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Long life cycle and lack of efficient and robust virus inoculation technique are the major technical challenges for studying virus infection in perennial woody plants such as fruit trees. Biolistic technology also called particle bombardment is a physical approach that can directly introduce virions or viral full-length cDNA infectious clones into target cells and tissues by high velocity microcarrier particles. The flexibility and high efficiency of the biolistic inoculation method facilitate research on fruit tree virology and the screening and identification of fruit tree germplasms resistant to viruses. Here, we describe a detailed protocol for the biolistic inoculation of peach with of a cDNA infectious clone of Plum pox virus (PPV) using the Helios gene gun, a biolistic particle delivery system., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana . As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus -infecting viruses.

In this study, we demonstrate a novel pro-viral role for the Nicotiana benthamiana ARGONAUTE 1 (AGO1) in potyvirus infection. AGO1 strongly enhanced potato virus A (PVA) particle production and benefited the infection when supplied in excess. We subsequently identified the potyviral silencing suppressor, helper-component protease (HCPro), as the recruiter of host AGO1. After the identification of a conserved AGO1-binding GW/WG motif in potyviral HCPros, we used site-directed mutagenesis to introduce a tryptophan-to-alanine change into the HCPro (HCProAG) of PVA (PVAAG) and turnip mosaic virus (TuMVAG). AGO1 co-localization and co-immunoprecipitation with PVA HCPro was significantly reduced by the mutation suggesting the interaction was compromised. Although the mutation did not interfere with HCPro's complementation or silencing suppression capacity, it nevertheless impaired virus particle accumulation and the systemic spread of both PVA and TuMV. Furthermore, we found that the HCPro-AGO1 interaction was important for AGO1's association with the PVA coat protein. The coat protein was also more stable in wild type PVA infection than in PVAAG infection. Based on these findings we suggest that potyviral HCPro recruits host AGO1 through its WG motif and engages AGO1 in the production of stable virus particles, which are required for an efficient systemic infection., Competing Interests: The authors have declared that no competing interests exist.

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1-97), the core (amino acids 98-245), and the C-terminus (amino acids 246-288). We found that deletion of CP or its segments amino acids 51-199, amino acids 200-283, or amino acids 265-274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6-50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional., (© 2020 Her Majesty the Queen in Right of Canada Molecular Plant Pathology © 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd. Reproduced with the permission of the Minister of Agriculture and Agri-food Canada.)

A successful viral infection requires complex, compatible molecular interactions between the invading virus and the host. A better understanding of such interactions may assist in the development of novel approaches to control viral diseases for sustainable crop production. In the past decade, the cell biology of virus-host and virus-vector interactions has been one of the most exciting areas of research in the molecular plant-microbe field. This is partially attributed to the availability of powerful cell biology techniques, including imaging tools like confocal microscopy and electron microscopy and tomography. As a result, there has been an unprecedented increase in knowledge in the areas of the bi- and tripartite interactions of virus, host, and vector. We now have a much clearer picture of viral virulence mechanisms, virus-induced host defenses, viral counteracting strategies, and viral circulations in the insect vectors. This Focus Issue highlights molecular virus-plant and virus-vector interactions in the areas of cell biology and closely related disciplines and explores biotechnology-based antiviral strategies using knowledge generated from these research areas.

RNA silencing is a fundamental, evolutionarily conserved mechanism that regulates gene expression in eukaryotes. It also functions as a primary immune defense in microbes, such as viruses in plants. In addition to RNA silencing, RNA decay and RNA quality-control pathways are also two ancestral forms of intrinsic antiviral immunity, and the three RNA-targeted pathways may operate cooperatively for their antiviral function. Plant viruses encode viral suppressors of RNA silencing (VSRs) to suppress RNA silencing and facilitate virus infection. In response, plants may activate a counter-counter-defense mechanism to cope with VSR-mediated RNA silencing suppression. In this review, we summarize current knowledge of RNA silencing, RNA decay, and RNA quality control in antiviral defense, and highlight the mechanisms by which viruses compromise RNA-targeted immunity for their infection and survival in plants., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

Bromoviridae is a family of plant viruses with tri-segmented, positive-sense, single-stranded RNA genomes of about 8 kb in total. Genomic RNAs are packaged in separate virions that may also contain subgenomic, defective or satellite RNAs. Virions are variable in morphology (spherical or bacilliform) and are transmitted between hosts mechanically, in/on the pollen and non-persistently by insect vectors. Members of the family are responsible for major disease epidemics in fruit, vegetable and fodder crops such as tomato, cucurbits, bananas, fruit trees and alfalfa. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Bromoviridae, which is available at www.ictv.global/report/bromoviridae.

