Construction of an Infectious Chimeric Geminivirus by Molecular Cloning Based on Coinfection and Recombination (original) (raw)
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Transmission Specificity and Coinfection of Mastrevirus with Begomovirus
International Journal of Agriculture and Biology, 2017
Coat protein (CP) of geminivirus is not requisite for replication of viral DNA yet it is multifunctional and have crucial role in virus transmission by insect vector, systemic infection and virion development. Although in geminivirus, systemic infection rely both on function of CP and specific host-geminivirus-vector interaction but in bipartite begomoviruses CP is dispensable for systemic infection and progression of symptoms. Nevertheless its role in spreading systemic infection cannot be neglected in monopartite viruses' viz. mastrevirus, begomoviruses and curtoviruses, therefore any alteration in CP gene upshot in a new epidemiological adaptation hence worth to study. So the study was designed to present a better picture to view regarding coinfection within different Geminiviruses belonging to mastrevirus and begomovirus particularly due to vector inspecificity consequently resulted in revolutionary recombination events, which ultimately yield new viruses, have more drastic affects on crops. Moreover, co-evolution of both genera may open new horizons in understanding the complexity of cotton leaf curl disease and then to chalk out strategies to counter this emerging threat in Pakistan.
Journal of Phytopathology, 2015
Whitefly transmitted begomoviruses (family Geminiviridae) are the major reason for significant yield losses of dicotyledonous crops in tropics and subtropics. Okra (Abelmoschus esculentus) is one of the important vegetable crops, and leaf curl disease caused by geminiviruses is the most important limiting factor for its production in Pakistan. Here, we report a new species of okra-infecting begomovirus in south-eastern region of Pakistan and the name Okra enation leaf curl virus (OELCuV) complex is proposed. This okra enation leaf curl disease complex (OELCuD) in Pakistan is found to be associated with Ageratum conyzoides symptomless alphasatellite (ACon-SLA). All efforts to clone the betasatellite were unsuccessful. Comprehensive sequence analyses suggest that intermalvaceous recombination between okra and cotton-infecting begomoviruses resulted in the evolution of the new species. Surprisingly, Bhendi yellow vein mosaic virus (BY-VMV) which has not been reported previously from Pakistan is the major parent while Cotton leaf curl Multan virus (CLCuMV) acts as a distant parent of the virus. Comparative recombination analysis also reveals that okrainfecting begomoviruses from south and north-western India is causing OELCuD in the Pakistan by recombining with CLCuMV at the Rep (1964-1513 nts). Recombination is common among geminiviruses and recombining of BYVMV and CLCuMV resulted in a new species: OELCuV. To the best of our knowledge, this evolution of a new species of okra-infecting begomovirus is the first report of intermalvaceous recombination where Rep acts as the target region.
Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan
Archives of Virology, 2008
Most mastreviruses (family Geminiviridae) infect monocotyledonous hosts and are transmitted by leafhopper vectors. Only two mastrevirus species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, have been identified whose members infect dicotyledonous plants. We have identified two distinct mastreviruses in chickpea stunt disease (CSD)-affected chickpea originating from Pakistan. The first is an isolate of BeYDV, previously only known to occur in South Africa. The second is a member of a new species with the BeYDV isolates as its closest relatives. A PCR-based diagnostic test was developed to differentiate these two virus species. Our results show that BeYDV plays no role in the etiology of CSD in Pakistan, while the second virus occurs widely in chickpea across Pakistan. A genomic clone of the new virus was infectious to chickpea (Cicer arietinum L.) and induced symptoms typical of CSD. We propose the use of the name Chickpea chlorotic dwarf Pakistan virus for the new species. The significance of these findings with respect to our understanding of the evolution, origin and geographic spread of dicot-infecting mastreviruses is discussed.
