Tracking the green invaders advances in imaging virus infection in plants

Biochem.J.(2010)430,21–37(Printed in Great Britain)

doi:10.1042/BJ20100372

21

REVIEW ARTICLE

Tracking the green invaders:advances in imaging virus infection in plants

Jens TILSNER*and Karl J.OPARKA ?1

*Plant Pathology Department,Scottish Crop Research Institute,Invergowrie,Dundee DD25DA,U.K.,and ?Institute of Molecular Plant Sciences,University of Edinburgh,May?eld Road,Edinburgh EH93JH,U.K.

Bioimaging contributes signi?cantly to our understanding of plant virus infections.In the present review,we describe technical advances that enable imaging of the infection process at previously unobtainable levels.We highlight how such new advances in subcellular imaging are contributing to a detailed dissection of all stages of the viral infection process.Speci?cally,we focus on:(i)the increasingly detailed localizations of viral proteins enabled by a diversifying palette of cellular markers;(ii)approaches using ?uorescence microscopy for the functional analysis of proteins in vivo ;(iii)the imaging of

viral RNAs;(iv)methods that bridge the gap between optical and electron microscopy;and (v)methods that are blurring the distinction between imaging and structural biology.We describe the advantages and disadvantages of such techniques and place them in the broader perspective of their utility in analysing plant virus infection.

Key words:correlative microscopy,?uorescent protein,in vivo interaction,membrane topology,RNA imaging,super-resolution.

INTRODUCTION

In order to infect a host cell and to spread systemically,viral pathogens have to usurp,manipulate and overwhelm numerous biological processes within cells.They often achieve this with a minimal set of multifunctional virus-encoded proteins,as well as through the recruitment of host factors [1–5].Most of these ‘hijacked’processes are compartmentalized within the host cell,and thus the virus and its gene products become localized to speci?c subcellular compartments during the appropriate stages of infection,often in a highly dynamic manner.The ability to visualize these events and their ensuing effect on the host cell have greatly enhanced our understanding of the infection process.

Since the 1950s,EM (electron microscopy),later coupled to immunogold detection of speci?c proteins and RNA,has been the mainstay of virological approaches,but is restricted to static snapshots of the infection process.Furthermore,the limited accessibility of antigenic epitopes in samples embedded and sectioned for EM makes it dif?cult to detect less-abundant proteins or to quantitatively assign visible structures to speci?c biomolecules [6].The discovery of FPs (?uorescent proteins)re-introduced LM (light microscopy)as a major tool for cell biology in the early 1990s,and today ?uorescence microscopy is one of the cornerstones of plant virology.Initially,virus-expressed FPs were simply used to monitor the spread of infection [7,8],but plant virologists quickly started to employ them as fusions to virus-encoded proteins.Nowadays,?uorescence microscopy is used increasingly to permit not just localization,but also

functional analysis of proteins in vivo ,and new approaches have allowed optical-based microscopy to break the diffraction barrier,long held as a ?xed limit to resolution [9–11].At the same time,EM has also made important advances,in particular through improved sample preservation using high-pressure freezing/freeze substitution [12]and the extension into 3D (three-dimensional)imaging using ET (electron tomography)[13,14].Consequently,correlative approaches that combine LM and EM are currently being developed in many laboratories [15,16].Cryo-EM tomography and AFM (atomic force microscopy)are starting to blur the boundary between imaging and structural biology,and are opening the door to a truly molecular understanding of the viral infection process.In the present review,we summarize some of these recent developments with a particular focus on the challenges and advantages that are speci?c to imaging plant virus infection.

ADVANCEMENT OF PROTEIN LOCALIZATION STUDIES

Since the ?rst use of GFP (green FP)as a reporter of virus infection in plant cells [7],the palette of auto?uorescent proteins available for localization studies has increased exponentially through the discovery of new natural source proteins and the engineering of new varieties.Generally,new FPs are selected to increase the number of spectral variants that can be separated in co-localization

experiments,and to overcome problems with photostability,brightness,oligomerization and fusion tolerance [17,18].For plant virus studies,however,brightness and photostability are often less important considerations than spectral properties because

Abbreviations used:3D,three-dimensional;AFM,atomic force microscopy;BiFC,bimolecular ?uorescence complementation;CFP ,cyan ?uorescent protein;CLEM,correlative light and electron microscopy;COP ,coatamer protein;CP ,capsid protein;DAB,diaminobenzidine;dsRNA,double-stranded RNA;EM,electron microscopy;ER,endoplasmic reticulum;ET,electron tomography;EYFP ,enhanced yellow ?uorescent protein;FlAsH,?uorescein arsenical helix binder;FLIM,?uorescence lifetime imaging;FLIP ,?uorescence loss in photobleaching;FP ,?uorescent protein;FPC,C-terminal fragment of split FP;FPN,N-terminal fragment of split FP;FRAP ,?uorescence recovery after photobleaching;FRET,?uorescence resonance energy transfer;GFP ,green ?uorescent protein;LM,light microscopy;MP ,movement protein;N,nucleocapsid;ORF ,open reading frame;PA,photoactivatable;PALM,photoactivation localization microscopy;pc-FP ,photoconvertible ?uorescent protein;PD,plasmodesma(ta);PSF ,point-spread function;PUMHD,Pumilio homology domain;PVX,potato virus X;Qdot,quantum-dot;ReAsH,resoru?n arsenical helix binder;RFP ,red ?uorescent protein;RNP ,ribonucleoprotein particle;ROI,region of interest;SIM,structured illumination microscopy;STED,stimulated emission depletion;STORM,stochastic optical reconstruction microscopy;TGB,triple gene block;TMV ,tobacco mosaic virus;VRC,viral replication centre;vRNA,viral RNA;YFP ,yellow ?uorescent protein.1

To whom correspondence should be addressed (email karl.oparka@https://www.doczj.com/doc/ea18242552.html,).

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transient expression from agrobacteria,particle bombardment or viral genomes is the most common experimental approach. As a result,researchers usually deal with problems related to overexpression rather than detectability or dimness,and FPs for plant virus studies can simply be chosen based on their spectral separation.Because virus-expressed FP constructs can accumulate at extremely high levels,sequential image acquisition is preferable for co-localizations,at least as a control.Several sets of expression vectors are now available that enable fast generation of multiple FP-fusions from a single clone of the gene of interest based on Invitrogen’s proprietary Gateway in vitro recombination system[19–31],and additional vectors using more recently developed FP variants are continuously being developed[30,31]. As knowledge of cell biology increases,so does the number of available?uorescent markers for subcellular compartments and transport pathways[32,33],and plant virologists are taking advantage of this to determine the location of viral proteins in detail,and to follow their traf?cking pathways throughout the cell.For instance,the6K protein of potyviruses,which functions to establish viral replication sites,was known previously to target the ER(endoplasmic reticulum)[34].Wei and Wang[35]have shown that the speci?c location of the6K protein is the ER exit site,where6K accumulation depends on both the COP(coatamer protein)I and COPII vesicle traf?cking machineries,and that the 6K-containing membrane compartments travel onwards through the secretory pathway to chloroplast membranes where they accumulate to form the main replication sites[36](Figures1a and 1b).In another study,Taliansky and co-workers used a variety of markers of subnuclear bodies to follow the path of the ORF3(open reading frame3)protein of the umbravirus groundnut rosette virus,as it enters the nucleus[37,38].Here it interacts with Cajal bodies,fuses them with the nucleolus and recruits the nucleolar RNA-binding protein?brillarin for export into the cytoplasm and incorporation into viral movement complexes(see Figure3c). Considerations speci?c to localizing viral proteins

As experiments designed to track the subcellular localizations of virus-encoded proteins become more sophisticated,careful interpretation of the results also becomes more critical.Previous reviews have covered such issues in a more general context[39–41],but for studies of viral proteins a number of additional considerations apply(Figure2).Three important factors are: (i)expression levels;(ii)potential interaction partners during infection(nucleic acids as well as proteins);and(iii)steric interference of the FP tag with protein localization and function. For endogenous plant proteins,the‘gold standard’control for all three criteria is to complement a knockout mutation of the gene of interest with the FP-fusion expressed from the gene’s native promoter.The equivalent to this for a virus-encoded protein would be to replace the original ORF with the FP-fused gene in the viral genome.However,this is often not possible.Viral genomes have been selected for extremely economic use of genomic capacity. Often ORFs overlap,or are expressed as polyproteins that are cleaved in tightly regulated processing pathways.The gene of interest may also overlap with(subgenomic)promoters or other regulatory sequence elements[42,43].Viral genome sizes are often limited by virion packaging mechanisms,constraining FP insertions,and in RNA viruses,high recombination rates can quickly eliminate insertions.With the additional potential for FP-fusions to disrupt the(multi)functionality of viral proteins,it is not surprising that approaches in which the FP-fusion functionally replaces the wild-type gene in the viral genome(e.g.[44,45])are the exception rather than the rule.Instead,various compromises have been used such as insertion under the control of alternative viral promoters(e.g.[7,34,46]),self-cleaving FP-fusions using a linker derived from the2A protein of foot and mouth disease virus to release suf?cient native viral protein for functional complementation[47]or expression from viral satellite genomes (e.g.[48]).Such viral expression strategies,together with agroin?ltration and particle bombardment,all comprise transient expression assays,which are convenient and produce fast results. They are also somewhat appropriate,as viral proteins in most cases are only‘transiently’expressed during a natural infection, and often to very high levels.Additionally,transient expression can be advantageous compared with transgenic plants stably ex-pressing viral proteins,as the latter are often resistant to infection. Where localization studies differ most signi?cantly between viral and plant proteins is the nature of the virus as a self-contained‘closed’system.Redundancy between functionally similar genes or lack of mutant phenotypes is not usually a problem in virus systems.On the other hand,expression of individual viral proteins can never be representative for the context of the actual infection and,by whatever means,localizations should be studied in infected tissue[49].Most likely,the viral protein of interest will be affected by interactions with other viral or infection-speci?c factors.If non-viral expression is the only option,35S-driven viral replicons can be a useful tool to spatially and temporally control co-introduction of the ?uorescent reporter with the virus.This can also facilitate studies of movement-de?cient or replication-attenuated viral mutants. However,some viral proteins may only function(and localize correctly)when expressed in cis.But the ability to compare localizations between uninfected and infected tissue provides an additional tool to dissect these interactions and functions,similar to genetic approaches using knockout mutations(Figure2). Although high transient expression levels are not problematic themselves,localizations should consider the timing and expression levels during infection.For example,plant virus MPs (movement proteins)are often expressed early and at low levels, whereas CPs(capsid proteins)are typically expressed later and at very high levels.Therefore prolonged overexpression of an MP–FP may need to be interpreted with more caution than a CP–FP fusion.After particle bombardment,FP?uorescence can often be observed after a few hours,and in agroin?ltration and viral expression systems,imaging may be possible as early as 1day after inoculation.In view of the fact that many viruses replicate and move intercellularly within less than1day[50–53],but may continue to accumulate progeny virions until the host cell’s resources are exhausted,the localization of a viral protein should be studied as early as possible as well as at later stages to observe changes in localization and function during the infection cycle.Within a viral lesion,the differences in localizations between cells at the leading edge(=early)and the centre(=late)of an infection site offer a valuable reference in this regard.Owing to the maturation time of FPs and requirements for replication or subgenomic RNA production,virus-expressed FP-fusions are generally unsuitable to study the earliest events during the infection process.Virus-independent transient expression can be a way to circumvent this problem,as the protein of interest can be present prior to infection,for instance by allowing a virus infection to enter an area already expressing the FP-fusion of interest.Again,the potential requirement for expression in cis needs to be kept in mind.

In transient experiments using non-native expression,relative ratios of potential interaction partners also have to be considered. For example,the overlapping ORFs of the second and third‘triple gene block’MPs of different viruses are expressed from a single subgenomic messenger RNA,with the downstream TGB3(triple gene block3)ORF translated by leaky ribosome scanning after

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Figure1Various applications of live-cell?uorescence microscopy suitable for plant virus imaging

I.Detailed localizations within the context of the host cell.(a)Potyviral6K replication protein induces ER-derived vesicles that co-localize with the ER-to-Golgi exit site marker Sar1(i,ii).A dominant-negative Sar1mutation abolishes6K vesicle formation(iv,v).At later stages,the6K-induced vesicles transfer to the chloroplast envelope(iii),the putative viral replication site.Transport depends on the actin cytoskeleton,shown by reduced plastid labelling after overexpression of a dominant-negative fragment of the motor protein myosin IX-K(vi)(reproduced from[35,36]with permission from American Society for Microbiology).II.Study of protein dynamics.(b)FRAP of PD-localized TMV MP shows that MP is delivered actively to PD:the metabolic inhibitor azide(lower panels)blocks?uorescence recovery(modi?ed from[83]with permission from John Wiley and Sons).(c)Use of the green-to-red photoswitching protein Kaede to localize translation sites.At time 0min,a patch of Kaede-labelled potassium channel Kv1.1in a neuronal dendrite(outlined)is converted into red.Within60min,newly synthesized green Kv1.1-Kaede has accumulated,which is again converted into red at time60min.Only green?uorescence is shown(modi?ed from[89].Reprinted with permission from AAAS).III.Detection of protein–protein interactions.(d)FLIM–FRET of tomato spotted wilt virus replication proteins.N protein co-localizes with both GP and Gn,but only interacts directly with GP,as shown by reduced CFP?uorescence lifetime(co-localization and FLIM–FRET images show different cells)(modi?ed from[189]c 2009,with permission from Elsevier).IV.Topology analysis of integral membrane proteins.(e)BiFC-based membrane

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24J.Tilsner and K.J.

Oparka

Figure 2Complementing approaches for studying the subcellular locations of plant viral FP-fusion proteins in the context of infection

initiation at the upstream TGB2ORF.This results in an excess of TGB2protein over TGB3[54].Recently,Lim et al.[48,55]have shown that the TGB2/TGB3ratio profoundly in?uences the TGB3-dependent localization of the TGB2protein of barley stripe mosaic virus.

Finally,the choice of host system needs to be https://www.doczj.com/doc/ea18242552.html,pared with animal virologists,researchers working on plant viruses are in the advantageous position of having both intact plants and isolated cells for infection,but both come with certain limitations.In some cases,for instance with fusion-intolerant viral proteins (often replicases)or speci?c RNAs (discussed below),immuno?uorescence is a better option than FP-fusions.However,to make intracellular epitopes accessible to antibody labelling,?xation and membrane permeabilization are usually insuf?cient in plant tissues due to the presence of the cell wall,requiring additional embedding and sectioning.The use of protoplasts can overcome this limitation and offer the additional bene?t of permitting synchronized infection of an entire culture,i.e.de?ned infection timing [56].Protoplasts are also more accessible for invasive delivery methods such as

membrane permeabilization or microinjection that may open new avenues in the investigation of early infection events (see below).However,even when prepared from fresh tissue rather than cell culture,they represent profoundly altered non-native host cells.Aditionally,many membranous organelles,which are important in viral processes such as replication and movement,are strongly perturbed by ?xation and embedding.For imaging,especially in vivo ,intact tissue should be the preferred choice,with protoplast systems reserved for more specialized experimental problems.