Areca palm ( Areca catechu ), one of the two most important commercial crops in Hainan, China, has been severely damaged by a variety of pathogens and insects. Here, we report a new disease, tentatively referred to as areca palm necrotic ringspot disease (ANRSD), which is highly epidemic in the main growing regions in Hainan. Transmission electron microscopy observation and small RNA deep sequencing revealed the existence of a viral agent of the family Potyviridae in a diseased areca palm plant (XC1). The virus was tentatively named areca palm necrotic ringspot virus (ANRSV). Subsequently, the positive-sense single-stranded genome of ANRSV isolate XC1 was completely determined. The genome annotation revealed the existence of two cysteine proteinases in tandem (HC-Pro1 and HC-Pro2) in the genomic 5' terminus of ANRSV. Sequence comparison and phylogenetic analysis suggested the taxonomic classification of ANRSV into the recently proposed genus Arepavirus in the family Potyviridae . Given the close relationship of ANRSV with another newly reported arepavirus (areca palm necrotic spindle-spot virus), the exact taxonomic status of ANRSV needs to be further investigated. In this study, a reverse transcription polymerase chain reaction assay for ANRSV-specific detection was developed and a close association between ANRSV and ANRSD was found.

Background: Pear ring rot, caused by Botryosphaeria species, is responsible for substantial economic losses by causing severe recession of pear tree growth in China. Mycovirus-mediated hypovirulence in plant pathogenic fungi is a crucial biological method to control fungal diseases., Methods: We conducted a large-scale and comprehensive transcriptome analysis to identify mRNA in B. dothidea in response to mycovirus. De novo sequencing technology from four constructed libraries of LW-C (Botryosphaeria dothidea chrysovirus 1, BdCV1), LW-P (Botryosphaeria dothidea partitivirus 1, BdPV1), LW-CP (LW-1 strain infection with BdCV1 and BdPV1), and Mock (free virus) was used to investigate and compare gene expression changes in B.dothidea strains infected with mycovirus., Results: In total, 30,058 Unigenes with an average length of 2128 bp were obtained from 4 libraries of B. dothidea strains. These were annotated to specify their classified function. We demonstrate that mRNAs of B. dothidea strains in response to mycovirus are differentially expressed. In total, 5598 genes were up-regulated and 3298 were down-regulated in the LW-CP group, 4468 were up-regulated and 4291 down-regulated in the LW-C group, and 2590 were up-regulated and 2325 down-regulated in the LW-P group. RT-qPCR was used to validate the expression of 9 selected genes. The B. dothidea transcriptome was more affected by BdCV1 infection than BdPV1. We conducted GO enrichment analysis to characterize gene functions regulated by B. dothidea with mycovirus infection. These involved metabolic process, cellular process, catalytic activity, transporter activity, signaling, and other biological pathways. KEGG function analysis demonstrated that the enriched differentially expressed genes are involved in metabolism, transcription, signal transduction, and ABC transport. mRNA is therefore involved in the interaction between fungi and mycovirus. In addition, changes in differential accumulation levels of cp and RdRp of BdCV1 and BdPV1 in B. dothidea strains were evaluated, revealing that the accumulation of BdCV1 and BdPV1 is related to the phenotype and virulence of B. dothidea strain LW-1., Conclusions: The identification and analysis of mRNAs from B. dothidea was first reported at the transcriptome level. Our analysis provides further insight into the interaction of B. dothidea strains infection with chrysovirus 1 (BdCV1) and partitivirus 1 (BdPV1) at the transcriptome level.

Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5'-3' RNA decay pathway gene-encoded proteins (5'RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5'RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5'RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5'RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection., Competing Interests: The authors have declared that no competing interests exist.

