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Detection and sequence analyses of a begomovirus-encoded C6 protein. a Detection of unique C6 peptides by mass spectrometry analysis in Nicotiana benthamiana plants infected with ToLCCNV/ToLCCNB. b MS spectrum identification of the unique-peptides 74 MLDKLQVLNCEHTPKTK 90 and 1 MGRLAHAFDFDMAGAIR 17 in the ToLCCNV-C6 protein. c Schematic representation of the genome organization of ToLCCNV. d Western blot showing GFP protein accumulation at 2 days post-inoculation (dpi). Agrobacterium tumefaciens clones harboring pCambia-GFP or pC6 (556)-GFP at an OD 600 = 1.0, or 35S-GFP at an OD 600 = 0.05 were individually inoculated in N. benthamiana leaves. Actin serves as a loading control. e A. tumefaciens clone harboring pC6(556)-GFP was inoculated in N. benthamiana leaves in the presence of ToLCCNV/ToLCCNB or pBinplus. At 3 dpi, GFP fluorescence was measured and processed with the same settings. Scale bars: 25 μm. f Western blot showing GFP protein accumulation at 3 dpi. A. tumefaciens clone harboring pC6 (556)-GFP was co-infiltrated with ToLCCNV/ToLCCNB infectious clones or pBinplus vector in N. benthamiana leaves. Ponceau S staining of Rubisco shows protein loading. Numbers indicate the average GFP accumulation of three independent biological replicates
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Begomoviruses cause significant losses to a wide range of crops worldwide, and a great progress has been made in characterizing some noncanonical proteins encoded by begomoviruses. In the present study, a novel viral protein, C6, was detected in Nicotiana benthamiana plants infected with tomato leaf curl China virus (ToLCCNV). Sequence analyses rev...
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... the total protein of Nicotiana benthamiana plants infected with ToLCCNV (GenBank: AJ558119) alongside with its betasatellite ToLCCNB (GenBank: AJ704612), we recovered unique peptides corresponding to a novel virus protein through mass spectrometry (MS) analysis (Fig. 1a, b). The identified ORF, designated as ToL-CCNV C6, is in the complementary sense of the virus genome, partially overlaps with the CP and V2 ORFs, and encodes a polypeptide of 97 amino acids (Fig. ...
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... ToLCCNB (GenBank: AJ704612), we recovered unique peptides corresponding to a novel virus protein through mass spectrometry (MS) analysis (Fig. 1a, b). The identified ORF, designated as ToL-CCNV C6, is in the complementary sense of the virus genome, partially overlaps with the CP and V2 ORFs, and encodes a polypeptide of 97 amino acids (Fig. ...
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... gene. An Agrobacterium tumefaciens strain harboring the resulting construct (pC6 (556)-GFP) was infiltrated in N. benthamiana leaves with a promoter-less negative control (pCambia-GFP) and a cauliflower mosaic virus 35S promoter positive control (35S-GFP). Western blot analysis showed that the C6 upstream sequence could drive GFP expression (Fig. 1d). To better determine the promoter activity of C6 upstream sequence in the context of the virus infection, N. benthamiana leaves were infiltrated with A. tumefaciens harboring pC6 (556)-GFP together with ToLCCNV/ToLCCNB infectious clones or empty vector (pBinplus). Confocal microscopy showed enhanced GFP fluorescence in N. benthamiana ...
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... upstream sequence in the context of the virus infection, N. benthamiana leaves were infiltrated with A. tumefaciens harboring pC6 (556)-GFP together with ToLCCNV/ToLCCNB infectious clones or empty vector (pBinplus). Confocal microscopy showed enhanced GFP fluorescence in N. benthamiana leaves co-infiltrated with pC6 (556)-GFP and ToLCCNV/ ToLCCNB (Fig. 1e). Consistent with this, Western blot analysis showed higher GFP protein accumulation in N. benthamiana leaves co-infiltrated with pC6 (556)-GFP and ToLCCNV/ToLCCNB compared with that in the control (co-infiltrated with pBinplus and pC6 (556)-GFP), indicating a strengthened C6 promoter activity in the presence of the virus (Fig. 1f ...
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... ToLCCNB (Fig. 1e). Consistent with this, Western blot analysis showed higher GFP protein accumulation in N. benthamiana leaves co-infiltrated with pC6 (556)-GFP and ToLCCNV/ToLCCNB compared with that in the control (co-infiltrated with pBinplus and pC6 (556)-GFP), indicating a strengthened C6 promoter activity in the presence of the virus (Fig. 1f ...
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... study the subcellular localization of ToLCCNV C6 protein in plant cells, GFP was fused to either the N-terminus (GFP-C6) or the C-terminus of C6 (C6-GFP), and the resulting fusion protein was transiently expressed in N. benthamiana leaves. The GFP fluorescence signal for C6, as detected by confocal microscopy, was co-localized with Mito Tracker Red (a mitochondrial fluorescent dye) and AtICP55(N100)-mCherry (a mitochondrial marker) to mitochondria (Carrie et al. 2015) (Fig. 3a, b and Additional file 2: Figure S1a). To further confirm the mitochondrial localization of the C6 protein, immunogold staining of N. benthamiana leaf tissues was performed. ...
