Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus (TYLCV) is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China. In this study, we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibit that of viral DNA and virion-sense strand mRNAs. However, two other resistant lines did not have such virus inhibition pattern. Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes, as compared to the susceptible line, suggesting a diversity of tomato TYLCV-resistance mechanisms.

A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of New World begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component.

Olea Europaea Geminivirus: A Novel Bipartite Geminivirid Infecting Olive Trees

In 2014, high-throughput sequencing of libraries of total DNA from olive trees allowed the identification of two geminivirus-like contigs. After conventional resequencing of the two genomic DNAs, their analysis revealed they belonged to the same viral entity, for which the provisional name of Olea europaea geminivirus (OEGV) was proposed. Although DNA-A showed a genome organization similar to that of New World begomoviruses, DNA-B had a peculiar ORF arrangement, consisting of a movement protein (MP) in the virion sense and a protein with unknown function on the complementary sense. Phylogenetic analysis performed either on full-length genome or on coat protein, replication associated protein (Rep), and MP sequences did not endorse the inclusion of this virus in any of the established genera in the family Geminiviridae. A survey of 55 plants revealed that the virus is widespread in Apulia (Italy) with 91% of the samples testing positive, although no correlation of OEGV with a disease or specific symptoms was encountered. Southern blot assay suggested that the virus is not integrated in the olive genome. The study of OEGV-derived siRNA obtained from small RNA libraries of leaves and fruits of three different cultivars, showed that the accumulation of the two genomic components is influenced by the plant genotype while virus-derived-siRNA profile is in line with other geminivirids reported in literature. Single-nucleotide polymorphism (SNP) analysis unveiled a low intra-specific variability.

Plant-virus-insect tritrophic interactions: insights into the functions of geminivirus virion-sense strand genes

The genome of the plant-infecting viruses in the family Geminiviridae is composed of one or two circular single stranded DNA of approximately 2.7–5.2 kb in length. These viruses have emerged as the most devastating pathogen infecting a large number of crops and weeds across the continents. They code for fewer open reading frames (ORFs) through the generation of overlapping transcripts derived from the bidirectional viral promoters. Members of geminiviruses code for up to four ORFs in the virion-sense strand, and their gene expression is regulated by various cis -elements located at their promoters in the intergenic region. These viral proteins perform multiple functions at every stage of the viral life cycle such as virus transport, insect-mediated virus transmission and suppression of host defence. They impede the host's multi-layered antiviral mechanisms including gene silencing (at transcriptional and post-transcriptional levels) and hypersensitive response. This review summarizes the essential role of virion-sense strand encoded proteins in transport of viral genomes within and between plant cells, countering defence in hosts (both plants and the insects), and also in the ubiquitous role in vector-mediated transmission. We highlight the significance of their pro-viral activities in manipulating host-derived innate immune responses and the interaction with whitefly-derived proteins. We also discuss the current knowledge on virus replication and transcription within the insect body.

First Report of Tomato leaf curl Albatinah virus (ToLCABV) and its Associated Betasatellite Infecting Papaya in Oman

Leaf curl disease with severe curling, vein darkening, and vein thickening was observed on papaya plants in a field in Qurayat district of Oman during December 2013. Disease incidence ranged from 50 to 70%, particularly in young papaya plants. The presence of a large population of whiteflies and symptoms observed on papaya plants suggested that the causal agent could be begomoviruses (family Geminiviridae) and associated satellites. Four leaf samples with mild and severe leaf curling were collected from the field. Total nucleic acid extracted from symptomatic and healthy plants using the CTAB method were used as a template to amplify circular DNAs using Φ29 DNA polymerase, and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. BamHI yielded fragments of ~2.8 and 1.4 kb when the digested products were resolved by electrophoresis on a 1% agarose gel. These fragments were cloned and sequenced using a primer walking strategy in both directions. Sequencing results confirmed the exact sizes of 1,303, 1,358, and 2,765 bp; the sequences were deposited in GenBank under the accession numbers HG969296, HG969297, and HG969260, respectively. BLAST results showed that the first two sequences are Tomato leaf curl betasatellite (ToLCB; isolates Pap-2 and Pap-3) showing 97% sequence identity with a previously reported ToLCB sequence (Accession No. KF229728). Both satellites encode a single gene in the complementary sense strand referred to as βC1, which showed 97% sequence identity to ToLCB (HE800551). The viral sequence (isolate Pap-6) showed four genes in the complementary sense (the replication-associated protein [Rep] gene, the transcription-activator protein [TrAP] gene, the replication-enhancer protein [REn] gene, and the C4 gene) and two genes (pre-coat protein [V2] and coat protein [CP]) in virion-sense (2). BLAST analysis showed 95.2% sequence identity to Tomato leaf curl Albatinah virus (ToLCABV; FJ956700), reported earlier to infect tomato in Oman (3). Amino acid sequence comparison of the four predicted proteins (Rep, TrAP, Ren, and C4) encoded by Pap-6 shared 95, 96, 100, and 100% sequence identity, whereas virion-sense proteins (V1 and V2) shared 99% sequence identity with ToLCABV (FJ956700). According to the recommendations of the International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of ToLCABV (1). All infected samples showed the presence of ToLCABV, while no hybridization was observed in healthy control DNA using ToLCABV probe. These findings are indicative of the rapid spread of diseases involving Begomovirus and betasatellites, which often result in increased host range, as is evident from this study. References: (1) C. M. Fauquet et al. Arch. Virol. 148:405, 2003. (2) L. Hanley-Bowdoin et al. Crit. Rev. Plant Sci. 18:71, 1999. (3) A. J. Khan et al. Arch. Virol. 159:445, 2013.

