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Synthesis of viral DNA forms in Nicotiana plumbaginifolia protoplasts inoculated with cassava latent virus (CLV); evidence for the independent replication of one component of the CLV genome

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Abstract

Totipotent leaf mesophyll protoplasts of Nicotiana plumbaginifolia, Viviani were inoculated with cassava latent virus (CLV) or with full length copies of CLV genomic DNAs 1 and 2 excised from replicative forms of M13 clones. Virus specific DNAs began to appear 48–72h after inoculation with virus or cloned DNAs, coincident with the onset of host cell division. Infected cells accumulated supercoiled forms of DNAs 1 and 2 as well as progeny single-stranded (ss) virion (+) sense DNAs representing each component of the genome. Both supercoiled and ss molecules were synthesised by cells inoculated with cloned DNA 1 alone but DNA 2 failed to replicate independently.

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... This structural similarity can be extended further to a functional level in that both DNA 1 of cassava latent virus (CLV) and DNA A of tomato golden mosaic virus (TGMV) are capable of replicating independent of DNA 2 or DNA B. This has been demonstrated in protoplasts of Nicotiana plumbaginifolia (Townsend et al., 1986) or in plants (Rogers et al., 1986). Using a cell suspension culture, we have shown that the single component of the WDV genome contains all information for sustained replication in monocotyledonous cells. ...
... This linear WDV monomer becomes circularized by the action of cellular ligases and, hence, serves as a replicative intermediate. Previous work on the replication of CLV component 1 in mesophyll protoplasts of N. plumbaginifolia (Townsend et al., 1986) led to the suggestion that the viral DNA replication is linked to the cell division of the host cell. Similar observations were made with WDV replicating in 7. monococcum suspension culture cells. ...
Article
Wheat dwarf virus (WDV) is a geminivirus that infects monocotyledonous plants. To exploit the potential of WDV as a replicative gene vector, we developed a transient replication and expression system based on the transfection of protoplasts derived from Triticum monococcum suspension culture cells. Cloned genomic copies of various WDV isolates as well as mutants constructed in vitro were introduced into the protoplasts and assayed for their ability to replicate. As a result, regions of the WDV genome necessary or dispensable for the viral DNA replication could be defined. In addition, the gene encoding the viral capsid protein was replaced by three different bacterial marker genes, neomycin phosphotransferase, chloramphenicol acetyltransferase, and beta-galactosidase. The beta-galactosidase gene doubled the size of the WDV genome. The replication of the recombinant WDV genomes and the expression of these genes were monitored in suspension culture cells of T. monococcum. The potential of replicative expression vectors based on the WDV genome is discussed.
... The use of protoplasts allows the study of viral DNA replication and gene expression under more or less synchronous conditions, independent of systemic movement requirements (for review, see Motoyoshi, 1985). Transient replication of several geminiviruses has been demonstrated in protoplasts, including bean golden mosaic virus (Haber et a/., 1981) African cassava mosaic virus (ACMV, synonym cassava latent virus; Townsend et al., 1986) beet curly top virus (BCTV; Briddon eta/., 1989), TGMV (Bisaro eta/., 1990;) and wheat dwarf virus (WDV; Woolston eta/., 1989;Matzeit eta/., 1991). In the case of ACMV, BCTV, and WDV, replicating viral DNA can be detected 2-3 days after transfection. ...
... Viral DNA levels reached a maximum after 2-3 days and have been detected for up to 7 days. This contrasts with the results of transfection experiments with the geminiviruses ACMV, BCTV, and WDV, where replicating viral DNA forms were not observed until 2-5 days post-transfection, coinciding with the onset of protoplast cell division (Townsend et a/., 1986;Briddon et a/., 1989;Matzeit et a/., 1991). ...
Article
We have analyzed the replication kinetics of the DNA A and DNA B genome components of the geminivirus tomato golden mosaic virus (TGMV) in protoplasts derived from Nicotiana tabacum suspension culture. In addition, the kinetics of TGMV coat protein promoter activity, as measured by expression of a beta-glucuronidase (GUS) reporter, have been examined. In our protoplast system, double-stranded DNA forms of both viral genome components appeared by 18 hr post-transfection, while single-stranded DNA accumulated to detectable levels after 18-24 hr. Expression of GUS from the TGMV coat protein promoter did not require viral DNA replication, nor was it dependent on expression of AL1, the only viral gene necessary for DNA replication. However, maximal expression was achieved following AL1-mediated replication of DNA A. GUS activity from replicating templates exceeded that from nonreplicating templates by 60- to 90-fold. Expression of the GUS reporter gene from nonreplicating viral DNA templates was similar to GUS expression from the 35S promoter of cauliflower mosaic virus in N. tabacum protoplasts.
... DNA A encodes the coat protein . When introduced into protoplasts it is capable of self-replication to produce both ss and ds DNA forms typical of infection which showed that the genes responsible for its replication are in this component (Townsend et al., 1986). Because both components are required for systemic infection in plants (Stanley, 1983), DNA B gene products are implicated in spread of the virus or viral DNA throughout the plant (Townsend et al., 1986;Etessami et al., 1988). ...
... When introduced into protoplasts it is capable of self-replication to produce both ss and ds DNA forms typical of infection which showed that the genes responsible for its replication are in this component (Townsend et al., 1986). Because both components are required for systemic infection in plants (Stanley, 1983), DNA B gene products are implicated in spread of the virus or viral DNA throughout the plant (Townsend et al., 1986;Etessami et al., 1988). In contrast to ACMV, beet curly top virus (BCTV) has a single genomic component, organized similarly to ACMV DNA A . ...
Article
Agroinoculation of African cassava mosaic virus DNA A alone into Nicotiana benthamiana resulted in the occasional spread of viral DNA throughout the stem, leaves and root. The amount of DNA A reached 5 % of that associated with full infection following agroinocu- lation of both DNAs A and B, although the plants remained asymptomatic. Detection of virus particles in the upper leaves indicates that DNA B is not essential for virus assembly. The predominant form of the virus- specific DNA was single-stranded, which is possibly encapsidated within the virus particles. Double-strand- ed DNA forms could also be detected in the upper
... DNA-A is essential for replication and encapsidation [10][11][12] while DNA-B plays a role in systemic movement and symptom production [13,14]. The begomovirus replication cycles rely entirely on DNA intermediates and occur within the nucleus of the infected cell through two basic stages: conversion of ssDNA to dsDNA intermediates and rolling circle replication (RCR) [15]. ...
Article
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Begomoviruses (family Geminiviridae) cause a number of serious diseases of cultivated crops and they are considered as the major constrains for cultivation of several crops all over the world. Presently human activity and modern day agriculture are one of the key factors in the emergence of begomoviruses in various parts of the world. The available disease management options include the organisation of agricultural practice to reduce disease, the use of cultural control such as sanitation programmes, the control of the vector population through the use of insecticides, and the breeding and growing of resistant crop cultivars. Transgenic resistance against begomoviruses has shown limited success despite the use of a number of strategies. Ability of begomoviruses to evolve rapidly by recombination and mutations is the major limitation to all these strategies. In modern-day biotechnology, focus is required on engineering begomovirus resistance through transgenic approach. Expression of various full length or truncated or defective proteins of the virus has been effective in achieving pathogen-derived resistance. Antisense RNA and RNAi technology have also been used with some success. This review focus on both conventional and nonconventional management strategies methods have been suggested and described over the years for management of begomoviruses diseases in economically important plants.
... The host cell is thought to play a role in the regulation of geminivirus replication (Townsend et al., 1986). Despite the fact that the complementary sense promoter of WDV must be active on input DNA in einkorn protoplasts (to generate viral replicons), the onset of virion sense promoter activity appeared to be delayed compared with the CaMV 35s promoter ( Figures 3A and 3B), and WDV2CAT replicon DNA was not detected immediately after transfection (Figure 2A). ...
