Biochemical properties of the pathogenesis-related proteins from tobacco

This review describes the discovery and identification of the pathogenesis-related proteins (PRs) from tobacco. In crude leaf extracts the PRs are distinguished from the proteins in uninfected plants by their solubility at pH 3, resistance to a range of proteases, and mobility in polyacrylamide gels upon electrophoresis (PAGE) in non-denaturing conditions. PAGE has been used as a qualitative and semi-quantitative assay for PRs, and their migration in gels made from different acrylamide concentrations has been used to identify charge and size isomers and electrophoretically identical PRs in different tobacco cultivars. The subunit composition and molecular weight (mol. wt) of the four PRs identified first in Xanthi-nc were determined by SDS-PAGE; staining the gels has shown that these same four proteins in Samsun NN did not contain carbohydrate, lipid or nucleic acid, nor were they isozymic forms of twenty five enzymes known to increase in activity following infection with TMV. Evidence suggests that most of the PRs in Xanthi-nc and Samsun NN are extracellular.The purification of several PRs from Xanthi-nc, Samsun NN and other tobaccos is described, as well as their mol. wt, subunit and amino acid composition. PRs 1a, b and c consist of a single polypeptide and have similar mol. wt and amino acid compositions. Antisera prepared against purified Xanthi-nc b₁ protein have been used to determine serological relationships between PRs and form the basis of a very sensitive quantitative assay using ELISA. The regulation of synthesis of some PRs has been shown to involve translational control.     

Interaction between the helicase domain of the tobacco mosaic virus replicase and a tobacco arginine decarboxylase

In a yeast two-hybrid screening test for tobacco proteins that interact with TMV replicase using the helicase (H) domain as bait, a cDNA clone was selected that encodes a polyamine biosynthetic enzyme, arginine decarboxylase (ADC). In yeast cells, the C-terminal internal region of ADC interacted with the H domain. This observation was confirmed in vitro by far-Western blotting. Inhibition of the binding between the H domain and the IRnHEL (I region and N-terminus of helicase domain) region by ADC using a yeast three-hybrid assay suggested possible interference of the heterodimerization of 126K and 183K by ADC.The nucleotide sequence data of pADCF reported in this study is available in the DDBJ/EMBL/GenBank databases under accession number AB110952     

水杨酸和乙烯在烟草抗黑胫病中的作用

5mmol/L水杨酸或1g/L乙烯外源处理可诱导烟草对黑胫病的抗性，推迟植株死亡。不能积累水杨酸的转nahG基因植株和不表现乙烯应答反应的转etr1-1基因植株对黑胫病的抗性显著弱于野生型植株。组成性积累高水平水杨酸的转entC和pmsB基因植株对黑胫病的抗性明显强于野生型植株。这些结果说明水杨酸和乙烯增强烟草对黑胫病的抗性，减缓黑胫病的扩展。0．5mmol/L KCN连续浇灌植株4天或浸溃叶片2，4h均不能恢复转nahG基因植株丧失的对黑胫病的抗病性。此结果进一步支持关于KCN处理不能恢复转nahG基因植株丧失的对真菌病害抗病性的假说。     

Possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea

The possible involvement of salicylic acid in systemic acquired resistance ofCucumis sativus againstSphaerotheca fuliginea was studied. Cucumber plants were inoculated with tobacco necrosis virus on the cotyledons and the level of endogenous salicylic acid in the first true leaf was determined by gas chromatography. Salicylic acid increased continously from the second day after virus inoculation to the fifth day, when the same leaf was inoculated withSphaerotheca fuliginea. In healthy plants, the efficiency of exogenous salicylic acid in inducing resistance was assayed by applying aqueous solutions at different times beforeSphaerotheca fuliginea inoculation. To evaluate the level of induced resistance, the following parameters were examined by light microscopy: percentage of conidial germination, length of the hyphae derived from single conidia, number of haustoria, percentage of epidermal cells with lignified walls and of necrotic cells underlying fungal hyphae. In treated plants conidial germination was reduced, the total length of the hyphae was shorter, the number of haustoria was lower and the haustorium-containing epidermal cells had more frequently lignified walls. Moreover, an evident increase in callose deposition was observed leading to the formation of oversized papillae around the penetration pegs. These results indicate that the application of salicylic acid before inoculation withSphaerotheca fuliginea reduces the intensity of the infectious process and that salicylic acid is involved in the expression of systemic resistance in cucumber challenged by the biotrophic pathogenSphaerotheca fuliginea.     

