A Turnip crinkle virus Isolate Lacking the CP Counter‐Defence Protein Gene Providing Protection Against the Wild‐Type Strain is Associated with Highly Localized RNA Silencing

Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.     

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus‐like particles

Plant expression systems based on nonreplicating virus‐based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant‐based production and assembly of Bluetongue virus‐like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus–based HyperTrans (CPMV‐HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV‐8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways.     

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

Background

Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use.

Results

We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10–14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date.

Conclusion

These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.     

Thiopurine Prodrugs for Plant Chemotherapy Purposes

Thiopurine prodrugs are antiviral chemicals used in medical therapy whose mechanisms of action are associated with inhibition of purine biosynthesis. In terms of plant chemotherapy, previous research of 6‐mercaptopurine (MP) administration in tobacco tissue culture infected by Tobacco mosaic virus (TMV) showed no inhibition of virus activity. Currently, not enough data exist to confirm thiopurine drug ineffectiveness against viruses in the plant kingdom. This paper presents a screening of MP, 6‐methylmercaptopurine riboside (MMPR), 6‐thioguanine (6‐TG) and 1‐amino‐6‐mercaptopurine (1A‐MP) against TMV and Cucumber mosaic virus (CMV) in in vitro tobacco explants and against Grapevine leafroll‐associated virus 3 (GLRaV 3) in in vitro grapevine explants. ELISA and RT‐PCR were used to evaluate antiviral activity. Higher toxicity levels of MP derivatives, compared to MP, were noted in tobacco and grapevine explants. 1A‐MP or 6‐TG treatment resulted CMV and GLRaV 3 virus‐eradicated explants as obtained with Inosine 5′‐monophosphate dehydrogenase inhibitors, whereas TMV was not eradicated by any of the studied drugs.     

A Basic Approach Towards the Development of Bioelectric Bacterial Biosensors for the Detection of Plant Viruses

There is a growing need for virus sensors with improved sensitivity and dynamic range for disease diagnosis, pharmaceutical research, agriculture and homeland security. Membrane‐engineered animal cells bearing antibodies against viral antigens have been previously used for biorecognition biosensors for the ultrarapid (3 min), sensitive (1 ng/ml) detection of plant viruses, such as the cucumber mosaic virus. We here report a new approach for the construction of cell‐based sensors for virus detection, based on membrane (antibody)‐engineered bacteria. The novel method was applied for the detection of tobacco mosaic virus (TMV) and cherry leaf roll virus (CLRV) using sensors containing modified Escherichia coli XL‐1Blue MRF’ bacteria. E. coli membranes have been engineered with electro‐inserted, virus‐homologous antibodies. The detection principle was based on the measurement of changes in the bacterial membrane potential as a result of virus–antibody binding. After optimization of the membrane‐engineering process, the virus detection limit for TMV and CLRV with the bacteria‐based biosensor system was 1 pg/ml, representing a 1000‐fold improvement over currently available methods. Although the novel biosensor is still in its proof‐of‐concept stage of development, its sensitivity and speed (assay time: 60–100 s) could make it a very promising tool for high throughput, field‐based virus screening.     

Detection of tobacco mosaic virus and tomato mosaic virus in pepper seeds by enzyme linked immunosorbent assay (ELISA)

Pepper seed samples were tested for the infection of tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV) by enzyme linked immunosorbent assay (ELISA). Out of 26 pepper seed samples tested, 17 were infected with TMV and ToMV in ELISA. About 34.7% of pepper seed samples were found to be healthy. Infections of TMV or ToMV were recorded to be 61.53% and 11.5%, respectively of the total tested seed samples.     

Genetic diversity and population structure of Sorghum mosaic virus infecting Saccharum spp. hybrids

Sugarcane mosaic disease is widespread in many countries and has been identified to be caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV) and Sugarcane streak mosaic virus (SCSMV). Viral surveys of SCMV, SrMV and SCSMV were performed from 104 leaf samples of Saccharum spp. hybrid growing in China and two leaf samples in Myanmar. Sorghum mosaic virus was a major causal agent for sugarcane mosaic disease in China whereby 72.1% (75/104) of samples had SrMV infection alone, 6.7% (7/104) were mixed with SCMV and 17.3% (18/104) were mixed with SCSMV. Sugarcane streak mosaic virus infection alone occurred in 3.8% (4/104) of samples, but no single infections were observed for SCMV. Two viruses (SrMV and SCSMV) were detected in sugarcane mosaic samples in Myanmar. Phylogenetic analysis revealed that all of the SrMV isolates were clustered into three major lineages encompassing six phylogroups/genotypes based on the CP sequences (825 nucleotides) of 113 Chinese and 2 Burmese isolates from this study and 73 isolates reported worldwide. Six clearly distinct SrMV phylogroups (G1–G6) were formed and shared 74.3–94.1% nucleotide identity and 84.7–98.1% amino acid identity of CP sequences. SrMV‐G5 was identified to be new distinct phylogroup that was restricted to the Fujian and Guangxi provinces. The unique SrMV‐G6 phylogroup only occurred in Yunnan province. Insertion/deletion mutations, negative selection and frequent gene flow are factors driving the genetic evolution and population structure of SrMV in China.     

