Rewiring carotenoid biosynthesis in plants using a viral vector

[EN] Plants can be engineered to sustainably produce compounds of nutritional, industrial or pharmaceutical relevance. This is, however, a challenging task as extensive regulation of biosynthetic pathways often hampers major metabolic changes. Here we describe the use of a viral vector derived from Tobacco etch virus to express a whole heterologous metabolic pathway that produces the health-promoting carotenoid lycopene in tobacco tissues. The pathway consisted in three enzymes from the soil bacteria Pantoea ananatis. Lycopene is present at undetectable levels in chloroplasts of non-infected leaves. In tissues infected with the viral vector, however, lycopene comprised approximately 10% of the total carotenoid content. Our research further showed that plant viruses that express P. ananatis phytoene synthase (crtB), one of the three enzymes of the heterologous pathway, trigger an accumulation of endogenous carotenoids, which together with a reduction in chlorophylls eventually result in a bright yellow pigmentation of infected tissues in various host-virus combinations. So, besides illustrating the potential of viral vectors for engineering complex metabolic pathways, we also show a yellow carotenoid-based reporter that can be used to visually track infection dynamics of plant viruses either alone or in combination with other visual markers.We thank Veronica Aragones and M. Rosa Rodriguez-Goberna for excellent technical assistance. This research was supported by Spanish Ministerio de Economia y Competitividad (MINECO) grants BIO2014-54269-R to J.-A.D., and BIO2014-59092-P and BIO2015-71703-REDT to M. R.-C. Financial support from the Generalitat Valenciana (PROMETEOII/2014/021), the Programa Iberoamericano de Ciencia y Tecnologia para el Desarrollo (Ibercarot 112RT0445), and the Generalitat de Catalunya (2014SGR-1434) is also acknowledged. E.M. is the recipient of a pre-doctoral fellowship (AP2012-3751) from the Spanish Ministerio de Educacion, Cultura y Deporte. B.L. is supported by a postdoctoral fellowship (FPDI-2013-018882) from MINECO.Majer, E.; Llorente, B.; Rodríguez-Concepción, M.; Daros Arnau, JA. (2017). Rewiring carotenoid biosynthesis in plants using a viral vector. Scientific Reports. 7. https://doi.org/10.1038/srep41645S7O’Connor, S. E. Engineering of secondary metabolism. Annu. Rev. Genet. 49, 71–94 (2015).Sainsbury, F. & Lomonossoff, G. P. Transient expressions of synthetic biology in plants. Curr. Opin. Plant Biol. 19, 1–7 (2014).Gleba, Y. Y., Tusé, D. & Giritch, A. Plant viral vectors for delivery by Agrobacterium. Curr. Top. Microbiol. Immunol. 375, 155–192 (2014).Chen, Q., He, J., Phoolcharoen, W. & Mason, H. S. Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants. Hum. Vaccin. 7, 331–338 (2011).Pogue, G. P., Lindbo, J. A., Garger, S. J. & Fitzmaurice, W. P. Making an ally from an enemy: plant virology and the new agriculture. Annu. Rev. Phytopathol. 40, 45–74 (2002).Peyret, H. & Lomonossoff, G. P. When plant virology met Agrobacterium: the rise of the deconstructed clones. Plant Biotechnol. J. 13, 1121–1135 (2015).Bedoya, L. C., Martínez, F., Orzáez, D. & Daròs, J. A. Visual tracking of plant virus infection and movement using a reporter MYB transcription factor that activates anthocyanin biosynthesis. Plant Physiol. 158, 1130–1138 (2012).Majer, E., Daròs, J. A. & Zwart, M. P. Stability and fitness impact of the visually discernible Rosea1 marker in the Tobacco etch virus genome. Viruses 5, 2153–2168 (2013).Bedoya, L., Martínez, F., Rubio, L. & Daròs, J. A. Simultaneous equimolar expression of multiple proteins in plants from a disarmed potyvirus vector. J. Biotechnol. 150, 268–275 (2010).Kelloniemi, J., Mäkinen, K. & Valkonen, J. P. Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths. Virus Res. 135, 282–291 (2008).Carrington, J. C., Haldeman, R., Dolja, V. V. & Restrepo-Hartwig, M. A. Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo . J. Virol. 67, 6995–7000 (1993).Li, X. H. & Carrington, J. C. Complementation of tobacco etch potyvirus mutants by active RNA polymerase expressed in transgenic cells. Proc. Natl. Acad. Sci. USA 92, 457–461 (1995).Fraser, P. D. & Bramley, P. M. The biosynthesis and nutritional uses of carotenoids. Prog. Lipid Res. 43, 228–265 (2004).Meléndez-Martínez, A. J., Mapelli-Brahm, P., Benítez-González, A. & Stinco, C. M. A comprehensive review on the colorless carotenoids phytoene and phytofluene. Arch. Biochem. Biophys. 572, 188–200 (2015).Rodríguez-Concepción, M. & Boronat, A. Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol. 130, 1079–1089 (2002).Giuliano, G. Plant carotenoids: genomics meets multi-gene engineering. Curr. Opin. Plant Biol. 19, 111–117 (2014).Cazzonelli, C. I. & Pogson, B. J. Source to sink: regulation of carotenoid biosynthesis in plants. Trends Plant Sci. 15, 266–274 (2010).Ruiz-Sola, M. A. & Rodríguez-Concepción, M. Carotenoid biosynthesis in Arabidopsis: a colorful pathway. Arabidopsis Book 10, e0158 (2012).Nisar, N., Li, L., Lu, S., Khin, N. C. & Pogson, B. J. Carotenoid metabolism in plants. Mol. Plant 8, 68–82 (2015).Misawa, N. et al. Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli . J. Bacteriol. 172, 6704–6712 (1990).Hasunuma, T. et al. Biosynthesis of astaxanthin in tobacco leaves by transplastomic engineering. Plant J. 55, 857–868 (2008).Lu, Y., Rijzaani, H., Karcher, D., Ruf, S. & Bock, R. Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons. Proc. Natl. Acad. Sci. USA 110, E623–632 (2013).Mann, V., Harker, M., Pecker, I. & Hirschberg, J. Metabolic engineering of astaxanthin production in tobacco flowers. Nat. Biotechnol. 18, 888–892 (2000).Wurbs, D., Ruf, S. & Bock, R. Contained metabolic engineering in tomatoes by expression of carotenoid biosynthesis genes from the plastid genome. Plant J. 49, 276–288 (2007).Cordero, M. T. et al. Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana . Mol. Plant-Microbe Interact. doi: 10.1094/MPMI-11-16-0239-R (2016).Ye, X. et al. Engineering the provitamin A (b-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science 287, 303–305 (2000).Ravanello, M. P., Ke, D., Alvarez, J., Huang, B. & Shewmaker, C. K. Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production. Metab. Eng. 5, 255–263 (2003).Fujisawa, M. et al. Pathway engineering of Brassica napus seeds using multiple key enzyme genes involved in ketocarotenoid formation. J. Exp. Bot. 60, 1319–1332 (2009).Ohara, K., Ujihara, T., Endo, T., Sato, F. & Yazaki, K. Limonene production in tobacco with Perilla limonene synthase cDNA. J. Exp. Bot. 54, 2635–2642 (2003).Gutensohn, M. et al. Cytosolic monoterpene biosynthesis is supported by plastid-generated geranyl diphosphate substrate in transgenic tomato fruits. Plant J. 75, 351–363 (2013).Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H. & Misawa, N. Metabolic engineering for production of beta-carotene and lycopene in Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem. 58, 1112–1114 (1994).Bahieldin, A. et al. Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter. Plasmid 72, 18–28 (2014).Xie, W., Lv, X., Ye, L., Zhou, P. & Yu, H. Construction of lycopene-overproducing Saccharomyces cerevisiae by combining directed evolution and metabolic engineering. Metab. Eng. 30, 69–78 (2015).Li, Y., Cui, H., Cui, X. & Wang, A. The altered photosynthetic machinery during compatible virus infection. Curr. Opin. Virol. 17, 19–24 (2016).Tilsner, J. & Oparka, K. J. Tracking the green invaders: advances in imaging virus infection in plants. Biochem. J. 430, 21–37 (2010).Kumagai, M. H. et al. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc. Natl. Acad. Sci. USA 92, 1679–1683 (1995).Kumagai, M. H., Keller, Y., Bouvier, F., Clary, D. & Camara, B. Functional integration of non-native carotenoids into chloroplasts by viral-derived expression of capsanthin-capsorubin synthase in Nicotiana benthamiana . Plant J. 14, 305–315 (1998).Zhai, S., Xia, X. & He, Z. Carotenoids in staple cereals: metabolism, regulation, and genetic manipulation. Front. Plant Sci. 7, 1197 (2016).Zhang, H. et al. A Narcissus mosaic viral vector system for protein expression and flavonoid production. Plant Methods 9, 28 (2013).Nielsen, A. Z. et al. Redirecting photosynthetic reducing power toward bioactive natural product synthesis. ACS Synth. Biol. 2, 308–315 (2013).Sainsbury, F., Saxena, P., Geisler, K., Osbourn, A. & Lomonossoff, G. P. Using a virus-derived system to manipulate plant natural product biosynthetic pathways. Methods Enzymol. 517, 185–202 (2012).Geisler, K. et al. Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants. Proc. Natl. Acad. Sci. USA 110, E3360–3367 (2013).Kanagarajan, S., Muthusamy, S., Gliszczynska, A., Lundgren, A. & Brodelius, P. E. Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana . Plant Cell Rep. 31, 1309–1319 (2012).Mozes-Koch, R. et al. Expression of an entire bacterial operon in plants. Plant Physiol. 158, 1883–1892 (2012).Thole, V., Worland, B., Snape, J. W. & Vain, P. The pCLEAN dual binary vector system for Agrobacterium-mediated plant transformation. Plant Physiol. 145, 1211–1219 (2007).Engler, C., Gruetzner, R., Kandzia, R. & Marillonnet, S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 4, e5553 (2009).Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).Cunningham, F. X. Jr., Chamovitz, D., Misawa, N., Gantt, E. & Hirschberg, J. Cloning and functional expression in Escherichia coli of a cyanobacterial gene for lycopene cyclase, the enzyme that catalyzes the biosynthesis of b-carotene. FEBS Lett. 328, 130–138 (1993).Shivprasad, S. et al. Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255, 312–323 (1999).Schürer, H., Lang, K., Schuster, J. & Mörl, M. A universal method to produce in vitro transcripts with homogeneous 3′ ends. Nucleic Acids Res. 30, e56 (2002).Lu, R. et al. High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J. 22, 5690–5699 (2003).Dickmeis, C., Fischer, R. & Commandeur, U. Potato virus X-based expression vectors are stabilized for long-term production of proteins and larger inserts. Biotechnol. J. 9, 1369–1379 (2014).Nakagawa, T. et al. Improved Gateway binary vectors: high-performance vectors for creation of fusion constructs in transgenic analysis of plants. Biosci. Biotechnol. Biochem. 71, 2095–2100 (2007).Bedoya, L. C. & Daròs, J. A. Stability of Tobacco etch virus infectious clones in plasmid vectors. Virus Res. 149, 234–240 (2010).Sparkes, I. A., Runions, J., Kearns, A. & Hawes, C. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat. Protoc. 1, 2019–2025 (2006).Llorente, B. et al. Tomato fruit carotenoid biosynthesis is adjusted to actual ripening progression by a light-dependent mechanism. Plant J. 85, 107–119 (2016)