Regulation of elicitin-induced ethylene production in suspension-cultured tobacco BY-2 cells

INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO₂. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

Increased activities of ribonuclease and protease after challenge in tobacco plants with induced systemic resistance

Inoculation of 3-4 lower leaves of tobacco with tobacco mosaic virus (TMV) increased ribonuclease (RNase) and protease activities in inoculated leaves. Little or no increase in the activities were apparent in upper noninoculated leaves prior to challenge. After challenge with TMV or Peronospora tabacina, RNase activities increased more rapidly in the upper leaves of induced plants as compared to those of noninduced plants. Protease activities in the leaves challenged with P. tabacina or TMV also increased after challenge, but little or no differences in the activities were apparent between induced and noninduced plants. The incubation of purified TMV with leaf extracts obtained from induced challenged, noninduced challenged and noninduced unchallenged plants prior to inoculation did not affect the number of local lesions formed on tobacco plants.     

Inhibitors of N-linked glycosylation induce systemic acquired resistance in cucumber

Localized treatment of cucumber (Cucumis sativus L. cv. Wisonsin) cotyledons with inhibitors of N-glycosylation such as tunicamycin or amphomycin resulted in systemic acquired resistance in the first leaf to the fungal pathogen Colletotrichum lagenarium. Resistance was maximal as early as 2 days after application and best results were observed when the inhibitor was used at 100 μ . The same treatment also induced salicylic acid accumulation as well as the expression of chitinase and a PR1-like protein. The systemic effect is not caused by the transport of tunicamycin, since tunicamycin was not detected in the leaves. Within 2 h after application tunicamycin inhibited N-glycosylation, but not protein synthesis as indicated by labelling experiments. The amount of large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase decreased after tunicamycin treatment and after pathogen inoculation and the expression of BiP, a protein localized in the endoplasmic reticulum was enhanced. The activation of defense reactions seems to be dependent and sensitive to N-linked glycosylation.     

Viruses and virus diseases in Dutch bulbous irises (Iris hollandica) in the Netherlands

The occurrence in Dutch bulbous irises (Iris hollandica) of two viruses — iris mild mosaic virus (IMMV) and iris severe mosaic virus (ISMV) — in association with two diseases — mosaic (mozaïek) and grey (grijs) — was reported so far. In the Netherlands, three virus diseases have been distinguished: mild mosaic (mozaïek), mild yellow mosaic (bont), and severe mosaic (grijs). These diseases were associated with IMMV (750 nm), IMMV plus iris mild yellow mosaic virus (IMYMV, a newly recognized virus; 660 nm), and IMMV plus ISMV (750 nm), respectively. The viruses are antigenically distinct and their presence could be established serologically. Tobacco mosaic virus (TMV), tobacco rattle virus (TRV), and tobacco ringspot virus (TRSV) were also detected in irises, but not in association with particular symptoms.Generally, the symptoms of the diseases can be distinguished early in the growing season, particularly in March. Later on, the distinctive symptoms mostly disappear on plants showing mild symptoms but not on severely affected plants. Growing and forcing conditions influence the symptoms. The IMYMV and the ISMV transmitted in May and early in June byMacrosiphum euphorbiae cause more severe symptoms than those induced by transmissions late in June and in July. Problems related to disease control in irises are discussed.Samenvatting Het virusonderzoek bij Hollandse irissen (Iris hollandica) in Nederland leidde tot het onderscheiden van drie ziekten, namelijk: het mozaïek (mild mosaic), het bont (mild yellow mosaic) en het grijs (severe mosaic). Het voorkomen van iris-mozaïek virus (deeltjeslengte 750 nm), iris-mozaïek virus plus iris-bontvirus (660 nm) en iris-mozaïekvirus plus iris-grijsvirus (750 nm), welke virussen serologisch zijn te onderscheiden, werd in verband gebracht met respectievelijk het mozaïek, het bont en het grijs. Geen verband werd gevonden tussen het voorkomen van tabaksmozaïekvirus, tabaksratelvirus en tabakskringvlekkenvirus en de genoemde ziekten.Op basis van de symptomen zijn de ziekten te velde vroeg in het voorjaar meestal wel te onderscheiden. Later in het groeiseizoen verdwijnt dit onderscheid veelal bij planten met milde symptomen, maar niet bij planten met ernstige symptomen. De symptomen van het mozaïek worden pas een paar maanden na de opkomst van de planten zichtbaar. De licht- en donkergroene mozaïeksymptomen doen zich duidelijk voor omstreeks de bloei. Het bont is bij opkomst te herkennen aan het geelgroene mozaïek, dat voornamelijk aan de bladranden voorkomt (Fig. 1). Na de lengtegroei van de planten worden de symptomen op de bloemscheden en op de brede bladgedeelten zichtbaar (Fig. 2). Het grijs (Fig. 2) uit zich met brede geel-en donkergroene strepen op de bladeren, die tot onder het grondniveau doorlopen en bij opkomst duidelijk zichtbaar zijn. Het geelgroene, soms streepvormige mozaïek blijft bij de lengtegroei van de planten duidelijk zichtbaar op de bladbases. Ernstige grijs-symptomen zijn bloembreking, gedraaide stand van smallere bladeren en dwerggroei van de planten. De duidelijkheid van de ziektebeelden, zowel van het mozaïek als van het bont en het grijs, is afhankelijk van de cultivar en van de teeltomstandigheden te velde en in de kas.Het iris-bontvirus en het iris-grijsvirus geven bij overdracht door de bladluis (Macrosiphum euphorbiae) in mei en in de eerste helft van juni in het volgende groeiseizoen ernstiger aangetaste planten dan bij latere overdracht.De mogelijkheden van de herkenning en de bestrijding van virusaantastingen in iriissen worden beschreven.     