LABELLING FUSION-INTOLERANT VIRAL PROTEINS WITH SMALL FLUORESCENT TAGS

Besides expression level and context,the most important factor in?uencing the accuracy of ?uorescence-based localizations is the effect of the ?uorescent tag on the protein of interest.FPs may mask functional domains,interfere with interactions with other macromolecules,or have a stabilizing or destabilizing effect.Simply increasing protein size by adding a ～27kDa GFP moiety

topology analysis of potato mop-top virus TGB2protein.YFPN (yellow FPN)fragment fused to the N-or C-termini of TGB2results in BiFC with cytoplasmic YFPC (yellow FPC)(i,iii),whereas YFPC introduced into the hydrophilic loop of TGB2complements ER-luminal YFPN (ii),leading to the topology models on the right.(modi?ed from [62]with permission from John Wiley and Sons).(f )Redox-sensitive GFP-based topology analysis.roGFP is more strongly excited by 405nm illumination in an oxidizing environment such as the ER lumen.Fusion of roGFP to the ends of transmembrane proteins results in distinct 405/488ratios depending on the cellular compartment which the protein terminus is facing.Reproduced from [121]with permission from John Wiley and Sons.V.RNA imaging.(g )PUMHD-BiFC vRNA imaging.In the VRC of TMV (left),RNA is localized to punctate ‘hot spots’,whereas in the PVX VRC (right),it is arranged in circular ‘whorls’.Red ?uorescence from virally expressed RFPs was used as infection markers [105].Scale bars:(a ,panels i,ii,iv and v),12μm;(a ,panels iii and vi),8μm;(b ),(d )and (e ),5μm;(c ),2μm;(f )and (g ),10μm.

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may in?uence localization,for instance a protein’s ability to move through PD(plasmodesmata)[57]or nuclear pores[58].Some FP-fusions are unstable,and proteolytic processing may release a signi?cant pool of the free FP resulting in nucleo-cytoplasmic labelling[7,39,59].When this is suspected,Western blotting against GFP should be carried out to test for degradation of the fusion protein.If mistargeting is suspected,valuable information may still be gleaned from the data.For instance,a secondary or transient localization may only become apparent when the main targeting site of a protein of interest is disrupted.This is especially relevant in the case of multifunctional viral proteins.In most cases, the orientation of the FP-fusion protein will have the greatest effect on localization.In a recent study of the TGB MPs of grapevine rupestris stem pitting-associated foveavirus,Rebelo et al.[60] systematically investigated the effects of FP-fusions to each TGB protein on both N-and C-termini.Such a thorough approach may not always be necessary,but with the available repertory of Gateway fusion vectors it can be done relatively quickly.If only one fusion orientation is used,publications should clearly state which,and for what reason it was selected.On some occasions,the best approach may be to introduce the?uorescent tag internally at a boundary between different protein domains[61].Few reports using such strategies have been published in plant virology to date (e.g.[62]),probably because there is surprisingly little structural information available for most plant viral proteins.An approach for rapid production of internal FP-fusions based on the Gateway technology has been described[61].

Some of these dif?culties might be overcome if smaller,less dis-ruptive?uorescent tags were available,which would additionally help to limit the detrimental effects of introducing the tag into viral genomes.One novel FP that meets some of these requirements is iLOV[63],derived from the light-,oxygen-or voltage-sensing LOV2domain of the Arabidopsis thaliana photoreceptor phototropin2.With a size of～300bp and17kDa,iLOV is approx.1.5–2-fold smaller than GFP and has been demonstrated to outperform GFP in some virus-speci?c contexts[63].For example,TMV(tobacco mosaic virus)expressing unfused or MP-fused iLOV moved faster cell-to-cell and systemically than the corresponding GFP constructs,indicating advantages both in terms of genetic load and functionality of the MP-fusion.

If a protein-speci?c antibody is available,immuno?uorescence microscopy can make tagging altogether unnecessary,but it is limited to?xed samples.A potential compromise permitting live-cell imaging could be to use a small non-?uorescent tag that is rendered?uorescent only by addition of a speci?c interacting compound(probe).A variety of such tag–probe labelling systems have been developed(reviewed in[64–66]),but unfortunately the majority are cell-impermeant and therefore unsuitable for plant studies.Of those which permit intracellular labelling,the proprietary SNAP TM and CLIP TM tag systems based on O6-alkylguanin-DNA alkyltransferase(New England Biolabs; [67,68]),HaloTag TM(Promega;[69]),based on haloalkane dehalogenase,and LigandLink TM,based on Escherichia coli dihydrofolate reductase(ActiveMotif;[70]),all utilize the reaction between small enzymes and substrate analogues to link a?uorophore covalently to a protein of interest,but provide only a modest size advantage over GFP(27kDa)with tag molecular masses between18and33kDa.Only HaloTag TM has been tested in plants,with good tissue permeability[71].A tag based on the human immunophilin FKBP12(FK506-binding protein12) [72,73]adds only12kDa,but has not been tested in plants yet.The smallest available tags with cell-permeant?uorescent probes are a38-amino-acid peptide that binds Texas Red with picomolar af?nity[74],which also has not yet been tested in plants,and the tetracysteine tag/biarsenical ligand system [75,76].The optimized,tetracysteine-containing peptide tag is of similar size as an epitope tag(12amino acids[77]).The biarsenical probes FlAsH(?uorescein arsenical helix binder)and ReAsH(resoru?n arsenical helix binder;[78])are membrane-permeant non-?uorescent?uorescein derivatives that become ?uorescent only when covalently bound to the tetracysteine tag. This‘switch on’is an advantage over the Texas Red-binding tag,and particularly useful as it eliminates the washing out of unbound dye,which is time-consuming and a problem when the tissue needs to be studied rapidly.Despite its promise,the tetracysteine tag/FlAsH system has seen little use in plants so far [79].The biarsenical probes can cause cytotoxic effects[76]and some background staining may result from non-speci?c reactions with other cysteine-containing proteins[64,80].From our own experience,the greatest problem in plants may be insuf?cient permeability through(and non-speci?c labelling of)the cell wall (P.Boevink,N.M.Christensen and K.J.Oparka,unpublished work).Tag–probe labelling systems could be an ideal approach to localize fusion-sensitive viral proteins,but so far none is entirely suited to plant systems.

TRACKING PROTEIN DYNAMICS AND LOCALIZING TRANSLATION SITES

Many proteins are not static in the cell and some may take different pathways to the same subcellular localization. Therefore signi?cant functional information can be gained from analysing protein dynamics.With standard FP-based?uorescence microscopy,this is possible only to a limited extent,e.g.by following protein localization over a time course.A range of techniques has been developed that permit more detailed insights into protein transport pathways,mobility and also the site of their synthesis.Tracking approaches are particularly valuable in the study of multifunctional viral proteins during an infection cycle, while determining the site of viral protein translation may help to link viral replication and protein function.

FRAP(?uorescence recovery after photobleaching)and FLIP

(?uorescence loss in photobleaching)

One way to gain dynamic information from FP-fusion localization studies is to use photobleaching approaches[81,82].High-excitation light intensities‘overload’the energy absorbtion of ?uorescent molecules and destroy them permanently,leading to photobleaching.With the spatially restricted light intensities that can be achieved with lasers,it is possible to locally bleach a speci?c?uorophore within a cell in a selected ROI (region of interest).Because all cellular proteins,including FP-fusions,participate in dynamic interactions with their environment,the non-?uorescent bleached molecules from the ROI will subsequently mix with their still-?uorescent counterparts elsewhere in the cell.The kinetics of this process can be measured and used to draw conclusions about the mobility and transport pathways of FP-fusion proteins.In FRAP experiments,the return of?uorescence to the bleached area is monitored.If the FP-fusion protein is freely diffusive within the cell,?uorescence will return rapidly and with a simple kinetic based on cellular diffusion constants.If the protein has limited mobility,e.g.by interactions with other cellular macromolecules,?uorescence recovery will be slowed and will follow a more complicated kinetic.For instance, Goodin et al.[49]were able to distinguish two intranuclear pools of the N(nucleocapsid)protein of Sonchus yellow net rhabdovirus.One pool of N in the nucleoplasm showed fast FRAP kinetics and was probably present in a soluble form.The other pool

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was membrane-associated and showed very slow FRAP kinetics, indicating that the protein is immobilized.N interacts with another viral protein,P,but FRAP experiments on N/P-co-expressing uninfected cells showed similar kinetics to soluble N,indicating that viral factors other than P are responsible for immobilizing N on membranes.If the bleached pool of an FP-fusion is compartmentally isolated,no?uorescence recovery may occur at all.Goodin et al.[49]used this FRAP approach to show that the membranous intranuclear replication sites of Sonchus yellow net virus are continuous with the nuclear envelope.Both the replication sites and the inner nuclear envelope were labelled by a GFP-fusion of the human lamin B receptor,and photobleached replication sites quickly recovered their?uorescence,indicating redistribution from the connected cellular endomembranes. FRAP can also give information on the traf?cking pathway a FP takes to reach the bleached area.Wright et al.[83]combined FRAP of the PD-localized TMV30K MP with cellular inhibitors to analyse the intracellular route of the protein to PD.Microtubule-depolymerizing drugs had no effect on30K PD targeting to PD,whereas actin cytoskeleton inhibitors signi?cantly inhibited MP targeting(Figure1c).Also,high concentrations of brefeldin A suf?cient to distort the cortical tubular ER network and treatment with the general metabolic inhibitor sodium azide both signi?cantly reduced?uorescence recovery at PD,indicating that the30K protein is actively transported along the cortical ER/actin network.

In contrast with FRAP,the depletion of?uorescence from the surrounding non-bleached cell areas is monitored in FLIP experiments.Similar to FRAP,this allows conclusions to be drawn about protein-traf?cking pathways as well as connectivity between compartments.

Fluorescent highlighter proteins and tag–probe pulse–chase labelling

FRAP and FLIP are not suitable for continuous tracking of the same protein pool or for distinguishing newly synthesized from pre-existing protein.The latter is of particular interest to the study of virus infections because the location of viral protein synthesis is of considerable functional relevance.The native location of viral proteins in the course of infection may result from both their intrinsic properties and their site of synthesis.For instance,plant virus MPs must associate with the genome they are transporting, but so far none has been characterized as sequence-speci?c nucleic acid binders[4].In some cases,speci?c interactions between the MP and other viral proteins,such as CPs,may aid speci?city (e.g.[84]),but the spatial proximity of the MPs to progeny genomes,i.e.‘co-compartmentation’,may also play a role which could be achieved by synthesis and retention of the MP in the vicinity of the replication site.Clear identi?cation of a speci?c subpopulation of proteins in a cell requires differential labelling. This can be achieved using either FPs whose spectral properties can be changed in vivo,so-called photoconvertible or‘highlighter’FPs(pc-FPs;reviewed in[18,64,85,86]).Alternatively,tag–probe systems can be employed that permit pulse–chase labelling with differently coloured probes.Both approaches permit the selective highlighting of the most recently synthesized protein pool and can thus identify translation sites.The ability to change the colour of the?uorescently labelled protein locally in a subcellular ROI and track its movement through the cell is,however,limited to pc-FPs.

One group of pc-FPs,including PA(photoactivatable)-GFP and PA-mCherry,is switched irreversibly from a near-non-?uorescent dark state to green or red?uorescence respectively by strong UV illumination[18,64,85,86].Activatable pc-FPs permit selective tracking of subcellular molecule populations,but do not allow pulse–chase labelling.Additionally,they are dif?cult to detect prior to photoactivation,which therefore has to be done‘blindly’or using other cellular markers for orientation.A different group of pc-FPs are‘switched’on and off reversibly,and include the variants of Dronpa,Padron,KPF1(Kindling),rsFastlime and rsCherry,IrisFP and DsRed Timer(see reviews[18,64,85,86] for comparisons of spectral properties).These proteins permit repeated on/off switching(in some cases many cycles),and can be used for repetitive FRAP or tracking experiments within the same cell.They are also important for new subdiffraction-resolution microscopy approaches(discussed below).

The most relevant group of pc-FPs for pulse–chase labelling are true photoconverters,i.e.irreversibly colour-changing proteins. These include PS-CFP2(where CFP is cyan FP),which switches from cyan to green?uorescence,and numerous green-to-red switching pc-FPs:Kaede,KikGR(Kikume),EosFP variants and Dendra2;also KFP1(kindling FP1)can be green-into-red converted irreversibly(for details of spectral properties,see [18,64,85,86]).Additionally,it was recently discovered that many red FPs can be photoswitched,including Katushka,mKate and HcRed1(from red to green)and mOrange1and2(from red to far-red),making them suitable as highlighters[87].A conversion into cyan?uorescence has also been described for the YFPs(yellow ?uorescent proteins)EYFP(enhanced YFP),Venus and Citrine [87,88].

Colour-changing pc-FPs are ideally suited for both tracking and pulse–chase labelling experiments.The label is easily visible before photoconversion,and the colour change leaves both switched and unswitched protein pools visible simultaneously. Movement of selected particles,organelles etc.can thus be followed,whereas newly synthesized protein can be visualized by?rst(irreversibly)photoswitching the entire pc-FP population in a cell and then observing reappearance of the unswitched colour due to de novo translation.This approach has been used to identify localized translation sites of a potassium channel in neuronal dendritic appendages[89](Figure1d).Translation inhibitors such as cycloheximide and puromycin can be used for controls.Also, both FRAP and PA-GFP were used to show that the NS4B and NS5A proteins of hepatitis C virus remain associated with putative viral replication sites when expressed from the virus,but are motile in uninfected cells[90,91].One potential problem with the use of pc-FPs in plants may be that the experimental organism has to be kept in the light.Since natural daylight contains a UV component,this can lead to undesired photoswitching(some pc-FPs are also switched by visible wavelengths).A potential solution is to keep the plants in the dark for some time before microscopy.

