4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells

Background

Synthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.

Results

RNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.

Conclusions

This first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.     

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.     

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.     

The serine/threonine/tyrosine kinase STY46 defends against hordeivirus infection by phosphorylating γb protein

Protein phosphorylation is a common post-translational modification that frequently occurs during plant–virus interaction. Host protein kinases often regulate virus infectivity and pathogenicity by phosphorylating viral proteins. The Barley stripe mosaic virus (BSMV) γb protein plays versatile roles in virus infection and the coevolutionary arms race between plant defense and viral counter-defense. Here, we identified that the autophosphorylated cytosolic serine/threonine/tyrosine (STY) protein kinase 46 of Nicotiana benthamiana (NbSTY46) phosphorylates and directly interacts with the basic motif domain (aa 19–47) of γb in vitro and in vivo. Overexpression of wild-type NbSTY46, either transiently or transgenically, suppresses BSMV replication and ameliorates viral symptoms, whereas silencing of NbSTY46 leads to increased viral replication and exacerbated symptom. Moreover, the antiviral role of NbSTY46 requires its kinase activity, as the NbSTY46_T436A mutant, lacking kinase activity, not only loses the ability to phosphorylate and interact with γb but also fails to impair BSMV infection when expressed in plants. NbSTY46 could also inhibit the replication of Lychnis ringspot virus, another chloroplast-replicating hordeivirus. In summary, we report a function of the cytosolic kinase STY46 in defending against plant viral infection by phosphorylating a viral protein in addition to its basal function in plant growth, development, and abiotic stress responses.

The cytosolic serine/threonine/tyrosine kinase STY46 negatively regulates Barley stripe mosaic virus replication by phosphorylating and interacting with cb protein.     

The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

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.     

Virus-induced changes in photosynthetic parameters and peroxidase isoenzyme contents in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis> L.) plants

Tomato samples were collected from the field of Absheron peninsula in Azerbaijan in order to evaluate the incidence of main Tobamoviruses. According to results of serological and molecular tests, Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Pepper mild mottle virus (PMMoV) were detected as single and mixed infections (TMV + PMMoV; ToMV + PMMoV) in various tomato samples. It was found that Tobamovirus infection caused an increase in the content of malondialdehyde, alterations in the activities of peroxidase enzymes and quantitative and qualitative changes in their molecular isoforms. A comparison of thylakoid membrane polypeptides from virus-infected leaves indicated a decrease in the content of the thylakoid membrane polypeptides with molecular masses of 123, 55, 47, 33, 28–24, 17, and 15 kD. PSII efficiency and the content of chlorophylls (a and b) were significantly lower in the virus-infected leaves.     

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.     

Involvement of C4 Protein of Beet Severe Curly Top Virus (Family Geminiviridae) in Virus Movement

Background

Beet severe curly top virus (BSCTV) is a leafhopper transmitted geminivirus with a monopartite genome. C4 proteins encoded by geminivirus play an important role in virus/plant interaction.

Methods and Findings

To understand the function of C4 encoded by BSCTV, two BSCTV mutants were constructed by introducing termination codons in ORF C4 without affecting the amino acids encoded by overlapping ORF Rep. BSCTV mutants containing disrupted ORF C4 retained the ability to replicate in Arabidopsis protoplasts and in the agro-inoculated leaf discs of N. benthamiana, suggesting C4 is not required for virus DNA replication. However, both mutants did not accumulate viral DNA in newly emerged leaves of inoculated N. benthamiana and Arabidopsis, and the inoculated plants were asymptomatic. We also showed that C4 expression in plant could help C4 deficient BSCTV mutants to move systemically. C4 was localized in the cytosol and the nucleus in both Arabidopsis protoplasts and N. benthamiana leaves and the protein appeared to bind viral DNA and ds/ssDNA nonspecifically, displaying novel DNA binding properties.

Conclusions

Our results suggest that C4 protein in BSCTV is involved in symptom production and may facilitate virus movement instead of virus replication.     

Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

Background

Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.

Methodology

We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.

Conclusions

We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

Viral Envelope Protein 53R Gene Highly Specific Silencing and Iridovirus Resistance in Fish Cells by AmiRNA

Background

Envelope protein 53R was identified from frog Rana grylio virus (RGV), a member of the family Iridoviridae, and it plays an important role in the virus assembly. Although inhibition of iridovirus major capsid protein (MCP) by small hairpin RNAs (shRNAs) has been shown to cause resistance to viral infection in vitro, RNA interference (RNAi) to inhibit aquatic animal virus envelope protein gene product has not been reported.

