Movement Protein Research Articles

Association of Citrus Virus A to Citrus Impietratura Disease Symptoms.

Citrus impietratura disease (CID) is a graft transmissible, virus-like disease observed in old-line citrus trees; its characteristic symptom is the appearance of gum in the albedo of the affected fruits. To identify the causal agent of the disease, high-throughput sequencing (HTS) was performed on symptomatic orange fruits. The analysis of the obtained data revealed in all samples mixed infections of viroids commonly found in citrus trees together with the recently described citrus virus A (CiVA). Examination of additional symptomatic fruits with conventional reverse transcription PCR led to the identification of a single CiVA infection in one tree, which was verified by HTS. Indexing of the single CiVA-infected tree on indicator plants resulted in the appearance of characteristic symptoms in the leaves that were correlated with virus accumulation. Moreover, a comparative analysis among symptomatic and asymptomatic fruits derived from the same trees was performed and included the single CiVA-infected orange tree. The analysis revealed a positive correlation between the appearance of symptoms and the accumulation of CiVA RNAs. To facilitate CiVA detection during certification programs of propagation material, a quantitative RT-PCR targeting the movement protein of the virus was developed and evaluated for reliable and sensitive detection of the virus. To the best of our knowledge this is the first study that associates CiVA with the appearance of CID symptoms.

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions.

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.

The Tomato Brown Rugose Fruit Virus Movement Protein Overcomes Tm-22 Resistance in Tomato While Attenuating Viral Transport.

Tomato brown rugose fruit virus is a new virus species in the Tobamovirus genus, causing substantial damage to tomato crops. Reports of recent tomato brown rugose fruit virus (ToBRFV) outbreaks from around the world indicate an emerging global epidemic. ToBRFV overcomes all tobamovirus resistances in tomato, including the durable Tm-22 resistance gene, which had been effective against multiple tobamoviruses. Here, we show that the ToBRFV movement protein (MPToBRFV) enables the virus to evade Tm-22 resistance. Transient expression of MPToBRFV failed to activate the Tm-22 resistance response. Replacement of the original MP sequence of tomato mosaic virus (ToMV) with MPToBRFV enabled this recombinant virus to infect Tm-22-resistant plants. Using hybrid protein analysis, we show that the elements required to evade Tm-22 are located between MPToBRFV amino acids 1 and 216 and not the C terminus, as previously assumed. Analysis of ToBRFV systemic infection in tomato revealed that ToBRFV spreads more slowly compared with ToMV. Interestingly, replacement of tobacco mosaic virus (TMV) and ToMV MPs with MPToBRFV caused an attenuation of systemic infection of both viruses. Cell-to-cell movement analysis showed that MPToBRFV moves less effectively compared with the TMV MP (MPTMV). These findings suggest that overcoming Tm-22 is associated with attenuated MP function. This may explain the high durability of Tm-22 resistance, which had remained unbroken for over 60 years.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Iranian Strawberry crinkle cytorhabdovirus variation assessed using its movement protein (P3) gene.

Strawberry crinkle virus (SCV) is a member of the genus Cytorhabdovirus, family Rhabdovirida, and order Mononegavirales. SCV affects the production of various strawberry cultivars. In this study we investigated the genetic diversity of SCV in strawberry fields based on P3 (movement protein) gene. The samples were collected from strawberry fields in the Kurdistan Province, Iran. P3 gene from 20 SCV isolates, representing 18 nucleic acid haplotypes, is composed of 729 nucleotides, encoding a protein with 243 amino acids. SCV-P3 sequences shared 98.77%-99.86% nucleotideand 97.5%-100% amino acidsequence identity. Phylogenetic analyses of the new P3 sequences with two previously published SCV-P3 sequences from the Czech Republic showed that there are two major phylogroups (I and II) and three minor phylogroups in the body of the phylogeny, I-1, I-2, II-1. Comparisons of P3 gene sequences revealed a mutational bias, with more differences being transitions than transversions. The ratio of non-synonymous/synonymous nucleotide changes was < 1, indicating that SCV-P3 gene is under predominantly negative selection. Phylogenetic and sequence identity analyses showed that SCV isolates from Iran are closely related and have not diverged more than 2% based on P3 gene despite geographical separation and strawberry cultivar. This is the first report of the genetic diversity of SCV worldwide.