Article
Full-text available
We have investigated the relationship between viral DNA replication and virion sense gene expression in wheat dwarf virus (WDV), a member of the geminivirus group, by testing a series of deletion mutants in transfected Triticum monococcum (einkorn) protoplasts. Mutants contained a transcription fusion of the chloramphenicol acetyltransferase coding sequence to the virion sense promoter that replaced the viral coat protein coding sequence. The deletion analysis revealed that WDV replication and virion sense transcription can proceed independently and are controlled in part by nonoverlapping elements in the large intergenic region. These data and those from a C2 open reading frame (ORF) frameshift mutant also showed that the product of the C2 ORF (C1-C2 protein) is independently involved in both DNA replication and activation of the virion sense promoter. The amino acid sequences encoded by C2, which are highly conserved in the geminivirus group, show some homology to the DNA binding domain of the myb-related class of plant transcription factors. The possible involvement of the host in controlling the function of the C1-C2 protein and the implication of these data for the development of WDV-based gene vectors are discussed.
... One subgroup includes viruses that infect dicotyledonous plants, are transmitted by whiteflies and have genomes divided between two 2-5 to 2-7 kb DNA components, designated A and B. Members of this bipartite subgroup include tomato golden mosaic virus (TGMV; Hamilton et al., 1984), African cassava mosaic virus (ACMV; formerly cassava latent virus, Stanley & Gay, 1983) and tomato yellow leaf curl virus (TYLCV, Rochester et al., 1990). With the exception of TYLCV, both genome components are required for infectivity and symptom development (Hamilton et al., 1983;); however, the A genome component encodes all viral functions necessary for the replication and encapsidation of viral DNA (Rogers et al., 1986; Townsend et al., 1986; Sunter et al., 1987). Geminiviruses belonging to a second subgroup, which includes wheat dwarf virus (WDV; MacDowell et al., 1985 ), are transmitted by leafhoppers, have monopartite genomes 2.7 to 3-0 kb in size and infect monocotyledonous plants. ...
Article
In addition to ss and ds genomic DNA, agroinoculation of Nicotiana benthamiana plants with the Logan strain of the geminivirus beet curly top virus (BCTV) consistently resulted in de novo production of subgenomic DNAs on initial passage. Single-stranded and dsDNA forms representing at least seven size classes (0.8 to 1.8 kb) of subgenomic DNA were observed in total DNA extracts from inoculated plants. Extracts from infected sugar beet and tomato contained variable but usually smaller amounts of subgenomic DNAs, suggesting that their production may be influenced by the host species. Restriction endonuclease mapping and partial nucleotide sequencing of three independent clones of a 1.5 kb size class indicated that this subgenomic DNA is produced from the standard viral genome by two separate deletion events. One deletion of 941 bp includes portions of the leftward open reading frames (ORFs) L1, L2 and L3, while the other deletion of 579 bp encompasses portions of the intergenic region and the rightward ORFs R1, R2 and R3. The data indicate that the 1.5 kb BCTV subgenomic DNA is a defective DNA that has retained cis-elements essential for replication.
... It has a monopartite/bipartite genome, with the DNA-A component encoding all the proteins necessary for viral replication in a single cell, while the DNA-B component provides proteins for the movement function for systemic spread. DNA-A comprises of 8 ORFs, most of which are characterized by their functional attributes (Townsend et al. 1986, Hanley-Bowdoin et al. 1999). Begomoviruses do not obligatorily produce long double-stranded RNA (dsRNA) during their life cycles, but do so possibly through an aberrant read through transcription (Shivaprasad et al. 2005) or through overlapping transcripts, as they might trigger RNA silencing (Chellappan et al. 2004). ...
Article
Plant RNA silencing systems are organized into different networks with overlapping functions. Primarily, micro RNAs (miRNAs) help in regulating development and small interfering RNAs (siRNAs) regulate transgene induced or virus induced silencing functions at the post transcriptional level. Small RNA silencing, using potent RNAi silencing target sequences from viral genome of leaf curl virus, was designed via artificial micro RNAs (amiRNAs) and transgene derived siRNAs to generate leaf curl resistance in plants. The comparative analysis of both the RNAi silencing systems is discussed.
... Because geminiviruses depend on the replicative machinery of host cells it was originally anticipated that meristematic tissues could be an important site of viral DNA replication (Townsend et al., 1986;Stanley, 1991). Contrary to this idea, Horns and Jeske (1991) showed that AbMV does not enter A. sellovianum meristematic tissues. ...
Article
Abutilon mosaic virus (AbMV), a bipartite geminivirus of the genus Begomovirus, has been vegetatively propagated for many years in Abutilon sellovianum in which it is strictly phloem-restricted. Using in situ hybridization and immunological analyses, the tissue tropism of AbMV in the laboratory host Nicotiana benthamiana was compared with that of two other bipartite begomoviruses, African cassava mosaic virus (ACMV) and tomato golden mosaic virus (TGMV). Analysis of the first systemically infected leaves and longitudinal sections of axillary and flower buds revealed that all three viruses are initially confined to the vascular traces, although both ACMV and TGMV are later detectable in nearly all tissue types. In contrast, AbMV remained strictly phloem-limited in this host throughout the course of infection. The ability of ACMV and TGMV to move out of N. benthamiana phloem tissues is correlated with the development of severe symptoms in comparison with the mild symptoms associated with AbMV infection. It was also demonstrated that Sida micrantha mosaic virus, a virus that is closely related to AbMV, is phloem-limited in Malva parviflora even though it induces severe leaf curl, stunting and necrosis in this host. The present data demonstrate that bipartite begomoviruses can exhibit strikingly different patterns of tissue tropism.
... Modulating increases and decreases in [ZA] DNA-A and DNA-B titers in both plants systems may be a consequence of a cyclic replication pattern typical of a situation with bipartite viruses, such as [ZA], where two DNA components are interdependent on each other for different functions at different times, such as replication (DNA-A) and movement proteins (DNA-B). Increases in DNA-A levels later on during the infection period indicate a recovery in DNA-A replication which may be predicted since DNA-A (AC1 and AC3 ORFs transcribe for the Rep-associated and replication enhancing proteins, respectively) is the rate-limiting factor for both its own autonomous replication and is required by both DNA-B and the DI for trans-replication [123,129]. Symptom amelioration suggests that efficient high replication of the DI by the helper virus is a prerequisite for symptom amelioration. However trans-replication and movement of DIs also requires high levels of cognate helper virus-encoded Rep and movement proteins, and therefore production of DNA-A and DNA-B components. ...
Article
Full-text available
The family Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite (two circular ssDNA components called DNA-A and DNA-B), many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.
... DNA-A is capable of autonomous replication in host cells (Evans and Jeske, 1993;Regers et al., 1986;Townsend et al., 1986) whereas the DNA-B provides movement functions (Noueiry et al., 1994;Frischmuth et al., 2007;Sanderfoot and Lazarowitz, 1995). The phenomenon of independent infection by DNA-A was attributed to the method of inoculation (using Agrobacterium) since it did not occur following inoculation by the biolistic method. ...
