A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF βC1 in satellite DNA β which are predicted to function as silencing suppressors. In the present study suppressor function of ORF βC1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB–[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing βC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with βC1 gene from ToLCBB. However, in the case of 35S-βC1 CLCuMB and 35S-βC1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which βC1 interfere in RNA-silencing pathway is different.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

Exploitation of nucleic acid packaging signals to generate a novel influenza virus-based vector stably expressing two foreign genes

At the final step in viral replication, the viral genome must be incorporated into progeny virions, yet the genomic regions required for this process are largely unknown in RNA viruses, including influenza virus. Recently, it was reported that both ends of the neuraminidase (NA) coding region are critically important for incorporation of this vRNA segment into influenza virions (Y. Fujii, H. Goto, T. Watanabe, T. Yoshida, and Y. Kawaoka, Proc. Natl. Acad. Sci. USA 100:2002-2007, 2003). To determine the signals in the hemagglutinin (HA) vRNA required for its virion incorporation, we made a series of deletion constructs of this segment. Subsequent analysis showed that 9 nucleotides at the 3' end of the coding region and 80 nucleotides at the 5' end are sufficient for efficient virion incorporation of the HA vRNA. The utility of this information for stable expression of foreign genes in influenza viruses was assessed by generating a virus whose HA and NA vRNA coding regions were replaced with those of vesicular stomatitis virus glycoprotein (VSVG) and green fluorescent protein (GFP), respectively, while retaining virion incorporation signals for these segments. Despite the lack of HA and NA proteins, the resultant virus, which possessed only VSVG on the virion surface, was viable and produced GFP-expressing plaques in cells even after repeated passages, demonstrating that two foreign genes can be incorporated and maintained stably in influenza A virus. These findings could serve as a model for the construction of influenza A viruses designed to express and/or deliver foreign genes.     

Molecular diagnosis of begomovirus associated with Chilli leaf curl disease in Jeddah,Saudi Arabia

Chilli (Capsicum annum L.) is well known as ‘wonder spice’. This is a very valuable cash crop grown as a vegetable globally. Chilli leaf curl disease is a major threat and global concern for the cultivation of Chilli by farmers and growers. In this work, the molecular diagnosis, genetic diversity, phylogenetic relationship, and begomovirus association with Chilli leaf curl disease have been discussed. The infected leaves were randomly harvested from the Chilli field, at Jeddah, Saudi Arabia. A group of begomovirus vector, whiteflies were also observed on the Chilli crop and infected weeds growing in the neighboring field. The begomovirus was confirmed by coat protein gene specific primer, dot blot hybridization, sequencing and sequence analysis. The full coat protein gene was found to have 774 nucleotides. The nucleotide sequences analysis shared the highest identity with Tomato yellow leaf curl virus reported earlier infecting tomato from Saudi Arabia, and the lowest identity was observed with Tomato yellow leaf curl virus Oman isolate. The overall sequence identity ranged from more than ninety percent among the analyzed sequences. The phylogenetic relationship analysis formed the major three clusters and showed the closed clustering with Tomato yellow leaf curl virus isolates. The natural spread of the Tomato yellow leaf curl virus on the Chilli crop from other crops poses an important and serious threat to Chili cultivation in the Kingdom of Saudi Arabia. Based on the literature review and current evidence, this is the first report of leaf curl disease of Chilli from Saudi Arabia.     

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.     