Genetic diversity of begomoviruses affecting diverse host plants in Periurban areas of Lahore
2019
Certificate Declaration certificate Dedications Acknowledgments Table of contents List of tables i List of figures iv List of research papers published from thesis ix List of abbreviations x Viruses and associated DNA-satellites (acronyms) xiv Abstract xviii 1 INTRODUCTION AND LITERATURE REVIEW 1.1 Viruses 1 1.2 Classification of viruses 1.13 Justification and likely benefits 1.14 Objectives 2 MATERIALS AND METHODS 2.1 Sample collection 2.2 Plants genomic DNA extraction 2.3 Quantification of DNA 2.4 Amplification of DNA 2.4.1 DNA amplification through PCR 2.4.2 Rolling circle amplification 2.4.3 Begomovirus DNA and their associated DAN satellite amplification 2.5 Cloning of PCR products 2.6 Transformation of competent cells 2.6.1 Transformation of Escherichia coli cells 2.6.2 Transformation of Agrobacterium tumefaciens competent cells 2.7 Preparation of competent cells 2.7.1 Preparation of heat shock competent Escherichia coli cells 2.7.2 Preparation of Agrobacterium tumefaciens electro competent cells 2.8 Plasmid isolation 2.9 Plasmid DNA digestion 2.10 Electrophoresis of agarose gel 2.11 Preparation of glycerol stocks 2.12 Purification of DNA 2.12.1 Gel extraction purification 2.12.2 Phenol chloroform treatment of DNA 2.13 Sequencing and sequence analysis 2.14 Recombination analysis 2.15 Agro inoculation 2.16 Plant growing conditions 2.17 Gene gun method 2.17.1 RCA product preparation for bombardment 2.17.2 Preparation of RCA product with tungsten particles 2.17.3 Tungsten particle coating 2.17.4 Plant preparation for inoculation 2.17.5 Hand held gene gun set up 2.17.6 Projectiles shooting 2.17.7 Gene gun shutting down 2.18 Confirmation analysis of infectivity 2.18.1 PCR 2.18.2 Southern blot hybridization analysis 2.18.3 Quantification by real-time PCR 2.18.3.1 Calculation for DNA-A standard 2.18.3.2 Calculation for betasatellite standard 2.19 Statistix-8.1 computer software 3 RESULTS 3 Samples collection of diverse host plants and their primarily results 3.1 Cherry tomato leaf curl virus isolated from congress grass (Parthenium hysterophorus) LIST OF FIGURES Figure No. Title Page No. 1.1 Family Geminiviridae genome organization consists of nine genera Begomovirus, Becurtovirus, Capulavirus, Curtovirus, Eragrovirus, Mastrevirus, Grablovirus, Turncurtovirus and Topocuvirus describe in (Varsani et al., 2017). Majority Old World begomoviruses have monopartite genome (DNA-A) associated with DNA-satellites and some have bipartite genome, New World begomoviruses contain two genomic components (DNA-A, DNA-B) both contain conserve region (CR) and intergenic region (IR; A, B). NW bipartite begomoviruse lack V2 ORF in DNA-A. The ORFs are in DNA-A components coat protein/CP, pre-coat protein/V2 on virion sens strand replication associated protein/Rep/C1, replication enhancer protein/REn/C2, transcription activator protein/TrAP/C3 on complimentary stand. DNA-B encodes movement (MP) and nuclear shuttle protein (NSP). 5 viii percent bootstrap values higher than 70 percent (1000 replicates). All isolates used for comparison are represented by their respective accession numbers in the trees. All acronyms for begomoviruses are according to Brown et al., 2012. 3.18 Structures of clone produced in the binary vector pBin19 for agroinoculation of Mesta yellow vain mosaic virus. Restriction endonuclease sites used to produce the constructs, approximate sizes of fragments, the position of the hairpin structure, the left (LB) and right borders (RB) of the T-DNA in the pBin19 (binary vector) are indicated. 3.19 Symptoms exhibited by N. benthamiana, (a) healthy (b) MeYVMV; MeYVMV alone systemically infected tissues showed vein thickening symptoms. (c, d) MeYVMV with CLCuMB, showed systemically severe vein-thickening and yellowing, mosaic and necrotic margin symptoms in new emerging leaves. 3.20 Southern hybridization of blots probed with MeYVMV (DNA-A) and CLCuMuB, 10ug of genomic DNA of N. benthamiana was loaded in each well. N. benthamiana inoculated with (lane 1) positive control, (lanes 2-3) MeYVMV alone, (lanes 4-5) MeYVMV with CLCuMuB, (lane 6) mock inoculation. Plants were photographed after 28 days per inoculation (dpi). 