Pre-existing and newly synthesized protein pools can also be pulse–chased using tag–probe labelling systems.The existing protein pool is?rst labelled with one?uorophore,then subsequent labelling with a different?uorophore selectively highlights only the most recently synthesized protein,identifying the translation sites.The tetracysteine tag/biarsenical ligand system has been used to localizeβ-actin translation sites in vivo with FlAsH/ReAsH pulse–chase labelling[92].Among the other tags which can be used intracellularly[64–66],the HaloTag TM[69], SNAP/CLIP-tag TM[67,68]and LigandLink TM[70]systems also offer different probe colours for pulse–chase labelling.However, a disadvantage over FlAsH/ReAsH is that these probes are permanently?uorescent.Non-?uorescent probes are available for blocking the pre-existing protein pool instead of changing the label colour,but the tissue still has to be washed to remove non-speci?c?https://www.doczj.com/doc/ea18242552.html,ng et al.[71]have reported washing

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Imaging plant virus infections27

times of between4and18h for HaloTag TM,the only one of these systems tested in plants.Unless the tissue is?xed,these times are too long for detecting translation sites.Lin et al.[93] recently introduced another alternative technique,TimeSTAMP, for selective labelling of translation sites.The protein of interest is tagged with an epitope tag via a linker that encodes both the NS3protease from hepatitis C virus and its NS4A/B cleavage site.Autoproteolysis constantly removes the tag until a speci?c membrane-permeant protease inhibitor is added.Any protein synthesized after addition of the inhibitor will remain labelled and can be localized by immuno?uorescence microscopy.The permeability of the inhibitor through plant tissue remains to be tested.

DETECTION OF PROTEIN–PROTEIN INTERACTIONS

Subcellular localization of a protein needs to be put into a spatial and functional context to permit conclusions about its role during infection.Co-localizations with other proteins including organelle markers can provide such a context.However,co-localizations do not provide proof of macromolecular interactions.The resolution limit of confocal microscopes in the focal plane is approx.200–250nm and even lower in the z-axis.A spherical volume with a diameter of200nm may contain up to140000GFP molecules that could be co-localized without any physical interaction[94]. Only in special situations can co-localization be a suf?cient indicator of protein interactions,for instance when one FP-fusion causes a clear redistribution of another,or when this is coupled to a functional phenotype.Therefore?uorescence microscopy applications have been developed to more speci?cally identify protein–protein interactions in vivo.

BiFC(bimolecular?uorescence complementation)

BiFC,reviewed in[39,94–96],is the reconstitution of a FP from two non-?uorescent fragments.BiFC can be used to detect protein–protein interactions because FP reconstitution depends on the FP halves being in close proximity long enough for refolding,a process thought to require up to several minutes.To detect interactions,the split-FP halves are translationally fused to two proteins of interest,whose interaction then facilitates BiFC,thereby‘switching on’?uorescence.In contrast with other protein fragment complementation approaches such as the yeast-two-hybrid system,BiFC not only allows direct microscopic visualization of the interaction but also provides additional information on the localization of the interaction within the cell.As for conventional FP-fusions,several sets of plant BiFC vectors compatible with restriction cloning[97–101]or Gateway technology[30,31,98,102]have been developed to facilitate rapid and easy experiments.

FPs are commonly split at either of two suitable splitting positions,between amino acids154and155or172and173 respectively(actual positions may vary slightly depending on the FP variant)[95].In both cases,the N-terminal fragment(FPN) is larger and contains the side chains forming the?uorophore. The smaller FPC(C-terminal fragment of split FP)is probably required to close theβ-barrel tertiary structure that protects the ?uorophore and allows its maturation.There do not seem to be any obvious bene?ts or disadvantages between the154/155 and172/173splitting positions and they are being used rather indiscriminately at this point.However,FPN172fragments can be complemented by FPC154counterparts,whereas FPN154 fragments cannot be complemented by FPN173halves[103], indicating that the refolded FP structure tolerates a small peptide ‘overlap’,but not a gap.This is of practical relevance as cross-complementation between different split-FP variants leads to the formation of FPs with intermediate spectral properties and thus allows simultaneous detection of different protein–protein interactions in the same cell[103].Multicolour BiFC can,for instance,be used to test two putative or competing interaction partners(fused to different FPN fragments)of the same bait protein(fused to an FPC).Cross-complementation competencies and resulting spectral properties of the hybrid FPs are reviewed in [95,103].Notably,GFP-derived FPC does not complement any of the other FP variants tested.Multicolour-BiFC has been used in plants,and suitable vector sets are available for both restriction-based[100,101]and Gateway[31,102]cloning,so this developing technique awaits exploitation by plant virologists.

Because of its relatively straighforward use,BiFC has quickly become the most popular technique to detect protein–protein interactions in vivo,and has been used in numerous plant virus studies.It is therefore worth being aware of the method’s complications and pitfalls,which are also reviewed in[94]and [95].A general drawback of BiFC is that the FP reconstitution is practically irreversible,making the technique unsuitable for monitoring dynamic interactions.This irreversibility potentially increases BiFC sensitivity,although it can also result in the accumulation of non-speci?c background?uorescence.For several experimental systems,no?uorescence was reconstituted when unfused FPN and FPC fragments were co-overexpressed. However,for some FPs,background?uorescence has been observed(e.g.[62,94]).Li et al.[104]have recently found that shortening the N-terminal fragment of YFP split at position154to YFP-N152eliminates non-speci?c background,albeit at the cost of reduced signal intensity.In any case,unfused split-FP fragments are not a suf?cient control in BiFC experiments,as the fusion to proteins of interest may in?uence their folding and stability so that non-speci?c?uorescence occurs for fused FP fragments even if no background was observed with unfused FPN+FPC[94,105]. Therefore fusions to known non-interacting proteins are a more adequate control.Additionally,the splitting position and the orientation of the split-FP-fusions may in?uence both undesired background signal and the detection of genuine interactions. The sterical constraints imposed on the FP reconstitution by fused interacting partners appears to be complex(see[94]). Sometimes one orientation will yield?uorescence,whereas another will not[62,97],and sometimes fusion orientations have no effect.Including information on splitting positions and fusion orientations in publications may help to improve the comparability between different experiments.Finally,both non-speci?c FP complementation and relative brightness of the BiFC signal will be increased by molecular crowding,i.e.if the FP-fusions are con?ned to a small subcellular compartment.Studies of such compartments require particularly rigorous controls,and the use of strong promoters is best avoided in BiFC experiments. However,even non-speci?c background?uorescence in BiFC can be used to obtain valuable information about the topology of integral membrane proteins(see below).

FRET(?uorescence resonance energy transfer)

FRET is the direct,nonradiative transmission of excitation energy from one?uorophore(FRET donor)to another(FRET acceptor), resulting in?uorescence of the acceptor after excitation of the donor.For this,the?uorophores have to come into very close proximity( 10nm),as FRET ef?ciency decreases with the sixth power of distance.This makes it ideally suited to study molecular interactions(reviewed in[39,94,106]).For this purpose,similar to BiFC,the FRET partners are linked to the macromolecules whose interaction is to be studied.In contrast with BiFC,which is based

c The Authors Journal compilation c 2010Biochemical Society

28J.Tilsner and K.J.Oparka

on the properties of a speci?c protein tertiary structure,FRET can occur between any kind of?uorophore,and is therefore much more versatile.For instance,it can be used to study interactions between proteins and other molecules such as nucleic acids [107].FRET can also occur intramolecularly between different ?uorophores attached to the same macromolecule,allowing microscopic study of conformation changes(e.g.[108]).Perhaps most importantly for in vivo applications,the interaction between the FRET?uorophores themselves remains transient so that no non-speci?c background signal can accumulate.However,non-speci?c FRET can result from random collisions of?uorophores, and this probability increases with?uorophore concentrations and decreasing experimental volume.FRET measurements therefore always require adequate controls.

In the most straightforward method for FRET detection, the donor is excited with an appropriate wavelength,and ?uorescence emission is then detected in the spectral window of the acceptor(‘sensitized emission’).For this approach, well-separated excitation and emission spectra respectively of donor and acceptor are desirable to avoid bleedthrough(donor ?uorescence in the acceptor channel or direct excitation of the acceptor by donor-‘speci?c’wavelengths).On the other hand, ef?cient FRET requires suf?cient overlap between the donor emission and acceptor excitation spectra.Controls that are good practice for FRET experiments of this type include measurement of FRET ef?ciency for the non-interacting(unfused)FRET pair (background level due to random collision),?uorescence intensity measurements of the individually expressed FP-fusions with both donor-and acceptor-speci?c?lters(to quantify bleedthrough)and ideally also a positive control,usually a direct fusion between donor and acceptor FPs.An alternative approach,which can also be used as an additional control in sensitized emission experiments,is acceptor photobleaching.Here,the quenching (reduction)of donor?uorescence by interaction with the FRET acceptor is measured.Selective photobleaching of the acceptor restores the full?uorescence intensity of the FRET donor (unquenching),allowing quanti?cation of FRET ef?ciency.For this,the spectral properties of the FRET pair need to permit selective bleaching of only the acceptor.All of these methods depend on the measurement of light intensities,i.e.photon counts, and are therefore dependent on?uorophore concentrations. However,reproducible,consistent levels of the same construct are dif?cult to achieve in independent in vivo experiments,leading to a certain level of inaccuracy in all intensity-based FRET measurements.

The most accurate method for FRET detection is FLIM (?uorescence lifetime imaging)of the FRET donor[109,110]. Energy transfer to the FRET acceptor shortens the average time that a donor molecule remains in the excited state,which for FPs is usually in the order of nanoseconds.Reduction of donor?uorescence lifetime is independent of?uorophore concentrations and depends only on the FRET pair characteristics and?uorophore distance.FLIM requires sophisticated special equipment:for‘time-domain’FLIM,where donor?uorescence decay is measured directly,a pulsed laser capable of producing bursts of excitation light suf?ciently shorter than the decay times is required,as well as similarly fast time-resolved photon detection.‘Frequency domain’FLIM measurements use an excitation laser that produces continuous light with a sinuosidally modulating intensity.Fluorescence lifetime is deduced from the phase-shift between the intensity curves of excitation and emission?uores-cence,again requiring specialized detection equipment.Because of these technical demands and prohibitive costs,FLIM–FRET, although considered the‘gold standard’for microscopic detection of protein–protein interactions,is not yet widely available.

FRET experiments are also dependent on the choice of FRET pair.Many FP pairs have been used for FRET,and a complete listing is beyond the scope of the present review(see [17,18,39,94,106,111]instead).The most commonly used FRET pair,CFP/YFP,has a number of problems.YFP has a small Stokes shift(distance between excitation and emission maxima), making it harder to avoid CFP bleedthrough.Additionally, EYFP can change into a CFP-like blue form in response to intense UV light during acceptor photobleaching,leading to overestimation of the FRET contribution to CFP unquenching [88].This effect has been disputed[112],but was recently also reported for the YFP variants Venus and Citrine[87].CFP is comparatively dim and its?uorescence decay curve follows a double exponential,complicating FLIM quanti?cation[113]. Brighter CFPs with single-exponential?uorescence decay are available[94,114].Recently,TagGFP/TagRFP(where RFP is red FP)has been promoted as an optimized FRET pair[115]. Importantly,in contrast with several other RFPs,TagRFP shows no photoconversion under strong UV illumination,avoiding potential artefacts during acceptor photobleaching[87].Also of note is a non-?uorescent YFP variant,REACh[116],which permits FLIM-and intensity-based FRET measurements with a GFP donor,free of any spectral bleedthrough.

Perhaps due to the technical sophistication required for FRET experiments,BiFC has so far proven the more popular method to study protein interactions in vivo among plant virologists. Recently,both approaches have been combined to study ternary protein interactions in planta[117].However,because of its reliance on transient molecular interactions and applicability to any type of?uorophore,FRET has tremendous potential for the functional study of plant virus replication,including protein–protein interactions within the replication site(e.g. [118]),protein–RNA interactions([107,119])and potentially even RNA secondary structure changes(discussed below).Ideally,both BiFC and FRET protein–protein interaction experiments should be veri?ed by genetic approaches such as yeast two-hybrid or split-ubiquitin complementation assays.

ANALYSIS OF PROTEIN MEMBRANE TOPOLOGY

Many plant viruses encode integral membrane proteins,including various MPs[4].So far,very few molecular structure data are available for these proteins.Of particular interest is information on the topology of viral transmembrane proteins,to gain insights into the position of functionally important groups with regard to the cellular surroundings and thus potential interaction partners.Integral membrane proteins may have one or several hydrophobic transmembrane helices and,depending on their mode of membrane insertion,the N-terminus can be directed towards the cytoplasm or towards the lumen of the ER (corresponding to the apoplast for plasma membrane proteins), also determining the orientation of other transmembrane domains and connecting loop regions.Membrane topology can be analysed by immuno?uorescence microscopy.A tag is fused to either end of the transmembrane protein(or internally)and its accessibility to antibody labelling at the cell surface(non-permeabilized)and intracellularly(membrane-permeabilized) is compared(e.g.[120]).However,this is unsuitable for intracellular endomembrane-resident proteins.Additionally,it requires?xation and permeabilization.Analogous biochemical approaches,https://www.doczj.com/doc/ea18242552.html,ing protease accessibility,require disruption of the cell.None of these approaches permits analyses in vivo. Two new live-cell imaging methods have been developed for analysis of membrane topology.Zamyatnin Jr et al.[62]used the

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Imaging plant virus infections29

non-speci?c?uorescence that occurs in BiFC assays:if one half of a split FP is fused to a transmembrane protein,the complementing split FP fragment then has to be localized to the same compartment for restoration of?uorescence.They demonstrated that a FPN fragment fused to either of the N-or C-termini of the potato mop-top virus TGB2protein,which has two transmembrane domains, reconstituted?uorescence only in combination with cytosolic FPC.On the other hand,an internal fusion of FPC within the loop region connecting the transmembrane helices led to BiFC only in combination with FPN targeted to the ER lumen.Accordingly, both N-and C-termini are located on the cytosolic face of the ER and the loop domain in the lumen(Figure1f).This approach could also be modi?ed for plasma membrane transmembrane proteins:only their cytoplasmic domains will enable BiFC with intracellular split FP fragments.The authors did?nd,however, that unfused cytoplasmic FPN was able to trap the smaller ER-targeted FPC in the cytoplasm,resulting in cytoplasmic ?uorescence.Therefore they used only the larger ER-targeted N-terminal YFP fragment to detect ER-luminal BiFC[62].

An alternative method uses the redox-sensitivity of a GFP variant together with redox differences in the cytoplasmic and ER-luminal compartments,and is therefore limited to endomembrane-resident transmembrane proteins(ReTA,redox-based topology analysis;[121]).In this approach,the transmembrane protein of interest is fused to the redox-sensitive GFP variant roGFP2 [122],which is strongly excited at405nm in its oxidized form, but only very weakly excited in the reduced state.The ratio of ?uorescence after405nm and488nm excitation can thus be used to determine the position of roGFP2in the cytoplasm(more reducing,low405/488nm excitation ratio)or the ER lumen(more oxidizing,high405/488nm excitation ratio).When fused to a membrane protein of interest,the405/488nm excitation ratio reveals the orientation of the fused protein domain in the ER or cytosol(Figure1g).These new tools,together with the analysis of protein–protein interactions in vivo will greatly facilitate our understanding of the membrane-associated protein complexes involved in plant virus infections.

