MiR827 positively regulates the resistance to chilli veinal mottle virus by affecting the expression of FBPase in Nicotiana benthamiana.

MicroRNA(miRNA) is a class of non-coding small RNA that plays an important role in plant growth, development, and response to environmental stresses. Unlike most miRNAs, which usually target homologous genes across a variety of species, miR827 targets different types of genes in different species. Research on miR827 mainly focuses on its role in regulating phosphate (Pi) homeostasis of plants, however, little is known about its function in plant response to virus infection. In the present study, miR827 was significantly upregulated in the recovery tissue of virus-infected Nicotiana tabacum. Overexpression of miR827 could improve plants resistance to the infection of chilli veinal mottle virus (ChiVMV) in Nicotiana benthamiana, whereas interference of miR827 increased the susceptibility of the virus-infected plants. Further experiments indicated that the antiviral defence regulated by miR827 was associated with the reactive oxygen species and salicylic acid signalling pathways. Then, fructose-1,6-bisphosphatase (FBPase) was identified to be a target of miR827, and virus infection could affect the expression of FBPase. Finally, transient expression of FBPase increased the susceptibility to ChiVMV-GFP infection in N. benthamiana. By contrast, silencing of FBPase increased plant resistance. Taken together, our results demonstrate that miR827 plays a positive role in tobacco response to virus infection, thus providing new insights into understanding the role of miR827 in plant-virus interaction.

An Agent-Based Model Shows How Mixed Infections Drive Multiyear Pathotype Dynamics in a Plant-Virus System.

Mathematical models are widely used to understand the evolution and epidemiology of plant pathogens under a variety of scenarios. Here, we used this approach to analyze the effects of different traits intrinsic and extrinsic to plant-virus interactions on the dynamics of virus pathotypes in genetically heterogeneous plant-virus systems. For this, we propose an agent-based epidemiological model that includes epidemiologically significant pathogen life-history traits related to virulence, transmission, and survival in the environment and allows for integrating long- and short-distance transmission, primary and secondary infections, and within-host pathogen competition in mixed infections. The study focuses on the tobamovirus-pepper pathosystem. Model simulations allowed us to integrate pleiotropic effects of resistance-breaking mutations on different virus life-history traits into the net costs of resistance breaking, allowing for predictions on multiyear pathotype dynamics. We also explored the effects of two control measures, the use of host resistance and roguing of symptomatic plants, that modify epidemiological attributes of the pathogens to understand how their populations will respond to evolutionary pressures. One major conclusion points to the importance of pathogen competition within mixed-infected hosts as a component of the overall fitness of each pathogen that, thus, drives their multiyear dynamics.

Ability of Non-Hosts and Cucurbitaceous Weeds to Transmit Cucumber Green Mottle Mosaic Virus.

Cucumber green mottle mosaic virus (CGMMV) is a Tobamovirus of economic importance affecting cucurbit crops and Asian cucurbit vegetables. Non-host crops of CGMMV, including capsicum (Capsicum annum), sweetcorn (Zea mays), and okra (Abelmoschus esculentus), were tested for their susceptibility to the virus, with field and glasshouse trials undertaken. After 12 weeks post-sowing, the crops were tested for the presence of CGMMV, and in all cases, no CGMMV was detected. Commonly found within the growing regions of cucurbits and melons worldwide are weeds, such as black nightshade (Solanum nigrum), wild gooseberry (Physalis minima), pigweed (Portulaca oleracea), and Amaranth species. Several weeds/grasses were tested for their ability to become infected with CGMMV by inoculating weeds directly with CGMMV and routinely testing over a period of eight weeks. Amaranthus viridis was found to be susceptible, with 50% of the weeds becoming infected with CGMMV. To further analyse this, six Amaranth samples were used as inoculum on four watermelon seedlings per sample and tested after eight weeks. CGMMV was detected in three of six watermelon bulk samples, indicating that A. viridis is a potential host/reservoir for CGMMV. Further research into the relationship between CGMMV and weed hosts is required. This research also highlights the importance of proper weed management to effectively manage CGMMV.