Thesis
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The legume yellow mosaic viruses (LYMVs) are members of the proposed sub-genus ―Legumovirus‖ within the genus Begomovirus of the family Geminiviridae; single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. The legumoviruses are evolutionarily distinct from all other begomoviruses and are of interest for this reason as well as for the losses they cause to leguminous crops across southern Asia. There are four LYMVs (Mungbean yellow mosaic virus [MYMV], Mungbean yellow mosaic India virus [MYMIV], Dolichos yellow mosaic virus [DoYMV] and Horsegram yellow mosaic virus [HgYMV]) that have been shown to be responsible for yellow mosaic disease (YMD) of legumes across southern Asia. An analysis of the genetic diversity of LYMVs across Pakistan was conducted. Samples were collected from 11 districts across Pakistan and 48 full-length begomovirus components (25 DNA-A, 21 DNA-B) were cloned and sequenced in their entirety. Analysis of these sequences showed that MYMIV is the most prevalent causal agent of YMD in legume crops in Pakistan and shows phylogeographic segregation; no other virus species was shown to cause YMD of leguminous crops. MYMV, which is the major pathogen responsible for YMD of legumes in southern and western India, was also identified in Pakistan but this was identified only in a leguminous weed, Rhynchosia capitata. In addition a novel begomovirus, with less than 70% nucleotide sequence identity to all other begomoviruses, was isolated from another leguminous weed, Rhynchosia minima. This newly identified begomovirus was shown to belong to the LYMV cluster and was tentatively named Rhynchosia yellow mosaic virus (RhYMV). As well as the LYMV components, two virus species not commonly identified in legumes (Pedilanthus leaf curl virus [PedLCV] and Papaya leaf curl virus [PaLCuV]) as well as a betasatellite (Tobacco leaf curl betasatellite [TbLCB]) were isolated from some legumes infected with MYMIV and showing typical YMD symptoms. Constructs for the Agrobacterium-mediated inoculation of representative isolates of all begomovirus species and two isolates of MYMIV were produced. The MYMV was shown to infect blackgram, inducing very mild symptoms. RhYMV was shown to be infectious to some lines of soybean but not any of the other leguminous crops tested. The limited host range of these two viruses possibly explains their absence in crops. In contrast, two isolates of MYMIV, isolated from soybean (MYMIV-Sb) and mungbean (MYMIV-Mg) showed differing infectivities to legumes. The soybean isolate showed high levels of infectivity to soybean but low levels in blackgram, whereas the mungbean isolate was highly infectious to blackgram but poorly infectious to mungbean. This suggests that isolates of this virus are adapted to distinct hosts. None of the LYMVs examined was infectious to the non-legume Nicotiana benthamiana, a species which is commonly used as an experimentally host for all other dicot-infecting begomoviruses for which infectivity has been investigated. This is the first time this lack of infectivity to N. benthamiana has been reported. Similarly the viruses were not infectious to N. tabacum. The identification of a betasatellite in legumes is of grave concern due to the possibility of it increasing disease severity. TbLCB was shown to have the capacity to be maintained by MYMIV, MYMV and RhYMV, the first time a betasatellite has been shown to be trans-replicated by a LYMV, and to extend the host range of these viruses to N. benthamiana. Although DNA-B of all three viruses had some role to play in such infections (co-inoculation of DNA-B or expression of the DNA-B encoded MP under the control of the 35S promoter increased infectivity of MYMIV DNA-A and TbLCB from 60% to 100%), in the absence of DNA-B, TbLCB complemented the usual functions of DNA-Bs of all three viruses. This ability of TbLCB to complement DNA-B functions was shown to be a function of the only gene product encoded by betasatellites, βC1. Expression of TbLCB βC1 from PVX or transiently under the control of the 35S promoter allowed MYMIV to move systemically in N. benthamiana. However, when βC1 was expressed transiently using the 35S promoter, virus levels in systemically infected tissues were low and no symptoms ensued, suggesting that the βC1 function that assists MYMIV infection acts only at the site of inoculation and does not spread. The results obtained indicate that the lack of the infectivity of MYMIV to N. benthamiana is due to a lack of adaptation of the DNA-B-encoded products to this host. Thus when complemented by TbLCB, or by one of several monopartite begomoviruses (including PedLCV), MYMIV was able to efficiently spread systemically. In addition, plant host-defense mediated by RDR6 was shown to play a small role in limiting infection in N. benthamiana. However, silencing of this gene by VIGS did not allow MYMIV to induce a symptomatic infection. At this time the transformation of many legumes, particularly the grain legumes, is problematic, precluding the use of legume-transformation for the study of pathogen derived resistance to the LYMVs. Using a novel system, based upon the complementation of MYMIV movement using TbLCB, N. benthamiana was shown to potentially be a useful model host for such studies. Using transient expression of an antisense Rep gene construct, the infectivity of MYMIV (in the presence of TbLCB) was reduced by 90%. This indicates that RNAi may be a useful tool in reducing losses to LYMVs across Asia and that the betasatellite assisted infectivity system provides a means of selecting the most efficient constructs prior to efficient transformation protocols for local legume species becoming available.
... Although both DNA components are required to infect plants, it has been shown by constructing transgenic plants with dimeric inserts of TGMV DNA components that DNA A, but not DNA B, can replicate and produce virus particles in the absence of the other DNA species (Rogers et al., 1986; Sunter et al., 1987). Similarly it has been shown that DNA 1 of ACMV (equivalent to DNA A of TGMV) can replicate independently of DNA 2 (equivalent to DNA B of TGMV) in protoplasts (Townsend et al., 1986). Thus DNA A encodes the coat protein and proteins required for virus DNA replication and DNA B is required for cell-to-cell spread of virus particles, but the roles of the translation products of individual ORFs in these processes are not known. ...
Article
SUMMARY Insertion mutations introduced in vitro into cloned DNA of tomato golden mosaic virus that considerably shortened the length of the open reading frames (ORFs) ALl, AL2/AL3, BL1 or BRI, abolished the ability of the DNA to infect Nicotiana benthamiana seedlings. Mutants in which ORF AR1 was similarly shortened by an insertion or a 28 bp deletion were infectious, showing that the formation of coat protein or virions is not required for replication and systemic spread of virus DNA, although the appearance of symptoms was delayed in infections with the deletion mutant. Mutants with larger deletions (178 bp to 603 bp) in ORF AR1 were not infectious. Infections could be initiated with mixtures of ALl and AL2/AL3 mutants, or BL1 and BRI mutants, primarily as a result of complementation, although a low proportion of wild-type DNA A molecules regenerated by recombination or reversion was detected in the progeny of infection with the DNA A mutants.
... Perhaps the introduced T G M V B D N A does not physically survive of cytoplasm-to-nucleus traverse in these ceils. A n o t h e r possibility is that, since the replication of geminiviruses must occur in actively growing, dividing cells [22] , the T G M V B D N A is seldom introduced into these competent cells. A final simple explanation would be that the cells in which the T G M V A D N A replicates are rapidly killed. ...
Article
Full-text available
We have adapted the "agroinfection" procedure of Grimsley and co-workers [4,5] to develop a simple, efficient, reproducible infectivity assay for the insect-transmitted, split-genome geminivirus, tomato golden mosaic virus (TGMV). Agrobacterium T-DNA vectors provide efficient delivery of both components of TGMV when used in mixed inoculation of wild-type host plants. A greater increase in infection efficiency can be obtained by Agrobacterium delivery of the TGMV A component to "permissive" transgenic plants. These "permissive" plants contain multiple tandem copies of the B component integrated into the host genome. An inoculum containing as few as 2000 Agrobacterium cells can produce 100% infection under these conditions. Further, our results show that there is a marked effect of the configuration of the TGMV A components within the T-DNA vector on time of symptom development. We have also found that transgenic plants carrying tandem copies of the A component do not complement the B component. Possible mechanisms to explain these results and the potential use of this system to further study the functions of the geminivirus components in infection are discussed.
... ulating gene expression. Evidence that AV2 the second viral sense ORF encodes a movement protein (MP) has been demonstrated byPadidam et al. (1995b) who observed only very low levels of ss and dsDNA in N. benthamiana plants inoculated with infectious ToLCV clones. By contrast inoculated protoplasts accumulated both ss and dsDNA to wild-type levels.Townsend et al. (1986) demonstrated using ACMV/KE that virus replication is controlled from DNA-A, subsequent studies have shown that REP is required for WTG ...