New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Identification of genetic determinants of tomato brown rugose fruit virus that enable infection of plants harbouring the Tm-22 resistance gene.

Tomato cultivars containing the Tm‐22 resistance gene have been widely known to resist tobacco mosaic virus (TMV) and tomato mosaic virus. Tomato brown rugose fruit virus (ToBRFV), a new emerging tobamovirus, can infect tomato plants carrying the Tm‐22 gene. However, the virulence determinant of ToBRFV that overcomes the resistance conferred by the Tm‐22 gene remains unclear. In this study, we substituted the movement protein (MP) encoding sequences between ToBRFV and TMV infectious clones and conducted infectivity assays. The results showed that MP was the virulence determinant for ToBRFV to infect Tm‐22 transgenic Nicotiana benthamiana plants and Tm‐22 ‐carrying tomato plants. A TMV MP chimera with amino acid residues 60–186 of ToBRFV MP failed to induce hypersensitive cell death in the leaves of Tm‐22 transgenic N. benthamiana plants. Chimeric TMV containing residues 60–186 of ToBRFV MP could, but chimeric ToBRFV containing 61–187 residues of TMV MP failed to infect Tm‐22 transgenic N. benthamiana plants, indicating that 60–186 residues of MP were important for ToBRFV to overcome Tm‐22 gene‐mediated resistance. Further analysis showed that six amino acid residues, H67, N125, K129, A134, I147, and I168 of ToBRFV MP, were critical in overcoming Tm‐22 ‐mediated resistance in transgenic N. benthamiana plants and tomato plants. These results increase our understanding of the mechanism by which ToBRFV overcomes Tm‐22 ‐mediated resistance.

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 have a bipartite genome with molecular signatures typical of the members of the genus Coguvirus (family Phenuiviridae).

Watermelon crinkle leaf-associated virus 1 and watermelon crinkle leaf-associated virus 2 (WCLaV-1 and WCLaV-2), two unclassified members of the order Bunyavirales, are phylogenetically related to members of the genus Coguvirus (family Phenuiviridae). The genome of both viruses was reported previously to be composed of three RNA segments. However, the terminal sequences of two genomic RNA segments, namely those encoding the putative movement protein (MP) and the nucleocapsid (NP) protein, remained undetermined. High-throughput sequencing of total RNA and small RNA preparations, combined with reverse transcription PCR amplification followed by sequencing, revealed that the WCLaV-1 and WCLaV-2 possess a bipartite genome consisting of a negative-sense RNA1, encoding the RNA-dependent RNA polymerase, and an ambisense RNA2, encoding the putative movement (MP) and nucleocapsid (NP) proteins. The two open reading frames of RNA2 are in opposite orientations and are separated by a long AU-rich intergenic region (IR) that may assume a hairpin conformation. RNA1 and RNA2 of both viruses share almost identical 5' and 3' termini, which are complementary to each other up to 20 nt. This genome organization is typical of members of the genus Coguvirus, with which WCLaV-1 and WCLaV-2 also share similar terminal 5' and 3' sequences of RNA1 and RNA2. These molecular features, together with phylogenetic reconstructions support the classification of WCLaV-1 and WCLaV2 as members of two new species in the genus Coguvirus.

Dynamic subcellular distribution of begomoviral nuclear shuttle and movement proteins

The Abutilon mosaic virus (AbMV) encodes a nuclear shuttle protein (NSP), and a movement protein (MP) which cooperatively accomplish viral DNA transport through the plant. Subcellular distribution patterns of fluorescent protein-tagged NSP and MP were tracked in Nicotiana benthamiana leaves in presence or absence of an AbMV infection using light microscopy. NSP was located within the nucleus and associated with early endosomes in the presence of MP. MP appeared at the plasma membrane, plasmodesmata and in motile vesicles, trafficking along the endoplasmic reticulum in an actin-dependent manner. MP and NSP did not co-localize and employed separate cellular pathways. Correspondingly, Förster resonance energy transfer analysis did not support physical interaction between NSP and MP. Time lapse movies illustrate the cellular dynamics of both proteins on their way around the nucleus and to the cell periphery and provide a first hint for the nuclear egress of NSP complexes.