3.21 Structures of clones produced in the binary vector pBin19 for agroinoculation of Mastrevirus. Restriction endonuclease sites used to produce the constructs, approximate sizes of fragments, the position of the hairpin structure, the left (LB) and right borders (RB) of the T-DNA in the binary vector are indicated. 3.22 N. benthamiana, plants leaves exhibiting vein thickening, downward and upward leaf curling with cup formation. 3.23 Southern hybridization of blots probe was prepared by using PCR primer; MV27BCPF/ MV27BCPR product to confirm the infectivity of Chickpea chlorotic dwarf virus (CpCDV). 10ug of genomic DNA of N. benthamiana was loaded in each well. N. benthamiana inoculated with (lane 1) a positive control (plasmid), (lane 2-3) Chickpea chlorotic dwarf virus clone (CpCDV) and lane 4 with mock inoculation. Plants were photographed after 28dpi ix
Plant Science, 1997
The Geminiviridae are a large family of plant viruses which have small single stranded circular DNA genomes. The minichromosome-like double stranded replicative form of the viral genome exists at very high copy number in the nuclei of infected cells. This feature of geminiviruses has attracted particular interest from molecular biologists, because of the potential for their use as plasmid-like extrachromosomal replicons in plant cells. Most research on applications of geminiviruses in applied plant molecular biology has focused on the mainly bipartite, whitefly-transmitted Begomoviruses, all of which infect dicotyledonous plants. In this article, we critically review the applications of the monopartite, mainly cereal-infecting Mastreviruses for solving problems in plant biotechnology and molecular biology. We discuss the use of Mastreviruses as markers and vectors for gene transfer, as transient or infectious gene vectors, and as episomal gene amplification systems for enhancing gene expression in transgenic plants. We also examine the potential for use of geminiviral genetic elements such as promoters and transcription factors for manipulating gene expression in transgenic plants.
Virus Genes, 2010
Diseased cotton plants showing typical leaf curl symptoms were collected from experimental plot of Agriculture Research Station—Sriganganagar, Rajasthan. Complete DNA-A component from samples taken from two areas were amplified through rolling circle amplification (RCA) using templiphi kit (GE Healthcare) and characterized. DNA-A of one isolate consists of 2751 nucleotides and second isolate of 2759 nucleotide. Both sequences comprised six ORF’s. Genome organization of DNA-A of one isolate shows high sequence similarity with other characterized local begomovirus isolates of Rajasthan, while other isolate shows high sequence similarity with CLCuV reported from Pakistan. The maximum similarity of first isolate, CLCuV-SG01, shows highest sequence identity with Cotton leaf curl Abohar (Rajasthan) virus, and second isolate, CLCuV-SG02, shows highest sequence identity with cotton leaf curl virus from Pakistan. Both isolates showed 85% similarities with each other. The sequence data revealed probable infiltration of some strains of Cotton leaf curl virus from Pakistan to India, or co-existence of different isolates under similar geographical conditions. While CLCuV-SG01 shows highest nt sequence similarity with CLCuV Rajasthan (Abohar), nt identity of V1 ORF (encoding coat protein) of SG01 shows the highest nt identity (100%) with CLCuV Multan (Bhatinda) and Abohar virus while AC1 region also showed difference. Complete nucleotide sequence of SG01 shows only 86% similarity with CLCuV Multan virus. Similarity search revealed significant difference in AV1 and AC1 regions with respect to DNA-A suggesting an evolutionary history of recombination. Computer based analysis, recombination detection Program (RDP) supports the recombination hypothesis, indicated that recombination with other begomoviruses had taken place within V1 ORF and AC1 ORF of CLCuV-SG01 and AC1 ORF of CLCuV-SG02 and also in noncoding intergenic region (IR).
Abstract: Recombination is one of the keys factor in evolutionary processes, involved in shaping the architecture of genomes and consequent phenotype. Understanding the recombination phenomenon especially among viruses will help in disease management. The present study aimed for in-silico analysis of recombination phenomenon among Begomoviruses. Particularly emphasizing on viruses strains reported from India and neighboring countries. A total of 956 virus sequences have been used in this study. The Tomato yellow leaf curl China viruses, namely gi|29825986|; gi| 283468151|; gi| 190559151| and gi|61652782| were identified with the highest number of recombination event (1273). However, the Mung bean yellow mosaic India virus (gi|66351988|) was found to have 1170 recombination event. The phylogenic analysis among the highly recombinant sequences was carried to get an insight of the evolution among viral sequences in this class of plant viruses. The phylogenetic analysis revealed a pattern in diversity among these virus strains and a split tree analysis showed diversity in the range of 0.049128335 to 10.269852. This in silico analysis may pave way for a greater understanding of recombination phenomenon in Ggeminiviruses and it might be helpful for strategic plant viral disease management. Key words: Geminivirus, Begomovirus, Recombination, Tomato yellow leaf curl China virus, split tree analysis.
Journal of General …, 2000
Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric ; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.
Interspecies Recombination-Led Speciation of a Novel Geminivirus in Pakistan
Viruses
Recombination between isolates of different virus species has been known to be one of the sources of speciation. Weeds serve as mixing vessels for begomoviruses, infecting a wide range of economically important plants, thereby facilitating recombination. Chenopodium album is an economically important weed spread worldwide. Here, we present the molecular characterization of a novel recombinant begomovirus identified from C. album in Lahore, Pakistan. The complete DNA- A genome of the virus associated with the leaf distortion occurred in the infected C. album plants was cloned and sequenced. DNA sequence analysis showed that the nucleotide sequence of the virus shared 93% identity with those of the rose leaf curl virus and the duranta leaf curl virus. Interestingly, this newly identified virus is composed of open reading frames (ORFs) from different origins. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the sequences. Th...
Identification of an Australian-like dicot-infecting mastrevirus in Pakistan
Archives of Virology, 2014
Although members of five distinct viral species in the genus Mastrevirus (family Geminiviridae) infect dicotyledonous plants in Australia, in the remainder of the world, only a single dicot-infecting mastrevirus, chickpea chlorotic dwarf virus (CpCDV) has ever been identified. This virus has been found infecting leguminous hosts in Africa, the Middle East and the Indian subcontinent. To further explore the diversity of CpCDV in Pakistan, ten full mastrevirus genome sequences from chickpea and lentil plants were determined. Eight of these genomes were from previously described CpCDV strains and included the first reported strain D and H isolates in Pakistan. Two other genomes derived from infected chickpea plants are more closely related to dicot-infecting mastreviruses found in Australia than they are to CpCDV. These two divergent genomes shared less than 75 % genome-wide nucleotide sequence identity with other characterised mastreviruses and therefore are likely to belong to a second species of dicot-infecting mastreviruses outside of Australia. We propose naming this species Chickpea yellow dwarf virus. We discuss how the presence of chickpea yellow dwarf virus (CpYDV) in Pakistan weakens the hypothesis that Australia is the geographical origin of the dicotinfecting mastreviruses. Keywords Geminivirus Á Mastrevirus The family Geminiviridae consists of plant-infecting viruses with circular, single-stranded DNA (ssDNA) genomes of *2.5-5.4 kb. This family is currently comprised of seven genera; Begomovirus, Curtovirus, Topocuvirus, Becurtovirus, Eragrovirus, Turncurtovirus, Mastrevirus [31]. Mastreviruses infect both monocotyledonous and dicotyledonous plants and are transmitted by leafhoppers (Homoptera: Cicadellidae) [21]. They have been identified in Australia, Africa, Europe, the Middle East, Asia, the Far East, and the Indian subcontinent. Mastrevirus genomes consist of a single circular *2.7-kb ssDNA molecule that Electronic supplementary material The online version of this article (