Relationship between Tomato yellow leaf curl viruses and the whitefly vector

Tomato Yellow Leaf Curl Virus (TYLCV) and Tomato Leaf Curl Virus (ToLCV) are the currently known begomoviruses in Uganda. The relationship with their whiteflies (Bemisia tabaci) vector and its management were not known in Uganda. A direct relationship was expected between these begomoviruses and whiteflies at Buwama in Mpigi district of Uganda. Farmer practices were expected to have limited efficacy. To investigate this, a completely randomised block design was used for all trials with six treatments replicated three times. Treatments evaluated were farmer whitefly pest management practices. These included both chemical pesticide and non-pesticide applications. Data on whitefly population and tomato yellow leaf curl virus disease incidence was recorded weekly, and analysed using SAS and SPSS statistical programmes for ANOVA, and correlations. Ranked means, coefficients of variation and standard errors were noted. Virus-vector relationship field studies established that virus occurrence varied in space and time, and with management practices, crop development stage, and weather conditions. A negative relationship (R = -0.14, p 0.04) was established between number of plants infected with TYLCV (sensu lato) and percentage marketable tomato yield. Tomato maturity was inversely propotional to whitefly infestation (R = -0.5, p 0.0001). Uprooting and application of dimethoate was most effective of the six treatments. Tomato leaf curl virus diseases and whitefly management options were established in Uganda.     

Detection of tomato leaf curl geminivirus in its vector Bemisia tabaci

Geminiviruses are single-stranded DNA plant infecting viruses that cause major losses in important crops in tropical and subtropical countries. Tomato leaf curl virus (TLCV) belonging to the genera Begomovirus, is a whitefly-transmitted geminivirus that causes a severe leaf curl disease in tomato (Lycopersicon esculentum). The importance of this disease has prompted a great need for a rapid identification of TLCV in its hosts and vector. Polymerase chain reaction (PCR) is the most sensitive approach to detect a minute amount of viral nucleic acid. It is the most ideal method to amplify geminiviruses as they replicate via a double-stranded, circular DNA form. In this study, geminivirus specific degenerated primers were employed to detect TLCV occurring in its vector whitefly Bemisia tabaci by PCR based approach. One primer pair, amplified TLCV DNA fragment of about 1.1 Kb representing partly replicase gene, intergenic region and partly coat protein gene was used. When a set of primer targeted to the core region of the coat protein gene of geminivirus was used, a PCR amplified fragment of about 0.5 Kb was obtained. This approach is highly useful for an early detection of TLCV occurring in very small amount in the vector B. tabaci. Its implications in geminivirus management strategies and their differentiation and being discussed.     

Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.     

Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South-east Coast of China

An epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.     

Host range, vector relations and serological relationships of cotton leaf curl virus from southern India

In 1989 to 1991, leaf curl disease was observed in cotton (Gossypium bar-badense cv. Local) grown in kitchen gardens in five districts in Karnataka State, India, and in 1994 it was recorded in G. hirsutum cv. Sharada in two districts. Symptoms consist of leaf curling, vein thickening, leaf enations, and stunting and distortion of plants. The disease is caused by cotton leaf curl virus (CLCuV-K), which was transmitted by the whitefly Bemisia tabaci to 24 plant species in six families. Hosts include bean (Phaseolus vulgaris), pepper, tobacco, tomato and several weeds, almost all of which developed leaf curl, with or without vein thickening. CLCuV-K was transmitted from cotton to cotton by adult B. tabaci after an acquisition access period of 1 h, could be inoculated in 5 min, had a minimum latent period of 8 h and was retained by viruliferous insects for up to 9 days. Female B. tabaci transmitted more frequently than males. CLCuV-K is a whitefly-transmitted geminivirus. It reacted with two out of 17 monoclonal antibodies (MAbs) raised to African cassava mosaic virus and five out of 10 MAbs raised to Indian cassava mosaic virus. CLCuV-K isolates from different locations in Karnataka had similar epitope profiles. As judged by these profiles, CLCuV-K is closely related to Indian tomato leaf curl virus from Karnataka, is distinguishable from several other whitefly-transmitted geminiviruses found in India and is still more distantly related to those, including cotton leaf crumple virus from the USA, found in other continents. CLCuV-K infected all cultivars tested of G. barbadense and one of six cultivars of G. hirsutum but none of G. arboreum or G. herbaceum.     

