A single amino acid substitution in the movement protein enables the mechanical transmission of a geminivirus

Begomoviruses of the Geminiviridae are usually transmitted by whiteflies and rarely by mechanical inoculation. We used tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, to address this issue. Most ToLCNDV isolates are not mechanically transmissible to their natural hosts. The ToLCNDV-OM isolate, originally identified from a diseased oriental melon plant, is mechanically transmissible, while the ToLCNDV-CB isolate, from a diseased cucumber plant, is not. Genetic swapping and pathological tests were performed to identify the molecular determinants involved in mechanical transmission. Various viral infectious clones were constructed and successfully introduced into Nicotiana benthamiana, oriental melon, and cucumber plants by Agrobacterium-mediated inoculation. Mechanical transmissibility was assessed via direct rub inoculation with sap prepared from infected N. benthamiana. The presence or absence of viral DNA in plants was validated by PCR, Southern blotting, and in situ hybridization. The results reveal that mechanical transmissibility is associated with the movement protein (MP) of viral DNA-B in ToLCNDV-OM. However, the nuclear shuttle protein of DNA-B plays no role in mechanical transmission. Analyses of infectious clones carrying a single amino acid substitution reveal that the glutamate at amino acid position 19 of MP in ToLCNDV-OM is critical for mechanical transmissibility. The substitution of glutamate with glycine at this position in the MP of ToLCNDV-OM abolishes mechanical transmissibility. In contrast, the substitution of glycine with glutamate at the 19th amino acid position in the MP of ToLCNDV-CB enables mechanical transmission. This is the first time that a specific geminiviral movement protein has been identified as a determinant of mechanical transmissibility.     

Serological studies on the accumulation and localisation of three tomato leaf curl geminiviruses in resistant and susceptible Lycopersicon species and tomato cultivars

Accessions of wild Lycopersicon spp. and selected Fl hybrid tomato cultivars were compared for their resistance to three whitefly-transmissible geminiviruses: Indian tomato leaf curl virus (ITmLCV) and tomato yellow leaf curl viruses from Sardinia (TYLCV-Sar) and Senegal (TYLCV-Sen). The resistance of different plant lines was expressed in different ways but in most instances a given line reacted similarly to graft inoculation with the three viruses. L. pimpinellifolium LA1478 produced as much virus antigen, assessed by triple antibody sandwich-ELISA, as the susceptible cv. Moneymaker but developed only very mild symptoms and is therefore tolerant of infection. In L. hirsutum LA1777 and L. peruvianum CMV-INRA, very mild or no symptoms developed but antigen concentrations were substantially less than in Moneymaker. L. chilense LA1969 remained symptomless and its antigen concentration was < 1% of that in Moneymaker. Symptoms were mild or barely evident in the Fl hybrid cultivars. Cultivars Tyking and Fiona had antigen concentrations about 5–10% of those of Moneymaker, whereas TY20, Top 21 and Tyger had intermediate antigen concentrations. In a few instances, the extent to which virus accumulation was restricted depended on the challenge virus. Accumulation of TYLCV-Sen in TY20, Top 21 and Tyger was less affected than that of the other two viruses, and accumulation of TYLCV-Sar in accessions LA1777 and CMV-INRA was less affected than that of TYLCV-Sen or ITmLCV. Tissue-printing tests showed that ITmLCV and TYLCV-Sen antigens were confined to phloem tissue. In Tyking, the number of virus antigen-containing phloem traces and the antigen content of individual traces were less than in Moneymaker but the partitioning of antigen between internal and external phloem was unaffected.     

Engineering resistance against tomato yellow leaf curl virus (TYLCV) using antisense RNA

One of the most severe diseases of cultivated tomato worldwide is caused by tomato yellow leaf curl virus (TYLCV), a geminivirus transmitted by the whitefly Bemisia tabaci. Here we describe the application of antisense RNAs to interfere with the disease caused by TYLCV. The target of the antisense RNA is the rare messenger RNA of the Rep protein, encoded by the C1 gene. Transgenic Nicotiana benthamiana plants expressing C1 antisense RNA were obtained and shown to resist infection by TYLCV. Some of the resistant lines are symptomless, and the replication of challenge TYLCV almost completely suppressed. The transgenes mediating resistance were shown to be effective through at least two generations of progeny.     

