493 research outputs found
Antiviral roles of plant ARGONAUTES
[EN] ARGONAUTES (AGOs) are the effector proteins functioning in eukaryotic RNA silencing pathways. AGOs associate with small RNAs and are programmed to target complementary RNA or DNA. Plant viruses induce a potent and specific antiviral RNA silencing host response in which AGOs play a central role. Antiviral AGOs associate with virus-derived small RNAs to repress complementary viral RNAs or DNAs, or with endogenous small RNAs to regulate host gene expression and promote antiviral defense. Here, we review recent progress towards understanding the roles of plant AGOs in antiviral defense. We also discuss the strategies that viruses have evolved to modulate, attenuate or suppress AGO antiviral functions.We thank members of the Carrington lab for useful and crucial discussions, and apologize to those colleagues whose work could not be cited because of space and reference limitations. This work was supported by grants from the National Science Foundation (MCB-1231726 and MCB-1330562) and National Institutes of Health (AI043288) to James C Carrington, and from the European Commission (H2020-MSCA-IF-2014-655841) to Alberto Carbonell.Carbonell, A.; Carrington, JC. (2015). Antiviral roles of plant ARGONAUTES. Current Opinion in Plant Biology. 27:111-117. https://doi.org/10.1016/j.pbi.2015.06.013S11111727Meister, G. (2013). Argonaute proteins: functional insights and emerging roles. Nature Reviews Genetics, 14(7), 447-459. doi:10.1038/nrg3462Poulsen, C., Vaucheret, H., & Brodersen, P. (2013). Lessons on RNA Silencing Mechanisms in Plants from Eukaryotic Argonaute Structures. The Plant Cell, 25(1), 22-37. doi:10.1105/tpc.112.105643Martínez de Alba, A. E., Elvira-Matelot, E., & Vaucheret, H. (2013). Gene silencing in plants: A diversity of pathways. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 1829(12), 1300-1308. doi:10.1016/j.bbagrm.2013.10.005Csorba, T., Kontra, L., & Burgyán, J. (2015). viral silencing suppressors: Tools forged to fine-tune host-pathogen coexistence. Virology, 479-480, 85-103. doi:10.1016/j.virol.2015.02.028Vaucheret, H. (2008). Plant ARGONAUTES. Trends in Plant Science, 13(7), 350-358. doi:10.1016/j.tplants.2008.04.007Morel, J.-B., Godon, C., Mourrain, P., Béclin, C., Boutet, S., Feuerbach, F., … Vaucheret, H. (2002). Fertile Hypomorphic ARGONAUTE (ago1) Mutants Impaired in Post-Transcriptional Gene Silencing and Virus Resistance. The Plant Cell, 14(3), 629-639. doi:10.1105/tpc.010358Qu, F., Ye, X., & Morris, T. J. (2008). Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proceedings of the National Academy of Sciences, 105(38), 14732-14737. doi:10.1073/pnas.0805760105Wang, X.-B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W.-X., … Ding, S.-W. (2011). The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes in Arabidopsis thaliana
. The Plant Cell, 23(4), 1625-1638. doi:10.1105/tpc.110.082305Dzianott, A., Sztuba-Solińska, J., & Bujarski, J. J. (2012). Mutations in the Antiviral RNAi Defense Pathway Modify Brome mosaic virus RNA Recombinant Profiles. Molecular Plant-Microbe Interactions®, 25(1), 97-106. doi:10.1094/mpmi-05-11-0137Garcia-Ruiz, H., Carbonell, A., Hoyer, J. S., Fahlgren, N., Gilbert, K. B., Takeda, A., … Carrington, J. C. (2015). Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection. PLOS Pathogens, 11(3), e1004755. doi:10.1371/journal.ppat.1004755Harvey, J. J. W., Lewsey, M. G., Patel, K., Westwood, J., Heimstädt, S., Carr, J. P., & Baulcombe, D. C. (2011). An Antiviral Defense Role of AGO2 in Plants. PLoS ONE, 6(1), e14639. doi:10.1371/journal.pone.0014639Jaubert, M., Bhattacharjee, S., Mello, A. F. S., Perry, K. L., & Moffett, P. (2011). ARGONAUTE2 Mediates RNA-Silencing Antiviral Defenses against Potato virus X in Arabidopsis
. Plant Physiology, 156(3), 1556-1564. doi:10.1104/pp.111.178012Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Gilbert, K. B., Montgomery, T. A., Nguyen, T., … Carrington, J. C. (2012). Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants
. The Plant Cell, 24(9), 3613-3629. doi:10.1105/tpc.112.099945Zhang, X., Zhang, X., Singh, J., Li, D., & Qu, F. (2012). Temperature-Dependent Survival of Turnip Crinkle Virus-Infected Arabidopsis Plants Relies on an RNA Silencing-Based Defense That Requires DCL2, AGO2, and HEN1. Journal of Virology, 86(12), 6847-6854. doi:10.1128/jvi.00497-12Ma, X., Nicole, M.-C., Meteignier, L.-V., Hong, N., Wang, G., & Moffett, P. (2014). Different roles for RNA silencing and RNA processing components in virus recovery and virus-induced gene silencing in plants. Journal of Experimental Botany, 66(3), 919-932. doi:10.1093/jxb/eru447Takeda, A., Iwasaki, S., Watanabe, T., Utsumi, M., & Watanabe, Y. (2008). The Mechanism Selecting the Guide Strand from Small RNA Duplexes is Different Among Argonaute Proteins. Plant and Cell Physiology, 49(4), 493-500. doi:10.1093/pcp/pcn043Hamera, S., Song, X., Su, L., Chen, X., & Fang, R. (2011). Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities. The Plant Journal, 69(1), 104-115. doi:10.1111/j.1365-313x.2011.04774.xBhattacharjee, S., Zamora, A., Azhar, M. T., Sacco, M. A., Lambert, L. H., & Moffett, P. (2009). Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. The Plant Journal, 58(6), 940-951. doi:10.1111/j.1365-313x.2009.03832.xRaja, P., Sanville, B. C., Buchmann, R. C., & Bisaro, D. M. (2008). Viral Genome Methylation as an Epigenetic Defense against Geminiviruses. Journal of Virology, 82(18), 8997-9007. doi:10.1128/jvi.00719-08Raja, P., Jackel, J. N., Li, S., Heard, I. M., & Bisaro, D. M. (2013). Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses. Journal of Virology, 88(5), 2611-2622. doi:10.1128/jvi.02305-13Scholthof, H. B., Alvarado, V. Y., Vega-Arreguin, J. C., Ciomperlik, J., Odokonyero, D., Brosseau, C., … Moffett, P. (2011). Identification of an ARGONAUTE for Antiviral RNA Silencing in Nicotiana benthamiana
. Plant Physiology, 156(3), 1548-1555. doi:10.1104/pp.111.178764Ghoshal, B., & Sanfaçon, H. (2014). Temperature-dependent symptom recovery in Nicotiana benthamiana plants infected with tomato ringspot virus is associated with reduced translation of viral RNA2 and requires ARGONAUTE 1. Virology, 456-457, 188-197. doi:10.1016/j.virol.2014.03.026Iki, T., Yoshikawa, M., Nishikiori, M., Jaudal, M. C., Matsumoto-Yokoyama, E., Mitsuhara, I., … Ishikawa, M. (2010). In Vitro Assembly of Plant RNA-Induced Silencing Complexes Facilitated by Molecular Chaperone HSP90. Molecular Cell, 39(2), 282-291. doi:10.1016/j.molcel.2010.05.014Schuck, J., Gursinsky, T., Pantaleo, V., Burgyán, J., & Behrens, S.-E. (2013). AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system. Nucleic Acids Research, 41(9), 5090-5103. doi:10.1093/nar/gkt193Zhu, H., Duan, C.-G., Hou, W.-N., Du, Q.-S., Lv, D.-Q., Fang, R.-X., & Guo, H.-S. (2011). Satellite RNA-Derived Small Interfering RNA satsiR-12 Targeting the 3’ Untranslated Region of Cucumber Mosaic Virus Triggers Viral RNAs for Degradation. Journal of Virology, 85(24), 13384-13397. doi:10.1128/jvi.05806-11Cao, M., Du, P., Wang, X., Yu, Y.-Q., Qiu, Y.-H., Li, W., … Ding, S.-W. (2014). Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs inArabidopsis. Proceedings of the National Academy of Sciences, 111(40), 14613-14618. doi:10.1073/pnas.1407131111Smith, N. A., Eamens, A. L., & Wang, M.-B. (2011). Viral Small Interfering RNAs Target Host Genes to Mediate Disease Symptoms in Plants. PLoS Pathogens, 7(5), e1002022. doi:10.1371/journal.ppat.1002022Shimura, H., Pantaleo, V., Ishihara, T., Myojo, N., Inaba, J., Sueda, K., … Masuta, C. (2011). A Viral Satellite RNA Induces Yellow Symptoms on Tobacco by Targeting a Gene Involved in Chlorophyll Biosynthesis using the RNA Silencing Machinery. PLoS Pathogens, 7(5), e1002021. doi:10.1371/journal.ppat.1002021Navarro, B., Gisel, A., Rodio, M. E., Delgado, S., Flores, R., & Di Serio, F. (2012). Small RNAs containing the pathogenic determinant of a chloroplast-replicating viroid guide the degradation of a host mRNA as predicted by RNA silencing. The Plant Journal, 70(6), 991-1003. doi:10.1111/j.1365-313x.2012.04940.xMiozzi, L., Gambino, G., Burgyan, J., & Pantaleo, V. (2012). Genome-wide identification of viral and host transcripts targeted by viral siRNAs inVitis vinifera. Molecular Plant Pathology, 14(1), 30-43. doi:10.1111/j.1364-3703.2012.00828.xDe Ronde, D., Pasquier, A., Ying, S., Butterbach, P., Lohuis, D., & Kormelink, R. (2013). Analysis ofTomato spotted wilt virus NSs protein indicates the importance of the N-terminal