Harpin-elicited hypersensitive cell death and pathogen resistance require the NDR1 and EDS1 genes

Plants sprayed with harpin, a bacterial protein that induces hypersensitive cell death (HCD), develop systemic acquired resistance (SAR) without macroscopic necrosis. HCD sometimes accompanies the development of resistance conferred by resistance (R) genes. In Arabidopsis, some R genes require one or both of the signalling components NDR1 and EDS1 for function. This study addresses whether HCD, NDR1 and EDS1 are required for induction of SAR by harpin. When Arabidopsis and tobacco leaves were sprayed with harpin, microscopic hypersensitive response (micro-HR) lesions developed. Systemic expression of PR genes and the development of resistance were accompanied by micro-HR, except in the ndr1-1 mutant, in which harpin induced micro-HR without the development of resistance or expression of the PR-1 gene. Cell death and resistance did not occur following treatment with harpin in plants that could not accumulate salicylic acid. Harpin also failed to induce resistance in Arabidopsis eds1-1 mutants. Therefore, harpin-induced resistance seems to develop concomitantly with cell death and resistance requires NDR1 and EDS1.     

超敏蛋白对烟草黄瓜花叶病毒的诱导抗性及生长、品质的影响

为明确超敏蛋白对烟草黄瓜花叶病毒(Cucumber mosaic virus,CMV)的诱导抗性及其生长、品质的影响,采用半叶枯斑法和田间试验研究了10%超敏蛋白对枯斑三生烟和普通烟的诱导抗性及促生作用,并分析了对烤烟经济性状和内在质量的影响。结果表明,10%超敏蛋白在活体外对CMV无钝化作用,但预防、治疗效果显著,接种前48 h喷施预防效果达75.70%,接种后24 h喷施治疗效果达65.45%,均显著高于对照;10%超敏蛋白处理后烟草的株高、茎围、最大叶面积均高于其它处理,单位产量比20%吗啉胍·乙铜可湿性粉剂处理增产211.8 kg/hm2,增值3 519.2元/hm2;经10%超敏蛋白处理后的烤烟化学成分均处于优质烟的适宜质量分数范围内。表明超敏蛋白不仅有利于提高烟草抗性和促进烟株生长,且可以明显提高烟叶经济效益和烟叶内在质量。     

Degree of RNA silencing and the ability of a viral suppressor vary depending on the cell species in a protoplast system

RNA silencing is a sequence-specific defense mechanism against viruses. As a counterdefense, viruses evolved silencing suppressors to interfere with host silencing. In analyses using protoplasts prepared from cultured cells (BY-2) and mesophyll cells of Nicotiana tabacum and N. benthamiana, viral suppressors differentially functioned in different cell types. This phenomenon has not been discussed in earlier papers on protoplast systems and RNA silencing. In investigations of the cellular activities of viral suppressors and their role in the RNA-silencing pathway, assays with host protoplasts offer many advantages and can complement other in planta assays such as Agrobacterium-mediated transient expression.     