RNA IMAGING

Since the advent of molecular cloning in the1970s,the functional investigation of viral genomes,including sequence determination, analysis of expression strategies and identi?cation of regulatory sequence elements[42,43],has been at the forefront of modern virology.The link between such data and the current wave of FP studies is the location of the viral genome in relation to its encoded proteins and host cell structures.Where,and in what cellular context,do the genetically characterized infection events actually take place?In situ hybridization at the light and electron microscopic level can provide some of this information,but is experimentally demanding and reveals only snapshots of dynamic processes.RNA localizations by in situ hybridization have often been carried out on protoplast systems due to better permeability and because some temporal resolution can be achieved by stopping synchronized infected cultures at de?ned timepoints (e.g.[123,124]).However,all RNA viruses replicate on cellular membranes which they reorganize extensively into a VRC(viral replication centre)[3,5]and the sensitivity of cellular membranes to standard aldehyde?xation protocols commonly employed in in situ hybridization is another reason for making in vivo RNA tracking a preferred choice.

Tools for sequence-speci?c live-cell RNA detection have been available since1998(reviewed in[125,126]).The?rst such system,based on the coat protein of bacteriophage MS2[127], and a more recent version using a22-amino-acid peptide from the N protein of bacteriophageλ(λN22)[128],employ fusion of

a sequence-speci?c RNA-binding protein to GFP.The MS2CP

andλN22peptides recognize RNA hairpins with which the RNA

of interest needs to be tagged.In order to separate unbound and

RNA-bound reporter,the GFP-fusion is usually targeted to the

nucleus.Cytoplasmic?uorescence then indicates redistribution

in response to RNA binding.The MS2system has been used

in numerous studies,and MS2andλN22together have enabled

simultaneous tracking of two RNAs in the same cell[129].Zhang

and Simon[130]tracked turnip crinkle virus infection using the

MS2system,but did not localize the viral RNA at the subcellular

level,although the technique is principally suited to this.Sambade

et al.[131]showed that a nuclear expressed,MS2-tagged RNA

encoding the TMV MP formed motile granules in tobacco leaf

epidermal cells which co-localized with MP at PD.A limitation

of the MS2andλN22systems for virus imaging is the introduc-

tion of additional sequences with extensive secondary structure

into the viral RNA,which may affect infectivity,recombination

rates or RNA localization[43,126,129].Additionally,multiple

tandem copies of the hairpin tag have to be introduced to obtain

suf?cient sensitivity(commonly6–24,but up to96repeats have

been used;[125]).Zhang and Simon[130]used only a single

MS2hairpin to tag turnip crinkle virus,which was suf?cient for

relocalization of MS2CP–GFP from the nucleus to the cytoplasm,

but this may permit subcellular RNA localization for very highly

accumulating viruses.

Because of these limitations,a novel approach based on

the RNA-binding domain of the translational repressor human

Pumilio1[132]held promise for virus studies.The PUMHD

(Pumilio homology domain)has a modular structure of eight

imperfect repeats that each bind one nucleotide with just three

amino acids per repeat involved in the protein–RNA interaction.

This makes it possible to alter the sequence speci?city of the

PUMHD in a de?ned way with relatively little genetic engineering

of the protein[133,134].Instead of tagging the viral RNA,the

detection system can be modi?ed to recognize the unaltered RNA.

Additionally,the PUMHD has a higher af?nity(K d=0.48nM) to its target sequence than either MS2CP(6.2nM)orλN22

(22nM;reviewed in[126]),and binding neither requires nor

introduces RNA secondary structure.Also,the need for nuclear

sequestration of the unbound reporter was avoided by instead

using BiFC between two PUMHD variants engineered to bind

to closely adjacent target sites on the same RNA and fused to

the two halves of a split FP[132].This system enabled detailed

mapping of the distribution of unencapsidated RNA of TMV and

PVX(potato virus X)in their respective VRCs(Figure1h),as

well as the co-introduction of an RNA reporter and a35S-driven

TMV replicon to study early vRNA(viral RNA)distribution

beginning～6h after co-bombardment[105].The perinuclear

VRC of TMV contained granular RNA‘hot spots’,whereas

that of PVX showed circular RNA‘whorls’arranged around

aggregates of the viral MP TGB1,indicating that these structures

show considerably different arrangements in different viruses.

The PUMHD variants were modi?ed to recognize unaltered TMV

genomic RNA,but subsequent tagging of the PVX genome with

the same TMV-derived sequences demonstrated that this short

(21nt)non-structured tag had no disruptive effect on the virus

[105].The PUMHD-BiFC reporter has also been used to localize

potyviral RNA to replication sites in membrane invaginations of

the chloroplast envelope[36].

More recent structural studies of the PUMHD protein have

revealed that its sequence speci?city is not as high as initially

believed[134].A certain degree of binding promiscuity is

probably balanced by the requirement of BiFC on binding of two

PUMHD fusions to generate a signal,but the BiFC approach

c The Authors Journal compilation c 2010Biochemical Society

30J.Tilsner and K.J.Oparka

is itself not free of background signal.Additionally,altering PUMHD speci?city often results in a marked reduction in af?nity [132,133]and thus sensitivity.PUMHD-BiFC has enabled the most precise in vivo localizations of viral RNA genomes so far, but further developments are probably required to provide plant virologists with a generally applicable live-cell RNA reporter. Extremely high sensitivity and signal-to-noise ratios can be achieved by methods that require invasive delivery of the RNA reporter into cells.In direct RNA labelling,the viral genome itself is rendered?uorescent by incorporation of?uorophore-coupled nucleotides during in vitro transcription and then microinjected[135].Although low-throughput,this approach overcomes the temporal limitations inherent in virus-expressed FP reporters,and permits insights into the very early stages of infection.Microinjected TMV RNA was found to attach to the ER/actin network immediately after entry in a cap-dependent manner[135],possibly in connection with the host translation machinery[136].Microinjection of directly labelled viral RNA encapsidated in?uorescence-conjugated CP allows the visualization of virus unencapsidation in vivo,also revealing the cellular context in which this infection step happens[135,137]. It is conceivable that such approaches could be expanded further,utilizing FRET to study changing macromolecular interactions during early infection events[119].For instance, FRET between an RNA-intercalated dye and a protein FP-fusion was recently used to detect protein–RNA interactions in vivo[107].Similarly,directly labelled viral RNA could be microinjected into cells expressing an FP-fused replication factor to study early interaction events.Alternatively,molecular beacons could be used.Molecular beacons are short synthetic oligonucleotides linked to?uorophores that are used for sequence-speci?c RNA detection in vivo and in vitro(reviewed in [125,126,138]).Molecular beacon signal is limited to the target-bound form because in the unbound state secondary-structure formation of the beacon brings a?uorophore and a quencher group in close contact,preventing?uorescence emission[139]. The beacons could be microinjected to detect viral RNAs,but another approch would be to hybridize them to the viral genome in vitro and then microinject beacon-labelled vRNA.The binding of the molecular beacon is sensitive to secondary structure of the target RNA[125,126,138,140].Therefore disapperance of the?uorescent signal could be used to monitor displacement of the molecular beacon by RNA secondary-structure rearrangements or protein binding in vivo.Protoplasts would probably be the easiest system for such experiments.

For some questions,no in vivo imaging techniques are currently available.For instance,double-stranded RNA replication intermediates are a particularly good indicator of replication sites.Currently they can only be visualized by immuno?uorescence microscopy with dsRNA(double-stranded RNA)-speci?c antibodies(e.g.[123,124]),although the use of preferentially dsRNA-binding cell-permeant dyes may provide an in vivo alternative[137].Newly synthesized RNA can be visualized by incorporation of bromo-UTP and subsequent detection with speci?c antibodies(e.g.[123,124]),an approach that is also suitable for DNA viruses,in which bromo-dUTP is used instead(e.g.[53]).With the available toolbox of RNA-imaging techniques,genetic and microscopical plant virus studies can now be linked at the subcellular level.

TRACKING THE COMPLETE VIRAL LIFE CYCLE IN FOUR DIMENSIONS

For some animal viruses,the complete viral lifecycle can now be followed microscopically(e.g.[52,141]).All viral gene products can be localized throughout infection either in living cells or cell cultures?xed at speci?c time points.Viral nucleic acids can be visualized and the interactions of viral components with host cell factors and among each other can be detected in vivo,individual viral genomes or particles can be tracked[142],and even transient and complex events such as virus entry[137,143],packaging [144]and exit/cell–cell-transfer[145]can be observed.For plant systems,the same tools and methods are also available,with certain imaging limitations due to auto?uorescence from the cell wall and chloroplasts[64].It can be expected that the spatial and temporal relationships between plant viruses and their host cells will soon be understood in similar detail.

The earliest events in the viral infection cycle are the most dif?cult to https://www.doczj.com/doc/ea18242552.html,ually,only a few viral genomes and a very low level of their encoded proteins are present in the host cell.Additionally,virus-expressed?uorescent reporters only become detectable after a few hours,due to a lag in expression and?uorophore maturation.One way to study early events,such as unencapsidation of virions and the initiation of translation,is to label viral components in vitro and then introduce them invasively,which may not differ all that much from the‘natural’delivery of plant viruses via mechanic wounds or insect feeding tubes.If unincorporated?uorescent molecules are removed after labelling,in vitro labelling yields extremely high signal/noise ratios.Also,larger pools of virions can be introduced than would be the case in a natural infection.This may lead to artefacts in some cases,but also overcomes some of the limitations resulting from low virus levels.Studies such as those of Christensen et al.[135],which investigated the initial localization and unencapsidation of microinjected CP-and RNA-labelled TMV virions could be expanded to RNA–protein and protein–protein interaction dynamics by FRET,as discussed above.Alternatively,transfection of entire double-labelled virus particles into protoplasts might be possible using electroporation, poly(ethylene glycol)precipitation or membrane-permeabilizing agents.

Very early infections events can also be studied indirectly. Cotton et al.[146]infected the same cells with two different potyviral genomes expressing either GFP-or RFP-fusions to the6K protein that establishes the replication complex on the ER.The replication complexes were either green or red,rarely both,leading the authors to conclude that each replication site is established from a single infectious genome and that the p6 protein remains associated with the same replication site.Not all viral systems permit double infections,but where this is possible,35S promoter-driven infectious clones are particularly suitable as different viruses can be co-introduced into the same cell with nearly100%co-transformation ef?ciency through particle bombardment[147].

Large imaging gaps are also still to?ll in two areas where plant and animal viruses show the greatest discrepancies.These are virus entry and exit,both between neighbouring plant cells and between the host plant and vector organisms.Entry into and exit from the plant are usually achieved mechanically,i.e. through perforation of the cell wall,whereas cell-to-cell transport within the host organism occurs through the plasmodesmal pores traversing the walls.By contrast,the majority of animal viruses are membrane-coated and enter and leave cells by membrane fusion and budding events.Virus movement through PD is probably an early infection event for many plant viruses[50,51] and happens before virus-expressed?uorescent reporters can be imaged.Microinjected?uorescently labelled TMV vRNA was not detected in neighbouring cells even in MP transgenic plants although the infection had spread,as shown by a virus-expressed FP[135].Traf?cked genomes were either recruited

c The Authors Journal compilation c 2010Biochemical Society

Imaging plant virus infections31

exclusively from the(unlabelled)pool of progeny genomes after

replication,or the amounts of?uorescence that were transported

across the cell–cell boundary were too small for detection.Other

studies have found cell–cell transport of viral RNA when MPs

were co-injected(e.g.[148]),possibly due to pre-formation of

movement complexes in vitro.Imaging of cell–cell transport

through PD is also hindered by dif?culties in imaging the wall-

embedded PD,which is prone to the risk of optical artefacts due to

auto?uorescence[64]and re?ection[149].Systemic transport in

the phloem is even more dif?cult to image due to the embedding

of the vasculature deep within the plant tissue.But perhaps

most challenging is imaging the release and uptake events that

occur from and into vector organisms,as direct visualization

would require bringing both plant and vector under the lens of

a microscope at the time of virus exchange.This remains an area

where the ingenuity of researchers is most strongly challenged. FROM CELLS TO MOLECULES:BRIDGING THE IMAGING GAPS Electron tomography

Similar to LM,EM has made signi?cant advances in the

last two decades[14],including expansion into3D imaging

through ET(reviewed in[13,14,16,150]).In ET,relatively thick

specimens(100–500nm)are tilted over typically+?65?in1–2?increments along an axis perpendicular to the electron beam

during acquisition of50–100images.This set of images allows

computational3D reconstruction with a z-resolution of2–10nm,

removing the limitation set by section thickness(～20–100nm)

of conventional EM.The digitized3D model can then be reduced

to only selected structure outlines of interest.ET can thus enable

the analysis of3D parameters such as volume,surface area or

connectivity of subcellular structures at unprecedented resolution.

V olumetric analysis of isolated macromolecular complexes by

cryo-ET even achieves sub-nanometer resolutions suf?cient for

macromolecular structure determination,which is particularly

valuable when crystallization is problematic as in the case of

membrane proteins[151].

Electron tomographic studies have yielded remarkable insights

into the structure of animal viral replication sites(e.g.?ock

house virus[152];Figure3a)and SARS(severe acute respiratory

syndrome)-coronavirus[153].The?ock house virus study

permitted calculations of replication site stoichiometry:virus-

induced invaginations of the outer mitochondrial envelope

membrane each contain approx.100molecules of the replicative

transmembrane protein A,as well as an average of three RNA

replication templates,and remain connected to the cytoplasm

by a10nm membrane channel wide enough to permit export

of progeny RNA[152].Such information is highly valuable

as it connects directly to recent biochemical insights into

how the oligomeric organization of viral RNA-dependent RNA

polymerase complexes assist in their overall function[154],which

may ultimately lead to the discovery of new antiviral drug targets.

ET has also been used to study animal virus entry[155,156],

uncoating[157],encapsidation[158]and exit events[159],and

cryo-ET has facilitated the study of complex,membranous virus

particles[160].Most recently,successive sample abrasion with

a focused ion beam has been used as an alternative to a tilt

series for gaining3D information in EM.This approach revealed

the connectivity of intracellular membraneous compartments

containing HIV particles with the plasma membrane[161].

For plant viruses,ET studies are still missing,although EM-

tomography has been very successfully applied to the study of

the plant endomembrane system and cytokinesis(e.g.[162,163]),

and was also used to study rice dwarf virus in its insect vector [164,165].It can be expected that plant viruses,also,will soon be imaged within their plant hosts by ET.

