants.In this study,a chemical induction system-driven rice material for OsLsi1expression was developed,which lays a foundation for further study on the coordination of OsLsi1 expression level,rice Si content and rice basal resistance.On the basis of cloning of rice ssb/OsLsi1 gene,the effect of silicon deletion on the basal resistance of rice blast was preliminarily analyzed from the two levels of active defense and passive defense of rice innate immunity.The co-deposition of silicon on the extracellular body with cell wall component and silicified cuticle at the physical level provides a physical barrier against fungal invasion.Silicon prevents the intracellular and extracellular recognition of PAMPs and PRRs by maintaining the integrity of plant cell walls.The innate immunity of rice was fully activated,which affected the burst of ROS,the synthesis of phytolaxin,the expression of PR genes,It also affects hormone-regulated complex signal pathway and activates the expression of defense-related genes.The analysis of this mechanism will provide a technical scheme for the cultivation of broad-spectrum resistant rice.更多还原