Physical contact transmission of Cucumber green mottle mosaic virus by Myzus persicae.

Cucumber green mottle mosaic virus (CGMMV), a critical plant virus, has caused significant economic losses in cucurbit crops worldwide. It has not been proved that CGMMV can be transmitted by an insect vector. In this study, the physical contact transmission of CGMMV by Myzus persicae in Nicotiana benthamiana plants was confirmed under laboratory conditions. The acquisition rate increased with time, and most aphids acquired CGMMV at 72 h of the acquisition access period (AAP). Besides, the acquired CGMMV was retained in the aphids for about 12 h, which was efficiently transmitted back to the healthy N. benthamiana plants. More importantly, further experiments suggested that the transmission was mediated by physical contact rather than the specific interaction between insect vector and plant virus. The results obtained in our study contribute to the development of new control strategies for CGMMV in the field.

Argonaute 5 family proteins play crucial roles in the defence against Cymbidium mosaic virus and Odontoglossum ringspot virus in Phalaenopsis aphrodite subsp. formosana.

The orchid industry faces severe threats from diseases caused by viruses. Argonaute proteins (AGOs) have been shown to be the major components in the antiviral defence systems through RNA silencing in many model plants. However, the roles of AGOs in orchids against viral infections have not been analysed comprehensively. In this study, Phalaenopsis aphrodite subsp. formosana was chosen as the representative to analyse the AGOs (PaAGOs) involved in the defence against two major viruses of orchids, Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). A total of 11 PaAGOs were identified from the expression profile analyses of these PaAGOs in P. aphrodite subsp. formosana singly or doubly infected with CymMV and/or ORSV. PaAGO5b was found to be the only one highly induced. Results from overexpression of individual PaAGO5 family genes revealed that PaAGO5a and PaAGO5b play central roles in the antiviral defence mechanisms of P. aphrodite subsp. formosana. Furthermore, a virus-induced gene silencing vector based on Foxtail mosaic virus was developed to corroborate the function of PaAGO5s. The results confirmed their importance in the defences against CymMV and ORSV. Our findings may provide useful information for the breeding of traits for resistance or tolerance to CymMV or ORSV infections in Phalaenopsis orchids.

Rapid generation of inoculum of a plant RNA virus using overlap PCR.

We have developed an efficient method to rapidly generate infectious inoculum of a plant RNA virus and confirmed its infectivity by mechanical inoculation. The method takes advantage of overlap PCR to bypass the cloning steps, which makes it relatively simple, rapid, and inexpensive compared to the traditional methods. Using this approach, inoculum of a tobamovirus, Turnip vein clearing virus (TVCV), was generated. PCR products specific for the 35S promoter and TVCV genome were used as templates for overlap PCR to form a single product containing the full-length TVCV cDNA under the control of the double 35S promoter, and the entire process took only 8 h. This inoculum was infectious in Nicotiana benthamiana, and its infectivity was ca. 67% compared to 0% and 100% with negative and positive controls, respectively. Thus, this rapid method generates efficient infectious inoculum for a plant RNA virus.

The phylogeographic history of tomato mosaic virus in Eurasia.

Tomato mosaic virus (ToMV) is a tobamovirus affecting solanaceous crops worldwide. The process of its emergence, however, is poorly understood. Here, Bayesian phylogenetic framework was employed to reconstruct the phylogeography of ToMV in Eurasia. The results showed that the ToMV in Europe, Middle East and East Asia has been evolving at a rate of 4.05 × 10-4 substitutions/site/year (95% credibility interval 2.43 × 10-4 - 5.62 × 10-4). Their most recent common ancestor (MRCA), most probably first appeared in Europe, was dated to around 1757 Common Era. The first introduction of ToMV into Middle East occurred in 1920s, with Europe as the source, while the first introduction of ToMV into East Asia occurred shortly afterwards, with Middle East as the source. From about 1950 onwards, inter-regional migrations of ToMV between Europe, Middle East and East Asia have been common. Overall, these data provide a glimpse into the phylogeographic history of ToMV in Eurasia.

iTRAQ-Based Proteomic Analysis of Watermelon Fruits in Response to Cucumber green mottle mosaic virus Infection.