For highly resolving EM approaches,high-pressure freezing has become a standard technique that preserves sample structure much better than traditional aldehyde?xation protocols, especially in the case of membranous organelles[12].Under high pressure,samples up to a few hundred nanometers thick can be frozen suf?ciently fast to maintain cellular water in an amorphous(vitri?ed)state,preventing tissue damage by ice crystals.Frozen-hydrated samples can be sectioned (cryo-ultramicrotomy;reviewed in[13,14])and imaged directly (cryo-EM),with resolutions up to3–5nm on cellular samples, enough to identify known macromolecular complexes by their shape[166,167].However,unstained vitri?ed specimens provide only phase contrast to generate detail and are also extremely sensitive to the electron beam.This results in low signal/noise ratio and makes it very dif?cult to pre-select a ROI,so that tomograms sometimes have to be aquired‘blindly’[16].Therefore alternative or complementing approaches are required to increase contrast, and permit molecular identi?cation of cellular components. The cellular water in high-pressure frozen samples can be replaced with organic solvents and then embedding plastics without thawing the sample(freeze substitution).Freeze substitution permits the use of stains to increase contrast, and embedded samples are stabilized for further sectioning at room temperature as well as being less sensitive to the electron beam although mostly retaining their preservation[168]. Truly speci?c labelling of proteins in ET can be achieved by immunogold labelling and similar immunodetection of dsRNA [153]or of bromo-UTP-labelled newly synthesized RNA[152], is also possible.However,antibodies cannot be applied in organic solvents or on frozen samples.Therefore high-pressure frozen/freeze-substituted specimens commonly used for ET can only be treated after embedding and sectioning,effectively limiting immunogold labelling to the surface of the specimen. Conversely,pre-embedding labelling gives greater sensitivity and higher labelling levels[6],but requires chemical?xation.As a compromise,several approaches have been developed to combine ultrathin cryosectioning,which provides a better label/sample volume ratio,with immunolabelling.This requires thawing and therefore also chemical?xation at some stage.Fixation can be done prior to freezing(Tokuyasu method),after thawing or by thawing and rehydrating high-pressure frozen/freeze-substituted samples followed by re-freezing and cryosectioning (see[169]for comparison).All of these approaches lose some of the pristine sample preservation of direct cryo-EM/ET,and chemical?xation also generally inactivates a proportion of the accessible epitopes,preventing quantitative labelling[13,14].As a result,macromolecule-speci?c labelling techniques for EM/ET currently fall short of complementing the available spatial imaging precision.An EM-equivalent to FPs,i.e.a genetic tag than can be directly detected throughout an EM sample would be highly desirable[14].

CLEM(correlative light and electron microscopy)

and‘super-resolution’

An alternative,or even preferable,approach would be to combine the advantages of LM and EM(speci?c,quantitative protein labelling and high resolution respectively)by correlative imaging of the same sample(CLEM;reviewed in[15,16]).The promise of CLEM has increased even further with the recent development of various LM approaches that break the diffraction barrier, long viewed as a limit to resolution in LM[9–11,170].Because

c The Authors Journal compilation c 2010Biochemical Society

32J.Tilsner and K.J.

Oparka

Figure 3Bridging the imaging gaps

I.Extending EM to 3D by electron tomography.(a )FHV (?ock house virus)induces invaginations of the outer mitochondrial membrane,where it replicates (i).Tomographic 3D reconstruction of these invaginations (ii)shows them to be individual compartments,each of which is linked to the cytoplasm by a ～10nm channel (iii and iv).Using biochemical and structural information,the potential packing arrangement of the replicative viral protein A within the spherule can be modelled (v)(reproduced from [152]under the Creative Commons Attribution License).II.Correlative light ‘super-resolution’microscopy and EM.(b )Diffraction-limited TIRF (total internal re?ection ?uorescence)microscopy (i),‘super-resolution’PALM and transmission EM (iii)of the same cryosection showing mitochondria.Overlay of PALM and EM images (iv).Reprinted from [173]with permission from https://www.doczj.com/doc/ea18242552.html,bination of LM,EM and AFM (c )The ORF3protein of groundnut rosette virus localizes in cytoplasmic inclusions (i)that contain ?brillar material (ii).In EM cross-sections,the ?brillar material can be seen to consist of ring-like protein complexes encapsidating the viral RNA (inset in ii).Fluorescence microscopy of ORF3–GFP (iii)and anti-?brillarin immuno?uorescence (iv)shows that ORF3targets the nucleolus and leads to redistribution of nucleolar ?brillarin into the cytoplasmic inclusions (v).In vitro ,ORF3and ?brillarin are both necessary to encapsidate viral RNA (vi–ix),but can form ring-like complexes in the absence of RNA (viii).These are similar to the structures observed in infected tissue (ii,inset)and AFM reveals them to consist of alternating ORF3and ?brillarin subunits (x)(reprinted by permission from Macmillian Publishers Ltd:EMBO

Journal [37]c

2007,reproduced from [38]c 2007National Academy of Sciences,U.S.A.,reproduced from [187]with permission from American Society for Microbiology,reprinted from [188]c

2008with permission from Elsevier).c-RNP ,cytoplasmic RNP inclusion;DC,densely coated RNA;F,protein ?laments;N,nucleus;No,nucleolus;V,vacuole;XB,cytoplasmic inclusion.Scale bars:(a ,panels i and ii),100nm;(a ,panels iii and iv),25nm;(b ),1μm;(c ,panels i,iii,iv and v),5μm;(c ,panel ii),100nm;(c ,panel ii inset),25nm;(c ,panels vi,vii,viii and ix),100nm;(c ,insets in panels vi,vii,viii and ix),50nm;(c ,panel x),20nm.

light is subject to diffraction in all lens-based optical systems,a ?uorescent spot will appear as a PSF(point-spread function),i.e.enlarged.Two ?uorescent spots whose point spread functions overlap cannot be resolved in a conventional light microscope and according to the principle described by Ernst Abbe in 1873,

this limit to resolution is approximately half the wavelength

of the emitted light,～250nm in the focal plane (x –y )and ～500nm along the optical axis (z )for ?uorescence microscopy.A number of different approaches now achieve resolutions beyond the diffraction barrier and are collectively referred to

c The Authors Journal compilation c 2010Biochemical Society

Imaging plant virus infections33

as‘super-resolution’or‘far-?eld’microscopy or‘nanoscopy’. These include STED(stimulated emission depletion)microscopy, which increases x–y resolution to tens of nanometers by focusing a second laser beam on the excitation spot which is ring-shaped and of an appropriate wavelength to quench excited?uorophores, effectively restricting excitation to the dark centre of the quenching laser[171].SIM(structured illumination microscopy) accesses subdiffraction information by moving a diffraction grid over the sample and calculating additional spatial information from the different diffraction patterns resulting from different grid orientations[172].Both STED and SIM are suitable for in vivo imaging.PALM(photoactivation localization microscopy;[173]), and STORM(stochastic optical reconstruction microscopy)is the highest-resolving super-resolution technique[174],achieving up to5nm lateral resolution.Imaging of individual?uorophores without interference from overlapping point spread functions allows precise calculation of the position of the actual?uorescent spot within its PSF.In PALM/STORM,small numbers of photoswitchable?uorophores are activated for each image, localized and then bleached or switched off.This procedure is repeated until the population of?uorescent molecules has been exhausted and the collective image can be assembled from the localizations of individual?uorophores.Reversibly photoswitching pc-FPs such as Dronpa are particularly suited for this approach[175].The high resolution and the slow image acquisition make PALM suitable mostly for?xed specimens.In all these techniques,additional modi?cations are necessary to achieve super-resolution also along the light path in the axial dimension.Various approaches have been developed including the use of two opposing objectives(4Pi microscopy),z-dependent lateral distortion of the image or axial interference patterns,and z-resolutions of approx.50nm can be achieved(reviewed in [10,170]).In vivo4Pi microscopy has been correlated with EM to track transport of cargo molecules through single Golgi stacks and PALM has been combined with cryo-EM[173](Figure3b). The power of such correlative approaches is evident from the achievable labelling density:Betzig et al.[173]counted>5500?uorophores in a subset of mitochondria in a single PALM cryosection,whereas only10–20labels/section could be expected in a comparative immunogold experiment[86].

To image the same sample in CLEM,either live-cell imaging has to be carried out before?xation or the same?xed speci-men has to be used in both LM and EM.The?rst approach makes full use of the bene?ts of LM,and ideally would allow the cessation of cellular processes at a precise moment of interest.Cryosectioning is ideal for the preservation of both?uorophores and sample ultrastructure,but continuous maintenance of samples at vitreous temperatures(

Also suitable for direct LM/EM correlation are Qdot(quantum-dot)-labelled antibodies.Qdots are?uorescent nanocrystals which neither bleach[64]nor require molecular oxygen.Their spectral properties are de?ned by their size and they are large enough and suf?ciently electron-dense to be directly visible in EM[181].Use of Qdots for live-cell imaging has been hampered by their large size and incompatibility with biological molecules.However, CLEM may be the area where their full utility can be realized [15].It can be expected that the combined imaging power of super-resolution LM and EM will provide many new insights into previously inaccessible aspects of plant virus infections in the near future.

Combined EM and AFM

At the far end of the resolution scale,EM can also be combined with AFM,which can achieve true atomic resolution[182]. In AFM,a very?ne needle tip is used to scan the surface of the sample,either by touching it directly(contact mode) or by remaining at a small distance utilizing electromagnetic interactions(non-contact mode).Needle movements are measured with high accuracy and allow3D reconstruction of the sample surface.The most signi?cant contribution of AFM to plant virology so far has been in the study of isolated virions and viral RNPs(ribonucleoprotein particles).

Unlike EM,AFM does not require staining,which can obscure small features and is therefore useful for discovering assymetric structures of viral RNPs which can then be identi?ed by immuno-EM[183].Interestingly,assymetric binding of viral proteins to only one end of virions or viral ribonucleoprotein complexes has been found for a number of very different viruses(e.g.[84,183–186])and may represent a general principle by which virions functionally interact with host and/or vector organisms.AFM and immuno-EM were used to investigate the destablizing effect on the PVX virion of binding by the TGB1MP to its5 end [84,184].These experiments established that binding of TGB1 is one way to render the encapsidated viral RNA translation-competent and also identi?ed an in vitro-assembled complex of viral RNA partially encapsidated in CP,termed a‘single-tailed particle’as the potential transport form of this virus[84].Lim et al.[48]recently isolated viral RNPs from plants infected with a mutant form of barley stripe mosaic virus lacking the coat protein. The absence of CP prevents formation of true virions and these RNPs,which contained the TGB1MP and positive sense genomic and subgenomic ssRNAs,were assumed to be viral movement complexes.Low amounts of unindenti?ed high-molecular-mass protein components were also found,but the minor MPs TGB2 and3were not detectable,despite heterologous interactions between the TGBs[48].Combined AFM and immuno-EM imaging of the complexes might be a useful approach to elucidate the potential presence,identity and position in the RNP of minor protein components.Similar approaches could be adopted for other viruses that do not require their coat protein for local movement,e.g.TMV.

In the case of the recruitment of nucleolar?brillarin by the groundnut rosette virus ORF3protein[37,38],described above, combined EM and AFM showed that?brillarin forms part of the ring-like complexes that protect the viral RNA during

c The Authors Journal compilation c 2010Biochemical Society

34J.Tilsner and K.J.Oparka

long-distance transport[38,187,188].This was the?rst direct demonstration of a plant host protein participating in a viral movement complex(Figure3c).The study of viral RNPs by combined AFM and EM has provided some of the deepest insights into the elusive nature of putative plant viral movement complexes and will become even more valuable when more structural data on MPs become available.Collectively,the combined power of the individual and correlative imaging approaches now at the disposal of plant virologists hold the promise that we will be able to gain a truly molecular understanding of all stages of the viral infection cycle within the not-so-distant future. ACKNOWLEDGEMENTS

We apologize to all collegues whose work we were unable to cite due to space limitations. REFERENCES

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188Canetta,E.,Kim,S.H.,Kalinina,N.O.,Shaw,J.,Adya,A.K.,Gillespie,T.,Brown,J.W.S.

and Taliansky,M.(2008)A plant virus movement protein forms ringlike complexes with the major nucleolar protein,?brillarin,in vitro.J.Mol.Biol.376,932–937

189Ribeiro,D.,Borst,J.W.,Goldbach,R.and Kormelink,R.(2009)Tomato spotted wilt virus nucleocapsid protein interacts with both viral glycoproteins Gn and Gc in planta.

Virology383,121–130

Received12March2010/20May2010;accepted1June2010

Published on the Internet28July2010,doi:10.1042/BJ20100372

c The Authors Journal compilation c 2010Biochemical Society

世界十大运动鞋品牌排名 随着人们生活水平的提高,人们对于品牌的追求也上了一个档次;在大街上溜达一圈下来至少可以数出不下 5 种运动鞋品牌。很多人对于品牌都已经有了自己“最爱”!那么现在让我们来细数世界十大运动鞋品牌。 1.Nike 耐克 美国著名品牌 1972 年 NIKE 公司正式成立。其前身是由现任NIKE 总裁菲尔耐特以及比尔鲍尔曼教练投资的蓝带体育公司。 1973 年全美 2000 米到 10000米跑记录创造者佩里方庭成为第一个穿NIKE 运动鞋的田径运动员。 1978 年 NIKE 国际公司正式成立。 NIKE 鞋开始进入加拿大、澳大利亚、欧洲和南美等海外市场。 1979 年第一款运用 NIKE 专利气垫技术的 Thaiwind 跑步鞋诞生。第一条 NIKE 服装生产线开始上马。 1980 年 NIKE 进入中国 ,在北京设立了第一个NIKE 生产联络代表处。之后 ,NIKE 秉承“Local for Local 在哪”(里 ,为哪里的观念,不仅将先进技术引入中国 ,而且全心致力于本地人才、生产技术、销售观念的培 养 ,取之本地 ,用之本地 ,在中国取得了飞速进展。 1996 年正式在中国成立了全资子公司 NIKE( 苏州体育用品有限公司 ,总部设于上海 ,并在北京、广州设立分公司 (香港也于 2002 年 1 月作为分公司并入中国区。 2002 年 5 月 NIKE 开始在全国范围内举办NIKE 蝎斗 3 对 3 足球赛 ,数百支青少年球队在广州、上海、北京三地分别角逐14、16、18 岁三个级别的奖牌。这是NIKE 公司为中国青少年体育发展做出的又一盛举。

农田水利学课程考试试题及答案 姓名年级专业学号 一、名词解释（每小题2分共10分） 1.灌水率： 2.排涝模数： 3.平均排除法： 4.（排涝计算中的）设计内水位： 5.容泄区： 二．选择题（共10分） 1.灌溉设计标准是反映灌区效益达到某一水平的一个重要技术指标，一般以( )与( )表示？ A、灌溉设计保证率、抗旱天数。 B、水文年型、降水量。 C、设计灌溉用水量全部获得满足的年数、抗旱天数。 D、水源来水量、灌区需水量。 2.什么叫田间渠系的灌排相邻布置？（） A、灌溉渠道与排水沟道的规划布置。 B、田间各级渠道规划布置的形式。 C、田间灌排渠系并行相邻的布置形式。 D、田间灌排渠系交错的布置形式。

