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Bioscientific Review (BSR) BioScientific Review (BSR) Volume 2 Issue 4, 2020 ISSN(P): 2663-4198 ISSN(E): 2663-4201 Journal DOI: https://doi.org/10.32350/BSR Issue DOI: https://doi.org/10.32350/BSR.0204 Homepage: https://journals.umt.edu.pk/index.php/BSR Journal QR Code: Molecular Characterization of the Begomovirus Article: Associated Satellites Infecting Spinacia Oleracea and Capsicum Annum Plants in Kohat, Pakistan Ali Shah, Ayesha Ayub, Malik Nawaz Shuja, Taj Ali, Fazal Author(s): Akbar Article DOI: https://doi.org/10.32350/BSR.0204.05 Article QR Code: Shah A, Ayub A, Shuja MN et al. Molecular characterization of the begomovirus associated satellites infecting Spinacia To cite this article: Oleracea and Capsicum Annum Plants in Kohat, Pakistan. BioSci Rev. 2020;2(4):43–56. Crossref A publication of the Department of Life Sciences, School of Science University of Management and Technology, Lahore, Pakistan Molecular Characterization of the Begomovirus Associated Satellites Infecting Spinacia Oleracea and Capsicum Annum Plants in Kohat, Pakistan 1 2 1* 1 1 Ali Shah , Ayesha Ayub , Malik Nawaz Shuja , Taj Ali , Fazal Akbar 1Kohat University of Science & Technology, Kohat, Pakistan 2Mars Institute of Health Sciences for Women, Lahore, Pakistan *Corresponding author: [email protected] Abstract Begomovirus is a major and economically important genus of the Geminiviridae family. It comprises a wide range of viruses that infect a number of dicot plants including the horticulture crops, cereal crops, aromatic plants, vegetable crops, medicinal plants and weeds in various regions of the world. This study aims to investigate and correlate the various symptoms of begomovirus / satellites in different plants grown in the vicinity of Kohat, Pakistan. Furthermore, the characterization of the selected virus-associated satellites at the molecular level is also studied. Samples of suspected plants showing begomoviral infection were collected from the Kohat District. Genomic DNA was extracted from the infected plants and subjected to PCR using DNA-1/DNA-2 and Beta01/Beta02 for alpha satellites and beta satellites, respectively. The amplified PCR products were cloned and sequenced commercially. After sequencing, in silico sequence and phylogenetic analysis was also performed. Our study discovered that many plants in the Kohat District display begomovirus and satellite disease symptoms with mild to extreme disease severity. Disease incidence is especially high in okra. Beta satellites were isolated and sequenced from Spinacia oleracea and Capsicum annum plants and they showed more than 90% sequence similarity with chilli leaf curl and tomato leaf curl beta satellites. The existence of betasatellites in spinach and chilli plants was discovered for the first time in the Kohat region. Moreover, the distribution of these highly pathogenic variants of chilli leaf curl and tomato leaf curl betasatellites in the district Kohat has been reported previously. Keywords: alphasatellites, begomovirus, betasatellites, geminiviridae, pathogenic variants 1. Introduction [3]. They use the rolling circle replication (RCR) mechanism for the In terms of its economic significance, replication of the nuclei of the infected Geminiviridae is the second largest and host plant cells [4]. It is used as a model most important family of plant viruses for replication and transcription which and it infects the majority of depends on the machinery of the host monocotyledonous and dicotyledonous DNA. plants on the planet [1]. Gemniviruses are acknowledged as among the most Begomovirus is the most widespread and detrimental pathogens of food and other economically significant genus in the cash crops [2]. These viruses have a Geminiviridae family. Bean Golden circular ssDNA instead of a linear one Mosaic virus and Bean Golden Yellow Department of Life Sciences 44 Volume 2 Issue 4, 2020 Shah et al. Mosaic virus also belong to this family stem loop structure with a highly [5]. Begomoviruses have a much wider conserved sequence known as nona- engulfing ability, destroying the world’s nucleotide (TAATATT/AC), which acts most economically valued crops in as the replication base. tropical and sub-tropical areas. Most There are 5-6 open reading frames viruses in this genus are usually (ORFs) on the complementary sense of transmitted in a persistent, circulatory the bipartite begomovirus’ DNA-A manner by insect vectorsBegomoviruses component. There are two ORFs on the are a significant detriment to agricultural virion sense strand which encode coat productivity in many warmer parts of the (CP) and V2 proteins (V2). Sense and world including Pakistan due to a high complementary strands of DNA-B level of whitefly host species [6]. contains two ORFs encoding movement Previous research showed that protein (MP) and nuclear shuttle protein begomoviruses only infect (NSP). OW viruses are distinct from the dicotyledonous species including bean NW viruses as they lack the V2 gene on golden mosaic, cassava mosaic, cotton DNA-A part of the virion sense