Thesis
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The cassava mosaic disease (CMD) occurs in all cassava growing areas of Africa and the Indian sub-continent. CMD is caused by at least three different geminiviruses in Africa, African cassava mosaic virus (ACMV), East African cassava mosaic virus (ACMV) and South African cassava mosaic virus (SACMV) and by Indian cassava mosaic virus (ICMV) in the Indian sub-continent. These viruses are spread naturally by their whitefly (Bemisia tabaci Gennadius) vectors and through infected cuttings which are routinely used for cassava propagation. The viruses contain two genomic components, DNA-A and DNA-B and belong to the genus Begomovirus of the family Geminiviridae. Manihot spp. are the only known natural hosts of the cassava mosaic geminiviruses (CMGs). Yield losses due to infection by CMGs vary considerably and depend on cultivar, time of infection, and environmental conditions. The yield losses range from 5% to a total loss of the crop. The spread of CMD under differing conditions in Cameroon was studied by planting CMD-free cuttings of six cassava cultivars at Ekona (450 metre above sea level, masl), Njombe (80 masl) and Dschang (1300 masl). The spread of the disease was assessed by recording incidence per month while the whitefly populations were recorded weekly. Spread was most rapid in all cultivars at Ekona followed by Njombe, both in the lowland forest zone, whereas little spread occurred at Dschang in the savannah area. Improved (with resistance to CMGs) cultivars showed a comparatively higher level of resistance to CMD than the local cultivars at all the locations tested. However, the severity of the symptoms on infected plants was greater at Dschang compared with Ekona and Njombe. Spread was less in cassava intercropped with maize than when intercropped with cowpea or in monoculture due probably to a lower number of whiteflies in maize containing plots. However, intercropping did not influence symptom severity in infected cassava plants. Field experiments were conducted in contrasting ecological conditions at two sites in southern Cameroon to study the reversion phenomenon - the extent to which the virus causing CMD was absent from cuttings collected from CMD-affected plants. The proportions of disease-free plants developing from the different batches of cuttings were recorded at one month after planting to indicate the extent of reversion. This was v significantly less frequent in susceptible cultivars than in improved cultivars. Reversion was also significantly higher in cuttings collected from the youngest portions of the stem compared with older portions and with short cuttings compared with long ones. There was an inverse relationship between symptom severity in different cultivars and the extent of reversion. In order to identify viruses causing CMD, cuttings were collected from plants displaying CMD symptoms in Cameroon and tested using the polymerase chain reaction (PCR) with primers specific for the coat protein (CP) gene of ACMV and EACMV. Out of a total of 314 samples tested 280 were infected by ACMV/CM alone, 37 samples were co-infected by ACMV/CM and EACMV/CM and five samples were infected by EACMV/CM alone. All samples infected by EACMV/CM were collected in the rainforest part of the country and none of the samples collected in the savannah region was infected by EACMV/CM. The intergenic region of 13 isolates, the coat protein (CP) gene of 22 isolates and 11 full-length sequences of both viruses were determined. Sequence comparisons with published CMGs sequences showed that only ACMV/CM and EACMV/CM occur in Cameroon. Comparison of the full-length components of ACMV/CM isolates showed that ACMV/CM was highly similar to ACMV from Kenya (ACMV/KE) and Nigeria (ACMV/NG). However, evidence for a unique double nucleotide recombination in EACMV was noted; recombination in DNA-A was within the region containing AC2-AC3 open reading frames (ORFs), and in DNA-B also evidence for recombination was in the BC1 ORF. Cloned components of both ACMV/CM and EACMV/CM were infectious to Nicotiana benthamiana although EACMV/CM produced apparently asymptomatic phenotypes. Southern blot analysis of viral DNAs from N. benthamiana plants infected with ACMV/CM and/or EACMV/CM showed that there were significantly higher levels of accumulation of both ACMV/CM components, and to a lesser extent of EACMV/CM components, in mixed infected plants than in singly infected plants. This observation confirmed evidence of a synergistic interaction between the two viruses as displayed in mixed infected N. benthamiana and cassava plants. Pseudo-recombinants made between the two components of ACMV/CM and EACMV/CM were infectious to N. benthamiana suggesting trans-complementation vi between these two viruses, and therefore may at least partly, explain the synergism occurring between the two viruses. Infection of cassava by ACMV/CM and EACMV/CM was demonstrated by inoculating clones of both viruses independently to cassava plantlets (cultivar TMS 60444) using the biolistic gun procedure. The symptoms displayed were typical of CMD symptoms showing therefore that both viruses are causal agents of the CMD in Cameroon.
... 4 and 5 in Gardiner et al., 1988).Fig. 2, compare lane 3 with lanes 6 and 10).Rogers et al., 1986; Townsend et al., 1986; Elmer et al., 1988b), Etessami et al., 1989 ...
Article
SUMMARY We have investigated the behaviour of coat protein deletion mutants of the geminivirus African cassava mosaic virus (ACMV) when introduced into Nicotiana benthamiana by agroinoculation. In dividing callus tissue, replicating mutant DNA A, in the absence of DNA B, remained subgenomic in size. However, systemic infection of plants was associated with the rapid reversion of the deletion mutants to native component size, as happened when the mutants were introduced into the host by more conventional mechanical inoculation procedures. The results contrast with those reported for tomato golden mosaic virus (TGMV) for which similar mutants, agroinoculated into N. benthamiana, remained subgenomic. The results indicate that the inoculation route is not responsible for the different behaviour of ACMV and TGMV mutants and illustrate a significant difference between the interaction of these otherwise closely related geminiviruses with a common host that might be exploited for the investigation of the determinants of DNA replication and spread.
... The A component of bipartite genome geminiviruses encodes all of the viral proteins required for replication and encapsidation (Rogers et ai,, 1986;Townsend et 1986; tions necessary for the spread of infection in plants (Brough et al., 1988;Etessami et al., 1988). Two genes, AL7 and AL3, on the A component encode viral proteins involved in replication. ...
Article
Tomato golden mosaic virus (TGMV) and bean golden mosaic virus (BGMV) are closely related geminiviruses with bipartite genomes. The A and B DNA components of each virus have cis-acting sequences necessary for replication, and their A components encode trans-acting factors are required for this process. We showed that virus-specific interactions between the cis- and trans-acting functions are required for TGMV and BGMV replication in tobacco protoplasts. We also demonstrated that, similar to the essential TGMV AL1 replication protein, BGMV AL1 binds specifically to its origin in vitro and that neither TGMV nor BGMV AL1 proteins bind to the heterologous origin. The in vitro AL1 binding specificities of the B components were exchanged by site-directed mutagenesis, but the resulting mutants were not replicated by either A component. These results showed that the high-affinity AL1 binding site is necessary but not sufficient for virus-specific origin activity in vivo. Geminivirus genomes also contain a stem-loop sequence that is required for origin function. A BGMV B mutant with the TGMV stem-loop sequence was replicated by BGMV A, indicating that BGMV AL1 does not discriminate between the two sequences. A BGMV B double mutant, with the TGMV AL1 binding site and stem-loop sequences, was not replicated by either A component, indicating that an additional element in the TGMV origin is required for productive interaction with TGMV AL1. These results suggested that geminivirus replication origins are composed of at least three functional modules: (1) a putative stem-loop structure that is required for replication but does not contribute to virus-specific recognition of the origin, (2) a specific high-affinity binding site for the AL1 protein, and (3) at least one additional element that contributes to specific origin recognition by viral trans-acting factors.
... Agrobacterium-mediated application of geminivirus vectors At the beginning of geminivirus vector development, directly purified recombinant geminivirus vectors were used. The isolated recombinant geminivirus genomes were directly inoculated onto plants (Howell et al. 1980;Townsend et al. 1986). That was a rather complex process involving largescale in vitro viral genome preparation. ...
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Being a major class of single-stranded DNA viruses, geminiviruses are mostly studied due to their catastrophic infectious effect on crops. These DNA viruses are characteristic for their ability in quickly multiplying viral genetic materials without integrating into the genome of plants, which makes them ideal for developing viral vectors for plants bioengineering. Geminivirus-derived vectors can be classified into expression vectors and virus-induced gene silencing (VIGS) vectors. Details of the design, construction, application and improvements of these geminivirus vectors are summarized and discussed.
... B components constitute bipartite genome A is essential for replication Townsend et al., 1986;., 1987) while DNA-B plays a role in systemic movement and symptom production (Etessami et al., 1988; ., 1994). The begomovirus replication cycles rely tes and occur within the nucleus of the infected cell through two basic stages: conversion of ssDNA to dsDNA intermediates and rolling circle replication (RCR) (Gutierrez, 2002) ( (Fig. 1b). ...
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More than 80% of the known geminiviruses are transmitted by whiteflies (Bemisia tabaci Gennadius) and belong to the genus Begomovirus, which mostly have bipartite genomes designated as DNA-A and DNA-B and infect dicotyledenous plants although numerous begomovirus with a monopartite genome occur in the Old World and there are some of which a single components is not infectious yet no DNA-B component has been found. There have been several of reports of satellite molecules associated with begomoviruses. Genome ORFs are plays important roles for host range determination, virus symptom development & severity, virus movement and virus replication. The frequency with which new begomoviruses are appearing shows that these viruses are still evolving and pose a serious threat to sustainable agriculture, particularly in the tropics and sub-tropics. In recent years, some begomoviruses have also moved to temperate regions causing concern in the production of vegetables in greenhouses. In this review we have discus about the genome organization of begomovirus, its ORFs and their possible pathogenesis n the basis of research findings.
