Genome organization of satellite DNA beta molecule associated with Mungbean yellow mosaic India virus Gujarat, India

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Priya John¹ , Kotramma Addangadi ¹ , Gomathy Muthukrishnan² , Bhukya Srinivas³ , V.A. Patil⁴ , V.G. Malathi⁵

¹Department of Plant Pathology, N.M. College of Agriculture, Navsari Agricultural University, Navsari-396450, Gujarat, India.

²Agriculture College and Research Institute, Tamil Nadu Agricultural University, Killikulam- 628252, Tamil Nadu, India

³Department of Plant Pathology, College of Agriculture, PJTSAU, Rajendranagar-500 030, Telangana, India

⁴Department of Plant Pathology, MRC, SWMRU, NAU, Navsari-396450, Gujarat, India.

⁵ICAR-IARI, Shree Kumaran Hill Crest Apartment, Thondamuthur Road, Coimbatore, 641046, Tamil Nadu, India.

Corresponding Author Email: pjohn@nau.in

DOI : https://doi.org/10.51470/AGRI.2024.3.2.06

Abstract

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INTRODUCTION

Begomoviruses are known to be agreat threat to several agriculture crops. They include cotton, okra, tobacco, and tomato and yellow mosaic diseases in grain legumes in the tropics for a long time. Sincce the Green Revolution  (GR) period,  begomoviruses spread their  host range enormously and spreading to weeds also [1] [2]. Grain legumes cultivated as rainfed and irrigated crops in Gujarat are severely affected by whitefly-transmitted yellow mosaic disease (YMD) which is a bipartite begomovirus relicating in a rolling circle mechanism [3] belonging to the family Geminiviridae. Monopartite and begomoviruses are associated with a small satellite designated as DNA-β. Uptil now more than 200 satellite sequences have been established from variety of plants which includes a range of weeds, vegetables and fibre crops in Africa and Asia, reflecting their diversity and impact on agriculture throughout the Old World [4].  Basil Kassanis in 1962 used the term satellite virus for the 17 nm diameter particles associated with tobacco necrosis virus. There are three types of  circular ssDNA satellite molecules. They are alphasatellites, betasatellites, and deltasatellites are associated with diseases caused by begomoviruses. The satellites, in  general, may have originated from genetic elements that were once part of the viral genome  but eventually lost their ability to replicate independently [1] and also replicate similar rolling circle and recombination-dependent replication mechanisms [5]. As there is no report on the molecular characterization of beta satellite viruses causing YMD in legumes in Gujarat, the present study is focused on molecular cloning of genomic components of yellow mosaic viruses (YMV) infecting legumes and establishment of their relationship with other reported beta satellite viruses.

MATERIALS AND METHODS

MYMIV infected legume samples were used for  DNA using the modified CTAB method [6]. PCR amplification was done based on the sequence of DNA beta satellite region using universal primers [7] PCR products were cloned in pGEM-T Easy vector. Computer analysis of sequence was carried out using the software programme Bioedit version 5.0.9, CLASTALW, the neighbour-joining method by MEGA version 3.1.

Table 1:List of beta satellites associated with begomoviruses with their accession numbers from GenBank used for all sequence analysis

Results and discussion

The sequence analysis of the beta satellite of  Mungbean yellow mosaic India virus-[IN:Ana: CpMBKA25:041350] showed 1367 base pairs of nucleotides and their genome analysis is shown in Fig. 1-4 and Table 2. The sequence analysis showed the presence of iteron-like elements homologous which help in the replication.TAATATTAC is also present in the β satellite which is a common feature of geminiviruses. The Gujarat isolate showed the presence of  an adenine-rich region, a satellite-conserved region and a single ORF encoding βC1 protein. Several studies show that the symptom development depends upon  βC1 protein. The present study supports the presence of β satellite which is occurring mainly in Africa and Asia with maximum diversity from the Indian continent. The similarity identity matrix showed that the whole genome had 96 % similarity with Potato apical leaf curl virus-associated DNA beta and approximately 96  per cent identity with Potato apical leaf curl virus-associated DNA beta and  Tomato leaf curl virus associated DNA beta viruses (Karnataka isolate) in βC1 region. The amino acid sequence showed similarity with those of other viruses which consisted of extra amino acid residues V, C, N  in the βC1 protein at the carboxyl end. Whne all the isolates were compared with MYMIV-[IN:Ana: CpMBKA25:04] isolates it is found to be a distinct isolate belonging to the Papaya leaf curl virus. Begomoviruses family Geminiviridae causes viral diseases in economically important crop plants. Genomically begomoviruses possess two genomic components DNA A and DNA B. DNA β components do not show any homology to helper begomoviruses. But they depend upon them for replication, encapsidation and movement. DNA β play a role in the host-specific symptoms in plants. Previous studies show that DNA β components exist in nature. So present paper also reveals the presence of DNA β component present in the legumes. Usually, β satellites are found to be associated with monopartite begomoviruses. Nowadays they are found to be spreading in bipartite begomoviruses spreading in the old world too. Earlier it has been reported from MYMIV infecting cowpea [8], mungbean  [9] and tomato  [10], chilli [11] [12] and kenaf [13]. Other part of the world showed the association of  DNA  β in Sri Lankan cassava  mosaic virus [14], Tomato yellow leaf curl Thailand  virus [15], and the New World bipartite Cabbage leaf  curl begomovirus [16].The presence of β satellite associated with begomoviruses was found to play a significant role in the evolution of begomovirus. Initiation of replication as well as the control of complementary-sense gene expression in  DNA  β  is initiated by TAATATTAC [17]. TAATATTAC is a universally conserved region within the loop of a hairpin  structure and it is a nonanucleotide sequence.  Rep protein nicks at its downstream of the virion sense strand  [18] [19].Tobacco ring spot nepovirus was identified as the first satellite RNA in 1969 [20]. The first monopartite begomovirus satellite ToLCV-sat  was isolated from tomato plants [21]. Different studies and its compilation shows that  DNA satellites usually consists of 1350 nucleotides and they are called as DNA  β. They are ususally seen in the case of monopartite begomoviruses [7] [22] [23] [24] [25]. Betasatellites are usually the half of the genome size of the helper begomoviruses [26].Satellite virus they depend upon the helper virus for its replication.  Ageratum yellow vein virus (AYVV) infects ageratum plants when inoculated with the virus. But when it is inoculated in Nicotiana benthamiana, it produces asymptomatic infection and indicates the requirement of the presence of a helper virus like satellite molecule [27]. Cotton leaf curl Multan virus (CLCuMV) and  Tomato leaf curl virus (ToLCV) showed severe symptoms in the plants in the presence of the helper virus [28] [29] [30]. This shows the role of β satellites in the disease symptoms [31)], virus movement in plants [32], binds DNA in a sequence non-specific manner [33], is a suppressor of RNA silencing [34] [35],  forms homo-multimeric complexes in planta [35], inter feres with host gene expression [36] and has been  shown to interact with a variety of host factors [37] [38]. So similarly at present too MYMIV infects legumes and its ability to infect Nicotiana benthamana is yet to be explored with its presence of β satellite virus. In the present studies, components contain an A-rich region suggesting that they may have originated as a bonafide component of another pathogenic agent before being captured by the begomovirus necessitating a slight increase in size as shown by earlier studies [4].  In addition to the above mentioned A-rich region, β satellite virus components usually in common posees  a single gene βC1. It is a highly conserved sequence consisting of 80 nucleotideswhich is known as the satellite-conserved region (SCR) and  PCR primers based on this sequence have provided a simple and robust method for the isolation of DNA-β components [23]. During the past years, it has been found from the database that more than one hundred full-length DNA-β sequences have been deposited in from plant species belonging to the family Malvaceae and those associated with non-Malvaceae [39]. A similar trend also can be seen in the present study. The present study also shows its phylogenetic relationship showing as a distinct clade differing from the isolates of monopartite viruses like Tomato leaf curl virus and the Malvaceae family. Eralier studies showed that distinct betasatellite  species share a high level of sequence identity with  respect to βC1 gene which are responsible for inducing distinct symptoms. The whitefly performance is alaso dependenet on the  serine-33, a conserved phosphorylation site in betasatellite-encoded βC1 protein which suppresses the plant terpenoid-based defense[40]. The identification of a relatively high level of diversity of beta satellites in okra showed possibility of development of  resistance by both conventional breeding and transgenic approaches [26]. Previous studies interpret the possibility of  monopartite begomoviruses of the other host might move legumes aided by the associated betasatellites. Still a lot of research is yet required to know  association of betasatellites with MYMIV and MYMV will affect its host range, virulence and transmission  to contain the YMD spread and mitigate the yield loss [41].

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Conclusion

Satellite DNA beta found to be associated with Mungbean yellow mosaic India virus-[IN:Ana: CpMBKA25:04]. Present study shows the presence of  DNA-β components that pose a threat to agriculture throughout the Old World. The recent isolation of novel satellite components suggests that other disease complexes and subviral components may exist and its role in symptom and transmission requires much exploration. DNA-βand DNA-A and DNA-B can show the possibility of interacting with different begomoviruses.

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