domain for avirulence and RNA silencing suppression. Molecular Plant Pathology, 15(2), 185-195. doi:10.1111/mpp.12082Lacombe, S., Bangratz, M., Vignols, F., & Brugidou, C. (2010). The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing. The Plant Journal, 61(3), 371-382. doi:10.1111/j.1365-313x.2009.04062.xGuo, H., Song, X., Xie, C., Huo, Y., Zhang, F., Chen, X., … Fang, R. (2013). Rice yellow stunt rhabdovirus Protein 6 Suppresses Systemic RNA Silencing by Blocking RDR6-Mediated Secondary siRNA Synthesis. Molecular Plant-Microbe Interactions®, 26(8), 927-936. doi:10.1094/mpmi-02-13-0040-rOkano, Y., Senshu, H., Hashimoto, M., Neriya, Y., Netsu, O., Minato, N., … Namba, S. (2014). In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor
. The Plant Cell, 26(5), 2168-2183. doi:10.1105/tpc.113.120535Weinheimer, I., Jiu, Y., Rajamäki, M.-L., Matilainen, O., Kallijärvi, J., Cuellar, W. J., … Valkonen, J. P. T. (2015). Suppression of RNAi by dsRNA-Degrading RNaseIII Enzymes of Viruses in Animals and Plants. PLOS Pathogens, 11(3), e1004711. doi:10.1371/journal.ppat.1004711Baumberger, N., Tsai, C.-H., Lie, M., Havecker, E., & Baulcombe, D. C. (2007). The Polerovirus Silencing Suppressor P0 Targets ARGONAUTE Proteins for Degradation. Current Biology, 17(18), 1609-1614. doi:10.1016/j.cub.2007.08.039Bortolamiol, D., Pazhouhandeh, M., Marrocco, K., Genschik, P., & Ziegler-Graff, V. (2007). The Polerovirus F Box Protein P0 Targets ARGONAUTE1 to Suppress RNA Silencing. Current Biology, 17(18), 1615-1621. doi:10.1016/j.cub.2007.07.061Csorba, T., Lózsa, R., Hutvágner, G., & Burgyán, J. (2010). Polerovirus protein P0 prevents the assembly of small RNA-containing RISC complexes and leads to degradation of ARGONAUTE1. The Plant Journal, 62(3), 463-472. doi:10.1111/j.1365-313x.2010.04163.xFusaro, A. F., Correa, R. L., Nakasugi, K., Jackson, C., Kawchuk, L., Vaslin, M. F. S., & Waterhouse, P. M. (2012). The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation. Virology, 426(2), 178-187. doi:10.1016/j.virol.2012.01.026Derrien, B., Baumberger, N., Schepetilnikov, M., Viotti, C., De Cillia, J., Ziegler-Graff, V., … Genschik, P. (2012). Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway. Proceedings of the National Academy of Sciences, 109(39), 15942-15946. doi:10.1073/pnas.1209487109Azevedo, J., Garcia, D., Pontier, D., Ohnesorge, S., Yu, A., Garcia, S., … Voinnet, O. (2010). Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes & Development, 24(9), 904-915. doi:10.1101/gad.1908710Zhang, X., Yuan, Y.-R., Pei, Y., Lin, S.-S., Tuschl, T., Patel, D. J., & Chua, N.-H. (2006). Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes & Development, 20(23), 3255-3268. doi:10.1101/gad.1495506Duan, C.-G., Fang, Y.-Y., Zhou, B.-J., Zhao, J.-H., Hou, W.-N., Zhu, H., … Guo, H.-S. (2012). Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein. The Plant Cell, 24(1), 259-274. doi:10.1105/tpc.111.092718Giner, A., Lakatos, L., García-Chapa, M., López-Moya, J. J., & Burgyán, J. (2010). Viral Protein Inhibits RISC Activity by Argonaute Binding through Conserved WG/GW Motifs. PLoS Pathogens, 6(7), e1000996. doi:10.1371/journal.ppat.1000996Szabo, E. Z., Manczinger, M., Goblos, A., Kemeny, L., & Lakatos, L. (2012). Switching on RNA Silencing Suppressor Activity by Restoring Argonaute Binding to a Viral Protein. Journal of Virology, 86(15), 8324-8327. doi:10.1128/jvi.00627-12Pérez-Cañamás, M., & Hernández, C. (2015). Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing. Journal of Biological Chemistry, 290(5), 3106-3120. doi:10.1074/jbc.m114.593707Buchmann, R. C., Asad, S., Wolf, J. N., Mohannath, G., & Bisaro, D. M. (2009). Geminivirus AL2 and L2 Proteins Suppress Transcriptional Gene Silencing and Cause Genome-Wide Reductions in Cytosine Methylation. Journal of Virology, 83(10), 5005-5013. doi:10.1128/jvi.01771-08Soitamo, A. J., Jada, B., & Lehto, K. (2012). Expression of geminiviral AC2 RNA silencing suppressor changes sugar and jasmonate responsive gene expression in transgenic tobacco plants. BMC Plant Biology, 12(1), 204. doi:10.1186/1471-2229-12-204Zhang, Z., Chen, H., Huang, X., Xia, R., Zhao, Q., Lai, J., … Xie, Q. (2011). BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis
. The Plant Cell, 23(1), 273-288. doi:10.1105/tpc.110.081695Várallyay, É., Válóczi, A., Ágyi, Á., Burgyán, J., & Havelda, Z. (2010). Plant virus-mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation. The EMBO Journal, 29(20), 3507-3519. doi:10.1038/emboj.2010.215Várallyay, É., & Havelda, Z. (2013). Unrelated viral suppressors of RNA silencing mediate the control of ARGONAUTE1 level. Molecular Plant Pathology, 14(6), 567-575. doi:10.1111/mpp.1202
Small RNA-based antiviral defense in the phytopathogenic fungus Colletotrichum higginsianum
Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism
P-SAMS: a web suite for plant artificial microRNA and synthetic trans-acting small interfering RNA design
[EN] The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated
design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syntasiRNAs)
for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications,
P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both
applications is wizard-assisted, and the job runtime is relatively short. Both applications output the
sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for
cloning into `B/c¿ compatible vectors.This work was supported by the National Institutes of Health [grant number AI043288 to J.C.C.]; the National Science Foundation [grants numbers MCB-1231726, MCB-1330562 to J.C.C.]; and the United States Department of Agriculture [fellowship number MOW-2012-01361 to N.F.).Fahlgren, N.; Hill, ST.; Carrington, JC.; Carbonell, A. (2016). P-SAMS: a web suite for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics. 32(1):157-158. https://doi.org/10.1093/bioinformatics/btv534S157158321Ahmed, F., Dai, X., & Zhao, P. X. (2015). Bioinformatics Tools for Achieving Better Gene Silencing in Plants. Plant Gene Silencing, 43-60. doi:10.1007/978-1-4939-2453-0_3Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T., & Carrington, J. C. (2014). New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis. Plant Physiology, 165(1), 15-29. doi:10.1104/pp.113.234989Carbonell, A., Fahlgren, N., Mitchell, S., Cox, K. L., Reilly, K. C., Mockler, T. C., & Carrington, J. C. (2015). Highly specific gene silencing in a monocot species by artificial micro
RNA
s derived from chimeric
mi
RNA
precursors. The Plant Journal, 82(6), 1061-1075. doi:10.1111/tpj.12835Fahlgren, N., & Carrington, J. C. (2009). miRNA Target Prediction in Plants. Plant MicroRNAs, 51-57. doi:10.1007/978-1-60327-005-2_4Ossowski, S., Schwab, R., & Weigel, D. (2008). Gene silencing in plants using artificial microRNAs and other small RNAs. The Plant Journal, 53(4), 674-690. doi:10.1111/j.1365-313x.2007.03328.xSchwab, R., Ossowski, S., Riester, M., Warthmann, N., & Weigel, D. (2006). Highly Specific Gene Silencing by Artificial MicroRNAs inArabidopsis. The Plant Cell, 18(5), 1121-1133. doi:10.1105/tpc.105.039834Tiwari, M., Sharma, D., & Trivedi, P. K. (2014). Artificial microRNA mediated gene silencing in plants: progress and perspectives. Plant Molecular Biology, 86(1-2), 1-18. doi:10.1007/s11103-014-0224-7Zhang, Z. J. (2014). Artificial trans-acting small interfering RNA: a tool for plant biology study and crop improvements. Planta, 239(6), 1139-1146. doi:10.1007/s00425-014-2054-
Photocapacitance study of type-II GaSb/GaAs quantum ring solar cells
In this study, the density of states associated with the localization of holes in GaSb/GaAs quantum rings are determined by the energy selective charging of the quantum ring distribution. The authors show, using conventional photocapacitance measurements, that the excess charge accumulated within the type-II nanostructures increases with increasing excitation energies for photon energies above 0.9 eV. Optical excitation between the localized hole states and the conduction band is therefore not limited to the Γ(k = 0) point, with pseudo-monochromatic light charging all states lying within the photon energy selected. The energy distribution of the quantum ring states could consequently be accurately related from the excitation dependence of the integrated photocapacitance. The resulting band of localized hole states is shown to be well described by a narrow distribution centered 407 meV above the GaAs valence band maximum
ARGONAUTE PIWI domain and microRNA duplex structure regulate small RNA sorting in Arabidopsis.