Leaf Plasmodesmata Respond Differently to TMV, ToBRFV and TYLCV Infection.

Macromolecule and cytosolic signal distribution throughout the plant employs a unique cellular and intracellular mechanism called plasmodesmata (PD). Plant viruses spread throughout plants via PD using their movement proteins (MPs). Viral MPs induce changes in plasmodesmata’s structure and alter their ability to move macromolecule and cytosolic signals. The developmental distribution of a family member of proteins termed plasmodesmata located proteins number 5 (PDLP5) conjugated to GFP (PDLP5-GFP) is described here. The GFP enables the visual localization of PDLP5 in the cell via confocal microscopy. We observed that PDLP5-GFP protein is present in seed protein bodies and immediately after seed imbibition in the plasma membrane. The effect of three different plant viruses, the tobacco mosaic virus (TMV), tomato brown rugose fruit virus (ToBRFV, tobamoviruses), and tomato yellow leaf curl virus (TYLCV, begomoviruses), on PDLP5-GFP accumulation at the plasmodesmata was tested. In tobacco leaf, TMV and ToBRFV increased PDLP5-GFP amount at the plasmodesmata of cell types compared to control. However, there was no statistically significant difference in tomato leaf. On the other hand, TYLCV decreased PDLP5-GFP quantity in plasmodesmata in all tomato leaf cells compared to control, without any significant effect on plasmodesmata in tobacco leaf cells.

First report of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 infecting watermelon (Citrullus lanatus) in the United States.

Watermelon (Citrullus lanatus) and other cucurbits are cultivated globally, and Texas ranks among its top 5 producers in the U.S. In July 2020, plants with virus-like disease symptoms consisting of mild leaf crinkling and yellow mosaic patterns were observed in a 174-ha watermelon field in Burleson Co., TX; disease incidence was visually estimated at 5%. Total nucleic acids were extracted from leaf tissues of 5 randomly sampled plants (Dellaporta 1983) and their equimolar amounts were made into a composite sample that was used for cDNA library construction with TruSeq Stranded Total RNA with Ribo-Zero Plant Kit (Illumina). The cDNA library was sequenced on the Illumina NextSeq 500 platform, generating ~37M single-end reads (each 75 nt), which were analyzed as per Al Rwahnih et al. (2018). Of these, 58,200 and 27,500 reads mapped to the genomes of watermelon crinkle leaf-associated virus 1 (WCLaV-1) and WCLaV-2 (Xin et al. 2017), respectively, along with 4 other virus-specific reads (data not shown). The near complete RNA1-RNA3 segments of WCLaV-1 (354-652X) and WCLaV-2 (144-258X) were generated from the mapped reads and they shared ≥96% nt identities with published RNA segments of both viruses. The results were verified by RT-PCR using newly designed primers WCLaV-1vRP: 5'-GGTGAGTTAGTGTGTCTGAAGG/WCLaV-1cRP: 5'-GAGGTTGCCTGAGGTGATAAG to target 881 bp of the RNA1-encoded RNA-dependent RNA polymerase (RdRP), WCLaV-1vMP: 5'-GAAGGTTTGCTCCCTTGAAATG/WCLaV-1cMP: 5'-GACTGTGGCTGAAGAGTCTATG target 538 bp of the RNA2-encoded movement protein (MP), and WCLaV-1vNP: 5'-CGAATAGACTCTGGAGGGTAGA/WCLaV-1cMP: 5'-GAAAGCAAGAAAGCTGGCTAAA target 786 bp of the RNA3-encoded nucleoprotein (NP). Similarly, the WWCLaV-2-specific primers WCLaV-2vRP: 5'-GTCTCACATTCCTGCACTAACT/WCLaV-2cRP: 5'-ATCGGTCCTGGGTTATTTGTATC target 968 bp of the RdRP, WCLaV-2vMP: 5'-GACTTCAGAACCTCAACATCCA/WCLaV-2cMP: 5'-CAAGGGAGAGTGCTGACAAA target 562 bp of the MP, and WCLaV-2vNP: 5'-ATTCCCAGTGAGAGCAACAA/WCLaV-2cMP: 5'-GAGGTGGAGGTAGGAAAGAAAG target 449 bp of the NP. Fresh cDNA synthesized from the 5 samples with PrimeScript First Strand cDNA synthesis kit (Takara Bio) were tested by PCR with all 6 primer pairs using the PrimeSTAR GXL DNA Polymerase kit (Takara Bio). Three of the 5 samples were positive for both viruses and one sample was positive for each virus. The obtained products from 4 samples were cloned individually into pJET1.2/Blunt vector (Thermo Scientific, USA), followed by bidirectional Sanger-sequencing of the plasmids with the GenElute Five-Minute Plasmid Miniprep kit (Sigma-Aldrich). In pairwise comparisons, the partial RNA1-RNA3 sequences of WCLaV-1 (GenBank accession nos. MW559074-82) shared 100% nt/aa identities with each other and with corresponding sequences of WCLaV-1 isolate KF-1 from China (KY781184-86). The partial RNA1-RNA3 sequences of WCLaV-2 (MW559083-91) shared 97-100% nt/96-100% aa identities with each other and with corresponding sequences of WCLaV-2 isolate KF-15 from China (KY781187-89). This is the first report of WCLaV-1 and WCLaV-2 in Texas and the first record of both viruses in the U.S. and elsewhere outside of China. Both negative-sense, single-stranded RNA viruses represent a novel taxon in the family Phenuiviridae (order Bunyavirales) (Xin et al. 2017). While aspects of the biology of both viruses are yet to be elucidated, our results expand their geographical range. The detection primers developed here will be useful for screening cucurbits germplasm to avert their spread.

Integrated Proteomics and Transcriptomics Analyses Reveal the Transcriptional Slippage of P3N-PIPO in a Bymovirus.

P3N-PIPO (P3 N-terminal fused with Pretty Interesting Potyviridae ORF), the movement protein of potyviruses, is expressed as a translational fusion with the N-terminus of P3 in potyviruses. As reported in previous studies, P3N-PIPO is expressed via transcriptional slippage at a conserved G2A6 slippery site in the genus Potyvirus. However, it is still unknown whether a similar expression mechanism of P3N-PIPO is used in the other genera of the family Potyviridae. Moreover, due to the extremely low expression level of P3N-PIPO in natural virus-infected plants, the peptides spanning the slippery site which provide direct evidence of the slippage at the protein level, have not been identified yet. In this study, a potato virus X (PVX)-based expression vector was utilized to investigate the expression mechanism of P3N-PIPO. A high expression level of the P3N-PIPO(WT) of turnip mosaic virus (TuMV, genus Potyvirus) was observed based on the PVX expression vector. For the first time, we successfully identified the peptides of P3N-PIPO spanning the slippery site by mass spectrometry. Likewise, the P3N-PIPO(WT) of wheat yellow mosaic virus (WYMV, genus Bymovirus) was also successfully expressed using the PVX expression vector. Integrated proteome and transcriptome analyses revealed that WYMV P3N-PIPO was expressed at the conserved G2A6 site through transcriptional slippage. Moreover, as revealed by mutagenesis analysis, Hexa-adenosine of the G2A6 site was important for the frameshift expression of P3N-PIPO in WYMV. According to our results, the PVX-based expression vector might be used as an excellent tool to study the expression mechanism of P3N-PIPO in Potyviridae. To the best of our knowledge, this is the first experimental evidence dissecting the expression mechanism of a bymovirus P3N-PIPO in the experimental host Nicotiana benthamiana.