A plant virus vector for systemic expression of foreign genes in cereals

Inserts bearing the coding sequences of NPT II and beta-glucuronidase (GUS) were placed between the nuclear inclusion b (NIb) and coat protein (CP) domains of the wheat streak mosaic virus (WSMV) polyprotein ORF. The WSMV NIb-CP junction containing the nuclear inclusion a (NIa) protease cleavage site was duplicated, permitting excision of foreign protein domains from the viral polyprotein. Wheat, barley, oat and maize seedlings supported systemic infection of WSMV bearing NPT II. The NPT II insert was stable for at least 18-30 days post-inoculation and had little effect on WSMV CP accumulation. Histochemical assays indicated the presence of functional GUS protein in systemically infected wheat and barley plants inoculated with WSMV bearing GUS. The GUS constructs had greatly reduced virulence on both oat and maize. RT-PCR indicated that the GUS insert was subject to deletion, particularly when expressed as a GUS-NIb protein fusion. Both reporter genes were expressed in wheat roots at levels comparable to those observed in leaves. These results clearly demonstrate the utility of WSMV as a transient gene expression vector for grass species, including two important grain crops, wheat and maize. The results further indicate that both host species and the nature of inserted sequences affect the stability and expression of foreign genes delivered by engineered virus genomes.     

Tomato yellow leaf curl virus differentially influences plant defence responses to a vector and a non‐vector herbivore

Plants frequently engage in simultaneous interactions with diverse classes of biotic antagonists. Differential induction of plant defence pathways by these antagonists, and interactions between pathways, can have important ecological implications; however, these effects are currently not well understood. We explored how Tomato yellow leaf curl virus (TYLCV) influenced the performance of its vector (Bemisia tabaci) and a non‐vector herbivore (Tetranychus urticae) occurring separately or together on tomato plants (Solanum lycopersicum). TYLCV enhanced the performance of B. tabaci, although this effect was statistically significant only in the absence of T. urticae, which adversely affected B. tabaci performance regardless of infection status. In contrast, the performance of T. urticae was enhanced (only) by the combined presence of TYLCV and B. tabaci. Analyses of phytohormone levels and defence gene expression in wild‐type tomatoes and various plant‐defence mutants indicate that the enhancement of herbivore performance (for each species) entails the disruption of downstream defences in the jasmonic acid (JA) pathway. For T. urticae, this disruption appears to involve antagonistic effects of salicylic acid (SA), which is cumulatively induced to high levels by B. tabaci and TYLCV. In contrast, TYLCV was found to suppress JA‐mediated responses to B. tabaci via mechanisms independent of SA.     

Transmission of tomato leaf curl geminiviruses by Bemisia tabaci: effects of virus isolate and vector biotype

Cultures of Bemisia tabaci from Ivory Coast (IC), Pakistan (PK) and USA (US B-type) were compared for the frequency with which they transmitted three tomato geminivirus isolates: Indian tomato leaf curl virus from Bangalore (ITmLCV), and tomato yellow leaf curl viruses from Nigeria (TYLCV-Nig) and Senegal (TYLCV-Sen). Frequency of transmission from tomato to tomato depended both on the whitefly culture and the virus isolate. US B-type and IC whiteflies transmitted TYLCV-Sen more frequently than ITmLCV whereas PK whiteflies transmitted ITmLCV more frequently than TYLCV-Sen. US B-type whiteflies transmitted both viruses four to nine times more frequently than IC whiteflies. TYLCV-Nig was transmitted rarely by US B-type and not at all by IC whiteflies. Previous work indicates that the geminivirus coat protein controls vector transmissibility. The differential adaptation of TYLCV-Sen to transmission by US B-type whiteflies and of ITmLCV to PK whiteflies was associated with a large difference in epitope profile of the coat proteins of the two viruses. Also, the readily transmissible TYLCV-Sen differed appreciably in epitope profile from the poorly transmissible TYLCV-Nig, which reached a consistently greater concentration in source tissues but lacked epitope 18. However, the lack of epitope 18 in ITmLCV did not prevent its transmission by US B-type whiteflies. Differences in frequency and specificity of geminivirus transmission by whitefly cultures from different countries therefore were associated with differences among epitope profiles of the coat proteins of the viruses, but the structural features of the proteins that control transmission remain to be determined.