... et al., (2000). DNA A is essential for replication and encapsidation (Rogers et al., 1986;Townsend et al., 1986). In DNA A (Table 2. ORFs; two ORFs, ORF AC1 and AC3, encode replication initiation protein (Rep) and ...
... DNA A and DNA B encode genes for different proteins necessary for viral encapsidation, replication and movement.Townsend et al., 1986). In DNA A (Table 2.6), viral sense strand has two ORFs, ORF AV2, pre-coat protein and coat protein, ORF AV1. In complementary sense, there are totally fiveORFs; two ORFs, ORF AC1 and AC3, encode replication initiation protein (Rep) and replication enhancer protein (REn) respectively (Elmer et al., 1988a;Etessami et al., 1991). One more ...
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Optimal conditions for electroporation have been determined using inoculation of brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV) and its RNA into protoplasts of Nicotiana tabacum and N. plumbaginifolia. The most satisfactory medium was 0.5-0.7 M mannitol; calcium ions were toxic and other electrolytes were not helpful during electroporation. Brief pulses (ca. 10 microsec) were less destructive to the protoplasts than longer ones (ca. 10 msec) and gave high percentage infections with CCMV RNA. RNA entered the protoplasts only if present during the voltage pulse. Optimal voltage depended on the sample size, interelectrode distance, and pulse duration. A 50-nF capacitor discharging a 5- to 10-microsec pulse through a 1-ml sample in 0.7 M mannitol with a 4-mm interelectrode distance gave maximum infection with minimal protoplast damage at 2.5 kV/cm. A single pulse was sufficient; multiple pulses slightly increased infection. Electroporation of viral RNA was at least as effective as inoculation in the presence of polyethylene glycol. Positively charged BMV also infected readily but negatively charged CCMV only poorly.
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The inability to transmit progeny virus resulting from the cloned components of an isolate of African cassava mosaic virus originating from Kenya (ACMV-K) has been shown to be due to defects in both genomic components. This was achieved by the production of infectious pseudorecombinants between ACMV-K and the cloned components of a whitefly-transmissible ACMV isolate originating from Nigeria (ACMV-NOg). The exchange of gene fragments between ACMV-K and ACMV-NOg has been used to demonstrate that the defects responsible for lack of transmissibility reside on the coat protein and DNA-B C1 gene of ACMV-K. The significance of these finding with respect to the present understanding of the function of these gene products are discussed.
Article
Two full length copies of Cassava latent virus (CLV) DNA1 were cloned in head to tail arrangement on a plant expression vector to evaluate the potential of CLV for the development of an extrachromosomal vector system in plants. After direct transfer of the plasmid into protoplasts of Nicotiana tabacum cv. Petit Havana SR1 extrachromosomal single-stranded (ss) and double-stranded (ds) forms of DNA1 appeared after the first cell division of protoplasts. The extrachromosomal copies could also be detected within transformants which has been regenerated from kanamycin-resistant calli. The CLV-harbouring transformants do not display any symptoms usually observed after CLV infection. Stable conservation of extrachromosomal DNA1 was observed in F1 plants derived from self-pollination and in plants regenerated from protoplasts of transformants. Our data show that dimer constructs of CLV DNA1 are attractive candidates for an extrachromosomal plant vector system.
Article
Geminiviruses have small, single-stranded DNA genomes that replicate through double-stranded intermediates in the nuclei of infected plant cells. Viral double-stranded DNA also assembles into minichromosomes and is transcribed in infected cells. Geminiviruses encode only a few proteins for their replication and transcription and rely on host enzymes for these processes. However, most plant cells, which have exited the cell cycle and undergone differentiation, do not contain the replicative enzymes necessary for viral DNA synthesis. To overcome this barrier, geminiviruses induce the accumulation of DNA replication machinery in mature plant cells, most likely by modifying cell cycle and transcriptional controls. In animals, several DNA viruses depend on host replication and transcription machinery and can alter their hosts to create an environment that facilitates efficient viral replication. Analysis of these viruses and their proteins has contributed significantly to our understanding of DNA replication, transcription, and cell cycle regulation in mammalian cells. Geminiviruses have the same potential for plant systems. Plants offer many advantages for these types of studies, including ease of transformation, well-defined cell populations and developmental programs, and greater tolerance of cell cycle perturbation and polyploidy. Our knowledge of the molecular and cellular events that mediate geminivirus infection has increased significantly during recent years. The goal of this review is to summarize recent research addressing geminivirus DNA replication and its integration with transcriptional and cell cycle regulatory processes.
Article
The replication of shuttle vectors derived from Wheat Dwarf Virus, a monopartlte geminivirus, was studied in cultured maize endosperm cells, and In the Black Mexican Sweet (BMS) maize cell line. Using In vivo labeling and DNA methylation analysis, we showed that replication was initiated within 24 hrs after transfection, and did not require cell division in both cell lines. Copy numbers of 30,000 ds DNA molecules per cell were observed in endosperm cells after three days. The replication protein was shown to act in trans, since the wild type gene of the shuttle vector enabled replication-deficient vectors carrying mutated genes to replicate. These properties suggest that WDV may have similar applications in plants as SV40 in mammalian cells.
Article
The replication-associated protein (Rep) of geminiviruses, single-stranded DNA viruses of higher plants, is essential for virus replication. Since these viruses do not encode their own polymerases, Rep induces differentiated plant cells to reenter the cell cycle by interacting with the plant homologues of retinoblastoma proteins in order to activate the host DNA synthesis machinery. We have used fission yeast (Schizosaccharomyces pombe) as a model organism to analyze the impact of ectopically expressed African cassava mosaic virus Rep protein on the cell division cycle in closer detail. Upon expression, Rep showed its characteristic DNA cleavage activity, and about 10% of the cells exhibited morphological changes. They were elongated threefold, on average, and possessed a single but enlarged and less compact nucleus in comparison to noninduced or vector-only control cells. Flow cytometry of Rep-expressing cultures revealed a distinct subpopulation of Rep protein-containing cells with aberrant morphology. The other 90% of the cells were indistinguishable from control cells, and no Rep was detectable. Rep-expressing cells exhibited DNA contents beyond 2C, indicating ongoing replication without intervening mitosis. Because a second open reading frame (ORF), AC4, is present within the Rep gene, the role of AC4 was examined by destroying its start codon within the AC1 ORF. The results confirmed that Rep is necessary and sufficient to induce rereplication in fission yeast. The unique potential of this well-investigated model for dissecting the cell cycle control by geminiviral proteins is discussed.
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Mutants of the monopartite geminivirus beet curly top virus have been screened for infectivity and symptom development in Nicotiana benthamiana and Beta vulgaris, for replication competence in N. benthamiana leaf discs, and for transmission by the leafhopper Circulifer tenellus. Disruption of open reading frame (ORF) V2 by the introduction of a termination codon resulted in symptomless infection of N. benthamiana associated with low levels of virus and reduced single-stranded (ss) DNA and prevented systemic infection of B. vulgaris. Reduced levels of ssDNA were produced by the mutant in N. benthamiana leaf discs, suggesting that V2 affects the synthesis or accumulation of this viral DNA form. Mutants in which ORF C2 had been truncated by the introduction of termination codons or by frame-shifting remained highly infectious and induced severe symptoms in both N. benthamiana and B. vulgaris. Similarly, a mutant containing a termination codon within ORF C3 was highly infectious and induced severe symptoms in N. benthamiana although infectivity in B. vulgaris was greatly reduced, symptoms were extremely mild, and virus levels were low. A synergistic effect of a double mutation in ORFs C2 and C3, manifested by the inability of mutants to systemically infect N. benthamiana and the production of reduced amounts of ssDNA in N. benthamiana leaf discs, suggests that both ORFs are functional in this host. A mutant containing a termination codon within the 5' terminus of ORF C4 produced severe symptoms in both N. benthamiana and B. vulgaris resembling those induced by wild-type virus. Comparison with the phenotypes of previously characterized ORF C4 mutants suggests that a conserved core sequence of this ORF is an important symptom determinant. ORF C2, C3, and C4 mutants produced virus particles and were transmitted by C. tenellus, eliminating agroinoculation as a contributory factor to the mutant phenotypes. Our results are compared with those derived from mutagenesis studies on related bipartite geminiviruses.