Cucumber green mottle mosaic virus (CGMMV) is an important viral pathogen on cucurbit plants worldwide, which can cause severe fruit decay symptoms on infected watermelon (usually called "watermelon blood flesh"). However, the molecular mechanism of this disease has not been well understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique to analyze the proteomic profiles of watermelon fruits in response to CGMMV infection. A total of 595 differentially accumulated proteins (DAPs) were identified, of which 404 were upregulated and 191 were downregulated. Functional annotation analysis showed that these DAPs were mainly involved in photosynthesis, carbohydrate metabolism, secondary metabolite biosynthesis, plant-pathogen interaction, and protein synthesis and turnover. The accumulation levels of several proteins related to chlorophyll metabolism, pyruvate metabolism, TCA cycle, heat shock proteins, thioredoxins, ribosomal proteins, translation initiation factors, and elongation factors were strongly affected by CGMMV infection. Furthermore, a correlation analysis was performed between CGMMV-responsive proteome and transcriptome data of watermelon fruits obtained in our previous study, which could contribute to comprehensively elucidating the molecular mechanism of "watermelon blood flesh". To confirm the iTRAQ-based proteome data, the corresponding transcripts of ten DAPs were validated by determining their abundance via quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). These results could provide a scientific basis for in-depth understanding of the pathogenic mechanisms underlying CGMMV-induced "watermelon blood flesh", and lay the foundation for further functional exploration and verification of related genes and proteins.

The expression of several pepper fatty acid desaturase genes is robustly activated in an incompatible pepper-tobamovirus interaction, but only weakly in a compatible interaction.

The replication of positive strand RNA viruses in plant cells is markedly influenced by the desaturation status of fatty acid chains in lipids of intracellular plant membranes. At present, little is known about the role of lipid desaturation in the replication of tobamoviruses. Therefore, we investigated the expression of fatty acid desaturase (FAD) genes and the fatty acid composition of pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation induced a hypersensitive reaction (incompatible interaction) while Pepper mild mottle virus (PMMoV) inoculation caused a systemic infection (compatible interaction). Changes in the expression of 16 FADs were monitored in pepper leaves following ObPV and PMMoV inoculations. ObPV inoculation rapidly and markedly upregulated seven Δ12-FADs that encode enzymes putatively located in the endoplasmic reticulum membrane. In contrast, PMMoV inoculation resulted in a weaker but rapid upregulation of two Δ12-FADs and a Δ15-FAD. The expression of genes encoding plastidial FADs was not influenced neither by ObPV nor by PMMoV. In accordance with gene expression results, a significant accumulation of linoleic acid was observed by gas chromatography-mass spectrometry in ObPV-, but not in PMMoV-inoculated leaves. ObPV inoculation led to a marked accumulation of H2O2 in the inoculated leaves. Therefore, the effect of H2O2 treatments on the expression of six tobamovirus-inducible FADs was also studied. The expression of these FADs was upregulated to different degrees by H2O2 that correlated with ObPV-inducibility of these FADs. These results underline the importance of further studies on the role of pepper FADs in pepper-tobamovirus interactions.

Signaling pathway played by salicylic acid, gentisic acid, nitric oxide, polyamines and non-enzymatic antioxidants in compatible and incompatible Solanum-tomato mottle mosaic virus interactions.