Host-parasite relationships in tobacco plants infected with a root-knot nematode (<Emphasis Type="Italic">Meloidogyne incognita</Emphasis>) population from the Azores

During a nematode survey, severe infections of tobacco feeder roots and heavy soil infestations byMeloidogyne incognita race 1 were found in S. Miguel (Azores islands, Portugal). This is the first record ofM. incognita infection of tobacco in Azores. Morphology of various life stages, analysis of the esterase electrophoretic pattern and differential host tests were used for nematode characterization and identification. Nematode-induced mature galls were spherical and/or ellipsoidal and usually contained more than one female, males and egg masses with eggs. Feeding sites were characterized by the development of giant cells that contained granular cytoplasm and many hypertrophied nuclei. Giant cell cytoplasm was aggregated along a thickened cell wall. Vascular tissues within galls appeared disorganized. The relationship between the initial nematode population density and growth of tobacco plants was tested in a glasshouse experiment in which inoculum levels varied from 0 to 512 eggs and juveniles (J₂) cm⁻³ of soil. Seinhorst’s model was fitted to height and top fresh weight data of the inoculated and control plants. Tolerance limits with respect to plant height and fresh top weight of tobacco cv. ‘Erzegovina’ plants toM. incognita race 1 were estimated as 1.25 eggs and J₂ cm⁻³ of soil. The maximum nematode reproduction rate was 404.7 at an initial population density of 4 eggs and J₂ cm⁻³ of soil. http://www.phytoparasitica.org posting March 2, 2004.     

壳寡糖季铵盐衍生物纳米银的合成及其诱导烟株抗烟草花叶病毒活性

为开发符合高效、低毒且环境友好的新型烟草花叶病毒(TMV)抑制剂,以具有季铵盐结构的新型壳寡糖季铵盐衍生物为还原剂和稳定剂,合成了壳寡糖季铵盐衍生物纳米银溶液,并研究了不同浓度该纳米银溶液诱导烟株产生抗TMV的活性。结果表明:所合成的壳寡糖季铵盐衍生物纳米银溶液在透射电镜(TEM)下显示分布较均匀,粒径主要集中在7~12 nm。在珊西烟上以25μg/m L的壳寡糖季铵盐衍生物纳米银溶液对枯斑的抑制效果最好,抑制率为74.0%,比50μg/m L壳寡糖溶液和2%宁南霉素水剂分别高41.5%和24.4%;对于普通烟K326,壳寡糖季铵盐衍生物纳米银溶液可缓解被病毒侵染的烟草叶绿素含量的下降幅度,提高叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(PPO)的活性,降低丙二醛(MDA)的含量,提高可溶性蛋白含量。表明壳寡糖季铵盐纳米银溶液可提高烟株对TMV的抗病性。     

A Survey of Viruses of Alstroemeria in the UK and the Characterisation of Carlaviruses Infecting Alstroemeria

Alstroemeria samples collected in the UK were tested for a range of viruses using ELISA. Alstroemeria mosaic virus (AlMV), alstroemeria carlavirus (AlCV), lily symptomless virus (LSV), cucumber mosaic virus (CMV) and tobacco rattle virus (TRV) were detected either singly or in combination in 67.5% of 203 samples. AlCV and LSV isolates from Alstroemeria and lily were studied and characterised serologically using existing antisera, and by PCR, using primers to an 11kDa open reading frame (ORF) unique to carlaviruses and to the coat protein gene of LSV. Sequences of isolates of AlCV and LSV from the coat protein gene were 94–99% similar and were 99% similar in the 11kDa ORF, supporting the view that these are strains of the same virus.     

四种杀菌剂对烟草灰霉病菌的毒力及对烟草灰霉病的抑制作用

为筛选防治烟草灰霉病的有效药剂,采用菌丝生长速率法和孢子萌发法测定氟啶胺、咪鲜胺、苯醚甲环唑及代森锰锌4种杀菌剂对烟草灰霉病菌Botrytis cinerea的毒力,同时通过离体叶片评价这4种杀菌剂对烟草灰霉病的保护和治疗作用。结果表明,氟啶胺和咪鲜胺对菌丝生长活性抑制最强,有效抑制中浓度ECjs50平均值分别为0.02、0.03 mg/L,苯醚甲环唑次之,代森锰锌最弱,EC_(js50)平均值分别为0.39、7.86 mg/L;氟啶胺对孢子萌发活性抑制最强,代森锰锌次之,有效抑制中浓度EC_(mf50)平均值分别为0.06、0.16 mg/L,咪鲜胺和苯醚甲环唑最弱,ECmf50平均值均大于25.00 mg/L。离体试验表明,氟啶胺对烟草灰霉病保护作用最强,浓度为50 mg/L时,防治效果为100.00%,咪鲜胺和苯醚甲环唑次之,防治效果分别为88.62%和76.46%,代森锰锌最弱,浓度为1 000 mg/L时防治效果仅为75.81%;氟啶胺对烟草灰霉病治疗作用最强,浓度为100 mg/L时的防治效果为85.75%,咪鲜胺和苯醚甲环唑次之,浓度为200 mg/L时的防治效果分别为85.47%和76.48%,代森锰锌最弱,浓度为1 000 mg/L时防治效果为70.24%。表明氟啶胺和咪鲜胺更适合烟草灰霉病的防治。     

