An efficient Foxtail mosaic virus vector system with reduced environmental risk

<p>Abstract</p> <p>Background</p> <p>Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of <it>Agrobacterium tumefaciens </it>has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment.</p> <p>Results</p> <p>In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from <it>Foxtail mosaic virus </it>to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, <it>Nicotiana benthamiana</it>. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as <it>N. benthamiana</it>, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low <it>Agrobacterium</it>-mediated transformation rate of monocots.</p> <p>Conclusions</p> <p>The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing.</p

Registration of 'Ok102' wheat

Peer reviewedPlant and Soil SciencesEntomology and Plant PathologyBiochemistry and Molecular Biolog

Phosphorylation of bamboo mosaic virus satellite RNA (satBaMV)-encoded protein P20 downregulates the formation of satBaMV-P20 ribonucleoprotein complex

Bamboo mosaic virus (BaMV) satellite RNA (satBaMV) depends on BaMV for its replication and encapsidation. SatBaMV-encoded P20 protein is an RNA-binding protein that facilitates satBaMV systemic movement in co-infected plants. Here, we examined phosphorylation of P20 and its regulatory functions. Recombinant P20 (rP20) was phosphorylated by host cellular kinase(s) in vitro, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and mutational analyses revealed Ser-11 as the phosphorylation site. The phosphor-mimic rP20 protein interactions with satBaMV-translated mutant P20 were affected. In overlay assay, the Asp mutation at S11 (S11D) completely abolished the self-interaction of rP20 and significantly inhibited the interaction with both the WT and S11A rP20. In chemical cross-linking assays, S11D failed to oligomerize. Electrophoretic mobility shift assay and subsequent Hill transformation analysis revealed a low affinity of the phospho-mimicking rP20 for satBaMV RNA. Substantial modulation of satBaMV RNA conformation upon interaction with nonphospho-mimic rP20 in circular dichroism analysis indicated formation of stable satBaMV ribonucleoprotein complexes. The dissimilar satBaMV translation regulation of the nonphospho- and phospho-mimic rP20 suggests that phosphorylation of P20 in the ribonucleoprotein complex converts the translation-incompetent satBaMV RNA to messenger RNA. The phospho-deficient or phospho-mimicking P20 mutant of satBaMV delayed the systemic spread of satBaMV in co-infected Nicotiana benthamiana with BaMV. Thus, satBaMV likely regulates the formation of satBaMV RNP complex during co-infection in planta

Formation of Complexes at Plasmodesmata for Potyvirus Intercellular Movement Is Mediated by the Viral Protein P3N-PIPO

Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants

Pollen tube energetics: respiration, fermentation and the race to the ovule

This review covers historical and recent progress in understanding how respiration, fermentation and mitochondria contribute to pollen tube growth. It also summarizes what is known about the energetic requirements of this growth. Molecular mechanisms are viewed in the context of pollen tube physiology, a necessary perspective to understanding pollen tube growth