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Two beet curly top virus (BCTV) mutants have been constructed in vitro that contain G-to-T transversions either at nucleotide 2682 or at nucleotide 2802 within the overlapping open reading frames (ORFs) C1 and C4. The mutations introduce termination codons in ORF C4 without affecting the amino acids encoded by ORF C1. When agroinoculated into Nicotiana benthamiana the mutants caused stunting and yellowing of the plant and downward leaf curl but not the vein swelling and upward leaf curl symptoms that are characteristic of wild-type BCTV infection in this host. Levels of viral single- and double-stranded DNA forms were similar in mutant and wild-type infections. Symptoms induced by one such mutant in Nicotiana clevelandii and Datura stramonium were less severe than those in wild-type infections and were again qualitatively distinct. The mutants caused symptomless infections in Beta vulgaris, contrasting with stunting, severe leaf curl, and vein swelling symptoms associated with wild-type infection of this host. The levels of mutant DNA in newly expanding asymptomatic leaves frequently reached those of wild-type virus in leaves showing severe symptoms. The results suggest that ORF C4 encodes a protein that is a major determinant of pathogenesis that might affect the hyperplastic response of the host to BCTV infection.
Article
Plant viruses encode proteins that mediate their movement through the host plant leading to the establishment of a systemic infection. We have analyzed the effect of tomato golden mosaic virus (TGMV) genes BL1 and BR1, which are thought to be involved in the process of virus movement, on the infectivity of African cassava mosaic virus (ACMV) in Nicotiana benthamiana. Recombinant genomes were constructed by replacing the ACMV coat protein coding sequence with those of either BL1 or BR1. Replication of recombinants containing BL1 and BR1 coding sequences in the sense orientation with respect to the coat protein promoter was detected in the inoculated leaves only when the constructs were co-inoculated, suggesting that both genes are being expressed and act in a cooperative manner. Co-inoculated recombinants induced localized symptoms on inoculated leaves but did not spread systematically, either because of a defect in BL1 and/or BR1 expression or due to the inability of the TGMV gene products to functionally complement their ACMV counterparts. Systemic spread of ACMV was inhibited when the recombinant containing the BL1 coding sequence in the sense, but not in the antisense, orientation was co-inoculated with ACMV DNA B. Disruption of the BL1 coding sequence by a frameshift mutation restored the ability of the recombinant to spread systemically, suggesting that the gene product is responsible for the inhibitory effect. The inhibitory phenotype was mimicked by a chimera containing amino-terminal sequences of TGMV BL1 and carboxy-terminal sequences of its ACMV homologue, BC1. The chimera has characteristics of a dominant negative mutant. We suggest that dominant negative mutants of virus movement genes may provide a novel source for virus resistance genes.
Article
Subgenomic viral DNA is accumulated in Nicotiana benthamiana and Beta vulgaris plants agroinoculated with the geminivirus beet curly top virus. The subgenomic DNA is more abundant in N. benthamiana and is distributed between two broad size groups in this host. Six unique examples, ranging in size from 887 to 1311 nucleotides, have been cloned from viral double-stranded DNA purified from N. benthamiana and analyzed by sequence determination. Deletions are distributed throughout most of the genome and only nucleotides 2946-410 are represented in all subgenomic DNAs. Comparison with a previously characterized subgenomic DNA suggests that cis-acting signals necessary for viral DNA replication are located in a predominantly intergenic region between nucleotides 2946-308.
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The virological situation of cassava in Africa is increasing in complexity due to the number and types of viruses isolated from different locations within the continent. Here, we report the complete nucleotide sequences of both A and B components of two geminivirus species infecting cassava in the Ivory Coast and review the current knowledge of the molecular and biological diversity of the African cassava geminiviruses. As a whole, newly obtained sequences are compared with those of the African cassava mosaic geminiviruses identified to date. Results indicate that all isolates of African cassava mosaic virus (ACMV), irrespective of their geographical origin are clustered together with little or no variation in their genomic sequence. On the contrary, the genomes of the East African cassava mosaic virus (EACMV) are more genetically diverse due to the frequent occurrence of recombinations within their two components. Indeed, the EACMV-like viruses vary so much that their classification is becoming problematic. In addition, there is also a large range of phenotypic symptom variation for each of these virus species, irrespective of the location of isolation. Furthermore, it has been shown that ACMV and EACMV can be synergistic in cassava, resulting in a greater DNA accumulation and consequently inducing severe symptoms. For all these reasons, this paper initiates a discussion concerning the species demarcation for cassava geminivirus.
Article
The geminiviruses are single-stranded (ss) DNA viruses with genomes comprising one or two circles of 2.5–3.0Kb. The biological and molecular properties of some members of the group have been reviewed recently (Harrison, 1985; Stanley and Davies, 1985; Stanley, 1985). The number of known viruses is increasing as a consequence of intensifying interest in the group. Three main areas of research offer particularly exciting possibilities.
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The dicot-infecting, whitefly-transmitted (WFT) geminiviruses have emerged as important plant viral pathogens during the last decade, resulting in pandemic disease conditions in subtropical and fringe-temperate locales. Substantial quantities of food and fiber are grown for fresh market and/or for processing in these areas, and these commodities are destined for either local consumption or for export. A heavy reliance on agricultural commodities for economic prosperity in many subtropical areas, and on a surplus production and subsequent export from certain subtropical and temperate locales, collectively, emphasize the serious threat posed by unprecedented geminivirus epidemics in a wide range of food and fiber crops on a global basis. Hence, reducing crop damage and economic losses due to WFT geminivirus epidemics has far reaching implications toward the sustainability of agriculture as a vital link toward creating opportunities and improving the quality of life worldwide.
Article
The movement of plant viruses from cell to cell is controlled by the action of virus‐encoded proteins. With the realization that understanding the mechanism involved will lead to a broad comprehension of virus disease and may open new ways to engineer virus resistance, this phenomenon has recently received much attention. Sequence homologies between different movement proteins indicates that they may share a common origin and some functional similarities. However, groupings based upon protein structure, function as determined by complementation in double infections or host range, are not correlated. The proteins influence plasmodesmatal structure to allow the passage of viral genomes or virus particles, and one protein has been shown to have single‐stranded nucleic acid binding properties. Transgenic plants, heterologous protein‐expression systems, and cell biological approaches will determine how these proteins interact with host components to elicit complex structural changes which result in systemic virus invasion.
Article
Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.
Article
When callus tissue was cultured from leaf pieces taken from a Nicotiana tabacum cv. Xanthi nc. plant systemically infected with tomato golden mosaic virus (TGMV), TGMV-specific DNA persisted for up to 6 months in culture. Analysis of TGMV-specific intracellular DNA forms indicated a decrease in double-stranded relative to single-stranded forms and an increase in sub-genomic relative to genomic single-stranded DNA species in the callus tissue compared to those in the original leaf explant. The implications of the results with regard to TGMV replication are discussed.
Article
Subterranean clover stunt virus (SCSV) was previously found to be representative of a new type of single-stranded DNA virus. Purified SCSV particles did not infect subterranean clover when various attempts were made to transmit them to this host using aphids (Aphis craccivora) which had previously been fed on preparations of the virus. To demonstrate that purified preparations of SCSV are capable of replication, pea and subterranean clover protoplasts were inoculated with the virus using PEG treatment or electroporation, and maintained for up to 13 days. Up to 40% of the protoplasts survived after PEG treatment but less than 10% survived after electroporation. Both enzyme-linked immunosorbent assay (ELISA) and nucleic acid hybridisation detected de novo synthesis of SCSV in the inoculated protoplasts. The amount of SCSV coat protein detected by ELISA decreased from day 0 to day 3 post-inoculation but increased thereafter over several days to a maximum at about 10 days. Nucleic acid hybridisation studies using strand-specific probes showed that the kinetics of synthesis of the virion and its complementary strand nucleic acid corresponded with the kinetics of accumulation of SCSV antigens. These results suggest that purified SCSV particles are capable of replication in transient protoplast systems. The protoplast assay could be used to characterise new or altered segments or functional domains of the SCSV genome and for the development of SCSV genome as a vector for foreign gene expression in plants.