One Is Enough: In Vivo Effective Population Size Is Dose-Dependent for a Plant RNA Virus

Effective population size (Ne) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. Experimental estimates of Ne for different plant viruses have, however, rendered diverging results. The independent action hypothesis (IAH) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. A corollary of IAH is that Ne must be dose dependent. A test of IAH for a plant virus has not been reported yet. Here we perform a test of an IAH infection model using a plant RNA virus, Tobacco etch virus (TEV) variants carrying GFP or mCherry fluorescent markers, in Nicotiana tabacum and Capsicum annuum plants. The number of primary infection foci increased linearly with dose, and was similar to a Poisson distribution. At high doses, primary infection foci containing both genotypes were found at a low frequency (<2%). The probability that a genotype that infected the inoculated leaf would systemically infect that plant was near 1, although in a few rare cases genotypes could be trapped in the inoculated leaf by being physically surrounded by the other genotype. The frequency of mixed-genotype infection could be predicted from the mean number of primary infection foci using the independent-action model. Independent action appears to hold for TEV, and Ne is therefore dose-dependent for this plant RNA virus. The mean number of virions causing systemic infection can be very small, and approaches 1 at low doses. Dose-dependency in TEV suggests that comparison of Ne estimates for different viruses are not very meaningful unless dose effects are taken into consideration

Effect of Host Species on the Distribution of Mutational Fitness Effects for an RNA Virus