Polymorphisms at the 3’end of the movement protein (MP) gene of grapevine Pinot gris virus (GPGV) affect virus titre and small interfering RNA accumulation in GLMD disease

Grapevine Leaf Mottling and Deformation (GLMD) is a grapevine disease that has been associated with a trichovirus, the grapevine Pinot gris virus (GPGV). A wide diversity in the severity of GLMD disease symptoms has been recorded worldwide, but the relationship of this diversity to the sequence variation in the GPGV genome is still a matter of debate. Results from comparative analysis of GPGV genomic sequences have suggested an association of polymorphisms at the 3’-end of the movement protein (MP) with GLMD severity. Here, the 3’-terminus of the MP gene of a GPGV infectious clone derived from an isolate from grapevine showing severe symptoms (fvg-12), was substituted with a 356 bp synthetic DNA fragment having a sequence resembling that of another GPGV isolate (fvg-15), recovered from an asymptomatic grapevine. The clone containing this chimeric construct was root-inoculated in virus-free Kober rootstocks along with the clones containing the fvg-12 and fvg-15 full length sequence. Remarkable differences in virus titre, accumulation of GPGV-derived small interfering RNAs (siRNAs), alterations in the gene expression of boron transporters and, to a lesser extent, in symptom expression were recorded among plants infected with either one of the three GPGV derived clones. In particular, the chimeric clone behaviour was indistinguishable from that of the donor of the small 356 bp fragment and significantly different from the other. Thus, this work experimentally confirmed the critical role of the GPGV-MP C-terminus in determining the fate of the infection, as it had been previously hypothesized on the basis of comparative sequence analysis.

Identification and Characterization of a Novel Emaravirus From Grapevine Showing Chlorotic Mottling Symptoms.

A novel negative-sense, single-stranded (ss) RNA virus was identified in a “Shennong Jinhuanghou” (SJ) grapevine showing severe chlorotic mottling symptoms by integrating high-throughput sequencing (HTS) and conventional Sanger sequencing of reverse transcription polymerase chain reaction (RT-PCR) products. The virus was provisionally named as “grapevine emaravirus A” (GEVA). GEVA had a genome comprising five genomic RNA segments, each containing a single open reading frame on the viral complementary strand and two untranslated regions with complementary 13- nt stretches at the 5′ and 3′ terminal ends. RNA1 (7,090 nt), RNA2 (2,097 nt), RNA3 (1,615 nt), and RNA4 (1,640 nt) encoded putative proteins P1–P4 that, based on their conserved motifs, were identified as the RNA-dependent RNA polymerase, glycoprotein, nucleocapsid protein, and movement protein, respectively. However, the functional role of protein P5 encoded by RNA5 (1,308 nt) could not be determined. Phylogenetic trees constructed based on amino acids of P1 to P4, allocated GEVA in clade I, together with other species-related emaraviruses. These data support the proposal that GEVA is a representative member of a novel species in the genus Emaravirus of the family Fimoviridae. Moreover, when GEVA was graft-transmitted to SJ and “Beta” grapevines, all grafted plants showed the same symptoms, similar to those observed in the source of the inoculum. This is the first report to our knowledge of an emaravirus infecting grapevine and its possible association with chlorotic mottling symptoms.

Molecular and serological detection of Parietaria mottle virus in Phytolacca americana, a new host of the virus

Parietaria mottle virus (PMoV) is an emerging virus in Mediterranean countries, responsible for severe disease in tomato and pepper crops in the field and protected cultivation. The principal wild reservoir of PMoV is Parietaria officinalis, and only few additional wild plants have been described as natural reservoirs of the virus. During field survey in southern Italy, several plants of Phytolacca americana showing virus-like symptoms were collected. Serological and molecular assays showed that these plants were infected by PMoV. Sequence comparison of the movement protein gene of the PMoV isolate from P. americana showed the greatest similarity to the corresponding sequence from tomato plants growing nearby. These results indicate that P. ameriacana is a new natural host of PMoV, and further investigation is warranted to establish the potential of this host as reservoir of the virus in the field.

Complete genome sequence analysis of a novel coguvirus isolated from Paris polyphylla var. yunnanensis.