Autophagy emerges as an essential immunity defense against intracellular pathogens. Here we report that turnip mosaic virus (TuMV) infection activates autophagy in plants and that Beclin1 (ATG6), a core component of autophagy, inhibits virus replication. Beclin1 interacts with NIb, the RNA-dependent RNA polymerase (RdRp) of TuMV, via the highly conserved GDD motif and the interaction complex is targeted for autophagic degradation likely through the adaptor protein ATG8a. Beclin1-mediated NIb degradation is inhibited by autophagy inhibitors. Deficiency of Beclin1 or ATG8a enhances NIb accumulation and promotes viral infection and vice versa. These data suggest that Beclin1 may be a selective autophagy receptor. Overexpression of a Beclin1 truncation mutant that binds to NIb but lacks the ability to mediate NIb degradation also inhibits virus replication. The Beclin1-RdRp interaction further extends to several RNA viruses. Thus Beclin1 restricts viral infection through suppression and also likely autophagic degradation of the viral RdRp.

Plasmodesmata (PD), unique to the plant kingdom, are structurally complex microchannels that cross the cell wall to establish symplastic communication between neighbouring cells. Viral intercellular movement occurs through PD. To better understand the involvement of PD in viral infection, we conducted a quantitative proteomic study on the PD-enriched fraction from Nicotiana benthamiana leaves in response to infection by Turnip mosaic virus (TuMV). We report the identification of a total of 1070 PD protein candidates, of which 100 (≥2-fold increase) and 48 (≥2-fold reduction) are significantly differentially accumulated in the PD-enriched fraction, when compared with protein levels in the corresponding healthy control. Among the differentially accumulated PD protein candidates, we show that an α-expansin designated NbEXPA1, a cell wall loosening protein, is PD-specific. TuMV infection downregulates NbEXPA1 mRNA expression and protein accumulation. We further demonstrate that NbEXPA1 is recruited to the viral replication complex via the interaction with NIb, the only RNA-dependent RNA polymerase of TuMV. Silencing of NbEXPA1 inhibits plant growth and TuMV infection, whereas overexpression of NbEXPA1 promotes viral replication and intercellular movement. These data suggest that NbEXPA1 is a host factor for potyviral infection. This study not only generates a PD-proteome dataset that is useful in future studies to expound PD biology and PD-mediated virus-host interactions but also characterizes NbEXPA1 as the first PD-specific cell wall loosening protein and its essential role in potyviral infection., (© 2017 Her Majesty the Queen in Right of Canada. The Plant Journal © 2017 John Wiley & Sons Ltd and Society for Experimental Biology.)

Rsv1, a single dominant resistance locus in soybean, confers extreme resistance to the majority of Soybean mosaic virus (SMV) strains, but is susceptible to the G7 strain. In Rsv1-genotype soybean, G7 infection provokes a lethal systemic hypersensitive response (LSHR), a delayed host defence response. The Rsv1-mediated LSHR signalling pathway remains largely unknown. In this study, we employed a genome-wide investigation to gain an insight into the molecular interplay between SMV G7 and Rsv1-genotype soybean. Small RNA (sRNA), degradome and transcriptome sequencing analyses were used to identify differentially expressed genes (DEGs) and microRNAs (DEMs) in response to G7 infection. A number of DEGs, DEMs and microRNA targets, and the interaction network of DEMs and their target mRNAs responsive to G7 infection, were identified. Knock-down of one of the identified DEGs, the eukaryotic translation initiation factor 5A (eIF5A), diminished the LSHR and enhanced viral accumulation, suggesting the essential role of eIF5A in the G7-induced, Rsv1-mediated LSHR signalling pathway. This work provides an in-depth genome-wide analysis of high-throughput sequencing data, and identifies multiple genes and microRNA signatures that are associated with the Rsv1-mediated LSHR., (© 2016 HER MAJESTY THE QUEEN IN RIGHT OF CANADA MOLECULAR PLANT PATHOLOGY © 2016 BSPP AND JOHN WILEY & SONS LTD.)

The Potyviridae is the largest family of RNA plant viruses, members of which have single-stranded, positive-sense RNA genomes and flexuous filamentous particles 680-900 nm long and 11-20 nm wide. There are eight genera, distinguished by the host range, genomic features and phylogeny of the member viruses. Genomes range from 8.2 to 11.3 kb, with an average size of 9.7 kb. Most genomes are monopartite but those of members of the genus Bymovirus are bipartite. Some members cause serious disease epidemics in cultivated plants. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Potyviridae, which is available at www.ictv.global/report/potyviridae.