The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India

Indian tomato leaf curl virus (ToLCV) (Geminiviridae: Sub-group III) was detected both in field-collected and laboratory-reared B. tabaci using a triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). ToLCV was detected in six of the 10 group samples of field collected B. tabaci. ToLCV was also identified in 13 weed species commonly found in Karnataka, both by symptom expression and TAS-ELISA. ToLCV from c. 61% of infected plants was transmitted successfully to tomato by B. tabaci. Tomato plots were planted at three locations on the University of Agricultural Sciences Campus, Bangalore. Indian tomato leaf curl virus disease (ToLCVD) incidence increased most rapidly when the tomato plot was situated adjacent to an older ToLCVD-infected tomato field. When the plots were positioned in a dryland or a wetland area, at least 500 m away from any infected tomato fields, the ToLCVD incidence increased less rapidly, although in all sites it was 100% by 11 wk after transplanting. The numbers of B. tabaci caught on yellow traps in all sites increased during weeks 1–3 after transplanting and thereafter remained at between 10–15 adults trap^-1 24 h^_1. Adult numbers recorded on tomato plants by direct counts remained approximately constant at 2–4 adults plant“”¹. Tomato fields were planted in three taluks (administrative areas) of Karnataka, that had different current and previous histories of tomato production. ToLCVD incidence increased most and least rapidly, respectively, in Kolar taluk where tomato is grown continuously and Doddaballapur tuluk where tomato was grown in the area for the first time. In Malur tuluk, where tomato was grown discontinuously (once a year), the incidence of ToLCVD increased at an intermediate rate. Weed host-plant species growing near the experimental sites had averages of between 1.5–10.0 B. tabaci nymphs per plant, whereas the tomato plants had only 0.3 nymphs per plant. The percentage parasitism of B. tabaci nymphs on tomato and weed species, respectively, was 0.7% and 2–6%. Nymphs and pupae were parasitised by an Encarsia sp. and Eretmocerus mundus Mercet. The relevance and implications of these findings for the epidemiology and management of ToLCVD in Karnataka State, South India is discussed.     

高抗番茄黄化曲叶病毒病番茄新品种浙粉702的选育及抗病性鉴定

高抗黄化曲叶病毒病番茄新品种浙粉702是以自育株系材料T7969F2-19-1-1-3为母本,T4078F2-3-3-3为父本,结合分子标记辅助技术选育的杂交一代粉红大果型番茄品种。母本‘T7969F2-19-1-1-3’系从以色列引进的耐贮运番茄品种‘NEMO-TAMMI’（F1）与抗叶霉病粉红株系材料‘T9179’杂交分离后代中经连续9代单株选择而成。父本‘T4078F2-3-3-3’系从荷兰引进的抗TYLCVD番茄品种‘奇诺亚’（F1）与粉红株系材料‘T9178’杂交分离后代中经连续8代单株选择而成。该品种2011年通过浙江省非主要农作物认定委员会认定。通过对浙粉702园艺性状、产量性状、品质性状和抗病性等进行研究,结果表明：浙粉702 品质优良,早熟,丰产,高抗番茄黄化曲叶病毒病,枯萎病,抗叶霉病和番茄花叶病毒病,适合我国喜食粉果地区种植,平均可达73.83 t.hm-2。     

Tomato yellow leaf curl virus infection of tomato does not affect the performance of the Q and ZHJ2 biotypes of the viral vector Bemisia tabaci