strand and tomato leaf curls [7]. Losses of up to [16]. This part (DNA-A) also encodes several billion dollars a year have been the proteins which are important for the reported [8]. Begomoviruses have encapsidation and replication of DNA emerged as a huge threat to several and other key factors responsible for the countries of the world in the form of the regulation of plant and viral genes [17]. reduced productivity in fruits, fibbers, The other component (DNA-B) is and ornamental crops. These viruses also important for the expression of viral affect other crops such as tomato, proteins needed for the transportation, squash, cotton, bean, cucurbits and many symptom growth and inter- and intra- more [8, 9, 10, 11]. cellular movement [18]. In case of the Begomoviruses of the family OW bipartite viruses, only DNA is Geminiviridae are divided into two important for systemic infection and groups based on their phylogenetic movement. However, in most of the NW analysis. Viruses from Africa, Asia, viruses, both genomic components are Australia, and Europe are known as the necessary for systemic infection and old world (OW) viruses, while those movement [19]. from the Americas are known as the new Except for a few bipartite world (NW) viruses [12, 13]. They are begomoviruses, the majority of the also classified as monopartite and begomoviruses identified from the OW bipartite based on their genomic are monopartite, meaning that they lack architecture. The genomes of NW a DNA-B molecule. Monopartite begomoviruses are composed of two begomoviruses have a single genomic distinctly encapsulated genomic features component that is similar to the bipartite referred to as DNA-A or/and DNA-B, begomoviruses’ DNA-A (DNA-A like) which are covalently closed, circular molecule. Monopartite genomes, ssDNA molecules with a size of 2.5 to including bipartite begomovirus DNA-A 3.0 kb. [14] Except for a 200-nucleotide molecules, encode all the necessary fragment of the intergenic region (IR) proteins for viral DNA replication, known as the common region, these transcription, and encapsidation [20]. components have no sequence similarity Monopartite begomoviruses’ pre-coat [15]. Both molecules share a hairpin BioScientific Review 45 Volume 2 Issue 4, 2020 Molecular Characterization of the Begomovirus… protein (V2) and coat protein (CP) SCR also has a replication origin site for perform the same transportation activity attaching the protein made by the helper as the bipartite begomoviruses’ DNA-B. begomovirus. As a result, replication Furthermore, the genomes of some begins inside this region by first creating monopartite begomoviruses have the a nick in the replication origin. The other power to induce wild type disease non-coding region is the A-rich region symptoms. Among these, tomato yellow which spans 200 nucleotides and is leaf curl virus (TYLCV), tomato leaf covered. [14] The aim of this study is to curl virus (TLCV) and tomato yellow characterize the begomovirus and its leaf curl Sardina virus (TYLCSV) are associated satellites that cause infections the most common [21]. in a variety of crops including medicinal plants, agricultural crops, aromatic One of the most important satellites plants, herbs, weeds and decorative associated with the helper plants in the Kohat region. begomoviruses is the betasatellite (formerly known as DNA-β). 2. Materials and Methods Betasatellite is a true class of satellites 2.1. Area of Study that cannot spread without the assistance of a helper virus and plays an important Among KP’s most important districts is role in disease production [22]. Kohat city. This area is a part of the Betasatellites are about 1.4 kb long Kohat Division which is further divided (approximately half of their helper virus) into five districts. There are two tehsils and belong to the tolecusatellitidae tribe. in this district (Kohat and Lachi). Kohat Encapsidation, transmission by insects, is located at latitudes 72-10 and 72-470 replication, and movement inside the E, and 34-09 and 34-430 N, respectively. host plants are all dependent on their The city of Peshawar is situated in the helper virus. As a result, a helper north of Kohat. The hills of Orakzai are begomvirus is needed for their survival situated to the west, while Bannu is and spread [12]. Since betasatellites situated to the south and the Indus river can’t make their own Rep protein, they flows to the east. From the sea level, depend on the helper virus’s encoded Kohat is 1768 feet high. However, the Rep protein [23]. elevation from the sea level varies, that is, Jalala Sir is 5,000 feet high in the Betasatellites are made of only one βC1 Cherat range (5,110 feet) and Molaghar gene that produces the βC1 protein. This is 7,060 feet high. It is a landmark hill in multifunctional protein has many Tira, 12 miles north-west of Kohat. To functions including acting as the the west, the Waziri hills are much lower suppressor of the host’s defense with Kafir Kot, the highest, being only mechanism and also as a pathogenicity 4,004 feet.
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