Molecular Analysis of Precursor Lesions in Familial Pancreatic Cancer

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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)

Arousal of Cancer-Associated Stroma: Overexpression of Palladin Activates Fibroblasts to Promote Tumor Invasion

Background: Cancer-associated fibroblasts, comprised of activated fibroblasts or myofibroblasts, are found in the stroma surrounding solid tumors. These myofibroblasts promote invasion and metastasis of cancer cells. Mechanisms regulating the activation of the fibroblasts and the initiation of invasive tumorigenesis are of great interest. Upregulation of the cytoskeletal protein, palladin, has been detected in the stromal myofibroblasts surrounding many solid cancers and in expression screens for genes involved in invasion. Using a pancreatic cancer model, we investigated the functional consequence of overexpression of exogenous palladin in normal fibroblasts in vitro and its effect on the early stages of tumor invasion. Principal Findings: Palladin expression in stromal fibroblasts occurs very early in tumorigenesis. In vivo, concordant expression of palladin and the myofibroblast marker, alpha smooth muscle actin (a-SMA), occurs early at the dysplastic stages in peri-tumoral stroma and progressively increases in pancreatic tumorigenesis. In vitro introduction of exogenous 90 kD palladin into normal human dermal fibroblasts (HDFs) induces activation of stromal fibroblasts into myofibroblasts as marked by induction of a-SMA and vimentin, and through the physical change of cell morphology. Moreover, palladin expression in the fibroblasts enhances cellular migration, invasion through the extracellular matrix, and creation of tunnels through which cancer cells can follow. The fibroblast invasion and creation of tunnels results from the development o