RNA silencing is a powerful technology for molecular characterization of gene functions in plants. A commonly used approach to the induction of RNA silencing is through genetic transformation. A potent alternative is to use a modified viral vector for virus-induced gene silencing (VIGS) to degrade RNA molecules sharing similar nucleotide sequence. Unfortunately, genomic studies in many allogamous woody perennials such as peach are severely hindered because they have a long juvenile period and are recalcitrant to genetic transformation. Here, we report the development of a viral vector derived from Prunus necrotic ringspot virus (PNRSV), a widespread fruit tree virus that is endemic in all Prunus fruit production countries and regions in the world. We show that the modified PNRSV vector, harbouring the sense-orientated target gene sequence of 100-200 bp in length in genomic RNA3, could efficiently trigger the silencing of a transgene or an endogenous gene in the model plant Nicotiana benthamiana. We further demonstrate that the PNRSV-based vector could be manipulated to silence endogenous genes in peach such as eukaryotic translation initiation factor 4E isoform (eIF(iso)4E), a host factor of many potyviruses including Plum pox virus (PPV). Moreover, the eIF(iso)4E-knocked down peach plants were resistant to PPV. This work opens a potential avenue for the control of virus diseases in perennial trees via viral vector-mediated silencing of host factors, and the PNRSV vector may serve as a powerful molecular tool for functional genomic studies of Prunus fruit trees., (© 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

A recently characterized calmodulin-like protein is an endogenous RNA silencing suppressor that suppresses sense-RNA induced post-transcriptional gene silencing (S-PTGS) and enhances virus infection, but the mechanism underlying calmodulin-like protein-mediated S-PTGS suppression is obscure. Here, we show that a calmodulin-like protein from Nicotiana benthamiana (NbCaM) interacts with Suppressor of Gene Silencing 3 (NbSGS3). Deletion analyses showed that domains essential for the interaction between NbSGS3 and NbCaM are also required for the subcellular localization of NbSGS3 and NbCaM suppressor activity. Overexpression of NbCaM reduced the number of NbSGS3-associated granules by degrading NbSGS3 protein accumulation in the cytoplasm. This NbCaM-mediated NbSGS3 degradation was sensitive to the autophagy inhibitors 3-methyladenine and E64d, and was compromised when key autophagy genes of the phosphatidylinositol 3-kinase (PI3K) complex were knocked down. Meanwhile, silencing of key autophagy genes within the PI3K complex inhibited geminivirus infection. Taken together these data suggest that NbCaM acts as a suppressor of RNA silencing by degrading NbSGS3 through the autophagy pathway.

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

Unlabelled: The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage., Importance: Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

MicroRNAs (miRNAs) play key roles in a variety of cellular processes through regulation of their target gene expression. Accumulated experimental evidence has demonstrated that infections by viruses are associated with the altered expression profile of miRNAs and their mRNA targets in the host. However, the regulatory network of miRNA-mRNA interactions during viral infection remains largely unknown. In this study, we performed small RNA (sRNA)-seq, degradome-seq and as well as a genome-wide transcriptome analysis to profile the global gene and miRNA expression in soybean following infections by three different Soybean mosaic virus (SMV) isolates, L (G2 strain), LRB (G2 strain) and G7 (G7 strain). sRNA-seq analyses revealed a total of 253 soybean miRNAs with a two-fold or greater change in abundance compared with the mock-inoculated control. 125 transcripts were identified as the potential cleavage targets of 105 miRNAs and validated by degradome-seq analyses. Genome-wide transcriptome analysis showed that total 2679 genes are differentially expressed in response to SMV infection including 71 genes predicted as involved in defense response. Finally, complex miRNA-mRNA regulatory networks were derived using the RNAseq, small RNAseq and degradome data. This work represents a comprehensive, global approach to examining virus-host interactions. Genes responsive to SMV infection are identified as are their potential miRNA regulators. Additionally, regulatory changes of the miRNAs themselves are described and the regulatory relationships were supported with degradome data. Taken together these data provide new insights into molecular SMV-soybean interactions and offer candidate miRNAs and their targets for further elucidation of the SMV infection process.