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Directed genetic modification of plants requires the transmission of specific foreign DNA to the host plant, where the DNA is incorporated and stably maintained in the plant nuclear genome. Although there are several DNA transmission techniques, the transfer of DNA mediated through the natural transmission of T-DNA of Ti plasmids of Agrobacterium tumefaciens has proven relatively efficient and practical. The initial processing and transmission of the T-DNA requires genes situated on the Ti plasmid and on the bacterial chromosome. The T-DNA processing genes on the Ti plasmid are located in the virulence (Vir) region. The virD2 gene of the virD operon, which appears to be involved in the formation of T-DNA intermediates by encoding a specific endonuclease, is constitutively synthesized in a permissive mutant ros of A. tumefaciens. Gene fusions to reporter genes such as the luciferase cassette of Vibrio fischeri made it possible to measure the expression of Vir genes during the on-going interaction between A. tumefaciens and carrot disks. Using phenolic inducers such as acetosyringone, virB, virC, virD and virE were identified as inducible operons of the Vir region in the presence of virA, virG and inducer. virC and virD are also controlled by the ros gene of the chromosome as mutations in the ros gene results in the constitutive expression of these operons. This has enabled us to study the formation of double stranded T-DNA intermediates in the absence of acetosyringone or other phenolic inducers. Integration of the T-DNA was directly observed in Haplopappus gracilis chromosome A by in situ hybridization analysis.
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Infectious clones of bean golden mosaic geminivirus (BGMV) can be inoculated onto the economically important host Phaseolus vulgaris, causing a reaction indistinguishable from that seen in the field. To elucidate the functions of the BGMV genome in beans, six clones, each with a point mutation, insertion, or deletion in the common region or BL1 open reading frame (ORF) of DNA-B of BGMV (Guatemalan isolate), were constructed. These clones were coinoculated with wild-type DNA-A of BGMV into bean radicles (P. vulgaris) by particle acceleration to determine the effect of the mutations on infectivity. Four mutants caused symptoms ranging from very mild mosaic to wild-type mosaic [...]
Article
The plant pathogenic single‐strand DNA‐containing geminiviruses have been the recent focus of intense investigation, owing both to their agronomic importance and to their potential as vectors for the expression of foreign genes in plants. Molecular genetic studies have provided detailed information on the genomic organization of many of these viruses. A greater genetic complexity has been demonstrated among the members of this viral family than had previously been suspected, as well as an apparently rapid rate of evolution of genetic diversity. We now recognize fundamental differences in the genome structure and organization of the whitefly‐ and leafhopper‐transmitted viruses, as well as among those geminiviruses infecting dicotyledonous or monocotyledonous hosts. This knowledge has provided new insights into the evolution of these viruses. The viral genes involved in replication and in systemic movement in the plant have been defined, and viral origins for single‐strand (ss) and double‐strand (ds) DNA replication have been mapped to small nucleotide regions. With the structural features of the viral genomes now well defined, current efforts are focused on elucidating the molecular aspects of viral gene regulation and interactions with host‐cell components that lead to the production of disease. Recent progress in determining the mechanism of replication and systemic movement and the contributions of these to symptom and disease development are discussed in the context of the potential for genetically engineering disease‐resistant plants.
Article
Geminiviruses are unique plant DNA viruses that frequently cause significant yield reductions in a wide range of cereal, vegetable and fibre crops. Encouraging progress has recently been made towards the control of geminiviruses that infect dicotyledonous plants. Under laboratory conditions, defective interfering (DI) DNA and antisense strategies, both directed against viral DNA replication, have been used to protect Nicotiana spp. against the bipartite geminivirus African cassava mosaic virus and tomato golden mosaic virus. The use of dominant negative mutants of virus systemic movement might also be adapted as a novel strategy for the control of these important pathogens.
Article
Tomato golden mosaic virus (TGMV), a member of the geminivirus group, has a genome consisting of two DNA molecules designated the A and B components. Both are required for infectivity in healthy plants, although the former has been shown to replicate independently in transgenic plants containing tandem direct repeats of the A genome component. In the studies presented here, petunia plants transgenic for either both components (A×B hybrids) or the A component alone were examined for the presence of virus particles and encapsidated, single stranded viral DNA. The results of DNase protection experiments and direct observation of extracts from transgenic plants by electron microscopy indicate that single stranded TGMV DNA is in both cases packaged into paired particles identical to those obtained from virus-infected plants. DNase-treated virions isolated from A×B hybrid petunia are infectious when inoculated onto healthy Nicotiana benthamiana. Likewise, virions obtained from transgenic A petunia are infectious for plants transgenic for the B component. Our observations of TGMV replication in transgenic plants indicate that TGMV A DNA encodes all viral functions necessary for the replication and encapsidation of viral DNA. The possible role of the B component in TGMV replication is discussed.
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Introduction Botany and Distribution Production and Use Constraints to Cassava Use and Cultivation Traditional Breeding and Biotechnology in Cassava Improvement Molecular Genetics Tissue Culture and Regeneration Genetic Transformation Applications and Potential Molecular Biology Future Prospects Literature Cited
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The geminiviruses are a unique group of infectious agents that replicate and cause disease in a wide variety of plant species. Because of their small DNA genomes and their ability to multiply to high copy number, these viruses have the potential to serve as important model systems for the processes of transcription and DNA replication in their hosts. Geminiviruses also have attracted considerable attention due to their potential to be modified for use as amplifiable vectors for stable plant transformation and transient expression assays. As a result, a great deal of effort has been directed toward understanding the molecular biology of these pathogens, and in the past few years considerable progress has been made with several group members. Many excellent reviews documenting this progress are available (Stanley, 1985; Harrison, 1985; Davies et al., 1987; Lazarowitz, 1987; Davies and Stanley, 1989). As this paper is based primarily on recent work done in this laboratory and on earlier work done in collaboration with the laboratory of Dr. Stephen G. Rogers, it will focus mainly on tomato golden mosaic virus and other bipartite geminiviruses. However, pertinent information about other group members will be discussed where appropriate.
Chapter
The first evidence of economic damage to vegetable crops caused by Bemisia tabaci Gennadius in Israel was recorded in 1931 (8), and since 1935 it has been a permanent pest, mainly in the Jordan Valley. The fluctuations in population size in different years were always directly correlated with the damage due to tomato yellow leaf curl virus (TYLCV). A TYLCV-like disease was first reported in Israel in 1939– 1940 and was associated with outbreaks of B. tabaci. The disease involved leaf curling, short internodes, leathery leaves, flower drop, and general dwarfing in comparison with healthy plants (8). Twenty years later, in 1959, when there was a heavy outbreak of B. tabaci in the Jordan and Bet She’an Valleys, the entire tomato crop was completely destroyed by a disease with the following syndrome: “Severe stunting of growth, the shoots are erect, and the leaflets are markedly smaller and misshapen. Those leaflets that appear soon after infection are cupped down and inwards, while subsequently developing leaves are strikingly chlorotic and show an upward curling of the leaflet margins. When young plants are attacked they lose vigor and hardly produce any marketable fruits” (25). The viral nature of the disease and its spread by B. tabaci were confirmed this time. The virus geminate shape was first observed in 1980 in electron microscope thin sections (82), but only in 1988 was TYLCV purified (32). From 1959 to date, TYLCV has spread to every region in Israel and become the major limiting factor in tomato production during both the summer and the winter.