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... label was detected in the mitochondria of N. benthamiana cells transiently expressing Flag-C6-GFP, while no background labeling was observed with the secondary antibody alone (Fig. 3c). In addition, co-infiltration with ToLCCNV/ToLCCNB had no obvious effect on the mitochondrial localization or protein accumulation of C6 protein at 3 days post-inoculation (dpi) (Additional file 2: Figure S1b). A C6 homologue in another begomovirus, tomato yellow leaf curl China virus (TYLCCNV, GenBank: AJ319675), was detected to be localized in mitochondria as well (Additional file 2: Figure S1c, d). ...
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... addition, co-infiltration with ToLCCNV/ToLCCNB had no obvious effect on the mitochondrial localization or protein accumulation of C6 protein at 3 days post-inoculation (dpi) (Additional file 2: Figure S1b). A C6 homologue in another begomovirus, tomato yellow leaf curl China virus (TYLCCNV, GenBank: AJ319675), was detected to be localized in mitochondria as well (Additional file 2: Figure S1c, d). ...
Citations
... The AC5 gene codes for a protein related to viral pathogenicity able to suppress the host post-transcriptional gene silencing [6]. Recently, new ORFs were identified in the DNA-A component, including ORF AV3, which codes for a 7.4 KDa protein without ascribed function [7]; ORF AC6, which codes for a protein that plays a role in targeting the host mitochondria [8]; and ORF AC7, which codes for a protein that interacts with AV2 and AC2 proteins, inhibiting RNA silencing and acting as a pathogenicity factor [9]. On the other hand, the DNA-B component comprises the ORF BV1 (=NSP) coding for the nuclear shuttle protein and ORF BC1 (=MP) coding for the movement protein of New World begomoviruses [10]. ...
The diversity of Geminiviridae and Alphasatellitidae species in tomatoes was assessed via high-throughput sequencing of 154 symptomatic foliar samples collected from 2002 to 2017 across seven Brazilian biomes. The first pool (BP1) comprised 73 samples from the North (13), Northeast (36), and South (24) regions. Sixteen begomoviruses and one Topilevirus were detected in BP1. Four begomovirus-like contigs were identified as putative novel species (NS). NS#1 was reported in the semi-arid (Northeast) region and NS#2 and NS#4 in mild subtropical climates (South region), whereas NS#3 was detected in the warm and humid (North) region. The second pool (BP2) comprised 81 samples from Southeast (39) and Central–West (42) regions. Fourteen viruses and subviral agents were detected in BP2, including two topileviruses, a putative novel begomovirus (NS#5), and two alphasatellites occurring in continental highland areas. The five putative novel begomoviruses displayed strict endemic distributions. Conversely, tomato mottle leaf curl virus (a monopartite species) displayed the most widespread distribution occurring across the seven sampled biomes. The overall diversity and frequency of mixed infections were higher in susceptible (16 viruses + alphasatellites) in comparison to tolerant (carrying the Ty–1 or Ty–3 introgressions) samples, which displayed 9 viruses. This complex panorama reinforces the notion that the tomato-associated Geminiviridae diversity is yet underestimated in Neotropical regions.
... CMB genomes consist of two DNA components designated as DNA-A and DNA-B that together encode 9-10 proteins (Li et al., 2015;Gong et al., 2021;Wang et al., 2022;Liu et al., 2023). Other studies have indicated that the coding capacity of begomoviruses is greater than the canonical open reading frames (ORFs) (Lin et al., 2017;Gong et al., 2021), and the number of CMB proteins is likely to be higher. ...
Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.
... Using ORF finder, we identified two putative ORFs, which we designated C5 and C6 based on sequence comparison with other putative begomovirus ORFs identified by BLAST analysis. Short putative ORFs present in the genome of monopartite geminiviruses (and DNA-A of bipartite begomoviruses) have recently [61][62][63][64][65][66]. Through in vivo ribosome profiling of tomato yellow leaf curl Thailand virus, a series of novel translation initiation sites (TISs) were identified including two which result in the translation of distinct functional isoforms of AV2. ...
... Both of our ToCSV variant C6 proteins were predicted to contain signal peptides and a Interestingly, Gong et al. [62] did not identify a transmembrane domain in their ORF3-encoded protein and theirs also lacked the N-terminal region containing the transmembrane domain present in the V30 and V22 C6 proteins. Expression of the tomato leaf curl China virus (ToLCCNV) C6 was recently confirmed and the C6 protein was shown to contain residues necessary for directing protein localisation to the mitochondria [65]. The authors found that C6 residues 30-97 were responsible for mediating this localisation. ...