TGBp3 triggers the unfolded protein response and SKP1‐dependent programmed cell death

The Potato virus X (PVX) triple gene block protein 3 (TGBp3), an 8‐kDa membrane binding protein, aids virus movement and induces the unfolded protein response (UPR) during PVX infection. TGBp3 was expressed from the Tobacco mosaic virus (TMV) genome (TMV‐p3), and we noted the up‐regulation of SKP1 and several endoplasmic reticulum (ER)‐resident chaperones, including the ER luminal binding protein (BiP), protein disulphide isomerase (PDI), calreticulin (CRT) and calmodulin (CAM). Local lesions were seen on leaves inoculated with TMV‐p3, but not TMV or PVX. Such lesions were the result of TGBp3‐elicited programmed cell death (PCD), as shown by an increase in reactive oxygen species, DNA fragmentation and induction of SKP1 expression. UPR‐related gene expression occurred within 8 h of TMV‐p3 inoculation and declined before the onset of PCD. TGBp3‐mediated cell death was suppressed in plants that overexpressed BiP, indicating that UPR induction by TGBp3 is a pro‐survival mechanism. Anti‐apoptotic genes Bcl‐xl, CED‐9 and Op‐IAP were expressed in transgenic plants and suppressed N gene‐mediated resistance to TMV, but failed to alleviate TGBp3‐induced PCD. However, TGBp3‐mediated cell death was reduced in SKP1‐silenced Nicotiana benthamiana plants. The combined data suggest that TGBp3 triggers the UPR and elicits PCD in plants.     

A dual gene‐silencing vector system for monocot and dicot plants

Plant virus‐based gene‐silencing vectors have been extensively and successfully used to elucidate functional genomics in plants. However, only limited virus‐induced gene‐silencing (VIGS) vectors can be used in both monocot and dicot plants. Here, we established a dual gene‐silencing vector system based on Bamboo mosaic virus (BaMV) and its satellite RNA (satBaMV). Both BaMV and satBaMV vectors could effectively silence endogenous genes in Nicotiana benthamiana and Brachypodium distachyon. The satBaMV vector could also silence the green fluorescent protein (GFP) transgene in GFP transgenic N. benthamiana. GFP transgenic plants co‐agro‐inoculated with BaMV and satBaMV vectors carrying sulphur and GFP genes, respectively, could simultaneously silence both genes. Moreover, the silenced plants could still survive with the silencing of genes essential for plant development such as heat‐shock protein 90 (Hsp90) and Hsp70. In addition, the satBaMV‐ but not BaMV‐based vector could enhance gene‐silencing efficiency in newly emerging leaves of N. benthamiana deficient in RNA‐dependant RNA polymerase 6. The dual gene‐silencing vector system of BaMV and satBaMV provides a novel tool for comparative functional studies in monocot and dicot plants.     

Pathogen-inducible CaUGT1 is involved in resistance response against TMV infection by controlling salicylic acid accumulation

Capsicum annuum L. Bugang exhibits a hypersensitive response against Tobacco mosaic virus (TMV) P₀ infection. The C. annuumUDP-glucosyltransferase 1 (CaUGT1) gene was upregulated during resistance response to TMV and by salicylic acid, ethephon, methyl viologen, and sodium nitroprusside treatment. When the gene was downregulated by virus-induced gene silencing, a delayed HR was observed. In addition, free and total SA concentrations in the CaUGT1-downregulated hot pepper were decreased by 52% and 48% compared to that of the control plants, respectively. This suggested that the CaUGT1 gene was involved in resistance response against TMV infection by controlling the accumulation of SA.     

Evidence that soilborne wheat mosaic virus moves long distance through the xylem in wheat

Summary Soilborne wheat mosaic virus (SBWMV) is a member of the genusFurovirus of plant viruses. SBWMV is transmitted to wheat roots by the plasmodiophorid vectorPolymyxa graminis. Experiments were conducted to determine the path for SBWMV transport from roots to leaves. The results of immunogold labeling suggest that SBWMV enters and moves long distance through the xylem. SBWMV may enter primary xylem elements before cell death occurs and then move upward in the plant after the xylem has matured into hollow vessels. There is also evidence for lateral movement between adjacent xylem vessels.Abbreviations SBWMV Soilborne wheat mosaic virus - TMV Tobacco mosaic virus - BMV Brome mosaic virus - PMTV Potato mop-top virus - BNYVV Beet necrotic yellow vein virus - WSSMV Wheat spindle streak mosaic virus - WSMV Wheat streak mosaic virus     

Tobacco mutants with reduced microtubule dynamics are less susceptible to TMV

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T‐DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, α‐tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus‐end marker GFP–EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell‐to‐cell movement of TMV encoding GFP‐tagged movement protein (MP‐GFP) is reduced in ATER2 accompanied by a reduced association of MP‐GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.     