Abstract To better understand the etiology of begomovirus epidemics in regions under invasion we need to know how indigenous and invasive whitefly vectors respond to virus infection. We investigated both direct and indirect effects of infection with Tomato yellow leaf curl virus (TYLCV) on the performance of the invasive Q biotype and the indigenous Asian ZHJ2 biotype of whitefly Bemisia tabaci. The Q biotype performed better than the ZHJ2 biotype on either uninfected or virus‐infected tomato plants. However, virus‐infection of host plants did not, or only marginally affected, the performance of either biotype of whiteflies in terms of fecundity, longevity, survival, development and population increase. Likewise, association of the vectors with TYLCV did not affect fecundity and longevity of the Q or ZHJ2 biotypes on cotton, a non‐host of TYLCV. These results indicate that the alien Q biotype whitefly, but not the indigenous ZHJ2 biotype, is likely to become the major vector of TYLCV in the field and facilitate virus epidemics.     

番茄玉米间套作对烟粉虱的屏障效应及控制番茄黄化曲叶病毒病的效果

为探讨番茄与玉米间套作对烟粉虱的趋避效应及控制番茄黄化曲叶病毒病的效果,设置番茄品种‘金山511'以固定株行距与玉米品种‘先玉335'分别以10、20、30 cm的播种株距进行间作套种,调查3种栽培方式对烟粉虱屏障效应及黄化曲叶病毒病发生的影响。结果表明: 与番茄单作相比,玉米植株建立起的自然屏障使番茄生长环境相对稳定,玉米播种株距为10、20、30 cm时,番茄植株在6:00—20:00的平均温度分别降低3.01、2.26和1.45 ℃,平均相对湿度分别提高13.0%、8.8%、6.0%,平均光照强度分别降低26.1%、20.4%和14.5%;在每日的高温时段可显著降低番茄温度,提高湿度,减少光照强度,有效缓解强光、高温和干旱等有利于病毒病发生的不良环境条件,且在玉米播种距离为10 cm时效果最好。番茄间作玉米对烟粉虱有趋避效应,能抑制番茄黄化曲叶病毒病的发生,玉米株距为10、20、30 cm时,烟粉虱虫口数分别比单作减少88.7%、82.0%、73.9%,对病毒病的抑制作用呈减弱趋势,间作病情指数分别显著降低67.3%、59.4%和44.5%。玉米/番茄间作的种植模式有利于番茄植株生长和坐果,可提升番茄产量,玉米种植密度越高,效果越好,本试验条件下以玉米种植株距10 cm效果最好。     

Reaction of some tomato cultivars to tomato leaf curl virus and evaluation of the endophytic colonisation with Beauveria bassiana on the disease incidence and its vector,Bemisia tabaci

Tomato line LA1478 and Pusa Ruby were resistant to tomato leaf curl virus (TLCV) disease. They registered higher plant height, number of branches, total phenol content and yield per plant than the other cultivars. Variety Peto 86 was tolerant to the disease while the other popular tomato cultivars, i.e. Ace, Early Pack, Money Maker, Prichard and Strain B were highly susceptible to the disease. Plant height and number of branches per plant revealed significantly positive association with fruit yield per plant. The disease index of TLCV exhibited significant negative correlations with plant height, total phenol content and fruit yield per plant – 0–4 and 5–25 adult whiteflies were observed on resistant susceptible cultivars. In the case of epiphytically colonisation by Beauveria bassiana conidia, not all developing hyphae on the leaf surface penetrated the whitefly cuticle. Many of the germ tubes elongated to a short distance before terminating its growth. On the other hand, the rapid staining of tomato tissues injected with B. bassiana conidial suspension indicates that the entomopathogenic fungus was established inside tomato tissues until the end time of the trial. The direct injection with the spore suspension yielded high post-colonisation, where the fungus was recovered from sites distant from the point of inoculation. This indicates that the fungus has the potential to move throughout the plant tissues. Laboratory bioassay of tomato whitefly feeding on tomato tissues containing B. bassiana conidial spores indicates that plant endophytic colonisation with entomopathogenic fungi may reduce insect survival on these plants. LT₅₀ values of the test diet were between three and four days. The mortality of Bemisia tabaci was high in the case of endophytically colonisation compared to epiphytically one (90.0% compared to 10.0% during three days) for whiteflies fed tomato tissues containing 1.5 × 10⁷ B. bassiana spores/ml. Application of B. bassiana as an artificial endophyte inside tomato plants can be an important component in the integrated control of tomato whiteflies. The endophytic colonising can achieve biocontrol effect based on induced disease resistance in plant tissues. According the available references, this is the first report on B. tabaci controlling by plant endophytic treatment.     

Association of tomato leaf curl Palampur virus with yellow mosaic disease of Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) in India

Natural occurrence of yellow mosaic disease was observed on Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) with disease incidences of ~36 and ~27%, respectively. Association of tomato leaf curl Palampur virus (ToLCPV) with the disease was investigated by Polymersae chain reaction (PCR) using begomovirus-specific primers. Full-length genome was amplified by rolling circle amplification (RCA) method from representative samples of C. melo and C. callosus. RCA products obtained were cloned and sequenced. Analyses of sequence data revealed the presence of full-length begomoviral genome of 2756 nucleotides with the gene arrangement of a typical begomovirus: HQ848383 (C. melo) and GU253914 (C. callosus). Both the isolates shared 99% sequence identity together and high 97–99% identities and the closest phylogenetic relationships with ToLCPV strains reported worldwide, hence identified as two new members of ToLCPV. Natural occurrence of ToLCPV on C. melo and C. callosus is the first report.     

Expression of the Full‐length Coat Protein Gene of Tomato leaf curl Taiwan virus is Not Necessary for Recovery Phenotype in Transgenic Tomato

Transgenic tomato plants expressing full‐length (CPV1) and truncated coat protein (CP) gene (CPV2) of Tomato leaf curl Taiwan virus (ToLCTWV) were generated by Agrobacterium‐mediated transformation. Transgene integration and expression was confirmed by PCR and Southern blotting and Northern analysis, respectively. Resistance was evaluated both in plants of T0 and T1 progenies using viruliferous whiteflies under two different inoculum pressures (10–15 and 40–50 whiteflies/plant). Upon inoculation with ToLCTWV using viruliferous whiteflies, various levels of phenotypic reaction were observed. No complete resistance was observed in any of the plants tested. The reaction of the transgenic tomato lines carrying full‐length and truncated CP gene to ToLCTWV phenotype was (i) susceptible as non‐transgenic control, (ii) delayed symptom expression, (iii) complete susceptible (from delayed symptom expression phenotype) and (iv) recovered phenotype (either plants from symptom expression as non‐transgenic plants or delayed symptom expression phenotype). Dot blot quantification of the ToLCTWV using the replicase gene as a probe revealed that the recovered phenotypes accumulated a low level of ToLCTWV, and virus concentration was gradually reduced from 10 to 14 weeks postinoculation. The possible mechanisms of CP‐mediated resistance are discussed.     

新疆地区烟粉虱生物型的区域分布及其携带的番茄黄化曲叶病毒检测

【目的】明确不同烟粉虱Bemisia tabaci (Gennadius)生物型在新疆地区的区域分布及其携带番茄黄化曲叶病毒的情况。【方法】本文利用烟粉虱mtDNA COⅠ基因片段作标记以及番茄黄化曲叶病毒(tomato yellow leaf curl virus, TYLCV)特异性引物, 鉴定新疆不同地区烟粉虱生物型, 并检测其携带TYLCV情况。【结果】石河子市、 博乐市、 岳普湖县、 泽普县、 叶城县、 莎车县、 墨玉县、 和田市与和田县烟粉虱属于B型烟粉虱, 且720 bp COⅠ基因序列一致性为100%; 阿克苏市、 哈密市、 鄯善县、 乌鲁木齐市、 克拉玛依市与伊宁市烟粉虱属于Q型烟粉虱, 720 bp COⅠ基因序列一致性为99.9%~100%; 吐鲁番市与库尔勒市兼有B型烟粉虱和Q型烟粉虱。伊宁市、 乌鲁木齐市、 鄯善县、 哈密市、 阿克苏市、 库尔勒市、 莎车县、 和田县和墨玉县的烟粉虱样本携带TYLCV, 除2011年底已经暴发危害的莎车县外, 其他区域均为番茄黄化曲叶病毒病发生的高风险区域。【结论】本研究为新疆地区烟粉虱及番茄黄化曲叶病毒病的科学防控提供依据。     