Plants are exposed to a vast array of pathogens. The interaction between them may be classified in compatible and incompatible. Polyamines (PAs) are involved in defense responses, as well as salicylic acid (SA), gentisic acid (GA) and nitric oxide (NO), which can increase the content of reactive oxygen species (ROS), creating a harsh environment to the pathogen. ROS can also damage the host cell and they can be controlled by ascorbate and glutathione. Among phytopathogens, one of the major threats to tomato crops is tomato mottle mosaic virus (ToMMV). Resistance against this virus probably involves the Tm-22 gene. This work aimed to analyze signaling and antioxidant molecules in the defense response against ToMMV in Solanum pimpinellifolium and in S. lycopersicum 'VFNT'. In S. pimpinellifolium plants inoculated with ToMMV, an increase in NO, SA, GA, ascorbate and oxidized glutathione and a decrease in the content of PAs were observed. Characteristic symptoms of diseased plants and high absorbance values in PTA-ELISA indicated a compatible interaction. In VFNT-inoculated plants, less significant differences were noticed. Symptoms and viral concentration were not detected, indicating an incompatible interaction, possibly associated with the effector-triggered immunity (ETI) response.

Insight Into Late Wilting Disease of Cucumber Demonstrates the Complexity of the Phenomenon in Fluctuating Environments.

Some diseases are caused by coinfection of several pathogens in the same plant. However, studies on the complexity of these coinfection events under different environmental conditions are scarce. Our ongoing research involves late wilting disease of cucumber caused by coinfection of Cucumber green mottle mosaic virus (CGMMV) and Pythium spp. We specifically investigated the role of various temperatures (18, 25, 32°C) on the coinfection by CGMMV and two predominant Pythium species occurring in cucumber greenhouses under Middle Eastern climatic conditions. During the summer months, Pythium aphanidermatum was most common, whereas P. spinosum predominated during the winter-spring period. P. aphanidermatum preferred higher temperatures while P. spinosum preferred low temperatures and caused very low levels of disease at 32°C when the 6-day-old seedlings were infected with P. spinosum alone. Nevertheless, after applying a later coinfection with CGMMV on the 14-day-old plants, a synergistic effect was detected for both Pythium species at optimal and suboptimal temperatures, with P. spinosum causing high mortality incidence even at 32°C. The symptoms caused by CGMMV infection appeared earlier as the temperature increased. However, within each temperature, no significant influence of the combined infection was detected. Our results demonstrate the complexity of coinfection in changing environmental conditions and indicate its involvement in disease development and severity as compared with infection by each of the pathogens alone.

Artificial microRNA-mediated resistance to cucumber green mottle mosaic virus in Nicotiana benthamiana.

MAIN CONCLUSION: We describe a Nicotiana benthamiana system for rapid identification of artificial microRNA (amiRNA) to control cucumber green mottle mosaic virus (CGMMV) disease. Although artificial miRNA technology has been used to control other viral diseases, it has not been applied to reduce severe cucumber green mottle mosaic virus (CGMMV) disease and crop loss in the economically important cucurbits. We used our system to identify three amiRNAs targeting CGMMV RNA (amiR1-CP, amiR4-MP and amiR6-Rep) and show that their expression reduces CGMMV replication and disease in virus-infected plants. This work streamlines the process of generating amiRNA virus-resistant crops and can be broadly applied to identify active antiviral amiRNAs against a broad spectrum of viruses to control disease in diverse crops.

Plant Protein-Mediated Inhibition of Virus Cell-to-Cell Movement: Far-Western Screening and Biological Analysis of a Plant Protein Interacting with a Viral Movement Protein.

Cell-to-cell movement via plasmodesmata is a crucial step for plant RNA viruses to determine their host ranges. Many viruses including Tomato mosaic virus (ToMV) encode one or more movement proteins (MPs) that are indispensable for cell-to-cell movement. During movement processes, MPs are thought to interact directly with many plant proteins that may be involved in supporting or inhibiting cell-to-cell movement of viruses. In order to understand the molecular mechanisms that regulate viral spread positively or negatively, it is important to discover such MP-interacting plant proteins and analyze their functions in viral cell-to-cell movement in efficient ways. In this chapter, we provide protocols of a radioisotope-based far-western screening strategy to construct a λ phage cDNA library from a nonhost Brassica campestris (syn. rapa) for ToMV and identify plant proteins that bind directly to the 32P-labeled probe of ToMV MP, and subsequently a biolistic bombardment method to examine whether a plant protein selected have a function as an inhibitory factor that can interfere with virus cell-to-cell movement.

