Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant βC1, located on the betasatellite associated with ...monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of βC1 in disease pathogenicity and to unveil potential interacting partners of βC1 protein in the model plant
The βC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of
and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and
protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families.
analyses were performed to validate the binding affinity of βC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of βC1 protein with NTF2 were identified by using PyMOL and Chimera.
The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the βC1. The NTF2 in
and
was found to be evolved from the
and
species. The
interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of βC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.
During the growing season of 2021-2022, a total of 145 symptomatic tomato leaf and fruit samples were collected from different locations in Riyadh Region, Saudi Arabia, showing a moderate-to-severe ...mosaic with dark green wrinkling, blistering, narrowing, and deformation with necrosis spot on tomato leaves, while irregular brown necrotic lesions, deformation, and yellowing spots rendering the fruits non-marketable were observed on tomato fruits. These samples were tested serologically against important tomato viruses using enzyme-linked immunosorbent assay (ELISA), and the obtained results showed that 52.4% of symptomatic tomato samples were found positive for Tomato brown rugose fruit virus (ToBRFV), wherein 12 out of 76 samples were singly infected; however, 64 out of 145 had mixed infection. A sample with a single infection of ToBRFV was used for mechanical inoculation into a range of different host plants; symptoms were observed weekly, and the presence of the ToBRFV was confirmed by ELISA and reverse transcription-polymerase chain reaction (RT-PCR). A total RNA was extracted from selected ELISA-positive samples, and RT-PCR was carried out using specific primers F-3666 and R-4718, which amplified a fragment of 1052 bp. RT-PCR products were sequenced in both directions, and partial genome nucleotide sequences were submitted to GenBank under the following accession numbers: MZ130501, MZ130502, and MZ130503. BLAST analysis of Saudi isolates of ToBRFV showed that the sequence shared nucleotide identities (99-99.5%) among them and 99-100% identity with ToBRFV isolates in different countries. A ToBRFV isolate (MZ130503) was selected for mechanical inoculation and to evaluate symptom severity responses of 13 commonly grown tomato cultivars in Saudi Arabia. All of the tomato cultivars showed a wide range of symptoms. The disease severity index of the tested cultivars ranged between 52% and 96%. The importance ToBRFV disease severity and its expanding host range due to its resistance breaking ability was discussed.
A survey was conducted in Al-Kharj governorate, Riyadh region to identify viruses causing variety of virus-like symptoms on tomato plants. A total of 135 samples were collected from symptomatic ...tomato plants. Symptoms included mottling, deformation, necrosis of leaves and fruits. Eighteen viruses were tested by DAS-ELISA. Tomato black ring virus (TBRV) was the virus of concern as it was not detected in Saudi Arabia before and was detected in 52.6% of the collected samples in this study. RT-PCR was used to confirm detection of TBRV and to sequence the amplified products to determine molecular characteristics of this virus. In the host range test study that was performed using a purified isolate of TBRV, sixteen out of the twenty two tested plants showed symptoms. Brassica oleracea was not infected by this virus. Gel electrophoreses (2% agarose) yielded fragments of 978 bp of coat protein gene of TBRV. Nucleotide sequences of purified RT-PCR products for three TBRV Saudi isolates were deposited in the GenBank with the following accession numbers MT274656, MT274657, and MT274658. These isolates of TBRV indicated a close Phylogenetic relationship of (99–100%) among themselves and with five isolates from Poland (95–98%) but a distant relationship of 85% with isolates from England and Lithuania deposited in the GenBank. This is the first report for detection and molecular characterization of TBRV infecting tomato plants in Saudi Arabia.
