SPINDLY在壳寡糖诱导拟南芥抗丁香假单胞菌中的功能

为了探讨拟南芥O-岩藻糖基转移酶(SPINDLY)在病原体相关分子模式诱导抗性中的作用,该研究以SPINDLY缺失拟南芥突变体spy-3为实验材料,从叶片表型、病情指数、病菌定殖量以及丁香假单胞菌(Pst DC3000)关键基因的表达水平等指标,系统考察了SPINDLY在壳寡糖诱导拟南芥抗Pst DC3000中的功能。结果显示:(1)spy-3突变体比野生型更易被Pst DC3000侵染。(2)与病菌侵染组相比,壳寡糖预处理明显缓解植株叶片黄化现象,显著降低Pst DC3000的定殖量。(3)壳寡糖预处理的spy-3植株中水杨酸和茉莉酸途径相关基因的表达量及水杨酸和茉莉酸含量均较病菌侵染组明显升高。(4)壳寡糖在spy-3中的诱抗效果与野生型相比无明显差别。研究表明,SPINDLY在植物先天免疫过程发挥重要作用,但并不影响壳寡糖的诱导抗性。     

青色荧光蛋白标记的禾谷镰孢转化子的构建

【背景】近年来玉米茎腐病在我国大部分玉米产区普遍重度发生,其中镰孢菌茎腐病不仅造成了重大的经济损失,而且镰孢菌产生的毒素给人体和动物的健康也带来严重威胁。【目的】玉米茎腐病的病原组成复杂,禾谷镰孢是其中的主要病原之一,该病原菌侵染寄主导致发病的机制急需深入研究。【方法】以pCAMBIA1300质粒为骨架,利用重叠PCR的方法构建表达青色荧光蛋白的质粒pCAMBIA1300-CFP-Kan,通过农杆菌介导的遗传转化技术,将青色荧光蛋白的编码基因整合到禾谷镰孢基因组中。【结果】经过PCR鉴定和荧光显微观察,确定获得了31株青色荧光标记的禾谷镰孢菌。【结论】侵染试验结果显示,激光共聚焦显微镜下禾谷镰孢在玉米茎秆组织中的定殖位置清晰可见,该结果为进一步研究不同镰孢菌在寄主中的定殖规律奠定了基础。     

激光共聚焦扫描显微镜在抗菌机理研究中的应用

激光共聚焦扫描显微镜采用激光光源、共聚焦技术和点扫描技术,使其分辨力较传统光学显微镜大为提高。与其他的生物学技术相配合,可定性、定量、定位地检测组织细胞内的多种生化成分。它具有的活细胞动态监测、断层扫描及三维图像重建等功能,使其在抗菌机理研究、尤其是抗生物膜研究中得到大量的应用。本文就激光共聚焦扫描显微镜在抗菌剂作用位点,抗菌剂对微生物细胞膜,微生物生理代谢,以及微生物生物膜形成与结构的影响等研究中的应用做一综述。     

两种瞬时表达体系研究拟南芥Profilin-1的亚细胞定位

使用两种瞬时表达方法研究Profilin-1(PRF1)的亚细胞定位,并比较了2种瞬时表达体系在亚细胞定位研究中的优缺点。利用拟南芥幼叶作为材料,提取叶片的RNA,采用特异性引物RT-PCR的方法克隆PRF1基因,连接到p CAMBIA1300-GFP的改造载体上,成功的构建p CAMBIA1300-GFP-PRF1的表达载体。然后分别利用PEG转化拟南芥原生质体、农杆菌浸染烟草叶片两种技术进行了瞬时表达,并在激光共聚焦显微镜下观察绿色荧光蛋白(GFP)融合蛋白的表达。研究结果表明,将PRF1基因导入拟南芥的原生质体和烟草表皮细胞后,融合蛋白绿色荧光均能被观察到,PRF1基因与GFP融合蛋白的产物在烟草表皮细胞中主要定位在细胞质和外周细胞器中,在拟南芥的原生质体中的细胞核和细胞质中都有定位。两种不同的瞬时表达体系中PRF1蛋白的定位出现了不同,这可能与同源或异源表达的植物的特性相关。     