Understanding the Transmissibility of Cucumber Green Mottle Mosaic Virus in Watermelon Seeds and Seed Health Assays.

Cucumber green mottle mosaic virus (CGMMV), an emerging tobamovirus, has caused serious disease outbreaks to cucurbit crops in several countries, including the United States. Although CGMMV is seed-borne, the mechanism of its transmission from a contaminated seed to germinating seedling is still not fully understood, and the most suitable seed health assay method has not been well established. To evaluate the mechanism of seed transmissibility, using highly contaminated watermelon seeds collected from CGMMV-infected experimental plants, bioassays were conducted in a greenhouse through seedling grow-out and by mechanical inoculation. Through natural seedling grow-out, we did not observe seed transmission of CGMMV to germinating seedlings. However, efficient transmission of CGMMV was observed using bioassays on melon plants through mechanical inoculation of seed extract prepared from CGMMV-contaminated seeds. Understanding the seed-borne property and the ease of mechanical transmission of CGMMV from a contaminated seed to seedling is an important finding. In comparative evaluation of various laboratory techniques for seed health assays, we found that enzyme-linked immunosorbent assay and loop-mediated isothermal amplification were the most sensitive and reliable methods to detect CGMMV on cucurbit seeds. Because CGMMV is a seed-borne and highly contagious virus, a new infection might not result in a natural seedling grow-out; it could occur through mechanical transmission from contaminated seeds. Therefore, a sensitive seed health test is necessary to ensure CGMMV-free seed lots are used for planting.

Expression profiling and regulatory network of cucumber microRNAs and their putative target genes in response to cucumber green mottle mosaic virus infection.

Cucumber green mottle mosaic virus (CGMMV) is an important pathogen of cucumber (Cucumis sativus). The molecular mechanisms mediating host-pathogen interactions are likely to be strongly influenced by microRNAs (miRNAs), which are known to regulate gene expression during the disease cycle. This study focused on 14 miRNAs (miR159, miR169, miR172, miR838, miR854, miR5658, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p, csa-miRn6-3p, csa-miRn7-5p and csa-miRn8-3p) and their target genes. The data collected was used to construct a regulatory network of miRNAs and target genes associated with cucumber-CGMMV interactions, which identified 608 potential target genes associated with all of the miRNAs except csa-miRn7-5p. Five of the miRNAs (miR159, miR838, miR854, miR5658 and csa-miRn6-3p) were found to be mutually linked by target genes, while another eight (miR169, miR172, csa-miRn1-3p, csa-miRn2-3p, csa-miRn3-3p, csa-miRn4-5p, csa-miRn5-5p and csa-miRn8-3p) formed subnetworks that did not display any connectivity with other miRNAs or their target genes. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to analyze the expression levels of the different miRNAs and their putative target genes in leaf, stem and root samples of cucumber over a 42-day period after inoculation with CGMMV. A positive correlation was found between some of the miRNAs and their respective target genes, although for most, the response varied greatly depending on the time point, indicating that additional factors are likely to be involved in the interaction between cucumber miRNAs and their target genes. Several miRNAs, including miR159 and csa-miRn6-3p, were linked to target genes that have been associated with plant responses to disease. A model linking miRNAs, their targets and downstream biological processes is proposed to indicate the roles of these miRNAs in the cucumber-CGMMV pathosystem.

Effects of deletion at the TTTSTTT motif of Hibiscus latent Singapore virus coat protein on viral replication and long-distance movement.

Hibiscus latent Singapore virus (HLSV) mutant HLSV-22A could not express coat protein (CP) nor infect plants systemically (Niu et al., 2015). In this study, a serine- and threonine-rich motif TTTSTTT at the C-terminus of HLSV CP was found to be involved in virus replication and systemic movement. Deletion the last amino acid residue in HLSV-22A led to a more rapid virus replication, but with delayed systemic movement. When the RNA structure in TTTSTTT motif was altered, while keeping its amino acids unchanged, mutants HLSV-87A-mmSL and HLSV-22A-mmSL showed no change in viral replication. These results indicated that the unique TTTSTTT motif is associated with virus replication and systemic movement. Deletion but not substitution of amino acid(s) at the C-terminus of TTTSTTT motif of HLSV CP with short internal poly(A) track enhanced virus replication, whereas the virus with a longer internal poly(A) tract of 87 A showed delayed systemic movement (147 words).