Lonicera latent virus,a new carlavirus serologically related to poplar mosaic virus: some properties and inactivation in vivo by heat treatment

A virus with elongate particles (656 nm) was isolated from severalLonicera species. This virus, apparently belonging to the carlavirus group, is serologically distantly related to shallot latent virus and closely related to poplar mosaic virus. The inability to infect poplar and two other hosts of poplar mosaic virus characterizes the virus fromLonicera as a new virus which was namedLonicera latent virus.The virus was easily sap-transmissible but was not transmitted byMyzus persicae.Dilution end-point was about 10^–3, thermal inactivation between 65°C and 80°C and ageing in vitro 1–6 days.Heat treatment, combined with tip-rooting appeared to be a good method to eliminate the virus from severalLonicera species and cultivars.Samenvatting In verschillende soorten en cultivars van het geslachtLonicera (kamperfoelie) blijkt een virus voor te komen dat gemakkelijk door sapinoculatie kan worden overgebracht op kruidachtige planten.Een tegen gezuiverd virus bereid antiserum had een titer van ca. 4096. Er kon mee worden aangetoond dat het virus van kamperfoelie serologisch nauw verwant is met populieremozaïekvirus (Tabel 1). Het virus van kamperfoelie is echter niet in staat om populier,Phaseolus vulgaris Bataaf enVigna sinensis te infecteren en wordt mede daarom als een afzonderlijk virus beschouwd. Het wordt aangeduid als latent kamperfoelievirus (Lonicera latent virus) en behoort evenals populieremozaïekvirus tot de carlavirusgroep (aardappelvirus-S-groep).Het virus blijkt vrij gemakkelijk te kunnen worden geëlimineerd door besmette kamperfoelieplanten gedurende ongeveer zes weken een warmtebehandeling (37°C) te geven en daarna de uiterste toppen (1 cm) te stekken. Van verschillende cultivars werd op deze wijze virusvrij uitgangsmateriaal verkregen.     

间作芹菜对辣椒抗烟粉虱特性的影响

为探索芹菜Apium graveolens对烟粉虱Bemisia tabaci的防控机理,以盆栽芹菜和辣椒Capsicum annuum为试材,以不同密度进行间作,分析间作芹菜对辣椒上烟粉虱的发育历期和存活率以及辣椒叶片中抗烟粉虱相关物质含量的影响。结果表明,间作芹菜对辣椒上烟粉虱若虫的发育历期和存活率有显著影响,芹菜密度越大,辣椒上烟粉虱若虫的发育历期越长,存活率越低,当辣椒与芹菜以2∶1、2∶2和2∶3的密度间作时,辣椒上烟粉虱从卵到伪蛹的发育历期分别较对照显著延长了5.47%、9.76%和16.64%,存活率分别较对照降低了20.85%、16.65%和54.15%。间作芹菜对辣椒体内抗烟粉虱相关生化物质的含量也存在显著影响,芹菜间作密度越大,辣椒叶片中可溶性糖、类黄酮和总酚的含量越高,脯氨酸含量则呈下降趋势,当辣椒与芹菜以2∶1、2∶2和2∶3的密度间作10 d后,辣椒叶片中脯氨酸含量分别较对照显著降低了20.23%、19.72%和29.98%,可溶性糖含量分别较对照升高了23.92%、30.20%和37.25%,类黄酮含量分别较对照升高了15.70%、24.92%和27.08%...     

Comparison of the nucleotide and amino acid sequences of parental and attenuated isolates of Zucchini yellow mosaic virus

To identify possible sites of viral attenuation, the complete nucleotide sequences of two isolates of Zucchini yellow mosaic virus (ZYMV) were determined; a severe isolate Z5-1 and an attenuated isolate from Z5-1 (designated ZYMV-2002). The viral genome of both isolates consisted of 9593 nucleotides in size and contained an open reading frame encoding a single polyprotein of 3080 amino acids. Comparison of the nucleotide sequences for Z5-1 and ZYMV-2002 revealed 14 nucleotide mutations, resulting in seven amino acid substitutions with four in the HC-Pro region, two in the CI region, and one in the NIb region. These results provide a genetic basis for future manipulation of the ZYMV reverse genetics system. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB188115 and AB188116