... We determined through deletion of the 35 C-terminal residues of V30 C6 that this region is not required for viral replication in N. benthamiana but may be implicated in modulating SV symptom severity via an unknown mechanism. Interestingly, Wang et al. [65] demonstrated through PVX-vector expression of ToLCCNV C6 and mutagenesis to generate a C6-null mutant that C6 is not a pathogenicity determinant in N. benthamiana and is not required for disease symptom expression. It is possible that the C6 protein may have a repertoire of accessory functions which could serve to fine-tune crucial infection events occurring at different regions within the cell. ...
Tomato production in South Africa is threatened by the emergence of tomato curly stunt virus (ToCSV), a monopartite Begomovirus transmitted by the whitefly vector Bemisia tabaci (Genn.). We investigated the role of sequence differences present in the 3' intergenic region (IR) and the V2 coding region on the differing infectivity of ToCSV sequence variant isolates V30 and V22 in the model host Nicotiana benthamiana. Using virus mutant chimeras, we determined that the development of the upward leaf roll symptom phenotype is mediated by sequence differences present in the 3' IR containing the TATA-associated composite element. Sequence differences present in the V2 coding region are responsible for modulating disease severity and symptom recovery in V22-infected plants. Serine substitution of V22 V2 Val27 resulted in a significant increase in disease severity with reduced recovery, the first study to demonstrate the importance of this V2 residue in disease development. Two putative ORFs, C5 and C6, were identified using in silico analysis and detection of an RNA transcript spanning their coding region suggests that these ORFs may be transcribed during infection. Additional virus-derived RNA transcripts spanning multiple ORFs and crossing the boundaries of recognised polycistronic transcripts, as well as the origin of replication within the IR, were detected in ToCSV-infected plants providing evidence of bidirectional readthrough transcription. From our results, we conclude that the diverse responses of the model host to ToCSV infection is influenced by select sequence differences and our findings provide several avenues for further investigation into the mechanisms behind these responses to infection.
Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop‐infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus‐transmitting arthropod vectors, and in‐depth interrogation of plant‐encoded resistance and susceptibility determinants. Notably, various plant virus‐based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.
The present study reports the complete genome of a novel monopartite begomovirus, named tentatively as "Citharexylum leaf curl virus" (CitLCuV), associated with leaf curl disease of Citharexylum spinosum in India. CitLCuV genome (2,767 nucleotide) contained the typical genome organization of Old World begomoviruses and shared the maximum nucleotide sequence identity of 89.2% with a rose leaf curl virus (RoLCuV) isolate. In addition, two small non-canonical open reading frames (C5 and C6) were determined in the complementary strand of CitLCuV genome. Phylogenetic analysis revealed the relatedness of CitLCuV to papaya leaf crumple virus and RoLCuV. Recombination analysis detected a possible recombination event in CitLCuV genome. Based on begomovirus species demarcation criteria, CitLCuV can be regarded as a novel begomoviral species.
Tomato yellow leaf curl virus (TYLCV) is a monopartite geminivirus, and one of the most devastating plant viruses in the world. TYLCV is traditionally known to encode six viral proteins in bidirectional and partially overlapping open reading frames (ORFs). However, recent studies have shown that TYLCV encodes additional small proteins with specific subcellular localizations and potential virulence functions. Here, a novel protein named C7, encoded by a newly-described ORF in the complementary strand, was identified as part of the TYLCV proteome using mass spectrometry. The C7 protein localized to the nucleus and cytoplasm, both in the absence and presence of the virus. C7 was found to interact with two other TYLCV-encoded proteins: with C2 in the nucleus, and with V2 in the cytoplasm, forming conspicuous granules. Mutation of C7 start codon ATG to ACG to block the translation of C7 delayed the onset of viral infection, and the mutant virus caused milder virus symptoms and less accumulations of viral DNAs and proteins. Using the potato virus X (PVX)-based recombinant vector, we found that ectopic overexpression of C7 resulted in more severe mosaic symptoms and promoted a higher accumulation of PVX-encoded coat protein in the late virus infection stage. In addition, C7 was also found to inhibit GFP-induced RNA silencing moderately. This study demonstrates that the novel C7 protein encoded by TYLCV is a pathogenicity factor and a weak RNA silencing suppressor, and that it plays a critical role during TYLCV infection.
Begomoviruses constitute an extremely successful group of emerging plant viruses transmitted by whiteflies of the Bemisia tabaci complex. Hosts include important vegetable, root, and fiber crops grown in the tropics and subtropics. Factors contributing to the ever-increasing diversity and success of begomoviruses include their predisposition to recombine their genomes, interaction with DNA satellites recruited throughout their evolution, presence of wild plants as a virus reservoir and a source of speciation, and extreme polyphagia and continuous movement of the insect vectors to temperate regions. These features as well as some controversial issues (replication in the insect vector, putative seed transmission, transmission by insects other than B. tabaci, and expansion of the host range to monocotyledonous plants) will be analyzed in this review.