Knowledge about the distribution of mutational fitness effects (DMFE) is essential for many evolutionary models. In recent years, the properties of the DMFE have been carefully described for some microorganisms. In most cases, however, this information has been obtained only for a single environment, and very few studies have explored the effect that environmental variation may have on the DMFE. Environmental effects are particularly relevant for the evolution of multi-host parasites and thus for the emergence of new pathogens. Here we characterize the DMFE for a collection of twenty single-nucleotide substitution mutants of Tobacco etch potyvirus (TEV) across a set of eight host environments. Five of these host species were naturally infected by TEV, all belonging to family Solanaceae, whereas the other three were partially susceptible hosts belonging to three other plant families. First, we found a significant virus genotype-by-host species interaction, which was sustained by differences in genetic variance for fitness and the pleiotropic effect of mutations among hosts. Second, we found that the DMFEs were markedly different between Solanaceae and non-Solanaceae hosts. Exposure of TEV genotypes to non-Solanaceae hosts led to a large reduction of mean viral fitness, while the variance remained constant and skewness increased towards the right tail. Within the Solanaceae hosts, the distribution contained an excess of deleterious mutations, whereas for the non-Solanaceae the fraction of beneficial mutations was significantly larger. All together, this result suggests that TEV may easily broaden its host range and improve fitness in new hosts, and that knowledge about the DMFE in the natural host does not allow for making predictions about its properties in an alternative host

Structural and Topographic Dynamics of Pulmonary Histopathology and Local Cytokine Profiles in Paracoccidioides brasiliensis Conidia-Infected Mice

Paracoccidioidomycosis (PCM), an endemic fungal infection of pulmonary origin resulting in severe disseminated disease, occurs in rural areas of most South American countries and presents several clinical forms. The infection is acquired by inhalation of specific fungal propagules, called conidia. Considering the difficulties encountered when studying the infection in humans, this work was done in mice infected by inhalation of infective fungal conidia thus mimicking the human natural infection. The lungs of mice were sequentially studied by histopathological and multiplex cytokine methods from 2 h to 16 weeks after infection to verify the course of the disease. The mycosis presented different morphologic aspects during the course of time, affecting several pulmonary compartments. Otherwise and based on the analysis of 30 cytokines, the immune response also showed heterogeneous responses, which were up or down regulated depending on the time of infection. By recognizing the different stages that correspond to the evolution of pulmonary lesions, the severity (benign, chronic or fibrotic) of the disease could be predicted and the probable prognosis of the illness be inferred

Bryostatin Modulates Latent HIV-1 Infection via PKC and AMPK Signaling but Inhibits Acute Infection in a Receptor Independent Manner

HIV's ability to establish long-lived latent infection is mainly due to transcriptional silencing in resting memory T lymphocytes and other non dividing cells including monocytes. Despite an undetectable viral load in patients treated with potent antiretrovirals, current therapy is unable to purge the virus from these latent reservoirs. In order to broaden the inhibitory range and effectiveness of current antiretrovirals, the potential of bryostatin was investigated as an HIV inhibitor and latent activator. Bryostatin revealed antiviral activity against R5- and X4-tropic viruses in receptor independent and partly via transient decrease in CD4/CXCR4 expression. Further, bryostatin at low nanomolar concentrations robustly reactivated latent viral infection in monocytic and lymphocytic cells via activation of Protein Kinase C (PKC) -α and -δ, because PKC inhibitors rottlerin and GF109203X abrogated the bryostatin effect. Bryostatin specifically modulated novel PKC (nPKC) involving stress induced AMP Kinase (AMPK) inasmuch as an inhibitor of AMPK, compound C partially ablated the viral reactivation effect. Above all, bryostatin was non-toxic in vitro and was unable to provoke T-cell activation. The dual role of bryostatin on HIV life cycle may be a beneficial adjunct to the treatment of HIV especially by purging latent virus from different cellular reservoirs such as brain and lymphoid organs

Admixture in Latin America: Geographic Structure, Phenotypic Diversity and Self-Perception of Ancestry Based on 7,342 Individuals

The current genetic makeup of Latin America has been shaped by a history of extensive admixture between Africans, Europeans and Native Americans, a process taking place within the context of extensive geographic and social stratification. We estimated individual ancestry proportions in a sample of 7,342 subjects ascertained in five countries (Brazil, Chile, Colombia, México and Perú). These individuals were also characterized for a range of physical appearance traits and for self-perception of ancestry. The geographic distribution of admixture proportions in this sample reveals extensive population structure, illustrating the continuing impact of demographic history on the genetic diversity of Latin America. Significant ancestry effects were detected for most phenotypes studied. However, ancestry generally explains only a modest proportion of total phenotypic variation. Genetically estimated and self-perceived ancestry correlate significantly, but certain physical attributes have a strong impact on self-perception and bias self-perception of ancestry relative to genetically estimated ancestry

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified