在小麦和叶锈菌互作过程中TCTP的表达特征

翻译控制肿瘤蛋白是调节植物生长发育、参与植物抵抗逆境胁迫的重要调节因子。本研究以叶锈菌生理小种260与4种含有不同抗叶锈基因的近等基因系分别组成亲和程度不同的组合为对象,采用RT-qPCR和Western Blotting方法,从转录和翻译两个水平探讨TCTP在小麦与叶锈菌互作过程中的表达模式。研究结果表明,无论是转录水平还是翻译水平上,TCTP在不亲和组合中均表现为表达量显著上调,但在不同的Lr基因背景下其表达模式有所不同;而在亲和组合中并未表现出明显的变化趋势。基于TCTP mRNA和蛋白水平的结果,发现在小麦与叶锈菌互作体系中TCTP在转录水平和翻译水平均受到调控,推测其在小麦抵抗叶锈菌侵染的防卫反应中可能发挥正调控作用,这将为进一步研究TCTP的功能及其作用机制奠定了基础。     

小麦CBL结合蛋白激酶基因TaCIPK16的克隆及特征分析

类钙调磷酸酶亚基B蛋白(calcineurin B-1ike protein,CBL)作为一类钙离子结合蛋白,通过与一类蛋白激酶(CBL-interacting protein kinase,ClPK)结合,从而在钙信号依赖的生理生化过程中发挥作用。该研究在条锈菌诱导的小麦叶片中克隆获得CIPK家族中1个基因TaCIPK16,并利用qRT-PCR技术、酵母双杂交技术及亚细胞定位技术分析了其功能特性。序列分析表明,TaCIPK16编码447个氨基酸,包含保守的激酶催化结构域及调控结构域,与水稻、拟南芥CIPK蛋白具有高度相似性。酵母双杂交分析验证显示,TaCIPK16与TaCBL4和TaCBL9存在强烈互作。定量分析表明,TaCIPK16受到条锈菌的诱导表达,在小麦与条锈菌互作过程中呈显著差异表达趋势。综上结果,TaCIPK16可能作为正调控因子参与了小麦对条锈菌的抗病防卫反应。     

利用酵母双杂交系统研究植物与病毒蛋白相互作用的进展

在长期进化中,植物形成了抵御病毒等病原微生物侵染的精细防御系统。在病毒侵染、复制和传播过程中,其编码的一些蛋白,如外壳蛋白、运动蛋白、复制酶类等能够与植物基因编码的蛋白发生相互作用。酵母双杂交系统是体外研究蛋白质间相互作用的有利工具,不但可以用于研究已知蛋白质的互作,还可以发现新蛋白,揭示特定蛋白互作网络与作用机制,在植物蛋白与病毒蛋白互作研究中已得到广泛的利用。本文主要综述利用酵母双杂交系统研究植物与病毒蛋白相互作用的国内外进展。     

采用酵母双杂交系统筛选橡胶树HbTCTP1互作蛋白

翻译控制肿瘤蛋白(translationally controlled tumor protein,TCTP)是一种在生物体中普遍存在和结构上高度保守的多功能蛋白。本研究以橡胶树Hb TCTP1基因的开放阅读框(open reading frame,ORF)作为诱饵,采用酵母双杂交系统构建筛选Hb TCTP1互作蛋白的诱饵表达载体p BD-GAL4-Hb TCTP1,并在p ADGAL4-2.1质粒构建的橡胶树胶乳cDNA文库中筛选Hb TCTP1互作蛋白。将获得的两个与Hb TCTP1互作的阳性克隆进行测序,比对NCBI非丰富蛋白数据库发现:它们分别是橡胶延长因子(rubber elongation factor,REF)和60S核糖体L44蛋白(60S ribosomal protein L44,RPL44)。本研究结果对进一步揭示Hb TCTP1在橡胶树中的作用具有重要意义。     

植物SnRK家族的研究进展

植物在自然界中面临各种环境侵害时候,如干旱、盐、低温和病菌袭击,会启动自身的抵御机制来适应各种侵害。蔗糖非发酵相关的蛋白激酶(sucrose non-fermenting-1-related protein kinase,SnRK)是广泛存在于植物中的一类Ser/Thr蛋白激酶,参与各种胁迫信号传导通路,对植物抵御不良环境起到重要作用。植物中蔗糖非发酵相关的蛋白激酶共有38个成员,可以分为3个亚家族:SnRK1、SnRK2和SnRK3。本文主要讨论SnRK家族的研究进展,揭示SnRK家族在植物抗逆中的重要作用。     

条形柄锈菌吸器特异效应蛋白Hasp68抑制寄主免疫并与小麦组织蛋白酶B互作

作为活体营养专性寄生真菌,条形柄锈菌（小麦条锈病）在侵染过程中通过形成吸器向寄主细胞释放效应蛋白,干扰寄主的防卫反应,促进其侵染与致病。因此,条形柄锈菌效应蛋白的鉴定与功能研究对揭示其毒性机理具有重要意义。本实验室前期完成了条形柄锈菌CYR31生理小种吸器转录组分析,从中鉴定得到一个吸器特异诱导表达分泌蛋白Hasp68,利用农杆菌侵染在烟草细胞中瞬时表达该基因,能够抑制小鼠促细胞凋亡蛋白Bax诱导的细胞程序性死亡,鉴定为条形柄锈菌候选效应蛋白。Hasp68基因全长318bp,编码105_aa,N-端包含20_aa的信号肽,无保守结构域。BlastX分析表明Hasp68为条形柄锈菌特有效应蛋白,在其他真菌中无同源蛋白,且在条形柄锈菌16个菌系中呈较低的序列多态性,表明其在条形柄锈菌的进化过程中相对保守。借助荧光假单胞菌EtHAn的三型分泌系统,在小麦细胞中过表达Hasp68能够抑制由非致病细菌引起的PTI（PAMP-triggered immunity）相关胼胝质的积累;同时,也能抑制小麦与无毒条形柄锈菌互作中ETI（effector-triggered immunity）相关的活性氧爆发和过敏性坏死反应,表明效应蛋白Hasp68具有抑制寄主免疫反应的功能。利用酵母双杂交系统筛选Hasp68在小麦中的互作蛋白,发现其与组织蛋白酶B（cathepsin B）TaCTSB互作,双分子荧光技术进一步验证二者在烟草细胞中共表达存在互作,初步揭示了效应蛋白Hasp68的互作靶标。     

小麦锌指蛋白基因TaLOL2的克隆及特征分析

通过电子克隆与RT-PCR相结合的方法,在条锈菌诱导的小麦叶片中克隆获得1个新的LSD1型锌指蛋白基因TaLOL2,并用qRT-PCR技术分析了其转录表达特征。结果显示:(1)小麦锌指蛋白基因TaLOL2的cDNA全长1 095bp,编码179个氨基酸。(2)TaLOL2含有3个典型的zf-LSD1型(CxxCxRxxLMYxxGASxVxCxxC)保守结构域,与水稻、拟南芥、大麦等植物LSD1型锌指蛋白序列具有高度相似性,其中与水稻OsLOL2相似度达86.0%。(3)进化树分析表明,TaLOL2与水稻、拟南芥和大麦中部分含有3个保守zf-LSD1锌指结构的基因亲缘关系较近,而与其它包含不同数目的zf-LSD1锌指结构的基因亲缘关系较远。(4)qRT-PCR定量分析表明,TaLOL2在条锈菌侵染前期呈上调表达,在亲和及非亲和反应中差异表达。研究表明,TaLOL2参与了条锈菌诱导的小麦抗病防卫反应,很可能作为正调控因子参与了小麦-条锈菌非亲和互作中对条锈菌的抗性信号途径。     

小麦F-box基因TaFBL14的克隆及表达分析

该研究利用同源克隆策略,获得了1条小麦F-box/LRR重复蛋白14基因(TaFBL14)。TaFBL14基因编码486个氨基酸,预测分子量为53.48kD,等电点为5.93,序列N端包含一个F-box结构域,C段包含7个LRR结构域。TaFBL14基因编码蛋白不具有信号肽及核定位信号序列,主要定位在细胞质中,二级结构以α-螺旋为主,呈球状。进化树分析表明,TaFBL14与粗山羊草和乌拉尔托小麦的FBL14蛋白亲缘关系较近。qRT-PCR分析结果显示,TaFBL14基因主要在小麦叶组织中表达,且受非亲和叶锈菌侵染后呈现上调表达趋势,说明该基因可能参与小麦抵御叶锈菌的侵染过程。     

利用酵母双杂交系统筛选与黄瓜花叶病毒外壳蛋白互作的寄主蛋白

利用酵母双杂交系统,以黄瓜花叶病毒(Cucumber mosaic virus,CMV)的外壳蛋白(coat protein,CP)为诱饵,从番茄叶片c DNA文库中筛选与其互作的蛋白。结果显示,诱饵载体pBT3-SUC-CMV-CP均能在酵母细胞中正确表达,无自激活活性而且对酵母无毒性;通过对酵母双杂交文库的筛选和回转验证,共获得了98个阳性克隆,分别编码67个可能与CMV-CP相互作用的蛋白,分别参与植物防御反应、光合作用、物质转运、信号转导、能量代谢、氨基酸代谢、细胞壁的形态建成、植物的激素代谢等。本研究结果表明,CMV CP可同时调控寄主的多个代谢过程,在CMV的致病过程中有多重功能。     

本生烟eIF4Es基因克隆及其酵母双杂交诱饵载体构建

由木薯褐色条斑病毒(Cassava brown steak virus, CBSV)和乌干达木薯褐色条斑病毒(Uganda Cassava brown steak virus, UCBSV)引起的木薯褐色条斑病毒病,是危害木薯的主要病害之一。目前尚无有效防治方法,并且特别是缺乏CBSV/UCBSV抗性育种材料。真核翻译起始因子4E (eukaryotie translation initiation factor4E, eIF4E)不仅参与蛋白质的翻译起始,并且Potyviruses的复制和翻译也依赖于eIF4E与病毒基因组连接蛋白(virus genome linked protein, VPg)的相互作用,目前发现的植物隐性抗病基因大部分都是eIF4E家族的等位基因。本研究利用生物信息学方法和RT-PCR技术获得本生烟(Nicotiana benthamiana) eIF4E家族的8个基因,聚类分析显示它们分别编码eIF4E、eIF4E的异构体和类似帽结合的蛋白。为筛选与CBSV/UCBSV相互作用的本生烟eIF4E家族蛋白,本研究进一步构建了这8个基因的Lex A-酵母双杂交系统的诱饵载体,并导入酵母细胞EGY48 (p8op-LacZ)进行细胞毒性和自激活检测。结果显示导入重组质粒的EGY48 (p8op-LacZ)酵母细胞在SD/-His/-Ura缺陷型培养基上生长良好,在SD/-Trp/-Ura缺陷型平板上不生长,并且在SD/Gal/Raf/-His/-Ura/X-Gal培养基上不显蓝色,说明重组质粒表达产物不仅对该酵母细胞无毒性,而且对下游报告基因无自激活作用,可用于该系统的互作蛋白筛选研究。本研究为利用酵母双杂交系统发现与CBSV/UCBSV相互作用的本生烟eIF4E基因,探索通过基因编辑与CBSV/UCBSV互作的寄主因子进行抗CBSV/UCBSV分子育种提供科学依据。     

A plant kinase plays roles in defense response against geminivirus by phosphorylation of a viral pathogenesis protein

The plant SNF1-related kinase (SnRK1) is the α-subunit of the SnRK1 heterotrimeric compleses. Although SnRK1 is widely known as a key regulator of plant response to various physiological processes including nutrient- and energy-sensing, regulation of global metabolism, and control of cell cycle, development, as well as abiotics stress, less is known about the function of SnRK1 during pathogen infection. Our previous work has demonstrated that a tomato SNF1-related kinase (SlSnRK1) can interact with and phosphorylate βC1, a pathogenesis protein encoded by tomato yellow leaf curl China betasatellite. Our results also showed that the plant SnRK1 can affect genimivirus infection in plant and reduce viral DNA accumulation. Phosphorylation of βC1 protein negatively impacts its function as a pathogenicity determinant. Here we provide more information on interaction between βC1 and SlSnRK1 and propose a mechanistic model for the SlSnRK1-mediated defense responses against geminiviruses and the potential role of SnRK1 in plant resistance to geminivirus.     

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.     

Metabolic signalling and carbon partitioning: role of Snf1-related (SnRK1) protein kinase

A protein kinase that plays a key role in the global control of plant carbon metabolism is SnRK1 (sucrose non-fermenting-1-related protein kinase 1), so-called because of its homology and functional similarity with sucrose non-fermenting 1 (SNF1) of yeast. This article reviews studies on the characterization of SnRK1 gene families, SnRK1 regulation and function, interacting proteins, and the effects of manipulating SnRK1 activity on carbon metabolism and development.     