3.渠道的输水损失包括以下四个部分：（） A、干渠、支渠、斗渠及农渠等四级渠道的水量损失。 B、渠床土质、地下水埋深、渠道的工作制度及输水时间。 C、自由渗流、顶托渗流、渠床土质、与渠道的工作制度等。 D、渠道水面蒸发损失、渠床渗漏损失、闸门漏水与渠道退水等。 4.什么叫渠道水的利用系数？（） A、灌溉渠系的净流量与毛流量的比值。 B、某一级渠道的净流量与毛流量的比值。 C、田间实际灌入的有效水量与末级渠道的供水量之比。 D、实际灌入农田的有效水量和渠首引入的水量之比。 5.在渠道规划设计中，渠道最小流量有何作用？（） A、用以校核对下一级渠道的水位控制条件。 B、用以校核渠道不淤条件。 C、用以确定修建节制闸的位置。 D、用以校核对下一级渠道的水位控制条件和确定修建节制闸的位置，并按最小流量验算渠道不淤条件。 6.什么叫雨水径流集蓄灌溉工程？（） A、导引、收集雨水径流，并把它蓄存起来加以有效灌溉利用的工程技术措施。 B、田面、坡面、路面及屋面庭院等各类集水工程。

式智能背包系统”。在过去的数年间，Deuter的网架透气系统背包成为所有透气式背包所争相仿效的对 象。 2003年Deuter 继续开发新的产品。新的“气触式智能背包系统”使产品达到了极点。 Marmot 公司网址：https://www.doczj.com/doc/ea18242552.html, 20多年前，三个加州的大学生一起爬山时决定自己创建一个户外产品公司，这个公司就是Marmot。Marmot的产品普遍采用优质材料，所以它在户外用户中的口碑很好。其中，Marmot的雪地服装和羽绒睡袋深受玩家的推崇，同时，它们的高山冲顶帐也是深受登山爱好者推崇的。和Mountain Hardware一样目前属于北美销量最好的品牌 之一。 Mountain Hardware 公司网址： https://www.doczj.com/doc/ea18242552.html,/www/action/Intro 说到MHW，这家公司和TNF渊源不浅。MHW的发起人是一些原TNF的雇员，它的服装和帐篷设计师都是原TNF的主要设计人员。据说他们是因为不满TNF管理层偷工减料的政策而分道扬镳的。不知传闻是否属实，MHW的产品质量普遍要比TNF好的多。MHW在产品细节上设计的更合理，它的质量倍受用户拥戴，而且它在产品创新方面遥遥领先，已经成了户外服装的领头羊。 Patagonia 公司网址：https://www.doczj.com/doc/ea18242552.html, Patagonia主要生产户外服装，而这也是它的优势。它的服装裁剪上比其它户外产品更好一些，细节方面也很优秀。比如说，身材比较高的人很难找到合适的户外裤子的，像TNF等公司的裤子全都不够长，而Patagonia是少数几个公司提供这种特殊尺码的。另外，Patagonia的抓绒衣也是非常受欢迎的。 The North Face 公司网址：https://www.doczj.com/doc/ea18242552.html, TNF的产品比较结实耐用，一些经典产品也深受户外爱好者的欢迎。但是，自从TNF上市以后，公司的经理层更注重如何降低成本增加利润。该公司的产品定位已经从高档探险器材转到了大众用品上。近几年TNF在产品创新方面比较落后，它的一些产品技术含量降低。不过TNF最近更换了管理层，公司正严抓质量关。公司的网站也已全面更新。从最近发布的几款相当不错的产品看，公司正努力重

世界十大顶级运动品牌 世界十大顶级运动品牌 专题简介 什么是品牌中的品牌？哪些品牌能够称为顶级的运动品牌？答案就在这里——世界十大顶级运动品牌。第一名: (耐克Nike)——美国第一名当之无愧是运动霸主：耐克公司（NIKE）1972 年NIKE公司正式成立。其前身是由现任NIKE总裁菲尔.耐特以及比尔.鲍尔曼教练投资的蓝带体育公司。 总部位于美国俄勒冈州Beaverton的耐克公司是全球著名的体育用品制造商。该公司生产的体育用品包罗万象：服装, 鞋类,运动器材等等。2002财年,公司的营业收入达到了创纪录的49.8亿[[美元]],比2001财年增长2%。 耐克公司用自身骄人的业绩印证着其创始人比尔·鲍尔曼曾说过的一句话："只要你拥有身躯,你就是一名运动员。而只要世界上有运动员,耐克公司就会不断发展壮大。" NIKE这个名字，在西方人的眼光里很是吉利，易读易记，

很能叫得响.耐克商标象征着希腊胜利女神翅膀的羽毛，代表着速度，同时也代表着动感和轻柔。耐克公司的耐克商标，图案是个小钩子，造型简洁有力，急如闪电，一看就让人想到使用耐克体育用品后所产生的速度和爆发力。首次以 “耐克”命名的运动鞋，鞋底有方形凸粒以增强稳定性，鞋身的两旁有刀形的弯勾，象征女神的翅膀。 第二名(阿迪达斯Adidas)——德国adidas 中文作阿迪达斯，德国运动用品制造商，是Adidas AG的 成员公司。阿迪达斯以其创办人阿道夫·阿迪·达斯勒(Adolf Adi Dassler)命名，在1920年于接近纽伦堡的赫佐格奥拉赫（Herzogenaurach）开始生产鞋类产品。1949年8月18日以adidas AG名字登记。阿迪达斯的服装及运动鞋设计通常都可见到3条平行间条，在其标志上亦可见，3条间条是阿迪达斯的特色。 阿迪达斯原本由两兄弟共同开设，在分道扬镳后，阿道夫的哥哥鲁道夫·达斯勒（Rudolf Dassler）开设了敌

世界十大运动鞋品牌排 名精编版 MQS system office room 【MQS16H-TTMS2A-MQSS8Q8-MQSH16898】

世界十大运动鞋品牌排名 随着人们生活水平的提高,人们对于品牌的追求也上了一个档次;在大街上溜达一圈下来至少可以数出不下5种运动鞋品牌。很多人对于品牌都已经有了自己“最爱”!那么现在让我们来细数世界十大运动鞋品牌。 耐克 美国着名品牌 1972年NIKE公司正式成立。其前身是由现任NIKE总裁菲尔耐特以及比尔鲍尔曼教练投资的蓝带体育公司。 1973年全美2000米到10000米跑记录创造者佩里方庭成为第一个穿NIKE运动鞋的田径运动员。 1978年NIKE国际公司正式成立。NIKE鞋开始进入加拿大、澳大利亚、欧洲和南美等海外市场。1979年第一款运用NIKE专利气垫技术的Thaiwind跑步鞋诞生。第一条NIKE服装生产线开始上马。1980年NIKE进入中国,在北京设立了第一个NIKE生产联络代表处。之后,NIKE秉承“LocalforLocal”(在哪里,为哪里的观念,不仅将先进技术引入中国,而且全心致力于本地人才、生产技术、销售观念的培养,取之本地,用之本地,在中国取得了飞速进展。 1996年正式在中国成立了全资子公司NIKE(苏州体育用品有限公司,总部设于上海,并在北京、广州设立分公司(香港也于2002年1月作为分公司并入中国区。 2002年5月NIKE开始在全国范围内举办NIKE蝎斗3对3足球赛,数百支青少年球队在广州、上海、北京三地分别角逐14、16、18岁三个级别的奖牌。这是NIKE公司为中国青少年体育发展做出的又一盛举。 2002年7月NIKE特邀被冠以“放客博士”之称的NBA巨星文斯?卡特来京,卡特此行的目的是为了支持中国青少年篮球事业,传播放客文化。 2002年8月耐克将会赞助一批代表美国自由篮球文化的“街头炫技篮球少年”来中国,跟中国的同龄人切磋球技。

农田水利学课程考试试题及答案 姓名 年级 专业 学号 一、名词解释（每小题 2 分 共 10 分） 1. 灌水率： 2. 排涝模数： 3. 平均排除法： 4. （排涝计算中的）设计内水位： 5. 容泄区： 二．选择题（共 10 分） 1. 灌溉设计标准是反映灌区效益达到某一水平的一个重要技术指标，一般以 （ ）与 （ ）表示？ A 、灌溉设计保证率、抗旱天数。 B 、水文年型、降水量。 C 、设计灌溉用水量全部获得满足的年数、抗旱天数。 D 、水源来水量、灌区需水量。 2. 什么叫田间渠系的灌排相邻布置？ （ ） 3. 渠道的输水损失包括以下四个部分： （ ） A 、干渠、支渠、斗渠及农渠等四级渠道的水量损失。 B 、渠床土质、地下水埋深、渠道的工作制度及输水时间。 C 、自由渗流、顶托渗流、渠床土质、与渠道的工作制度等。 D 、渠道水面蒸发损失、渠床渗漏损失、闸门漏水与渠道退水等。 4. 什么叫渠道水的利用系数？ （ ） A 、灌溉渠系的净流量与毛流量的比值。 B 、某一级渠道的净流量与毛流量的比值。 C 、田间实际灌入的有效水量与末级渠道的供水量之比。 D 、实际灌入农田的有效水量和渠首引入的水量之比。 5. 在渠道规划设计中，渠道最小流量有何作用？ （ ） A 、用以校核对下一级渠道的水位控制条件。 A 、灌溉渠道与排水沟道的规划布置。 B 、田间各级渠道规划布置的形 式。

B、用以校核渠道不淤条件。 C、用以确定修建节制闸的位置。 D、用以校核对下一级渠道的水位控制条件和确定修建节制闸的位置，并按最小流量验算渠道不淤条件。 6.什么叫雨水径流集蓄灌溉工程？（） A 、导引、收集雨水径流，并把它蓄存起来加以有效灌溉利用的工程技术措施。 B、田面、坡面、路面及屋面庭院等各类集水工程。 C、各类形式的水窖、水窑窖等蓄水工程。 D、各类最为有效节水的灌溉方式。 7.集流面的处理主要包括哪三类方法？（） A 、采用混凝土、水泥土、三七灰土进行表面处理。 B、采用塑料薄膜、或塑膜复沥青、复草泥。 C、植被管理；地表处理；化学处理。 D、采用钠盐、硅有机树脂及粗石蜡等化学处理方法。 8.蓄水工程有哪几种主要的类型？（） A 、引水渠沟或管道、入水口、拦污栅、沉沙槽、蓄水设施以及放水装置等。 B、涝池、旱井、田间蓄水池、水窖、水窑窖等。 C、引水渠、蓄水窑洞与放水暗管与放水暗渠。 D 、沉沙池、进水管、水窖等。 9.什么叫续灌方式？（） A 、类似于自来水管道可随机用水的供水方式。 B、输配水管道进行输水、配水和灌水的方式。 C、是指上一级管道按预先划分好的轮灌组分组向下一级管道配水的方式。 D 、是指上一级管道向所有的下一级管道同时配水的方式。 10.什么叫集水效率？（） A 、降水特征（次降雨量、降雨强度）和集水面质地、坡度、前期含水量与集水面尺寸。 B、集水面的处理材料、集水面积、集流路径和汇流时间。 C、随降水强度的增大而提高。 D、某时段内或某次降雨集水面的集水量占同一时期内的降雨量的比值。 三、简答题（每题6分，共30分） 1.四种地表取水方式的使用条件

美国十大顶级保健品品牌大盘点 GNC 健安喜 全球第一维生素品牌:在全球 40多个国家拥有超过 5000个连锁专卖店,是全世界最大的健康与营养食品连锁专卖店之一。曾连续十四年被着名杂志 《 Entrepreneur Magazine》评为美国维生素及营养食品特许经营商第一位。在亚洲地区, GNC 在新加坡、菲律宾、日本、马来西亚、印尼、泰国、台湾、香港等地均设有连锁店。 Puritan’s Pride 普瑞登 全球知名垂直营销保健品牌:创立于 1960年美国纽约长岛, 至今已有超过 40年的历史, 是美国垂直整合的营养食品生产商、特许经营商、分销商的航母企业 ----NBTY Inc.旗下的公司,也是美国最大最全的保健品公司之一。该公司从事维生素、矿物质、草药及有关健康、美容、减肥等健康食品的开发、研制、生产,销售超过1000种的营养品。 Nature Made 自然制造 美国顶级品牌 Nature Made:美国医师推荐品牌,高品质的象征。该公司成立于1971年, 所有产品均经美国食品与药品管理局的优良制造认证(GMP ,是美国及全球客户值得信赖的高端品牌。 Neocell 全球知名胶原保健食品品牌:致力于抗衰老及肌肤护理类产品的研发和生产, 尤其专利胶原蛋白的研制,更是畅销海内外。公司于 1997年成立于加州 Newport Beach,但其产品线生产的胶原蛋白早于 1986年出现于市场上。 Neocell 注重提供最专业和最全面的胶原保健食品给当今的健康和美容市场,同时对于客户的反馈非常敏感,提供 100%的满意度。 Martek纽曼斯

全球知名 DHA 保健品牌: MARTEK DHA系列产品为纯植物性 DHA ,按照美国现行 GMP 规范生产,在充满维生素 E 的环境下生产,完全杜绝了其他同类产品的氧化问题。 Neuromins DHA系列产品是采用美国 MARTEK 公司的专利技术,未经任何基因工程处理,按照美国现行 GMP 规范生产的专为孕妇和婴幼儿设计的,目前唯一通过美国食品和药品管理局(FDA 的食品最高级别安全认证(GRAS 的植物型纯天然绿色 DHA 营养食品。 Amway安利 全球知名直销保健品牌:安利公司所生产及销售的产品达 400多项, 种类齐全、多样化, 为每个家庭创造洁净、舒适的环境,并为个人带来健康与美丽。品质优异、效能卓越是所有安利产品的共同特色, 由原料挑选、配方测试到包装设计, 每项产品都必须经过严格的检验筛选方能出厂; 而将环保意识适切注入于产品制造的过程中, 更代表了安利追求高品质的极致表现。 Nature's bounty 自然之宝 自然之宝是美国 NBTY 集团旗下的子公司。 30多年来,自然之宝始终致力于用纯天然的膳食补充剂产品帮助人们改善健康, 提高生活品质。如今, 自然之宝已经发展成为全球最大的膳食补充剂公司 NBTY 旗下最为知名的经典品牌,销售于超过100,000个美国主流零售连锁店和各类健康食品商店。 Rainbow Light 国顶级保健品牌: 1981年成立于加州 Santa Cruza , 创造了顶级的基于食物链的保健食品, 提供超强消化酶和提供能量。 Rainbow Light 以其 100%的纯天然特性和绝佳的配方系统而着名, 其中每日一片男性维生素、每日一片女性维生素、每日一片孕妇维生素更是被评比为全美 6000多种产品的前 1%。 Rainbow Light 所有产品纯度和质量都是非常优秀的,全部经过 USP 、 GMP 、 NNFA 的认证。 . Organic