茶树CsPAL3基因的亚细胞定位及遗传转化

苯丙氨酸解氨酶(phenylalanine ammonia-lyase,PAL)由多基因家族编码,是花青素等多酚物质合成途径的起始酶,对其合成具有调控作用。以紫化茶树武夷奇种C18为材料,采用Gateway技术体系分别构建了茶树的CsPAL3过表达载体pGWB502:CsPAL3和pGWB505:CsPAL3:GFP,并成功将其转入根癌农杆菌GV3101。注射烟草瞬时表达激光共聚焦扫描显微镜可观察到GFP绿色荧光,结果表明CsPAL3主要集中在细胞核和细胞膜中。通过侵染拟南芥,筛选纯合子,获得稳定表达的转CsPAL3基因拟南芥。实时荧光定量PCR(qPCR)检测发现,CsPAL3在转CsPAL3基因拟南芥中的根部表达量显著高于叶片,且CsPAL3基因受光照调控。该结果为进一步研究茶树CsPAL3基因功能以及促进茶树花青素合成与积累的分子调控机理提供科学依据。     

植物microRNA功能瞬时验证体系的建立简

目的：建立植物microRNA（miRNA）功能的瞬时活体验证体系,并检验该体系的有效性。方法：选用双元表达载体pcAMBIA1200,并插入烟草花叶病毒双35s启动子,以驱动目标miRNA超表达;选用双元表达载体pFGC5941的绿色荧光蛋白（GFP）改造载体用于潜在的靶基因与GFP融合蛋白的超表达,以转入这2种载体的农杆菌侵染烟草叶片,观察GFP融合蛋白的荧光,作为验证miRNA对其潜在靶基因调控作用的瞬时验证体系。选取拟南芥已知功能的miR393及其靶基因A船3,分别构建pcAMBIA1200-35s-miR393和pFGc5941-GFP-AFB3载体,利用农杆菌注射烟草叶片进行2个载体共转化,并以pFGC5941-GFP-AFB3单转化作为对照,激光共聚焦显微镜下观察融合蛋白的表达。结果：只将A朋3导入烟草表皮细胞,可观察到绿色荧光;而将miR393与A期3同时导入烟草表皮细胞后,未能观察到绿色荧光。表明miR393抑制了A朋3的表达。结论：本瞬时表达体系可作为植物miRNA功能的活体瞬时验证体系,为miRNA调控靶基因表达功能提供简单、快速、有效的证据。     

植物microRNA功能瞬时验证体系的建立

目的:建立植物microRNA(miRNA)功能的瞬时活体验证体系,并检验该体系的有效性。方法:选用双元表达载体pCAMBIA1200,并插入烟草花叶病毒双35S启动子,以驱动目标miRNA超表达;选用双元表达载体pFGC5941的绿色荧光蛋白(GFP)改造载体用于潜在的靶基因与GFP融合蛋白的超表达,以转入这2种载体的农杆菌侵染烟草叶片,观察GFP融合蛋白的荧光,作为验证miRNA对其潜在靶基因调控作用的瞬时验证体系。选取拟南芥已知功能的miR393及其靶基因AFB3,分别构建pCAMBIA1200-35S-miR393和pFGC5941-GFP-AFB3载体,利用农杆菌注射烟草叶片进行2个载体共转化,并以pFGC5941-GFP-AFB3单转化作为对照,激光共聚焦显微镜下观察融合蛋白的表达。结果:只将AFB3导入烟草表皮细胞,可观察到绿色荧光;而将miR393与AFB3同时导入烟草表皮细胞后,未能观察到绿色荧光。表明miR393抑制了AFB3的表达。结论:本瞬时表达体系可作为植物miRNA功能的活体瞬时验证体系,为miRNA调控靶基因表达功能提供简单、快速、有效的证据。     

用于植物病原细菌标记的pBB-GFP载体构建及应用北大核心CSCD

扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到p HM1:P_(RiPAK)LacZ。携带pHM1:P_(RiPAK)LacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达。为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白。pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构。荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集。本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光。     

用于植物病原细菌标记的pBB-GFP载体构建及应用

扩增青枯劳尔氏菌RipAK基因启动子序列,与lacZ基因融合得到p HM1:P_(RiPAK)LacZ。携带pHM1:P_(RiPAK)LacZ的青枯劳尔氏菌在营养丰富和基本培养基中都有LacZ活性,表明RipAK启动子可以推动lacZ基因的表达。为构建用于标记植物病原细菌的绿色荧光表达载体,把RipAK启动子和gfp基因克隆到质粒pBBR1MCS-5,使得gfp基因在RipAK启动子的驱动下表达;构建的表达载体pBB-GFP在大肠杆菌中即可表达绿色荧光蛋白。pBB-GFP载体能有效标记青枯劳尔氏菌、番茄细菌性斑点病菌和柑橘溃疡病菌,在荧光显微镜下观察到3种植物病原细菌呈短杆状,青枯劳尔氏菌还可形成多个菌体串联的线状结构。荧光标记对3种病原菌在寄主植物上的致病力没有影响,将标记菌株分别滴加在寄主植物叶片的创伤处,可观察到大量的绿色荧光聚集。本研究构建的pBB-GFP载体能用于多种植物病原细菌的绿色荧光标记,标记后的病原细菌在液体培养及侵染寄主植物过程中都能观察到荧光。     