Identification of Cotton leaf curl Gezira virus in Papaya in Oman

Papaya is an important fruit crop in Oman covering some 130 ha with an annual production of 20 tonnes. In 2011, during surveys of farms in the Quriyat region of Oman, papaya plants were found severely affected by leaf curl disease. Leaves with severe curling, vein darkening, and vein thickening were collected for study. Disease incidence ranged from 30 to 50%, particularly in fields with young papaya. A begomovirus (family Geminiviridae) was suspected as the causal agent based on symptoms (1) and the presence of whiteflies in the field. Samples (four to five) were collected from three farms. Total nucleic acids extracted from symptomatic leaves using the CTAB method were used as templates to amplify circular DNAs using Φ29 DNA polymerase and products were digested with restriction enzymes to identify fragments of 2.6 to 2.8 kb typical of geminiviruses. PstI yielded a fragment of ~1.8 kb when the digested product was analyzed by electrophoresis on a 1% agarose gel. The fragment was cloned and sequenced using primer walking strategy in both directions. The sequencing confirmed the exact size (1,764 bp) and the sequence was deposited in GenBank (HE800524). The viral sequence from Oman (isolate Pap-2) showed four open reading frames (ORFs) in the complementary sense (replication associated protein [Rep] gene, the C2 gene, the replication enhancer protein [REn] gene, and the C4 gene) and the virion-sense ORFs (V1 and V2) were missing in the sequence. An initial comparison to NCBI database sequences using BLAST showed the clone from Oman had the highest level of sequence identity to Cotton leaf curl Gezira virus (CLCuGeV) (FJ868828) cloned from okra in Sudan. Subsequent pair wise sequence comparison was done using ClustalV algorithm. Full length sequences of CLCuGeV from database were trimmed according to the size and genomic coordinates of Pap-2 isolate. The Pap-2 isolate sequence was found to have 83.3 to 95.1% sequence identity to CLCuGeV sequences with maximum value to the Sudan isolate. Amino acid sequence comparison showed that the four predicted proteins (Rep, C2, REn, and C4) encoded by the Pap-2 isolate shared 95.3%, 97.8%, 97.7%, and 87.6% sequence identity, respectively, with the homologous proteins of CLCuGeV-SD (FJ868828). The absence of virion-sense protein sequences indicated it to be a subgenomic molecule of CLCuGeV. According to the recommendations of International Committee on Taxonomy of Viruses, these results indicate that the virus identified in association with papaya leaf curl disease in Oman is a variant of CLCuGeV. CLCuGeV is a begomovirus of African origin which is distinct from the begomoviruses of the Middle East and Asia. To our knowledge, this is the first report of CLCuGeV, or any other cotton infecting begomovirus, from papaya in Oman. The presence of a recombinant fragment of CLCuGeV in a Tomato yellow leaf curl virus isolate from Iran (2), and the association of CLCuGeV with cotton in Pakistan (3) and hollyhock in Jordan (GU945265) suggests this virus has moved into the Middle East and Asia from Africa. The identification of CLCuGeV in Oman shows the widespread occurrence of this virus species. This discovery is important since Oman, and other countries in the area, are a hub of international trade and travel, particularly by air and sea, meaning that the virus could spread further. References: (1) R. W. Briddon and P. G. Markham. Virus Res. 71:151, 2000. (2) P. Lefeuvre et al. PLoS Pathog. 6:e1001164, 2010. (3) M. N. Tahir et al. PLoS ONE 6:e20366, 2011.

Transcriptional Analysis of Complementary Sense Genes in Spinach curly top virus and Functional Role of C2 in Pathogenesis

Spinach curly top virus (SCTV), the fifth characterized Curtovirus species belonging to the family Geminiviridae, is an agriculturally significant plant pathogen representing an emerging disease threat in the southern United States. The SCTV genome comprises a single DNA chromosome of approximately 3.0 kb, with the potential to code for seven proteins larger than 10 kDa but which relies extensively on the host for replication and transcription of its genome. In this study, we have identified viral and complementary sense transcripts in SCTV-infected plants, confirming a bidirectional transcription strategy for SCTV. The most abundant RNA maps to the virion sense (1.1-kb transcript) and is comparable in size and location to that observed in Beet curly top virus (BCTV). Two complementary sense transcripts (1.7 and 0.7 kb) were identified in SCTV-infected plants. The large, 1.7-kb transcript is comparable in size and position to that identified in BCTV and several begomoviruses and most likely encodes the C1 protein. Both complementary sense RNAs could potentially direct expression of C2 and C3 from polycistronic mRNAs. A mutation in the C2 gene of SCTV results in expression of a truncated protein of 38 amino acids that is capable of interacting with two cellular kinases, AKIN11 and ADK2, and the resulting mutant virus remains highly infectious. A second mutant virus can only express the first three amino acids of the C2 protein and is unable to interact with the same kinases. However, this mutant virus still remains infectious, although a reduction in infectivity and symptom severity was seen in both Arabidopsis and spinach. A possible relationship between the interaction of C2 with AKIN11 and ADK2 and disease severity is presented.