农田水利学课程考试试题 姓名年级专业学号 一、名词解释（每小题2分共10分） 1、渠道设计流量： 2、灌溉水利用系数： 3、最小流量： 4、田间净流量:： 5、不冲流速： 二．单向选择题（共10分） 1.地下水临界深度是指? （） A、地下水埋藏深度。 B、在一定的自然条件和农业技术措施条件下，为了保证土壤不产生渍害，所要求保持的地下水最小埋深。 C、在一定的自然条件和农业技术措施条件下，为了保证土壤不产生盐碱化和作物不受盐害，所要求保持的地下水最小埋深。 D、在一定的自然条件和农业技术措施条件下，为了保证土壤不产生盐碱化和作物不受盐害，所要求保持的地下水最大埋深。 2.对于控制一定地下水位要求的农田排水系统，下列哪种说法是正确的？（） A、在同一排水沟深度的情况下，排水沟的间距愈大，地下水位下降速度愈快，在一定时间内地下水位的下降值愈大，在规定时间内地下水位的下降值也愈大。 B、在允许的时间内要求达到的地下水埋藏深度ΔH一定时，排水沟的间距愈大，需要的深度也愈大。 C、在允许的时间内要求达到的地下水埋藏深度ΔH一定时，排水沟的间距愈小，需要的深度也愈大。 D、在同一排水沟间距的情况下，排水沟的深度愈小，地下水位下降速度愈快，在一定时间内地下水位的下降值愈大，在规定时间内地下水位的下降值也愈大。

3.设计排涝标准时，需选择发生一定重现期的暴雨，一般选择标准是？（） A、1-5年。 B、5-10年。 C、10-15年。 D、15-20年。 4.对渍害最不敏感的作物是？（） A、小麦； B、玉米； C、高粱； D、水稻。 5.特别适宜防治土壤次生盐碱化的农田排水方式是？（） A、明沟排水； B、竖井排水； C、暗管排水； D、暗沟排水。 6.在进行排水沟设计时，用来校核排水沟的最小流速的设计流量是？（） A、排涝设计流量； B、排渍设计流量； C、日常排水设计流量； D、排涝模数。 7.农田长期渍水不会造成下列后果？ A、土壤的透气性很差。 B、土层都处于强烈的氧化状态。 C、利于硫化氢等硫化物的形成，对作物根系产生永久性伤害。 D、有机质矿化程度低，分解释放的有效养分少，不能满足作物生长的需要。 8.防治土壤盐碱化的水利技术不包括？（） A、明沟排水 B、井灌井排 C、灌水冲洗 D、放淤改良 9.什么叫计划用水？（） A、灌溉水量的分配方法。 B、就是按作物的需水要求与灌溉水源的供水情况，结合渠系工程状况，有计划地蓄水、引水、配水与灌水。 C、是指灌溉水在灌区各级渠系调配、管理的方式。 D、是指灌溉水通过各级渠道流入田间的方法。 10.灌区用水计划一般来说有哪四种主要类型？（） A、水源引水计划、渠系配水计划与田间的用水计划等。 B、水权集中、统筹兼顾、分级管理、均衡受益。 C、年度轮廓用水计划、某灌季全渠系用水计划、干支渠段用水计划及用水单位的用水计划。 D、上下结合、分级编制，统一调度、联合运用。

世界十大名牌运动鞋 1.Nike耐克 美国著名品牌 1972年NIKE公司正式成立。其前身是由现任NIKE总裁菲尔耐特以及比尔鲍尔曼教练投资的蓝带体育公司。 1973年全美2000米到100米跑记录创造者佩里方庭成为第一个穿NIKE运动鞋的田径运动员。 1978年NIKE国际公司正式成立。NIKE鞋开始进入加拿大、澳大利亚、欧洲和南美等海外市场。 1979年第一款运用NIKE专利气垫技术的Thaiwind跑步鞋诞生。第一条NIKE服装生产线开始上马。 1980年NIKE进入中国，在北京设立了第一个NIKE生产联络代表处。之后，NIKE秉承“Local for Local”(在哪里，为哪里)的观念，不仅将先进技术引入中国，而且全心致力于本地人才、生产技术、销售观念的培养，取之本地，用之本地，在中国取得了飞速进展。 1996年正式在中国成立了全资子公司NIKE(苏州)体育用品有限公司，总部设于上海，并在北京、广州设立分公司(香港也于 2002年1月作为分公司并入中国区)。 2.Adidas阿迪达斯 在运动用品的世界中，adidas一直代表着一种特別的地位象征，而这种象征有人称之为“胜利的三条线”。自1948年创立至今，adidas帮助过无数的运动选手缔造佳绩，成就了不少的丰功伟业。因此，adidas也可以说是集合了众人信赖及尊敬的最佳典范 “adidas”为德国人AdiDassler创办（现如今由上奇广告公司董事长——法国人路易斯狄经营）。本人不但是一位技术高超的制鞋家，同时也是一位喜好运

动的运动家，他的梦想就是“为运动家们设计制作出最合适的运动鞋”。在这个理念下，AdiDassler于1920年设计出第一双运动鞋，由于他不断的研发，使他所设计的运动鞋获得许多顶尖选手的喜爱，不仅在奥林匹克运动会中大放异彩，并从此在运动场上立下金牌口碑。在各界的肯定下，Adi Dassler于1948年创立了adidas品牌，并将他多年来制鞋经验中，得到利用鞋侧三条线能使运动鞋更契合运动员脚型的发现融入设计的新鞋中，于是adidas品牌第一双有三条线造型的运动鞋便在1949年呈现在世人面前。从此，人们便不断在运动场上见到“胜利的三条线”所创下的胜利画面，三条文也便成了adidas的标志今日，总部设在德国Herzogenaurach镇的adidas依然秉持AdiDassler完美制鞋的理念，不断的与世界级的顶尖运动家与教练交换心得与需求，经过一连串反覆的测试与考验，发展出符合人体工学的各项产品，不但能帮助各类专业运动家们提升运动表现、更能满足一般市场消费者对高品质运动商品的需求。近年來，adidas 不仅在设计上、功能上有新突破，代表性的三条线设计概念亦在流行趋势中掀起另一股风潮，席卷时下的年轻新世代形成流行新风格，带领全球运动商品迈向更多元化的远景 3.Reebok锐步 创始人： 科士达先生(英国) 成立年代：1895年 名字意义： 活泼的小羚羊(非洲羚羊) 商标： 跳跃中的小羚羊。 目前经营者： 由美国人费尔文经营。 现属国籍：

农田水利学计算题 1、某小型灌区作物单一为葡萄，某次灌水有600亩需灌水，灌水定额为25m 3/亩，灌区灌溉水利用系数为0.75，试计算该次灌水的净灌溉用水量和毛灌溉用水量。 解：W 毛=MA/η水=25*600/0.75=20000（m3） W 净=MA=25*600=15000（m3） 2、某灌区A =0.2万亩，A 蔬菜=0.16万亩，A 花卉=0.04万亩，m 蔬菜=20m 3/亩，m 花卉=15m 3/亩。求综合净灌水定额m 综及净灌溉用水量。 解：m 综=α1m 1+α2m 2=0.8*20+0.2*15=16+3=19（m3/亩） W=m 综*A=19*2000=38000（m3） 3、某小型提水灌区，作物均为果树，面积1000亩，用水高峰期最大灌水定额为25m3/亩，灌溉水利用系数为0.75，灌水延续4天，每天灌水20小时。试计算水泵设计流量。 解：Q 设= =25*1000/(3600*20*4*0.75)=0.12（m3/s ） 4、已知苏南某圩区，F=3.8Km2，其中旱地占20%，水田占80%。水田日耗水e=5mm/d ，水田滞蓄30mm ，旱地径流系数为0.6 。排涝标准采用1日暴雨200mm ，2天排除，水泵每天工作时间22小时。试求泵站设计排涝流量Q 和综合设计排涝模数q 。 解：R 水田=P-h 田蓄-eT=200-30-5*2=160（mm ） 水ηTt A m t T W Q k j j ij i i i 360036001∑==??=

R旱田=αP=0.6*200=120（mm） ∴Q=(R水田F水田+R旱田F旱田)/3.6Tt =(160*3.8*0.8+120*3.8*0.2)/3.6*2*22 =3.65(m3/s) ∴q =Q/F=3.65/3.8=0.96(m3/km2) 5、冬小麦播前土壤最大计划湿润层深度为0.6m，土壤平均孔隙率42.5%（占土壤体积百分比），土壤田间持水率为70%（孔隙百分比）。播前土壤含水率为45.0%（孔隙百分比）。计算冬小麦的播前灌水定额。解：M=667Hn(θmax-θ0)=667*0.6*0.425*(0.7-0.45) =42.5(m3/亩) 6、已知某渠系如图1-4-3所示，干、支渠采用续灌，设计灌水率q=0.78m3/（s·万亩），一支灌溉面积为2万亩，二支灌溉面积为2.4万亩，三支灌溉面积2.5万亩，支渠的灌溉水利用系数为0.82，干渠渠道水利用系数0.9。 【要求】 （1）计算各支渠设计流量； （2）计算干渠设计流量和灌区灌溉水利用系数。 解：（1）Q1=qA1/η支水=0.78*2/0.82=1.90(m3/s) Q2=qA2/η支水=0.78*2.4/0.82=2.28(m3/s) Q3=qA3/η支水=0.78*2.5/0.82=2.38(m3/s) （2）η水=η支水*η干=0.82*0.9=0.74 7、1）下图渠系干、支渠续灌

全世界顶级自行车品牌 1.Nicolai(尼古拉):德国，世界顶级山地车，号称脚踏车界的“劳斯莱斯”或者“悍马”，全世界铝合金后避震山地车架之王，一直致力于制造最顶级手工铝合金自行车，CNC机加工技术堪称行业巅峰，涂装艳丽强度超高性能卓越。 2.Specialized（闪电）：1974年由铁杆车友Mike Sinyard创立的美国单车品牌，堪称自行车界BMW。它的公路、山地系列做得尤其精湛。环法比赛用车。 3.Marmot（土拨鼠/旱獭）由美国高端自行车品牌CANNONDALE前任总裁等人联合创立，堪称自行车界的法拉利，是目前世界上生产运动型自行车最专业的自行车品牌之一，世界27.5寸/650B整车自行车始创者（2009年），该车型已成为全球主流产品。 4.Time（泰姆）:法国， LOOK创始人的女婿1987年创立的世界顶级品牌，它的公路车架系统、自锁系统的设计是世界一流水平，手工制作，拥有世界最高级的碳纤维车架，主销欧洲，有最领先的品牌碳纤维跑车车架和前叉之一。 4.Colnago（梅花）：诞生于1954年，意大利著名公路自行车厂家。C40是该厂的顶级型号，环意赛，世锦赛用车，梅花标志，以制造手工钢架而闻名。 5.BMC:瑞士，创立于1986年，主产山地车、公路车、计时赛用车，BMC与Easton 合作开发的纳米碳纤管材是闻名于自行车界的独特设计，创意、简洁的车架设计、精确的造工、及夺目的车花，车架轻盈采用最新的计算机模拟技术，碳纤车架是市场上最安全和最耐久的车架。 6.Trek（崔克）：美国著名品牌，赞助的Discover Channel车队曾连续七次赢得环法桂冠。目标是制造简单而且最好的自行车。 11.Cannondale：美国品牌，素以单臂著称，俗称“左撇子”，生产的公路自行车铝架CAAD9有"铝架之王"的美誉，并在自行车三大环赛中都有车队使用。12.Tyrell（泰勒）德国奢侈品牌，1912年创立，代表了自行车行业最高造车工艺，以经典纯手工打造为主，低调但设计高超性能卓越。 13.Pinarello（皮纳瑞罗）意大利 14.De Rosa（德罗莎）意大利，1953年，心型商标，手工打造极致公路车。16.Storck（斯道克）德国，1995年，超高强度重量比的车架特点，高科技运用与低调涂装。 17.Look（洛克）法国，顶级碳纤维车架。车架独特上管与后管72.5度角，爬坡省力。 10.Mongoose：美国的超级极限自行车品牌，中国BMX自行车国家队的赞助商18.Ellsworth（艾斯沃斯）美国三大顶级品牌之一，车架几乎全手工，已降速和全山地车著名。 19.SantaCruz（圣克鲁兹）美国三大顶级品牌之一，1993年创立，全山地车杰出代表。 7.Schwinn（施文）：美国知名品牌。旗下有以舒适著称的Sierra，质量上乘的Frontier，适合于公路骑行和旅游的Road等。 8.Huffy：美国山地车知名品牌之一，创立于1892年，他们生产的山地车舒适简单又坚固耐用，中高端入门级，非顶级车。 9.Marin (马林)：美国品牌，以山地车见长，rst的避震算是入门级。

河海大学2002年攻读硕士学位研究生入学考试试题 名称:农田水利学 一:名词解释（每小题4分） 1、作物需水量 2、喷灌强度 3、灌溉水利用系数 4、作物水分生产函数 5、排渍水位 6、排水承泄区 二、判断题（每小题2分，共20分） 1、土壤中的毛管水是不能被植物根系吸收利用的水分（） 2、灌水定额是灌区的单位面积在单位时间内的灌水量（） 3、水库取水方式适用于河道水位和流量都满足灌溉引水要求的情况（） 4、规划固定式喷灌系统时，支管轮灌方式是否合理对于管的设计流量有显著影响（） 5、设计灌溉渠道时，如果糙率系数取值偏小，就会使渠道断面偏小，从而影响渠道过水能力（） 6、田间排水沟的间距取决于土壤性质和排水要求，和排水沟的深度无关（） 7、排渍模数是排水渠单位面积的排渍流量（） 8、灌溉渠道实行轮灌的主要目的在于减少渠道渗漏损失（） 9、喷灌工程不适用于地面坡度变化复杂的农田使用（） 10、用平均排出法计算的排涝设计流量比可能出现的排涝设计流量偏大（） 三、问答题（每小题8分，共40分） 1、灌溉渠道的设计流量、加大流量、最小流量在渠道设计中各有什么用途？ 2、排水沟道系统的规划布置要考虑哪些原则？ 3、局部灌溉包括哪些类型？渠道衬砌有何优缺点？ 4、渠道的水量损失包括哪些方面？渠道衬砌有何优缺点？ 5、灌溉管道系统的工作制度包括哪些内容？各自的适用条件是什么？ 河海大学2001年攻读硕士学位入学考试试题 名称：农田水利学 一：名词解释（每小题三分） 1. 凋萎系数2、作物需水量3、灌溉设计保证率 4、灌溉制度5、日常水位6、排水承泻区7、排渍模数8、轮灌 二、判断题（每小题而分） 对以下概念，你认为正确的在括号内填“+”号，你认为错误的在括号内填“-”号。 1、土壤中的吸湿水是可以被作物根系吸收利用的水分（） 2、鉴定土壤水分对作物生长是否有效的主要标志是土壤含水量（） 3、制定作物灌溉制度的基本原理是水量平衡（） 4、从河道引水灌溉时，如果河道流量大于灌溉引水流量，但枯水期水位偏低，饮水不足，应修筑水库调节径流（） 5、设计灌溉渠道时，如果糙率系数取值偏小，就会失渠道断面过大而增加工程量（） 6、上层滞水是停留在包气带土壤中的重力水（） 7、地下水的流量、流速、水位等运动要素随时间而变化的运动叫做地下水非稳定流动（） 8、田间排水沟的间距取决于土壤性质和排水要求，与排水沟深度无关（） 9、制定旱作物灌溉制度是作物地下水利用量指的是地面以下土层的储水量（） 10、用平均排出法计算的排涝设计流量比可能出现的排涝设计流量偏大（） 三、问答题（每小题6分）