Rice stripe tenuivirus p2 may recruit or manipulate nucleolar functions through an interaction with fibrillarin to promote virus systemic movement

Rice stripe virus (RSV) is the type species of the genus Tenuivirus and represents a major viral pathogen affecting rice production in East Asia. In this study, RSV p2 was fused to yellow fluorescent protein (p2‐YFP) and expressed in epidermal cells of Nicotiana benthamiana. p2‐YFP fluorescence was found to move to the nucleolus initially, but to leave the nucleolus for the cytoplasm forming numerous distinct bright spots there at later time points. A bimolecular fluorescence complementation (BiFC) assay showed that p2 interacted with fibrillarin and that the interaction occurred in the nucleus. Both the nucleolar localization and cytoplasmic distribution of p2‐YFP fluorescence were affected in fibrillarin‐silenced N. benthamiana. Fibrillarin depletion abolished the systemic movement of RSV, but not that of Tobacco mosaic virus (TMV) and Potato virus X (PVX). A Tobacco rattle virus (TRV)‐based virus‐induced gene silencing (VIGS) method was used to diminish RSV NS2 (encoding p2) or NS3 (encoding p3) during RSV infection. Silencing of NS3 alleviated symptom severity and reduced RSV accumulation, but had no obvious effects on virus movement and the timing of symptom development. However, silencing of NS2 abolished the systemic movement of RSV. The possibility that RSV p2 may recruit or manipulate nucleolar functions to promote virus systemic infection is discussed.     

Biological and Molecular Characterization of a Crucifer Tobamovirus Infecting Oilseed Rape

In China, the tobamovirus that infects oilseed rape has been misdiagnosed as Tobacco mosaic virus (TMV) based on its morphological similarity and serological relatedness. Recently, a tobamovirus has been isolated from oilseed rape in China, which we named Youcai mosaic virus Br (YoMV-Br), according to its biological and molecular characteristics. It had strong infectivity to Cruciferae but less to Solanaceae, Leguminosae, and Cucurbitaceae, and its virion morphology was consistent with that of the tobamoviruses. At high concentrations, it serologically cross reacted with TMV antiserum. The 3′ terminal sequence (2,283 nucleotides) of YoMV-Br was determined, including the 3′ noncoding region, the CP and MP genes, and the C-terminal part of the replicase gene. Between the MP and CP genes, 77 nucleotides overlapped. Compared with homologous regions of 21 recognized species of Tobamovirus, YoMV-Br had a much higher identity to crucifer species than to other tobamoviruses. Phylogenetic analysis demonstrated that YoMV-Br was closely related to the YoMV cluster of tobamoviruses and distantly to TMV, so that they likely belong to different strains of the same species.     

Immunogenicity of plant‐produced African horse sickness virus‐like particles: implications for a novel vaccine

African horse sickness (AHS) is a debilitating and often fatal viral disease affecting horses in much of Africa, caused by the dsRNA orbivirus African horse sickness virus (AHSV). Vaccination remains the single most effective weapon in combatting AHS, as there is no treatment for the disease apart from good animal husbandry. However, the only commercially available vaccine is a live‐attenuated version of the virus (LAV). The threat of outbreaks of the disease outside its endemic region and the fact that the LAV is not licensed for use elsewhere in the world, have spurred attempts to develop an alternative safer, yet cost‐effective recombinant vaccine. Here, we report the plant‐based production of a virus‐like particle (VLP) AHSV serotype five candidate vaccine by Agrobacterium tumefaciens‐mediated transient expression of all four capsid proteins in Nicotiana benthamiana using the cowpea mosaic virus‐based HyperTrans (CPMV‐HT) and associated pEAQ plant expression vector system. The production process is fast and simple, scalable, economically viable, and most importantly, guinea pig antiserum raised against the vaccine was shown to neutralize live virus in cell‐based assays. To our knowledge, this is the first report of AHSV VLPs produced in plants, which has important implications for the containment of, and fight against the spread of, this deadly disease.     

Type I J-Domain NbMIP1 Proteins Are Required for Both Tobacco Mosaic Virus Infection and Plant Innate Immunity

Tm-2² is a coiled coil-nucleotide binding-leucine rich repeat resistance protein that confers durable extreme resistance against Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by recognizing the viral movement protein (MP). Here we report that the Nicotiana benthamiana J-domain MIP1 proteins (NbMIP1s) associate with tobamovirus MP, Tm-2² and SGT1. Silencing of NbMIP1s reduced TMV movement and compromised Tm-2²-mediated resistance against TMV and ToMV. Furthermore, silencing of NbMIP1s reduced the steady-state protein levels of ToMV MP and Tm-2². Moreover, NbMIP1s are required for plant resistance induced by other R genes and the nonhost pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In addition, we found that SGT1 associates with Tm-2² and is required for Tm-2²-mediated resistance against TMV. These results suggest that NbMIP1s function as co-chaperones during virus infection and plant immunity.     

Structural Lability of Barley Stripe Mosaic Virus Virions

Virions of Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.