基于农杆菌介导瞬时转化法的AtGLR1.4亚细胞定位分析

将拟南芥基因AtGLR1.4启动子驱动的AtGLR1.4基因与绿色荧光蛋白(GFP)基因融合后,利用根瘤农杆菌介导瞬时转化法(Fast Agro-mediated Seedling Transfomation,FAST)浸染拟南芥幼苗,对其进行亚细胞定位的研究。转基因植株通过激光共聚焦扫描显微镜的观察,发现GFP绿色荧光在叶片表皮细胞的细胞膜上特异表达,表明At-GLR 1.4蛋白定位于细胞质膜上,为其后续的功能研究提供了线索。     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

The wxacO gene of Xanthomonas citri ssp. citri encodes a protein with a role in lipopolysaccharide biosynthesis, biofilm formation, stress tolerance and virulence

Xanthomonas citri ssp. citri (Xcc) causes citrus canker, one of the most economically damaging diseases affecting citrus worldwide. Biofilm formation is important for the pathogen to survive epiphytically in planta prior to the induction of canker symptoms. In this study, two EZ-Tn5 transposon mutants of Xcc strain 306, affected in biofilm formation, were isolated; subsequent analyses led to the identification of a novel gene locus XAC3596 (designated as wxacO), encoding a putative transmembrane protein, and the rfbC gene, encoding a truncated O-antigen biosynthesis protein. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that lipopolysaccharide (LPS) biosynthesis was affected in both wxacO and rfbC mutants. The wxacO mutant was impaired in the formation of a structured biofilm on glass or host plant leaves, as shown in confocal laser scanning microscopy analysis of strains containing a plasmid expressing the green fluorescent protein. Both wxacO and rfbC mutants were more sensitive than the wild-type strain to different environmental stresses, and more susceptible to the antimicrobial peptide polymyxin B. The two mutants were attenuated in swimming motility, but not in flagellar formation. The mutants also showed reduced virulence and decreased growth on host leaves when spray inoculated. The affected phenotypes of the wxacO and rfbC mutants were complemented to wild-type levels by the intact wxacO and rfbC genes, respectively. This report identifies a new gene influencing LPS production by Xcc. In addition, our results suggest that a structurally intact LPS is critical for survival in the phyllosphere and for the virulence of Xcc.     

Non-host defense response in a novel Arabidopsis-Xanthomonas citri subsp. citri pathosystem

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gene transfer technology.     

Xanthomonas citri ssp. citri requires the outer membrane porin OprB for maximal virulence and biofilm formation

Xanthomonas citri ssp. citri (Xcc) causes canker disease in citrus, and biofilm formation is critical for the disease cycle. OprB (Outer membrane protein B) has been shown previously to be more abundant in Xcc biofilms compared with the planktonic state. In this work, we showed that the loss of OprB in an oprB mutant abolishes bacterial biofilm formation and adherence to the host, and also compromises virulence and efficient epiphytic survival of the bacteria. Moreover, the oprB mutant is impaired in bacterial stress resistance. OprB belongs to a family of carbohydrate transport proteins, and the uptake of glucose is decreased in the mutant strain, indicating that OprB transports glucose. Loss of OprB leads to increased production of xanthan exopolysaccharide, and the carbohydrate intermediates of xanthan biosynthesis are also elevated in the mutant. The xanthan produced by the mutant has a higher viscosity and, unlike wild‐type xanthan, completely lacks pyruvylation. Overall, these results suggest that Xcc reprogrammes its carbon metabolism when it senses a shortage of glucose input. The participation of OprB in the process of biofilm formation and virulence, as well as in metabolic changes to redirect the carbon flux, is discussed. Our results demonstrate the importance of environmental nutrient supply and glucose uptake via OprB for Xcc virulence.     

Construction of EGFP-Labeling System for Visualizing the Infection Process of Xanthomonas axonopodis pv. citri in planta

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.     

Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), and huanglongbing disease do not exist in the Stapleton Station area of the Northern Territory of Australia

Abstract  A series of specimens of the Asian citrus psyllid, Diaphorina citri , collected from the Northern Territory (NT) in 1915 was recently rediscovered in the Natural History Museum, London. Surveys were conducted in 2002 on suitable hosts in the locality of the 1915 collections to see if the infestation had persisted. These failed to detect either D. citri or the bacterium that it transmits and that causes huanglongbing disease in citrus. It is presumed that D. citri was eradicated fortuitously by the removal of all citrus plants above latitude 19°S during an eradication program for citrus canker in the NT from 1916 until 1922.     

Molecular characterization of copper resistance genes from Xanthomonas citri subsp. citri and Xanthomonas alfalfae subsp. citrumelonis

Copper sprays have been widely used for control of endemic citrus canker caused by Xanthomonas citri subsp. citri in citrus-growing areas for more than 2 decades. Xanthomonas alfalfae subsp. citrumelonis populations were also exposed to frequent sprays of copper for several years as a protective measure against citrus bacterial spot (CBS) in Florida citrus nurseries. Long-term use of these bactericides has led to the development of copper-resistant (Cu(r)) strains in both X. citri subsp. citri and X. alfalfae subsp. citrumelonis, resulting in a reduction of disease control. The objectives of this study were to characterize for the first time the genetics of copper resistance in X. citri subsp. citri and X. alfalfae subsp. citrumelonis and to compare these organisms to other Cu(r) bacteria. Copper resistance determinants from X. citri subsp. citri strain A44(pXccCu2) from Argentina and X. alfalfae subsp. citrumelonis strain 1381(pXacCu2) from Florida were cloned and sequenced. Open reading frames (ORFs) related to the genes copL, copA, copB, copM, copG, copC, copD, and copF were identified in X. citri subsp. citri A44. The same ORFs, except copC and copD, were also present in X. alfalfae subsp. citrumelonis 1381. Transposon mutagenesis of the cloned copper resistance determinants in pXccCu2 revealed that copper resistance in X. citri subsp. citri strain A44 is mostly due to copL, copA, and copB, which are the genes in the cloned cluster with the highest nucleotide homology (≥ 92%) among different Cu(r) bacteria.     

A novel Xanthomonas citri subsp. citri NADPH quinone reductase involved in salt stress response and virulence

BackgroundXanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker is maintained as an epiphyte on citrus leaves until entering the plant tissue. During epiphytic survival, bacteria may encounter low water availability that challenges the infection process. Proteomics analyses of Xcc under saline stress, mimicking the conditions found during epiphytic survival, showed increased abundance of a putative NAD(P)H dehydrogenase encoded by XAC2229.MethodsExpression levels of XAC2229 and a Xcc mutant in XAC2229 were analyzed in salt and oxidative stress and during plant-pathogen interaction. An Escherichia coli expressing XAC2229 was obtained, and the role of this protein in oxidative stress resistance and in reactive oxygen species production was studied. Finally, Xac2229 protein was purified, spectrophotometric and cofactor analyses were done and enzymatic activities determined.ResultsXAC2229 was expressed under salt stress and during plant-pathogen interaction. ΔXAC2229 mutant showed less number of cankers and impaired epiphytic survival than the wild type strain. ΔXAC2229 survived less in the presence of H₂O₂ and produced more reactive oxygen species and thiobarbituric acid-reactive substances than the wild type strain. Similar results were observed for E. coli expressing XAC2229. Xac2229 is a FAD containing flavoprotein, displays diaphorase activity with an optimum at pH 6.0 and has quinone reductase activity using NADPH as an electron donor.ConclusionsA FAD containing flavoprotein from Xcc is a new NADPH quinone reductase required for bacterial virulence, particularly in Xcc epiphytic survival on citrus leaves.General significanceA novel protein involved in the worldwide disease citrus canker was characterized.     

植物挥发物对柑橘木虱趋避机制的研究进展

柑橘木虱Diaphorina citri为半翅目Hemiptera木虱科Psyllidae,主要危害芸香科植物,是柑橘黄龙病（Citrus Huanglongbing, HLB）的主要昆虫传播媒介。HLB在柑橘产业中破坏性极大,柑橘木虱的及时防控可有效降低HLB传播风险。目前,柑橘木虱对化学药剂已产生一定的抗药性,而柑橘木虱在植物挥发物作用下的行为反应可为其绿色防控提供一定的参考。本文就往年研究中不同植物挥发物对柑橘木虱的引诱、驱避作用以及柑橘植株、HLB、柑橘木虱三者间通过植株挥发物产生的互作进行论述,旨在为柑橘木虱的科学高效绿色防控提供参考。