Begomovirus is the largest genus in the family Geminiviridae and constitutes more than 445 virus species. Begomoviruses are characterized by single-stranded circular genomes with monopartite or ...bipartite components and transmitted by whitefly (Bemisia tabaci). Begomoviruses cause severe diseases in many economically important crops throughout the world. Typical symptoms of a begomovirus infection including severe leaf curling, vein thickening, vein darkening and reduced leaf size were observed in papaya plants in the Dammam district of the Eastern Province of Saudi Arabia during the growing season in 2022. A total of 10 samples were collected, and total genomic DNA was isolated from naturally infected papaya tree samples and subjected to PCR amplification using universal diagnostic primers for begomoviruses and associated satellites. Three PCR-amplified genomic components of begomoviruses and betasatellite namely P61Begomo (645 bp), P62Begomo (341 bp) and P62Beta (563 bp) were sent for Sanger DNA sequencing to Macrogen Inc. These partial viral genome sequences were submitted to Genbank database and accession numbers ON206051, ON206052 and ON206050 were assigned to P61Begomo, P62Begomo and P62Beta respectively. Phylogenetic analysis and pairwise nucleotide sequence identity studies identified P61Begomo was identified as Tomato yellow leaf curl virus, P62Begomo as DNA A component of a bipartite begomovirus Watermelon chlorotic stunt virus and P62Beta as begomovirus associated betasatellite; Cotton leaf curl Gezira betasatellite. To the best of our knowledge, this is the first report of a begomovirus complex infecting papaya (Carica papaya) in the Kingdom of Saudi Arabia.
BACKGROUND: Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant ...yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed. FINDINGS: The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants. CONCLUSION: Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.
Traditional DNA sequencing methods are inefficient, lack the ability to discern the least abundant viral sequences, and ineffective for determining the extent of variability in viral populations. ...Here, populations of single-stranded DNA plant begomoviral genomes and their associated beta- and alpha-satellite molecules (virus-satellite complexes) (genus, Begomovirus; family, Geminiviridae) were enriched from total nucleic acids isolated from symptomatic, field-infected plants, using rolling circle amplification (RCA). Enriched virus-satellite complexes were subjected to Illumina-Next Generation Sequencing (NGS). CASAVA and SeqMan NGen programs were implemented, respectively, for quality control and for de novo and reference-guided contig assembly of viral-satellite sequences. The authenticity of the begomoviral sequences, and the reproducibility of the Illumina-NGS approach for begomoviral deep sequencing projects, were validated by comparing NGS results with those obtained using traditional molecular cloning and Sanger sequencing of viral components and satellite DNAs, also enriched by RCA or amplified by polymerase chain reaction. As the use of NGS approaches, together with advances in software development, make possible deep sequence coverage at a lower cost; the approach described herein will streamline the exploration of begomovirus diversity and population structure from naturally infected plants, irrespective of viral abundance. This is the first report of the implementation of Illumina-NGS to explore the diversity and identify begomoviral-satellite SNPs directly from plants naturally-infected with begomoviruses under field conditions.
A total of one-hundred and fourteen
Capsicum annum
L. samples were collected from Qassim and Al-Baha regions, Saudi Arabia in the growing season of 2021–2022, showing virus like symptoms including, ...mottling, mosaic, chlorosis, leaf distortion, stunted growth, and necrotic lesions, dwarfing of leaves and fruits. These samples were tested against important viruses infecting pepper using ELISA kits. According to ELISA results, 80/114 (70%) samples were found to be infected with one or more of the tested viruses. However, incidence of the PMMoV was observed in 18/21 (85.7%) and 23/59 (38.98%) from Al-Baha and Qassim regions respectively. Based on highly positive ELISA samples of PMMoV three samples were selected for biological detection using mechanical inoculation with sap prepared and the results showed that systemic mosaic, mottling, necrotic spots, yellowing on
N. tabacum, N. benthamiana, N. glutinosa, Solanum nigrum and
necrotic and chlorotic spots on
Chenopodium quinoa
and
C. amranticolor
, while local lesions symptoms were produced on
Datura stramonium.
According to the ELISA results, positive samples from Qassim and Al-Baha regions were amplified by RT-PCR analysis, and 474-bp fragment was amplified using PMMoV specific primers. Two directional sequencing was done for the amplified products and the sequences of the virus isolates were submitted in the NCBI database with the following accession numbers, OP723368, OP723369, OP723370, OP723371, OP723372. The phylogenetic analysis showed the close relationship our isolates with other isolation of PMMoV and pairwise nucleotide sequence identity analysis revealed that these PMMoV isolates shared their identity between 92.5 and 100% with PMMoV isolates available in the databases. This study provides comprehensive report regarding the diagnosis of chili infecting viruses through serological and characterization of PMMoV through serological, biological, and molecular methods in the kingdom of Saudi Arabia.
A survey was conducted to determine the status of
(LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic ...alfalfa, and seven
samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that
,
,
, and
are new host plant species for LTSV and that
being a good propagative host for it.
A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three ...hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it.
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