Background: MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures., Results: Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots., Conclusions: This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

Profiling small RNAs in soybean Williams 82 (rsv), susceptible to Soybean mosaic virus (SMV, the genus Potyvirus, family Potyviridae) strains G2 and G7, and soybean PI96983 (Rsv1), resistant to G2 but susceptible to G7, identified the microRNA miR168 that was highly overexpressed only in G7-infected PI96983 showing a lethal systemic hypersensitive response (LSHR). Overexpression of miR168 was in parallel with the high-level expression of AGO1 mRNA, high-level accumulation of miR168-mediated AGO1 mRNA cleavage products but with severely repressed AGO1 protein. In contrast, AGO1 mRNA, degradation products and protein remained without significant changes in G2- and G7-infected Williams 82. Moreover, knock-down of SGS3, an essential component in RNA silencing, suppressed AGO1 siRNA, partially recovered repressed AGO1 protein, and alleviated LSHR severity in G7-infected Rsv1 soybean. These results suggest that both miRNA and siRNA pathways are involved in G7 infection of Rsv1 soybean, and LSHR is associated with breakdown of AGO1 homeostasis., (Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.)

A successful infection by a plant virus results from the complex molecular interplay between the host plant and the invading virus. Thus, dissecting the molecular network of virus-host interactions advances the understanding of the viral infection process and may assist in the development of novel antiviral strategies. In the past decade, molecular identification and functional characterization of host factors in the virus life cycle, particularly single-stranded, positive-sense RNA viruses, have been a research focus in plant virology. As a result, a number of host factors have been identified. These host factors are implicated in all the major steps of the infection process. Some host factors are diverted for the viral genome translation, some are recruited to improvise the viral replicase complexes for genome multiplication, and others are components of transport complexes for cell-to-cell spread via plasmodesmata and systemic movement through the phloem. This review summarizes current knowledge about host factors and discusses future research directions.

Unlabelled: The plant reoviruses, plant rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent propagative manner. These viruses induce the formation of viral inclusions to facilitate viral propagation in insect vectors. The intestines of insect vectors are formed by epithelial cells that lie on the noncellular basal lamina surrounded by visceral muscle tissue. Here, we demonstrate that a recently identified plant reovirus, southern rice black-streaked dwarf virus (SRBSDV), exploits virus-containing tubules composed of virus-encoded nonstructural protein P7-1 to directly cross the basal lamina from the initially infected epithelium toward visceral muscle tissues in the intestine of its vector, the white-backed planthopper (Sogatella furcifera). Furthermore, such tubules spread along visceral muscle tissues through a direct interaction of P7-1 and actin. The destruction of tubule assembly by RNA interference with synthesized double-stranded RNA targeting the P7-1 gene inhibited viral spread in the insect vector in vitro and in vivo. All these results show for the first time that a virus employs virus-induced tubule as a vehicle for viral spread from the initially infected midgut epithelium through the basal lamina, facilitating the rapid dissemination of virus from the intestine of the insect vector., Importance: Numerous plant viruses are transmitted in a persistent manner by sap-sucking insects, including thrips, aphids, planthoppers, and leafhoppers. These viruses, ingested by the insects, establish their primary infection in the intestinal epithelium of the insect vector. Subsequently, the invading virus manages to transverse the basal lamina, a noncellular layer lining the intestine, a barrier that may theoretically hinder viral spread. The mechanism by which plant viruses cross the basal lamina is unknown. Here, we report that a plant virus has evolved to exploit virus-induced tubules to pass through the basal lamina from the initially infected midgut epithelium of the insect vector, thus revealing the previously undescribed pathway adapted by the virus for rapid dissemination of virions from the intestine of the insect vector., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Plum pox virus (PPV) causes the most economically-devastating viral disease in Prunus species. Unfortunately, few natural resistance genes are available for the control of PPV. Recessive resistance to some potyviruses is associated with mutations of eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. In this study, we used an RNA silencing approach to manipulate the expression of eIF4E and eIF(iso)4E towards the development of PPV resistance in Prunus species. The eIF4E and eIF(iso)4E genes were cloned from plum (Prunus domestica L.). The sequence identity between plum eIF4E and eIF(iso)4E coding sequences is 60.4% at the nucleotide level and 52.1% at the amino acid level. Quantitative real-time RT-PCR analysis showed that these two genes have a similar expression pattern in different tissues. Transgenes allowing the production of hairpin RNAs of plum eIF4E or eIF(iso)4E were introduced into plum via Agrobacterium-mediated transformation. Gene expression analysis confirmed specific reduced expression of eIF4E or eIF(iso)4E in the transgenic lines and this was associated with the accumulation of siRNAs. Transgenic plants were challenged with PPV-D strain and resistance was evaluated by measuring the concentration of viral RNA. Eighty-two percent of the eIF(iso)4E silenced transgenic plants were resistant to PPV, while eIF4E silenced transgenic plants did not show PPV resistance. Physical interaction between PPV-VPg and plum eIF(iso)4E was confirmed. In contrast, no PPV-VPg/eIF4E interaction was observed. These results indicate that eIF(iso)4E is involved in PPV infection in plum, and that silencing of eIF(iso)4E expression can lead to PPV resistance in Prunus species.