Host range and symptomatology of Pepino mosaic virus strains occurring in Europe

Pepino mosaic virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe when tested under different environmental conditions. To this end we compared the infectivity and symptom development of three, well characterized isolates belonging to three different PepMV strains, EU-tom, Ch2 and US1, by inoculating them on tomato, possible alternative host plants in the family Solanaceae and selected test plants. The inoculation experiments were done in 10 countries from south to north in Europe. The importance of alternative hosts among the solanaceous crops and the usefulness of test plants in the biological characterization of PepMV isolates are discussed. Our data for the three strains tested at 10 different European locations with both international and local cultivars showed that eggplant is an alternative host of PepMV. Sweet pepper is not an important host of PepMV, but potato can be infected when the right isolate is matched with a specific cultivar. Nicotiana occidentalis 37B is a useful indicator plant for PepMV studies, since it reacts with a different symptomatology to each one of the PepMV strains.Ravnikar, M.; Blystad, D.; Van Der Vlugt, R.; Alfaro Fernández, AO.; Del Carmen Cordoba, M.; Bese, G.; Hristova, D.... (2015). Host range and symptomatology of Pepino mosaic virus strains occurring in Europe. European Journal of Plant Pathology. 143(1):43-56. doi:10.1007/s10658-015-0664-1S43561431Alfaro-Fernández, A., Córdoba-Sellés, M. C., Herrera-Vásquez, J. A., Cebrián, M. C., & Jordá, C. (2009). Transmission of Pepino mosaic virus by the fungal vector Olpidium virulentus. Journal of Phytopathology, 158, 217–226.Charmichael, D. J., Rey, M. E. C., Naidoo, S., Cook, G., & van Heerden, S. W. (2011). First report of Pepino mosaic virus infecting tomato in South Africa. Plant Disease, 95(6), 767.2.Córdoba, M. C., Martínez-Priego, L., & Jordá, C. (2004). New natural hosts of Pepino mosaic virus in Spain. Plant Disease, 88, 906.Córdoba-Sellés, M. C., García-Rández, A., Alfaro-Fernández, A., & Jordá-Gutiérrez, C. (2007). Seed transmission of pepino mosaic virus and efficacy of tomato seed disinfection treatments. Plant Disease, 91, 1250–1254.Efthimiou, K. E., Gatsios, A. P., Aretakis, K. C., Papayannis, L. C., & Katis, N. I. (2011). First report of Pepino mosaic virus infecting greenhouse cherry tomato in Greece. Plant Disease, 95(1), 78.2.Fakhro, A., von Bargen, S., Bandte, M., Büttner, C., Franken, P., & Schwarz, D. (2011). Susceptibility of different plant species and tomato cultivars to two isolates of Pepino mosaic virus. European Journal of Plant Pathology, 129, 579–590.Gómez, P., Sempere, R. N., Elena, S. F., & Aranda, M. A. (2009). Mixed infections of Pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus. Journal of Virology, 83, 12378–12387.Hanssen, I. M., Paeleman, A., Wittemans, L., Goen, K., Lievens, B., Bragard, C., Vanachter, A. C. R. C., & Thomma, B. P. H. J. (2008). Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination. European Journal of Plant Pathology, 121, 131–146.Hanssen, I. M., Paeleman, A., Vandewoestijne, E., Van Bergen, L., Bragard, C., Lievens, B., Vanachter, A. C. R. C., & Thomma, B. P. H. J. (2009). Pepino mosaic virus isolates and differential symptomatology in tomato. Plant Pathology, 58, 450–460.Hanssen, I. M., Mumford, R., Blystad, D.-G., Cortez, I., Hasiów-Jaroszewska, B., Hristova, D., Pagán, I., Pereira, A.-M., Peters, J., Pospieszny, H., Ravnikar, M., Stijger, I., Tomassoli, L., Varveri, C., van der Vlugt, R., & Nielsen, S. L. (2010). Seed transmission of Pepino mosaic virus in tomato. European Journal of Plant Pathology, 126, 145–152.Hasiów-Jaroszewska, B., Borodynko, N., Jackowiak, P., Figlerowicz, M., & Pospieszny, H. (2010a). Pepino mosaic virus – a pathogen of tomato crops in Poland: biology, evolution and diagnostics. Journal of Plant Protection Research, 50, 470–476.Hasiów-Jaroszewska, B., Jackowiak, P., Borodynko, N., Figlerowicz, M., & Pospieszny, H. (2010b). Quasispecies nature of Pepino mosaic virus and its evolutionary dynamics. Virus Genes, 41, 260–267.Jeffries, C. J. (1998). FAO/IPGRI technical guidelines for the safe movement of germplasm no. 19. Potato. Food and agriculture organization of the United Nations, Rome/International Plant Genetic Resources Institute, Rome pp 177Jones, R. A. C., Koenig, R., & Lesemann, D. E. (1980). Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum). Annals of Applied Biology, 94, 61–68.Jordá, C., Lázaro Pérez, A., & Martínez Culebras, P. (2001). First report of Pepino mosaic virus on natural hosts. Plant Disease, 85, 1292.King, A. M. Q., Adams, M. J., Carstens, E. B., Lefkowitz, E. J., (eds). (2012). potexvirus, pp 912–915, in virus taxonomy, classification and nomenclature of viruses; ninth report of the international committee on taxonomy of viruses (p 1327) London, UK: Elsevier Academic PressLing, K.-S., & Zhang, W. (2011). First report of Pepino mosaic virus infecting tomato in Mexico. Plant Disease, 95(8), 1035.Martin, J., & Mousserion, C. (2002). Potato varieties which are sensitive to the tomato strains of Pepino mosaic virus (PepMV). Phytoma Défence Végétaux, 552, 26–28.Mehle, N., Gutierrez-Aguirre, I., Prezelj, N., Delić, D., Vidic, U., & Ravnikar, M. (2014). Survival and transmission of potato virus Y, pepino mosaic virus, and potato spindle tuber viroid in water. Applied and Environmental Microbiology, 80(4), 1455–1462.Moreno-Pérez, M. G., Pagán, I., Aragón-Caballero, L., Cáceres, F., Aurora Fraile, A., & García-Arenal, F. (2014). Ecological and genetic determinants of Pepino mosaic virus emergence. Journal of Virology, 88(6), 3359–3368.Noël, P., Hance, T., & Bragard, C. (2014). Transmission of the pepino mosaic virus by whitefly. European Journal of Plant Pathology, 138, 23–27.Pagan, I., Cordoba-Selles, M. D., Martinez-Priego, L., Fraile, A., Malpica, J. M., Jorda, C., & Garcia-Arenal, F. (2006). Genetic structure of the population of pepino mosaic virus infecting tomato crops in Spain. Phytopathology, 96, 274–279.Papayiannis, L. C., Kokkinos, C. D., & Alfaro-Fernández, A. (2012). Detection, characterization and host range studies of Pepino mosaic virus in Cyprus. European Journal of Plant Pathology, 132, 1–7.Pospieszny, H., Haslow, B., & Borodynko, N. (2008). Characterization of two Polish isolates of Pepino mosaic virus. European Journal of Plant Pathology, 122, 443–445.Salomone, A., & Roggero, P. (2002). Host range, seed transmission and detection by ELISA and lateral flow of an Italian isolate of Pepino mosaic virus. Journal of Plant Pathology, 84, 65–68.Samson, R. G., Allen, T. C., & Whitworth, J. L. (1993). Evaluation of direct tissue blotting to detect potato viruses. American Potato Journal, 70, 257–265.Schwarz, D., Beuch, U., Bandte, M., Fakhro, A., Büttner, C., & Obermeier, C. (2010). Spread and interaction of pepino mosaic virus (PepMV) and pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield. Plant Pathology, 59(3), 443–452.Shipp, J. L., Buitenhuis, R., Stobbs, L., Wang, K., Kim, W. S., & Ferguson, G. (2008). Vectoring of pepino mosaic virus by bumble-bees in tomato greenhouses. Annals of Applied Biology, 153, 149–155.Van der Vlugt, R. A. A. (2009). Pepino mosaic virus (review). Hellenic Plant Protection Journal, 2, 47–56.Van der Vlugt, R. A. A., & Stijger, C. C. M. M. (2008). Pepino mosaic virus. In B. W. J. Mahy & M. H. V. Van Regenmortel (Eds.), Encyclopedia of virology (5th ed., pp. 103–108). Wageningen: Oxford Elsevier.Van der Vlugt, R. A. A., Stijger, C. C. M. M., Verhoeven, J. T. J., & Lesemann, D.-E. (2000). First report of Pepino mosaic virus on tomato. Plant Disease, 84, 103.Van der Vlugt, R. A. A., Cuperus, C., Vink, J., Stijger, I. C. M. M., Lesemann, D.-E., Verhoeven, J. T. J., & Roenhorst, J. W. (2002). Identification and characterization of Pepino mosaic potexvirus in tomato. Bulletin EPPO/EPPO Bulletin, 32, 503–508.Verchot-Lubicz, J., Chang-Ming, Y., & Bamunusinghe, D. (2007). Molecular biology of potexviruses: recent advances. Journal of General Virology, 88(6), 1643–1655.Verhoeven, J. T. H. J., van der Vlugt, R., & Roenhorst, J. W. (2003). High similarity between tomato isolates of pepino mosaic virus suggests a common origin. European Journal of Plant Pathology, 109, 419–425.Werkman, A.W., & Sansford, C.E. (2010). Pest risk analysis for pepino mosaic virus for the EU. Deliverable Report 4.3. EU Sixth Framework project PEPEIRA. http:// www.pepeira.com .Wright, D., & Mumford, R. (1999). Pepino mosaic potexvirus (PepMV): first records in tomato in the United Kingdom. Plant disease notice (89th ed.). York, UK: Central Science Laboratory