Small RNAs (sRNAs) are loaded into ARGONAUTE (AGO) proteins to induce gene silencing. In plants, the 5'-terminal nucleotide is important for sRNA sorting into different AGOs. Here we show that microRNA (miRNA) duplex structure also contributes to miRNA sorting. Base pairing at the 15th nucleotide of a miRNA duplex is important for miRNA sorting in both Arabidopsis AGO1 and AGO2. AGO2 favours miRNA duplexes with no middle mismatches, whereas AGO1 tolerates, or prefers, duplexes with central mismatches. AGO structure modelling and mutational analyses reveal that the QF-V motif within the conserved PIWI domain contributes to recognition of base pairing at the 15th nucleotide of a duplex, while the DDDE catalytic core of AtAGO2 is important for recognition of the central nucleotides. Finally, we rescued the adaxialized phenotype of ago1-12, which is largely due to miR165 loss-of-function, by changing miR165 duplex structure which we predict redirects it to AGO2
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Evidence that the Potyvirus P1 Proteinase Functions in trans as an Accessory Factor for Genome Amplification
The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa,
HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral
infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine
if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire
P1 coding region (ΔP1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing β-glucuronidase
(GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts
and plants. The ΔP1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental
TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA
amplification. The ΔP1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but
at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a
processing-defective P1 proteinase and which was nonviable in plants. Both ΔP1 and S256A mutants were
complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification
of the ΔP1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level
of accumulation of the DP1 mutant was stimulated, although the rate of cell-to-cell movement was the same as
in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-
expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the
P1 proteinase functions in trans to stimulate genome amplification
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Debilitation of Plant Potyvirus Infectivity by P1 Proteinase-Inactivating Mutations and Restoration by Second-Site Modifications
Tobacco etch virus (TEV) encodes three proteinases that catalyze processing of the genome-encoded polyprotein.
The P1 proteinase originates from the N terminus of the polyprotein and catalyzes proteolysis between
itself and the helper component proteinase (HC-Pro). Mutations resulting in substitution of a single amino
acid, small insertions, or deletions were introduced into the P1 coding sequence of the TEV genome. Deletion
of the N-terminal, nonproteolytic domain of P1 had only minor effects on virus infection in protoplasts and
whole plants. Insertion mutations that did not impair proteolytic activity had no measurable effects regardless
of whether the modification affected the N-terminal nonproteolytic or C-terminal proteolytic domain. In
contrast, three mutations (termed S256A, F, and Δ304) that debilitated P1 proteolytic activity rendered the
virus nonviable, whereas a fourth proteinase-debilitating mutation (termed C) resulted in a slow-infection
phenotype. A strategy was devised to determine whether the defect in the P1 mutants was due to an inactive
proteinase domain or due simply to a lack of proteolytic maturation between P1 and HC-Pro. Sequences coding
for a surrogate cleavage site recognized by the TEV NIa proteinase were inserted into the genome of each
processing-debilitated mutant at positions that resulted in NIa-mediated proteolysis between P1 and HC-Pro.
The infectivity of each mutant was restored by these second-site modifications. These data indicate that P1
proteinase activity is not essential for viral infectivity but that separation of P1 and HC-Pro is required. The
data also provide evidence that the proteinase domain is involved in additional, nonproteolytic functions
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