The use of genetic resistance is considered to be the most effective and sustainable approach to the control of plant pathogens. Although most of the known natural resistance genes are monogenic dominant R genes that are predominant against fungi and bacteria, more and more recessive resistance genes against viruses have been cloned in the last decade. Interestingly, of the 14 natural recessive resistance genes against plant viruses that have been cloned from diverse plant species thus far, 12 encode the eukaryotic translation initiation factor 4E (eIF4E) or its isoform eIF(iso)4E. This review is intended to summarize the current state of knowledge about eIF4E and the possible mechanisms underlying its essential role in virus infection, and to discuss recent progress and the potential of eIF4E as a target gene in the development of genetic resistance to viruses for crop improvement., (© HER MAJESTY THE QUEEN IN RIGHT OF CANADA 2012. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

We determined the entire RNA1, 2 and 3 sequences of two prunus necrotic ringspot virus (PNRSV) isolates, Chr3 from cherry and Pch12 from peach, obtained from an orchard in the Niagara Fruit Belt, Canada. The RNA1, 2 and 3 of the two isolates share nucleotide sequence identities of 98.6%, 98.4% and 94.5%, respectively. Their RNA1- and 2-encoded amino acid sequences are about 98% identical to the corresponding sequences of a cherry isolate, CH57, the only other PNRSV isolate with complete RNA1 and 2 sequences available. Phylogenetic analysis of the coat protein and movement protein encoded by RNA3 of Pch12 and Chr3 and published PNRSV isolates indicated that Chr3 belongs to the PV96 group and Pch12 belongs to the PV32 group.

A naturally occurring Rsv4 resistance-breaking isolate (L-RB) and a closely related non-resistance-breaking isolate (L) of Soybean mosaic virus (SMV) were identified in soybean fields in London, Ontario, Canada. The viral genomes of L and L-RB were completely sequenced. Each isolate has a 9585-nucleotide genome with a single open reading frame encoding a polyprotein of approximately 350 kDa. L-RB and L have a very high sequence similarity (99.6%) at both the nucleotide and amino acid levels. Phylogenetic analysis showed that the two isolates belong to the G2 pathotype. Pathogenicity predictions of all virus/soybean combinations, based on the phylogenetic profile, were confirmed by pathogenicity tests using L and L-RB isolates and soybeans carrying different resistance genes, with an exception that L-RB infected a soybean cultivar carrying Rsv4 resistance. The temporal and spatial proximity of L and L-RB and their high sequence similarity suggest L-RB was likely derived from the SMV-L quasispecies. Recombination analysis did not reveal the evidence of genetic recombination for the emergence of L-RB. Mutations introduced by virus-encoded RNA-dependent RNA polymerase during viral genome replication and selection pressure probably contributed to the occurrence of L-RB.