Methodology

We devised artificial microRNAs (amiRNAs) that target a viral envelope protein gene RGV 53R. By incorporating sequences encoding amiRNAs specific to 53R of RGV into pre-miRNA155 (pSM155) vectors, which use the backbone of natural miR-155 sequence and could intracellularly express 53R-targeted pre-amiRNAs. The pre-amiRNAs could be processed by the RNase III-like enzyme Dicer into 21–25 nt amiRNAs (amiR-53Rs) in fish cell lines. The levels of 53R expression were analyzed through real-time PCR and RGV virions assembly were observed by electronic microscopy in fish cells transfected with or without amiR-53Rs at 72 h of RGV infection.

Conclusion/Significance

The results argue that viral envelope protein RGV 53R can be silenced and the virions assembly was deficient by amiR-53R-1, and further identified the first amiRNA of envelope protein gene from iridovirus that was able to cause resistance to virus infection in fish cells. The data demonstrate that the viral infection is efficiently suppressed (58%) by amiR-53R-1 targeting positon 36–57 of RGV 53R. Moreover, electron microscopic observations revealed virion assembly defect or reduced virions assembly capacity was closely correlated to expression of amiR-53R-1. Based on real time PCR of the Mx gene, we found no evidence of activation of IFN by amiR-53R-1.     

Global Analyses of Small Interfering RNAs Derived from Bamboo mosaic virus and Its Associated Satellite RNAs in Different Plants

Background

Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.

Methodology/Principal Findings

Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.

Conclusions/Significance

The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.     

Plant-made trastuzumab (herceptin) inhibits HER2/Neu+ cell proliferation and retards tumor growth

Background

Plant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.

Methodology/Principal Findings

We describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.

Conclusions/Significance

We conclude that PMT is active in suppression of cell proliferation and tumor growth.     

Effect of mitochondria-targeted antioxidant SkQ1 on programmed cell death induced by viral proteins in tobacco plants

Programmed cell death (PCD) is the main defense mechanism in plants to fight various pathogens including viruses. The best-studied example of virus-induced PCD in plants is Tobacco mosaic virus (TMV)-elicited hypersensitive response in tobacco plants containing the N resistance gene. It was previously reported that the animal mitochondrial protein Bcl-xL, which lacks a homolog in plants, effectively suppresses plant PCD induced by TMV p50 — the elicitor of hyper-sensitive response in Nicotiana tabacum carrying the N gene. Our studies show that the mitochondria-targeted antioxidant SkQ1 effectively suppresses p50-induced PCD in tobacco plants. On the other hand, SkQ1 did not affect Poa semilatent virus TGB3-induced endoplasmic reticulum stress, which is followed by PCD, in Nicotiana benthamiana epidermal cells. These data suggest that mitochondria-targeted antioxidant SkQ1 can be used to study molecular mechanisms of PCD suppression in plants.     

Cross-neutralization potential of native human papillomavirus N-terminal L2 epitopes

Background

Human papillomavirus (HPV) capsids are composed of 72 pentamers of the major capsid protein L1, and an unknown number of L2 minor capsid proteins. An N-terminal “external loop” of L2 contains cross-neutralizing epitopes, and native HPV16 virions extracted from 20-day-old organotypic tissues are neutralized by anti-HPV16 L2 antibodies but virus from 10-day-old cultures are not, suggesting that L2 epitopes are more exposed in mature, 20-day virions. This current study was undertaken to determine whether cross-neutralization of other HPV types is similarly dependent on time of harvest and to screen for the most effective cross-neutralizing epitope in native virions.

Methodology and Principal Findings

Neutralization assays support that although HPV16 L2 epitopes were only exposed in 20-day virions, HPV31 or HPV18 epitopes behaved differently. Instead, HPV31 and HPV18 L2 epitopes were exposed in 10-day virions and remained so in 20-day virions. In contrast, presumably due to sequence divergence, HPV45 was not cross-neutralized by any of the anti-HPV16 L2 antibodies. We found that the most effective cross-neutralizing antibody was a polyclonal antibody named anti-P56/75 #1, which was raised against a peptide consisting of highly conserved HPV16 L2 amino acids 56 to 75.

Conclusions and Significance

This is the first study to determine the susceptibility of multiple, native high-risk HPV types to neutralization by L2 antibodies. Multiple anti-L2 antibodies were able to cross-neutralize HPV16, HPV31, and HPV18. Only neutralization of HPV16 depended on the time of tissue harvest. These data should inform attempts to produce a second-generation, L2-based vaccine.