A cell cycle role for a plant sucrose nonfermenting-1-related protein kinase (SnRK1) is indicated by expression in yeast

Plant sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRK1s) have been shown to restore carbon catabolite derepression of gene expression in the yeast Saccharomyces cerevisiae when expressed in snf1 mutants. SNF1 has been implicated in the mediation of cell cycle control in response to nutrient levels and, in the present study, we show that expression of the rye (Secale cereale) SnRK1, RKIN1, in a yeast snf1 mutant has a dramatic effect on the size of cells growing on a minimal medium where SNF1 function is essential. The mean volume of the yeast cells which were expressing RKIN1 was two-thirds that of the whi1 mutant, the smallest viable cells known in S. cerevisiae, and the cells died after 3 days unless rescued onto complex medium. This is the first experimental evidence of a role for SnRK1s in plant cell cycle control.     

Regulation of spinach SNF1-related (SnRK1) kinases by protein kinases and phosphatases is associated with phosphorylation of the T loop and is regulated by 5''-AMP

Members of the SNF1-related protein kinase-1 (SnRK1) subfamily of protein kinases are higher plant homologues of mammalian AMP-activated and yeast SNF1 protein kinases. Based on analogies with the mammalian system, we surmised that the SnRK1 kinases would be regulated by phosphorylation on a threonine [equivalent to Thr175 in Arabidopsis thaliana SnRK1 (AKIN10)] within the 'T loop' between the conserved DFG and APE motifs. We have raised an antibody against a phosphopeptide based on this sequence, and used it to show that inactivation of two spinach SnRK1 kinases by protein phosphatases, and reactivation by a mammalian upstream protein kinase, is associated with changes in the phosphorylation state of this threonine. We also show that dephosphorylation of this threonine by protein phosphatases, and consequent inactivation, is inhibited by low concentrations of 5'-AMP, via binding to the substrate (i.e. the kinase). This is the first report showing that the plant SnRK1 kinases are regulated by AMP in a manner similar to their mammalian counterparts. The possible physiological significance of these findings is discussed.     

Domain fusion between SNF1-related kinase subunits during plant evolution

Members of the conserved SNF1/AMP-activated protein kinase (AMPK) family regulate cellular responses to environmental and nutritional stress in eukaryotes. Yeast SNF1 and animal AMPKs form a complex with regulatory SNF4/AMPKγ and SIP1/SIP2/GAL83/AMPKβ subunits. The β-subunits function as target selective adaptors that anchor the catalytic kinase and regulator SNF4/γ-subunits to their kinase association (KIS) and association with the SNF1 complex (ASC) domains. Here we demonstrate that plant SNF1-related protein kinases (SnRKs) interact with an adaptor-regulator protein, AKINβγ, in which an N-terminal KIS domain characteristic of β-subunits is fused with a C-terminal region related to the SNF4/AMPKγ proteins. AKINβγ is constitutively expressed in plants, suppresses the yeast Δsnf4 mutation, and shows glucose-regulated interaction with the Arabidopsis SnRK, AKIN11. Our results suggest that evolution of AKINβγ reflects a unique function of SNF1-related protein kinases in plant glucose and stress signalling.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Influence of the StubSNF1 kinase complex and the expression of the yeast TPS1 gene on growth and tuber yield in potato

Expression of the yeast trehalose-6-phosphate synthase-1 (TPS1) gene in potato results in growth aberrations and arrest of development. Recent studies have shown that this phenomenon could be related to the inhibitory effect of trehalose-6-phosphate on SnRK1s, a family of sucrose non-fermenting-1 (SNF1)-related protein kinases that link metabolic and stress signalling in plants. SnRK1s are heterotrimeric enzymes similar to yeast SNF1 and mammalian AMP-activated protein kinases (AMPKs). Previously, we showed that antisense repression of StubGAL83, one of the three subunits of the potato SnRK1 complex, results in a delay in rooting and increases sensitivity to salt stress. Here we report that StubGAL83 is a positive regulator of SNF1 kinase activity in potato and that repression of the kinase subunit of the SnRK1 complex, StubSNF1, reduces growth and tuber yield in potato plants. Co-repression of StubGAL83 and StubSNF1 at a certain level, however, can result in larger plants and increased tuber yield. We found that repression of StubGAL83, but not repression of StubSNF1 attenuated growth aberrations caused by TPS1 expression. We provide evidence that the increased plant size and yield in StubGAL83-StubSNF1 co-repressed plants as well as the attenuation of aberrations caused by TPS1 expression are related to increased nitrate reductase activity.     

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.