A novel negative-stranded (ns) RNA virus tentatively named "Yunnan paris negative-stranded virus" (YPNSV), was isolated from Paris polyphylla var. yunnanensis plants exhibiting leaf chlorosis and mosaic symptoms in Yunnan. Its complete genome sequence was determined using Illumina and Sanger sequencing. YPNSV has a bipartite genome that consists of a negative-stranded (ns) RNA1 encoding the viral RNA-dependent RNA polymerase (RdRp, p251), an ambisense RNA2 coding for the putative movement protein (MP, p46) and nucleocapsid protein (NP, p39), with the two open reading frames separated by a long intergenic region that is rich in A and U. Sequence comparisons showed that the RdRp, MP, and NP of YPNSV are most similar to those of watermelon crinkle leaf-associated virus 2 (WCLaV-2), with 69.1%, 50.4%, and 60.9% amino acid sequence identity, respectively. Phylogenetic analysis based on deduced amino acid sequences of RdRp and NP showed that YPNSV clustered in a clade with coguviruses and that its closest known relative is WCLaV-2. Based on the above results, YPNSV should be regarded as a new member of genus Coguvirus, family Phenuiviridae.

Receptor-like kinase BAM1 facilitates early movement of the Tobacco mosaic virus

Cell-to-cell movement is an important step for initiation and spreading of virus infection in plants. This process occurs through the intercellular connections, termed plasmodesmata (PD), and is usually mediated by one or more virus-encoded movement proteins (MP) which interact with multiple cellular factors, among them protein kinases that usually have negative effects on MP function and virus movement. In this study, we report physical and functional interaction between MP of Tobacco mosaic virus (TMV), the paradigm of PD-moving proteins, and a receptor-like kinase BAM1 from Arabidopsis and its homolog from Nicotiana benthamiana. The interacting proteins accumulated in the PD regions, colocalizing with a PD marker. Reversed genetics experiments, using BAM1 gain-of-function and loss-of-function plants, indicated that BAM1 is required for efficient spread and accumulation the virus during initial stages of infection of both plant species by TMV. Furthermore, BAM1 was also required for the efficient cell-to-cell movement of TMV MP, suggesting that BAM1 interacts with TMV MP to support early movement of the virus. Interestingly, this role of BAM1 in viral movement did not require its protein kinase activity. Thus, we propose that association of BAM1 with TMV MP at PD facilitates the MP transport through PD, which, in turn, enhances the spread of the viral infection.

Molecular Epidemiology of Citrus Leprosis Virus C: A New Viral Lineage and Phylodynamic of the Main Viral Subpopulations in the Americas.

Despite the importance of viral strains/variants as agents of emerging diseases, genetic and evolutionary processes affecting their ecology are not fully understood. To get insight into this topic, we assessed the population and spatial dynamic parameters of citrus leprosis virus C (CiLV-C, genus Cilevirus, family Kitaviridae). CiLV-C is the etiological agent of citrus leprosis disease, a non-systemic infection considered the main viral disorder affecting citrus orchards in Brazil. Overall, we obtained 18 complete or near-complete viral genomes, 123 complete nucleotide sequences of the open reading frame (ORF) encoding the putative coat protein, and 204 partial nucleotide sequences of the ORF encoding the movement protein, from 430 infected Citrus spp. samples collected between 1932 and 2020. A thorough examination of the collected dataset suggested that the CiLV-C population consists of the major lineages CRD and SJP, unevenly distributed, plus a third one called ASU identified in this work, which is represented by a single isolate found in an herbarium sample collected in Asuncion, Paraguay, in 1937. Viruses from the three lineages share about 85% nucleotide sequence identity and show signs of inter-clade recombination events. Members of the lineage CRD were identified both in commercial and non-commercial citrus orchards. However, those of the lineages SJP were exclusively detected in samples collected in the citrus belt of São Paulo and Minas Gerais, the leading Brazilian citrus production region, after 2015. The most recent common ancestor of viruses of the three lineages dates back to, at least, ∼1500 years ago. Since citrus plants were introduced in the Americas by the Portuguese around the 1520s, the Bayesian phylodynamic analysis suggested that the ancestors of the main CiLV-C lineages likely originated in contact with native vegetation of South America. The intensive expansion of CRD and SJP lineages in Brazil started probably linked to the beginning of the local citrus industry. The high prevalence of CiLV-C in the citrus belt of Brazil likely ensues from the intensive connectivity between orchards, which represents a potential risk toward pathogen saturation across the region.

Carrot mottle virus ORF4 movement protein targets plasmodesmata by interacting with the host cell SUMOylation system.