Development of a new tobamovirus-based viral vector for protein expression in plants.

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

Biological and Genomic Characterization of a Novel Tobamovirus Infecting Hoya spp.

Foliar symptoms suggestive of virus infection were observed on the ornamental plant hoya (Hoya spp.; commonly known as waxflower) in Florida. An agent that reacted with commercially available tobamovirus detection reagents was mechanically transmitted to Chenopodium quinoa and Nicotiana benthamiana. Rod-shaped particles ∼300 nm in length and typical of tobamoviruses were observed in partially purified virion preparations by electron microscopy. An experimental host range was determined by mechanical inoculation with virions, and systemic infections were observed in plants in the Asclepiadaceae, Apocynaceae, and Solanaceae families. Some species in the Solanaceae and Chenopodiaceae families allowed virus replication only in inoculated leaves, and were thus only local hosts for the virus. Tested plants in the Amaranthaceae, Apiaceae, Brassicaceae, Cucurbitaceae, Fabaceae, and Malvaceae did not support either local or systemic virus infection. The complete genome for the virus was sequenced and shown to have a typical tobamovirus organization. Comparisons of genome nucleotide sequence and individual gene deduced amino acid sequences indicate that it is a novel tobamovirus sharing the highest level of sequence identity with Streptocarpus flower break virus and members of the Brassicaceae-infecting subgroup of tobamoviruses. The virus, for which the name Hoya chlorotic spot virus (HoCSV) is proposed, was detected in multiple hoya plants from different locations in Florida.

Identifying optimal reference genes for the normalization of microRNA expression in cucumber under viral stress.

Cucumber green mottle mosaic virus (CGMMV) is an economically important pathogen and causes significant reduction of both yield and quality of cucumber (Cucumis sativus). Currently, there were no satisfied strategies for controlling the disease. A better understanding of microRNA (miRNA) expression related to the regulation of plant-virus interactions and virus resistance would be of great assistance when developing control strategies for CGMMV. However, accurate expression analysis is highly dependent on robust and reliable reference gene used as an internal control for normalization of miRNA expression. Most commonly used reference genes involved in CGMMV-infected cucumber are not universally expressed depending on tissue types and stages of plant development. It is therefore crucial to identify suitable reference genes in investigating the role of miRNA expression. In this study, seven reference genes, including Actin, Tubulin, EF-1α, 18S rRNA, Ubiquitin, GAPDH and Cyclophilin, were evaluated for the most accurate results in analyses using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gene expression was assayed on cucumber leaves, stems and roots that were collected at different days post inoculation with CGMMV. The expression data were analyzed using algorithms including delta-Ct, geNorm, NormFinder, and BestKeeper as well as the comparative tool RefFinder. The reference genes were subsequently validated using miR159. The results showed that EF-1α and GAPDH were the most reliable reference genes for normalizing miRNA expression in leaf, root and stem samples, while Ubiquitin and EF-1α were the most suitable combination overall.

Conferring virus resistance in tomato by independent RNA silencing of three tomato homologs of Arabidopsis TOM1.

The TOM1/TOM3 genes from Arabidopsis are involved in the replication of tobamoviruses. Tomato homologs of these genes, LeTH1, LeTH2 and LeTH3, are known. In this study, we examined transgenic tomato lines where inverted repeats of either LeTH1, LeTH2 or LeTH3 were introduced by Agrobacterium. Endogenous mRNA expression for each gene was detected in non-transgenic control plants, whereas a very low level of each of the three genes was found in the corresponding line. Small interfering RNA was detected in the transgenic lines. Each silenced line showed similar levels of tobamovirus resistance, indicating that each gene is similarly involved in virus replication.