顶级运动品牌背后的故事 北京奥运掀起了运动热潮，时下很多外贸服装甚至时装都有运动化的趋势，如果各位多补 习一点运动品牌的小花絮，在跟顾客的沟通中或许会有不错的效果，毕竟现在的年轻人， 即使不爱运动也很少有不爱穿运动装的，所以请好好看看这些你所不知的运动品牌背后的 故事。 REEBOK reebok（锐步）的宣传攻势受挫后，reebok决定改头换面重打鼓另开张，推出其倡导“以 高科技生产超卓效能的产品”的概念。而它的新商标，就是为配合这个概念而度身订制，只是在原来reebok运动鞋两侧的两条带子和交加的基础上稍作改动，这便是现在大家经常见到的图形。而它原来带有英国米字旗的商标则仍保留成为其经典系列和公司的官方标志。 NIKE 一个再熟悉不过的美国品牌。1971年蓝带体育用品公司的创办人菲尔·奈特（philkinpht） 为了拓展其亚洲市场，改善公司的形象，决定为公司改名。老板提出以“六度空间”为名， 但被公司职员否定。最后老板便要求职员在规定期限之前提出一个更好的名字，否则就坚 持以“六度空间”为名，而这个期限只有12个钟头。全公司惟一的一个全职职员——杰夫·约翰逊，利用两地的时差，拖延3个钟头，挖空心思，绞尽脑汁地想，但是进展并不大。累 得打起了瞌睡，喜爱古希腊文学的杰夫在梦里遇到了古希腊传说中掌握胜利的女神nike， 梦境中女神给他带来了灵感，于是他提出以nike（耐克）作为蓝带公司的新名字，得到老 板的认可。1978年，公司销售额突破1亿美元以后，蓝带体育公司才正式更名为耐克公司，而这个名字，今天则已成为亿万资产的代名词。至于nike商标那个著名的“钩子”状图形， 是花35美元买来的设计——一个亚特兰大设计系学生的创作。现在人们所见到的钩状图形要比原来的细小了许多，但却表达着更强烈的速度感与兴奋感。 FILA 意大利罗马fila（斐乐）运动用品公司在1972年聘用了一位日本的设计师来为fila设计商标。于是以字母“f”为主要元素的商标问世了，并且大受斐乐意大利老板的赞赏。这个“f”字母极具创意，利用了美丽丰满的几何形图案，具有浓厚的艺术美感，与意大利悠久的艺术 氛围相吻合。因此直到今天，fila公司仍没有打算更换商标。 CHAMPION “冠军”——champion是一家首创运动服的厂商，它和美国运动事业结下了不解之缘。以c 与“匡威”等品牌一样，自从美国有运动比赛时便存在。c字为主的商标和红蓝白三色的经典设计流传已久，现在已无法追寻它的起源，但它仍会继续使用下去，因为它本身就是运动 的化身，是美国运动历史的一部分。 CONVERSE 目前为止converse（匡威）已有90多年的历史，已在人们心目中有固定的形象。但是在1989年又在原来的商标上作了一些调整与改变：把方框里的“星”放在字母“o”的中间，同

Life Fitness(力健) 全球设备品牌中，力健以其独特的跑步机设计而闻名，超前的设计思路与独特的防震系统，划出了跑步机的专业与应用的分界线，跑步机夸张的表盘和立柱设计是一种霸气的表现。力健跑步机主要分两个系列，一个是Elevation超越系列中的93T、95T、97T。另一个是Integrity悦动系列。力量产品力健主要有Pro2的方管系列，Signature卓越系列，Optima奥体系列和Circuit炫系列。 Technogym(泰诺健) 国际一线品牌中唯一的一个欧洲品牌，却有着不同凡响的发展速度，有氧与力量设备都同时进入顶级品牌中的品牌。除了品牌与质量外，技术上的创新也是成功的。健身俱乐部商用设备中，太空的力量系列主要是Selection 系列和Element + 系列，免维护Pure Strength 系列，单车Group Cycle，跑步机主要是Excite +系列 Precor(必确) 必确是上世纪80年代初开始生产健身设备的，从台阶器开始。但真正让必确闻名于世的是他们在95年推出的椭圆机。在全球椭圆机专利上，谁都不可否认是Precor 发明了它。Precor的创新和专业精神，也使它成为有氧设备的鼻祖之一，毫无疑问Precor是椭圆机的先驱。跑步机主要是TRM8系列和TRM9系列，椭圆机主要是EFX5系列和EFX8系列，还有AMT系列的体适机。 Star Trac(星驰) 力健是上世纪60年代末70年代初开始做健身器材的企业，从跑步机开始生产，后来逐步完善了整个产品链。跑步机主要是E系列和S系列，E系列为高端，可配置内置液晶屏，有真人教学功能，S系列低端，可接外挂电视。力量设备星驰有Impact(冲击)方管系列，Instinct(直觉)系列和其高端的Inspiration(灵感)系列。 Nautilus(诺德士) Nautilus旗下的StairMaster(班霸)及Schwinn(思汶)都是很有历史意义的品牌，StairMaster 在1983年推出了台阶器，在1984年推出了靠背式自行车(磁控车)，至今StairMaster依然是全球最专业的台阶器品牌。Schwinn(思汶)同样是行业中的最优秀品牌之一，1968 年Schwinn生产了全球第一台动感单车(十字星)，是动感单车的鼻祖。

世界知名运动鞋品牌标志大全 1.Nike耐克—美国著名品牌 取名叫耐克 (Nike) ，这是依照希腊胜利之神 (Greek goddess of victory) 的名字而取的。耐克 (Nike) 鞋的标识是“Swoosh” (意为“嗖的一声”)，是由(Portland State University) 的图形设计学生卡罗琳·戴维森 (Carolyn Davidson) 于1971年设计的。Swoosh 极为醒目、独特、有动感，也就是大家现在熟悉的 NIKE 的那个对勾形标志。 2.Adidas阿迪达斯—德国

在运动用品的世界中， adidas一直代表着一种特别的地位象征，而这种象征有人称之为“胜利的三条线”。自1948年创立至今，adidas帮助过无数的运动选手缔造佳绩，成就了不少的丰功伟业。因此，adidas也可以说是集合了众人信赖及尊敬的最佳典范 3.Reebok锐步—英国 成立年代：1895年 名字意义：活泼的小羚羊(非洲羚羊) 商标：跳跃中的小羚羊。

4.Puma彪马—德国 Herzogenaurach(荷索金劳勒市)，位于德国南部的巴伐利亚州，在十九世纪末，还是个默默无闻的小镇，主要的经济传统的制衣业，然而今天，因为是世界闻名的体育用品---PUMA“彪马”公司的总部所在地，而被受人们关注。 5.Converse匡威—美国 Converse是美国名牌.1908年，Mr.Marquis Mills Converse在美国马萨诸塞州创建了CONVERSE(匡威)公司，专门生产运动鞋。世界上第一双篮球鞋由CONVERSE 制作。 6. UMBRO茵宝——英国

第1xx灌溉用水量 一、名词解释 1.吸湿系数 吸湿水达到最大时的土壤含水率称为吸湿系数。 2.凋萎系数 植物开始发生永久凋萎时的土壤含水率，也称凋萎含水率或萎蔫点。 3.田间持水率 农田土壤某一深度内保持吸湿水、膜状水和毛管悬着水的最大含水量。 4.作物需水量 指生长在大面积上的无病虫害作物，土壤水分和肥力适宜时，在给定的生长环境中能取得高产潜力的条件下为满足植株蒸腾、土壤蒸发及组成植株体所需的水量。 5.灌溉制度 按作物需水要求和不同灌水方法制定的灌水次数、每次灌水的灌水时间和灌水定额以及灌溉定额的总称。 6.灌水定额 一次灌水在单位灌溉面积上的灌水量。 7.灌溉定额 各次灌水定额之和。 8.灌水率(灌水模数) 单位灌溉面积上的灌溉净流量q 净。

9.灌溉设计保证率 是指灌区灌溉用水量在多年期间能够得到充分满足的机率，一般以正常供水的年数或供水不破坏的年数占总年数的百分比表示。 二、简答 1.简述农田土壤水分的存在形式。 按农田土壤水分存在的三种基本形式为地面水、土壤水和地下水。其中土壤水是与作物生长关系最密切的水分存在形式，按形态可分为气态水、吸着水、毛管水和重力水。 2.土壤含水量的表示方法有哪几种？它们之间的换算关系怎样？ 主要有四种： 质量百分数，以水分质量占干土质量的百分数表示；体积百分数，以土壤水分体积占土壤体积的百分数表示，或以土壤水分体积占土壤孔隙体积的百分数表示；相对含水率，以土壤实际含水率占田间持水率的百分数表示；水层厚度，将某一土层所含的水量折算成水层厚度，以mm计。 3.简述土壤水的有效性。 土壤水按是否能被作物利用而划分为无效水、过剩水和有效水。其中无效水是指低于土壤吸着水（最大分子持水率）的水分，过剩水是指重力水，有效水是指重力水和无效水之间的毛管水。 4.xx旱灾、洪灾、涝灾和渍害？ 农田水分不足引起作物产量减少或绝收的灾害称为旱灾。洪灾主要是指河、湖泛滥开形成的灾害。涝灾是指旱田积水或水田淹水过深，导致农业减产的现象。渍害是指由于地下水位过高或土壤上层滞水，因而土壤过湿，影响作物生长发育，导致农作物头疼或失收的现象。 5.何谓凋萎系数和田间持水率？两者各有什么用途？凋萎系数是指植物开始发生永久凋萎时的土壤含水率，也称凋萎含水率或萎蔫点。田间持水率是指农

世界顶级运动品牌的发展与营销经历 众所周知，阿迪达斯是当今世界着名的体育品牌之一，与耐克、锐步等品牌占据了全球体育用品消费的主要市场份额。阿迪达斯从1920年创立以来（“ADIDAS”商标注册于1948年），既有过成功的辉煌，也有过失败的教训。这些商战中的起落，与其品牌的定位和发展策略有着密切的关系。审视阿迪达斯的发展历史和品牌经营过程中的得与失，对于我国方兴未艾的体育用品产业的发展，无疑具有重要的借鉴价值。从初创到世界体育用品一流品牌——? 扩张性的品牌策略频频奏效? 阿迪达斯公司初创时，虽然还只是一个作坊式的小企业，但其眼光已瞄准了世界大市场。所以，在公司发展早期，阿迪达斯就将产品技术创新作为开拓市场、提高品牌知名度的动力。“功能第一”，“给运动员最好的”是公司品牌发展的原则。阿迪达斯的创始人阿迪·达斯勒不但是位田径运动员和体育爱好者，也是位推崇工艺、品质和热衷于创新的企业家和发明家，阿迪达斯运动鞋制作工艺中的许多技术突破都是由他实现的，他先后共获得700项的专利。同时，阿迪·达斯勒也是世界运动鞋制作领域的开先河者。1920年，阿迪就发明了世界上第一双训练用运动鞋，在他领导下的阿迪达斯诞生了世界上第一双冰鞋和胶铸足球钉鞋。阿迪达斯研制的旋入型钉鞋是个非常革命性的创新，人们甚至认为它为德国足球队1954年获得世界杯立下了汗马功劳。? 阿迪达斯品牌扬名世界始于1936年在其本土德国柏林举行的奥运会上。此届奥运会前夕，阿迪找到极为希望夺冠的美国短跑运动员杰西·欧文斯，并向他保证钉鞋对其比赛肯定大有帮助，但当时被欧文斯拒绝了。于是阿迪又建议他可以在赛前训练中试穿。结果，使用效果使欧文斯如获至宝，并在正式比赛

第1章灌溉用水量 一、名词解释 1.吸湿系数 吸湿水达到最大时的土壤含水率称为吸湿系数。 2.凋萎系数 植物开始发生永久凋萎时的土壤含水率，也称凋萎含水率或萎蔫点。 3.田间持水率 农田土壤某一深度内保持吸湿水、膜状水和毛管悬着水的最大含水量。 4.作物需水量 指生长在大面积上的无病虫害作物，土壤水分和肥力适宜时，在给定的生长环境中能取得高产潜力的条件下为满足植株蒸腾、土壤蒸发及组成植株体所需的水量。 5.灌溉制度 按作物需水要求和不同灌水方法制定的灌水次数、每次灌水的灌水时间和灌水定额以及灌溉定额的总称。 6.灌水定额 一次灌水在单位灌溉面积上的灌水量。 7.灌溉定额 各次灌水定额之和。 8."灌水率(灌水模数) 单位灌溉面积上的灌溉净流量q 净。

9.灌溉设计保证率 是指灌区灌溉用水量在多年期间能够得到充分满足的机率，一般以正常供水的年数或供水不破坏的年数占总年数的百分比表示。 二、简答 1.简述农田土壤水分的存在形式。 按农田土壤水分存在的三种基本形式为地面水、土壤水和地下水。其中土壤水是与作物生长关系最密切的水分存在形式，按形态可分为气态水、吸着水、毛管水和重力水。 2.土壤含水量的表示方法有哪几种？它们之间的换算关系怎样？ 主要有四种： 质量百分数，以水分质量占干土质量的百分数表示；体积百分数，以土壤水分体积占土壤体积的百分数表示，或以土壤水分体积占土壤孔隙体积的百分数表示；相对含水率，以土壤实际含水率占田间持水率的百分数表示；水层厚度，将某一土层所含的水量折算成水层厚度，以mm计。 3.简述土壤水的有效性。 土壤水按是否能被作物利用而划分为无效水、过剩水和有效水。其中无效水是指低于土壤吸着水（最大分子持水率）的水分，过剩水是指重力水，有效水是指重力水和无效水之间的毛管水。 4.何谓旱灾、洪灾、涝灾和渍害？ 农田水分不足引起作物产量减少或绝收的灾害称为旱灾。洪灾主要是指河、湖泛滥开形成的灾害。涝灾是指旱田积水或水田淹水过深，导致农业减产的现象。渍害是指由于地下水位过高或土壤上层滞水，因而土壤过湿，影响作物生长发育，导致农作物头疼或失收的现象。 5."何谓凋萎系数和田间持水率？两者各有什么用途？凋萎系数是指植物开始发生永久凋萎时的土壤含水率，也称凋萎含水率或萎蔫点。田间持水率是指