Plant virus movement proteins (MPs) facilitate virus spread in their plant hosts, and some of them are known to target plasmodesmata (PD). However, how the MPs target PD is still largely unknown. Carrot mottle virus (CMoV) encodes the ORF3 and ORF4 proteins, which are involved in CMoV movement. In this study, we used CMoV as a model to study the PD targeting of a plant virus MP. We showed that the CMoV ORF4 protein, but not the ORF3 protein, modified PD and led to the virus movement. We found that the CMoV ORF4 protein interacts with the host cell small ubiquitin-like modifier (SUMO) 1, 2 and the SUMO-conjugating enzyme SCE1, resulting in the ORF4 protein SUMOylation. Downregulation of mRNAs for NbSCE1 and NbSUMO impaired CMoV infection. The SUMO-interacting motifs (SIMs) LVIVF, VIWV, and a lysine residue at position 78 (K78) are required for the ORF4 protein SUMOylation. The mutation of these motifs prevented the protein to efficiently target PD, and further slowed or completely abolished CMoV systemic movement. Finally, we found that some of these motifs are highly conserved among umbraviruses. Our data suggest that the CMoV ORF4 protein targets PD by interacting with the host cell SUMOylation system.

Micro RNA-induced gene silencing strategy for the delivery of siRNAs targeting Meloidogyne incognita in a model plant Nicotiana benthamiana.

Occurrence of multiple biotic stresses on crop plants result in drastic yield losses which may have severe impact on the food security. It is a challenge to design strategies for simultaneous management of these multiple stresses. Hence, establishment of innovative approaches that aid in their management is critical. Here, we have introgressed a micro RNA-induced gene silencing (MIGS) based combinatorial gene construct containing seven target gene sequences of cotton leaf curl disease (CLCuD), cotton leaf hopper (Amrasca biguttula biguttula), cotton whitefly (Bemisia tabaci) and root-knot nematode (Meloidogyne incognita). Stable transgenic lines of Nicotiana benthamiana were generated with the T-DNA harboring Arabidopsis miR173 target site fused to fragments of Sec23 and ecdysone receptor (EcR) genes of cotton leaf hopper and cotton whitefly. It also contained C2/replication associated protein (C2/Rep) and C4 (movement protein) along with βC1 gene of betasatellite to target CLCuD, and two FMRFamide-like peptide (FLP) genes, Mi-flp14 and Mi-flp18 of M. incognita. These transgenic plants were assessed for the amenability of MIGS approach for pest control by efficacy evaluation against M. incognita. Results showed successful production of small interfering RNA (siRNA) through the tasiRNA (trans-acting siRNA) pathway in the transgenic plants corresponding to Mi-flp18 gene. Furthermore, we observed reduced Mi-flp14 and Mi-flp18 transcripts (up to 2.37 ± 0.12-fold) in females extracted from transgenic plants. The average number of galls, total endoparasites, egg masses and number of eggs per egg mass reduced were in the range 27-62%, 39-70%, 38-65% and 34-49%, respectively. More importantly, MIGS transgenic plants showed 80% reduction in the nematode multiplication factor (MF). This study demonstrates successful validation of the MIGS approach in the model plant, N. benthamiana for efficacy against M. incognita, as a prelude to translation to cotton. © 2021 Society of Chemical Industry.

Articles of Significant Interest in This Issue

Opium poppy mosaic virus (OPMV) is a single-stranded plus-sense RNA virus that does not encode a capsid protein and requires a helper virus. Ilyas et al. (e02109-20) found that OPMV generates two similarly sized subgenomic RNAs (sgRNAs), one of which is synthesized by the replicase and is the template for translation of viral movement proteins. The other is generated by an exoribonuclease-resistant sequence, a mechanism that normally produces small, noncoding sgRNAs. However, this sgRNA codes for a 30-kDa protein with a functional role in OPMV accumulation. The authors also determined that OPMV uses a Barley yellow dwarf virus-like translation element (BTE) as one of its 3′ cap-independent translation enhancers. While BTEs were thought to have little in common beyond a 17-nucleotide conserved sequence, analysis of the 14 known BTE structures showed that BTEs contain additional, strongly conserved sequences and structural features that include residues known to interact with translation initiation factor eIF4G.