RNA recombination is one of the two major factors that create RNA genome variability. Assessing its incidence in plant RNA viruses helps understand the formation of new isolates and evaluate the effectiveness of crop protection strategies. To search for recombination in Soybean mosaic virus (SMV), the causal agent of a worldwide seed-borne, aphid-transmitted viral soybean disease, we obtained all full-length genome sequences of SMV as well as partial sequences encoding the N-terminal most (P1 protease) and the C-terminal most (capsid protein; CP) viral protein. The sequences were analyzed for possible recombination events using a variety of automatic and manual recombination detection and verification approaches. Automatic scanning identified 3, 10, and 17 recombination sites in the P1, CP, and full-length sequences, respectively. Manual analyses confirmed 10 recombination sites in three full-length SMV sequences. To our knowledge, this is the first report of recombination between distinct SMV pathotypes. These data imply that different SMV pathotypes can simultaneously infect a host cell and exchange genetic materials through recombination. The high incidence of SMV recombination suggests that recombination plays an important role in SMV evolution. Obtaining additional full-length sequences will help elucidate this role.

Background: Virus infection induces the activation and suppression of global gene expression in the host. Profiling gene expression changes in the host may provide insights into the molecular mechanisms that underlie host physiological and phenotypic responses to virus infection. In this study, the Arabidopsis Affymetrix ATH1 array was used to assess global gene expression changes in Arabidopsis thaliana plants infected with Plum pox virus (PPV). To identify early genes in response to PPV infection, an Arabidopsis synchronized single-cell transformation system was developed. Arabidopsis protoplasts were transfected with a PPV infectious clone and global gene expression changes in the transfected protoplasts were profiled., Results: Microarray analysis of PPV-infected Arabidopsis leaf tissues identified 2013 and 1457 genes that were significantly (Q < or = 0.05) up- (> or = 2.5 fold) and downregulated (< or = -2.5 fold), respectively. Genes associated with soluble sugar, starch and amino acid, intracellular membrane/membrane-bound organelles, chloroplast, and protein fate were upregulated, while genes related to development/storage proteins, protein synthesis and translation, and cell wall-associated components were downregulated. These gene expression changes were associated with PPV infection and symptom development. Further transcriptional profiling of protoplasts transfected with a PPV infectious clone revealed the upregulation of defence and cellular signalling genes as early as 6 hours post transfection. A cross sequence comparison analysis of genes differentially regulated by PPV-infected Arabidopsis leaves against uniEST sequences derived from PPV-infected leaves of Prunus persica, a natural host of PPV, identified orthologs related to defence, metabolism and protein synthesis. The cross comparison of genes differentially regulated by PPV infection and by the infections of other positive sense RNA viruses revealed a common set of 416 genes. These identified genes, particularly the early responsive genes, may be critical in virus infection., Conclusion: Gene expression changes in PPV-infected Arabidopsis are the molecular basis of stress and defence-like responses, PPV pathogenesis and symptom development. The differentially regulated genes, particularly the early responsive genes, and a common set of genes regulated by infections of PPV and other positive sense RNA viruses identified in this study are candidates suitable for further functional characterization to shed lights on molecular virus-host interactions.

Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. To assess transcriptional changes in soybean plants infected with soybean mosaic virus (SMV), the first soybean trifoliate leaf, immediately above the SMV-inoculated unifoliate leaf, was sampled at 7, 14 and 21 days post-inoculation (p.i.) and subjected to microarray analysis. The identified changes in gene expression in soybean leaves with SMV infection at different time points were associated with the observed symptom development. By using stringent selection criteria (>or=2- or

The development of novel strategies against plant viral diseases relies on a better understanding of molecular virus-host interactions. Here, we report an easy, efficient and reproducible protocol for Arabidopsis protoplast isolation and transfection to study the infection and replication of a potyvirus, Plum pox virus (PPV). Macerozyme and cellulose were used to release protoplasts from Arabidopsis leaf tissues, and polyethylene glycol-mediated DNA uptake was employed for transfection of a PPV infectious clone. Protoplast viability was monitored by fluorescein diacetate staining, and transfection efficiency was estimated by transient expression of the green fluorescent protein. The protocol allowed production of 95% viable mesophyll protoplasts and a successful transfection rate of 35%. The system was used further in a time-course experiment to investigate PPV viral RNA accumulation. It was found that 3 h post-transfection (hpt) in the transfected protoplasts viral RNA increased by about 150-fold and progressively accumulated to reach the maximum at 12 hpt. Viral RNA then decreased dramatically at 24 hpt reaching 40% of its peak level. Considering the availability of the whole genome microarrays, and other genomic resources of Arabidopsis, the synchronized single-cell (protoplast) infection system will be useful for elucidating early molecular events associated with PPV infection.

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