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The yellow mosaic disease (YMD) is known to affect a number of legumes in the country. However, mungbean and urdbean are the most important grain legume crops which are widely affected by this disease. Symptoms of the disease are broadly similar not only in mungbean and urdbean but also in other host plants and hence it is quite often taken as caused by the same virus. However, in the last decade rapid advancement has been made towards understanding of viruses involved in YMD. At present three distinct viruses viz., Mungbean yellow mosaic India virus (MYMIV), Mungbean yellow mosaic virus (MYMV) and Horsegram yellow mosaic virus (HgYMV) are known to cause YMD in mungnean and urdbean. Another common virus, Dolichos yellow mosaic virus (DoYMV) is known to cause YMD in Dolichos beans only. These four viruses along with some recently described begomoviruses from leguminous plant species are together termed as Legume Yellow Mosaic Viruses (LYMVs). Mungbean and urdbean also known as greengram and blackgram, respectively are important food grain legume crops (pulse crops) grown throughout India. These two legumes along with other legumes serve as an important source of protein to a large portion of the Indian population that traditionally practices vegetarianism. Mungbean and urdbean are primarily cultivated as Kharif (rainy season) crop, occupying about 25% of the total area under all pulse crops. With the development of short duration varieties, these crops have been successfully introduced as spring/summer crops in northern plains. In peninsular India, these crops are cultivated not only as Kharif crop but also as Rabi and summer crops. Introduction of these crops especially of urdbean in vast tract of rice fallows in many parts of south India and parts of Orissa is expected to increase contribution of these crops to the national pulse basket. In India mungbean occupied 3.38 million hectares in 2013-14 with a production of 1.61 m/t and a productivity of 474 kg/h. Urdbean occupied 3.06 m hectares with a total production of 1.70 million tonnes and a productivity of 535 kg/hectare. These two crops are known to be affected by yellow mosaic disease (YMD), the single most important biotic constraint to the production of these crops in the country. In this review article, progress made on various aspects of YMD in mungbean and urdbean including diagnostics for the causal viruses has been analyzed.
Article
Three methods utilizing PCR technology for the detection of members of the Geminiviridae are described. As well as being a tool useful for detection, the products of the diagnostic PCR reactions are suitable for further characterization of the viruses. Firstly, a pair of degenerate primers has been used to allow the detection of geminiviruses infecting dicotyledonous plants by PCR amplification. Secondly, a set of specific primers has been used which discriminate between the economically significant maize streak geminivirus and closely related viruses which infect mainly grasses. Finally, a PCR-based method for the production of full-length, infectious geminivirus clones is described. The advantages and disadvantages of PCR compared with other methods of detection and characterization are discussed.
Article
The temporal and spatial patterns of pea seed-borne mosaic potyvirus (PSbMV) and pea early browning tobra-virus (PEBV) accumulation in pea embryos were analyzed using in-situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co-inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.
Article
Agroinoculation of blackgram (Vigna mungo) by the cloned genomic components (DNA-A and DNA-B) of a non-sap transmissible isolate of mungbean yellow mosaic geminivirus (MYMV) has been demonstrated. It has been shown that MYMV has a bipartite genome. Tandem dimeric constructs of DNA-A and DNA-B were infectious when inoculated together and induced systemic yellow mosaic symptoms. Agroinoculation of germinated seeds of blackgram was superior to inoculation of stems of seedlings. Infection of blackgram plants was confirmed by ELISA, dot blot and Southern hybridization. Agroinfected plants contained different forms of DNA characteristic of whitefly transmitted geminiviruses. The virus from agroinfected plants was transmissible by the whitefly Bemisia tabaci.
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It is widely accepted that all divided tendons other than the digital flexors, when sutured and with appropriate postoperative treatment, heal well and that a good return of function can be expected. Clinical and experimental evidence is presented. which indicates that digital flexor tendons also have the ability to heal well, with a good return of function, if suitably treated. The results of repair of 275 divided digital flexor tendons in man are presented.
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Publisher Summary Interest in the molecular biology of the group Geminiviruses has been intense subsequent to the demonstration of a circular single-stranded (ss) DNA genome for several members, imparting obvious advantages for their investigation over RNA-containing viruses with the use of modern techniques for DNA manipulation and analysis. This characteristic genome structure set the geminiviruses apart from all other known eukaryotic viruses, although the genome of porcine circovirus (PCV)—a small isometric animal virus—has since been shown to adopt a similar conformation. As geminivirus proliferation is mainly confined to the nuclei of infected cells, it is anticipated that a study of the replication cycle at the molecular level may not only be informative concerning features of molecular pathology of importance to future virus control—such as the determinants of host range, cell-to-cell spread, cross protection, and transmission vector specificity—but also ultimately contribute to the understanding of the regulation of plant gene expression. The geminiviruses have also attracted a great deal of interest concerning their potential contribution to the development of vectors for the transformation of plants, particularly cereal crops.
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In addition to the major encapsidated DNA species found in preparations of cassava latent virus (genomic DNAs 1 and 2) there are minor DNA populations of twice (dimeric) and approximately half genome length. Both minor species resemble the genomic DNAs in that they are composed of predominantly circular single-stranded DNA. All of these size groups have a corresponding covalently-closed circular double-stranded DNA form in infected tissue. Infectivity studies using cloned DNAs 1 and 2 show that dimeric DNA routinely appears, suggesting it to be an intermediate in the DNA replicative cycle that can be encapsidated at low efficiency. In contrast, half unit length DNA has not yet been detected after multiple passaging of virus derived from the cloned DNA inoculum. Half unit length DNAs appear to be derived exclusively from DNA 2 and consist of a population of molecules exhibiting a relatively specific deletion. As they have an inhibitory effect on virus multiplication, their encapsidated forms are analogous to defective interfering particles associated with other eukaryotic DNA containing viruses. Small primer molecules associated with the genomic single-stranded DNAs, as reported for another geminivirus, have not been detected in CLV.
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Extracts obtained from cells infected with the geminivirus tomato golden mosaic (TGMV) are shown to contain, in addition to viral single-stranded DNA, several novel species of virus-specific single- and double-stranded DNA (as and ds DNA). The results of nuclease studies and electron microscopy suggest that three of the intracellular DNAa are unit-genome length duplexes of closed circular, relaxed circular, and linear form. The remaining ds DNA species are of high molecular weight and appear to be concatamers consisting of two or more unit-length genones. A low molecular weight virus-specific DNA species was also detected. Restriction endonuclease digestion of unit-length circular ds TGMV DNA resulted in fragments whose combined size is twice the unit-genome length. Thus ds TGMV is composed of two components of nearly identical size but different nucleotide sequence.
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More than 10 different dominant transforming genes (oncogenes) have been identified in human tumours. A human bladder carcinoma oncogene, closely related in sequence to retroviral transforming genes, is split into four exons; the first encodes the N-terminal 37 residues of p21, a protein of unknown function. The oncogene is activated by a single point mutation (guanine to thymine) resulting in the change glycine to valine at position 12 of p21 (refs 3, 4). We report here that the amino acid sequence surrounding this residue is highly homologous to the beta-subunit of mitochondrial and bacterial ATP-synthase in the region of the polypeptide that is believed to contribute to nucleotide binding. Thus, p21 may form part of an enzyme that uses purine nucleotides in catalysis. This is consistent with the finding that an equivalent murine oncogene product binds GTP.
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The chapter discusses the potential of plant viruses as vectors for introducing foreign genes into plants. Understanding how viruses function and interact with their hosts may lead to means of controlling some economically important plant diseases; inserting foreign genes may enable a means of improving economically important crops. This chapter examines how molecular biological techniques, especially molecular cloning and in vitro mutation, are being applied to the study of plant viruses, what progress has been made so far, and how gene vectors might be derived from plant viruses. There are two main uses of genetic engineering with regard to plant viruses: cloning of viral sequences in prokaryotic systems and the development of vector systems for eukaryotes. Gaining expression of a foreign gene in a vector derived from a plant virus is still an unattained goal, but considerable progress is made in an area prerequisite to this—namely, understanding the mechanisms of replication, transcription, and translation of virus genes. The vector requires the necessary sequences for replication, transcription (promoters), and other regulatory signals. The successful vector may be a second-generation vector constructed of these basic requirements from different origins. It might comprise a replication signal from one plant virus, promoters from another (even animal) virus, parts of plant genes, and part of a bacterial plasmid to allow amplification and selection in bacteria.
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The nucleotide sequences of infectious cloned DNAs 1 and 2 of a Kenyan isolate of cassava latent virus (CLV) have been determined. Five virus-specific polyadenylated transcripts have been identified and mapped either to the viral or complementary sense DNAs of both components of the CLV genome, confirming that transcription is bidirectional on both DNAs. A major mRNA has been translated in vitro to yield a 30 000 mol. wt. product, which is precipitated by antibodies raised against whole virus, and has been mapped by both the S1 nuclease procedure and hybrid-arrested translation to the long open reading frame (ORF) in the viral sense of DNA 1 which encodes the coat protein. Other transcripts were of sufficient size and appropriate origin to encode at last five potential products.
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