辽宁省冬季温室内越冬粉虱伪蛹的种类鉴定及烟粉虱体内番茄黄化曲叶病毒的检测

为了揭示辽宁省冬季温室内越冬粉虱伪蛹的种类及烟粉虱Bemisia tabaci （Gennadius）携带番茄黄化曲叶病毒（tomato yellow leaf curl virus, TYLCV）情况, 于2012年1月份在辽宁省不同县市区的温室作物上采集了17份粉虱伪蛹样品（每样品含30头粉虱伪蛹） , 镜检鉴别粉虱种类并利用mtCOI基因对烟粉虱生物型进行了鉴定; 检测了烟粉虱携带TYLCV情况并对其PCR扩增产物进行了测序分析。结 果表明： 辽宁省冬季温室内存在越冬温室白粉虱Trialeurodes vaporariorum （Westwood）与烟粉虱。17份粉虱样品中, 11份样品为烟 粉虱样品, 6份样品为温室白粉虱和烟粉虱混合样品。混合样品中, 温室白粉虱仅在锦州凌海（LH）样品中占优势。17份烟粉虱样品（包 括混合样品）中, 仅有4份样品为B型与Q型混合样品, 其他13份样品烟粉虱生物型均为Q型。17份烟粉虱样品中有3份Q型烟粉虱样品检测 到TYLCV, 系统树分析进一步证实该病毒是TYLCV。调查结果为辽宁省粉虱与TYLCV的早期测报和防控提供了科学依据。     

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp,nonstructural proteins p16 and p23, playing a role in virus replication

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.     

新城疫病毒M蛋白在病毒毒力和复制中的作用研究进展

M蛋白是新城疫病毒(Newcastle disease virus,NDV)基因组编码的一种非糖基化膜相关蛋白,主要位于病毒囊膜内表面,构成病毒囊膜与核衣壳连接的支架.研究表明,M蛋白是一种细胞核-细胞质穿梭蛋白,在抑制细胞基因转录和蛋白质合成以及协助病毒粒子组装和出芽方面发挥了重要作用.目前,国内外对NDV毒力和复制...     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

Acidic dileucine motifs in the cylindrical inclusion protein of turnip mosaic virus are crucial for endosomal targeting and viral replication

Previously we reported that the multifunctional cylindrical inclusion (CI) protein of turnip mosaic virus (TuMV) is targeted to endosomes through the interaction with the medium subunit of adaptor protein complex 2 (AP2β), which is essential for viral infection. Although several functionally important regions in the CI have been identified, little is known about the determinant(s) for endosomal trafficking. The CI protein contains seven conserved acidic dileucine motifs [(D/E)XXXL(L/I)] typical of endocytic sorting signals recognized by AP2β. Here, we selected five motifs for further study and identified that they all were located in the regions of CI interacting with AP2β. Coimmunoprecipitation assays revealed that alanine substitutions in the each of these acidic dileucine motifs decreased binding with AP2β. Moreover, these CI mutants also showed decreased accumulation of punctate bodies, which enter endocytic-tracking styryl-stained endosomes. The mutations were then introduced into a full-length infectious clone of TuMV, and each mutant had reduced viral replication and systemic infection. The data suggest that the acidic dileucine motifs in CI are indispensable for interacting with AP2β for efficient viral replication. This study provides new insights into the role of endocytic sorting motifs in the intracellular movement of viral proteins for replication.     

Virus–vector Relationships,Host Range,Detection and Sequence Comparison of Chilli leaf curl virus Associated with an Epidemic of Leaf Curl Disease of Chilli in Jodhpur,India

An epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli‐growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.