Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features

[EN] Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host.We wish to thank Elvira Dominguez for technical assistance. This work was supported by grants BIO2010-18541 from the Spanish Ministerio de Educacion y Ciencia (MEC), SAL/0185/2006 from Comunidad de Madrid and KBBE-204429 from the European Union. B. S. was a recipient of a Formacion de Personal Investigador fellowship from MEC.Maliogka, VI.; Salvador, B.; Carbonell, A.; Saenz, P.; San Leon, D.; Oliveros, JC.; Delgadillo, MO.... (2012). Virus variants with differences in the P1 protein coexist in a Plum pox virus population and display particular host-dependent pathogenicity features. Molecular Plant Pathology. 13(8):877-886. https://doi.org/10.1111/j.1364-3703.2012.00796.xS877886138Adams, M. J., Antoniw, J. F., & Fauquet, C. M. (2004). Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology, 150(3), 459-479. doi:10.1007/s00705-004-0440-6Ayme, V., Petit-Pierre, J., Souche, S., Palloix, A., & Moury, B. (2007). Molecular dissection of the potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper. Journal of General Virology, 88(5), 1594-1601. doi:10.1099/vir.0.82702-0Biebricher, C. K., & Eigen, M. (s. f.). What Is a Quasispecies? Quasispecies: Concept and Implications for Virology, 1-31. doi:10.1007/3-540-26397-7_1Brantley, J. D., & Hunt, A. G. (1993). The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. Journal of General Virology, 74(6), 1157-1162. doi:10.1099/0022-1317-74-6-1157Charron, C., Nicolaï, M., Gallois, J.-L., Robaglia, C., Moury, B., Palloix, A., & Caranta, C. (2008). Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg. The Plant Journal, 54(1), 56-68. doi:10.1111/j.1365-313x.2008.03407.xChiang, C.-H., Lee, C.-Y., Wang, C.-H., Jan, F.-J., Lin, S.-S., Chen, T.-C., … Yeh, S.-D. (2007). Genetic analysis of an attenuated Papaya ringspot virus strain applied for cross-protection. European Journal of Plant Pathology, 118(4), 333-348. doi:10.1007/s10658-007-9130-zChung, B. Y.-W., Miller, W. A., Atkins, J. F., & Firth, A. E. (2008). An overlapping essential gene in the Potyviridae. Proceedings of the National Academy of Sciences, 105(15), 5897-5902. doi:10.1073/pnas.0800468105Domingo, E., & Holland, J. J. (1997). RNA VIRUS MUTATIONS AND FITNESS FOR SURVIVAL. Annual Review of Microbiology, 51(1), 151-178. doi:10.1146/annurev.micro.51.1.151EIGEN, M. (1996). On the nature of virus quasispecies. Trends in Microbiology, 4(6), 216-218. doi:10.1016/0966-842x(96)20011-3Hajimorad, M. R., Wen, R.-H., Eggenberger, A. L., Hill, J. H., & Maroof, M. A. S. (2011). Experimental Adaptation of an RNA Virus Mimics Natural Evolution. Journal of Virology, 85(6), 2557-2564. doi:10.1128/jvi.01935-10Holmes, E. C., & Moya, A. (2002). Is the Quasispecies Concept Relevant to RNA Viruses? Journal of Virology, 76(1), 460-462. doi:10.1128/jvi.76.1.460-462.2002Jenkins, G. M., Worobey, M., Woelk, C. H., & Holmes, E. C. (2001). Evidence for the Non-quasispecies Evolution of RNA Viruses. Molecular Biology and Evolution, 18(6), 987-994. doi:10.1093/oxfordjournals.molbev.a003900Jridi, C., Martin, J.-F., Marie-Jeanne, V., Labonne, G., & Blanc, S. (2006). Distinct Viral Populations Differentiate and Evolve Independently in a Single Perennial Host Plant. Journal of Virology, 80(5), 2349-2357. doi:10.1128/jvi.80.5.2349-2357.2006Li, H., & Durbin, R. (2010). Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics, 26(5), 589-595. doi:10.1093/bioinformatics/btp698López-Moya, J. J., & Garcı́a, J. A. (2000). Construction of a stable and highly infectious intron-containing cDNA clone of plum pox potyvirus and its use to infect plants by particle bombardment. Virus Research, 68(2), 99-107. doi:10.1016/s0168-1702(00)00161-1Moxon, S., Schwach, F., Dalmay, T., MacLean, D., Studholme, D. J., & Moulton, V. (2008). A toolkit for analysing large-scale plant small RNA datasets. Bioinformatics, 24(19), 2252-2253. doi:10.1093/bioinformatics/btn428Nakahara, K. S., Shimada, R., Choi, S.-H., Yamamoto, H., Shao, J., & Uyeda, I. (2010). Involvement of the P1 Cistron in Overcoming eIF4E-Mediated Recessive Resistance Against Clover yellow vein virus in Pea. Molecular Plant-Microbe Interactions®, 23(11), 1460-1469. doi:10.1094/mpmi-11-09-0277Ohshima, K., Akaishi, S., Kajiyama, H., Koga, R., & Gibbs, A. J. (2009). Evolutionary trajectory of turnip mosaic virus populations adapting to a new host. Journal of General Virology, 91(3), 788-801. doi:10.1099/vir.0.016055-0Pruss, G., Ge, X., Shi, X. M., Carrington, J. C., & Bowman Vance, V. (1997). Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. The Plant Cell, 9(6), 859-868. doi:10.1105/tpc.9.6.859Rajamäki, M.-L., Kelloniemi, J., Alminaite, A., Kekarainen, T., Rabenstein, F., & Valkonen, J. P. T. (2005). A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions. Virology, 342(1), 88-101. doi:10.1016/j.virol.2005.07.019Rohožková, J., & Navrátil, M. (2011). P1 peptidase – a mysterious protein of family Potyviridae. Journal of Biosciences, 36(1), 189-200. doi:10.1007/s12038-011-9020-6Sáenz, P., Riechmann, J. L., Dallot, S., Quiot, L., Garcı́a, J. A., Cervera, M. T., & Quiot, J.-B. (2000). Identification of a pathogenicity determinant of Plum pox virus in the sequence encoding the C-terminal region of protein P3+6K1. Journal of General Virology, 81(3), 557-566. doi:10.1099/0022-1317-81-3-557Sáenz, P., Quiot, L., Quiot, J.-B., Candresse, T., & García, J. A. (2001). Pathogenicity Determinants in the Complex Virus Population of a Plum pox virus Isolate. Molecular Plant-Microbe Interactions®, 14(3), 278-287. doi:10.1094/mpmi.2001.14.3.278Salvador, B., García, J. A., & Simón-Mateo, C. (2006). Causal agent of sharka disease: Plum pox virus genome and function of gene products. EPPO Bulletin, 36(2), 229-238. doi:10.1111/j.1365-2338.2006.00979.xSalvador, B., Delgadillo, M. O., Sáenz, P., García, J. A., & Simón-Mateo, C. (2008). Identification of Plum pox virus Pathogenicity Determinants in Herbaceous and Woody Hosts. Molecular Plant-Microbe Interactions®, 21(1), 20-29. doi:10.1094/mpmi-21-1-0020SALVADOR, B., SAÉNZ, P., YANGÜEZ, E., QUIOT, J. B., QUIOT, L., DELGADILLO, M. O., … SIMÓN-MATEO, C. (2008). Host-specific effect of P1 exchange between two potyviruses. Molecular Plant Pathology, 9(2), 147-155. doi:10.1111/j.1364-3703.2007.00450.xSoumounou, Y., & Laliberte, J.-F. (1994). Nucleic acid-binding properties of the P1 protein of turnip mosaic potyvirus produced in Escherichia coli. Journal of General Virology, 75(10), 2567-2573. doi:10.1099/0022-1317-75-10-2567Suehiro, N., Natsuaki, T., Watanabe, T., & Okuda, S. (2004). An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein. Journal of General Virology, 85(7), 2087-2098. doi:10.1099/vir.0.79825-0Valli, A., Martín-Hernández, A. M., López-Moya, J. J., & García, J. A. (2006). RNA Silencing Suppression by a Second Copy of the P1 Serine Protease ofCucumber Vein Yellowing Ipomovirus, a Member of the FamilyPotyviridaeThat Lacks the Cysteine Protease HCPro. Journal of Virology, 80(20), 10055-10063. doi:10.1128/jvi.00985-06Valli, A., López-Moya, J. J., & García, J. A. (2007). Recombination and gene duplication in the evolutionary diversification of P1 proteins in the family Potyviridae. Journal of General Virology, 88(3), 1016-1028. doi:10.1099/vir.0.82402-0Vancanneyt, G., Schmidt, R., O’Connor-Sanchez, A., Willmitzer, L., & Rocha-Sosa, M. (1990). Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Molecular and General Genetics MGG, 220(2), 245-250. doi:10.1007/bf00260489Verchot, J., & Carrington, J. C. (1995). Debilitation of plant potyvirus infectivity by P1 proteinase-inactivating mutations and restoration by second-site modifications. Journal of Virology, 69(3), 1582-1590. doi:10.1128/jvi.69.3.1582-1590.1995Verchot, J., & Carrington, J. C. (1995). Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification. Journal of Virology, 69(6), 3668-3674. doi:10.1128/jvi.69.6.3668-3674.1995Verchot, J., Koonin, E. V., & Carrington, J. C. (1991). The 35-kDa protein from the N-terminus of the potyviral polyprotein functions as a third virus-encoded proteinase. Virology, 185(2), 527-535. doi:10.1016/0042-6822(91)90522-

Virus variants with differences in the p1 protein coexist in a plum pox virus population and display particular host-dependent pathogenicity features

Subisolates segregated from an M-type Plum pox virus (PPV) isolate, PPV-PS, differ widely in pathogenicity despite their high degree of sequence similarity. A single amino acid substitution, K109E, in the helper component proteinase (HCPro) protein of PPV caused a significant enhancement of symptom severity in herbaceous hosts, and notably modified virus infectivity in peach seedlings. The presence of this substitution in certain subisolates that induced mild symptoms in herbaceous hosts and did not infect peach seedlings suggested the existence of uncharacterized attenuating factors in these subisolates. In this study, we show that two amino acid changes in the P1 protein are specifically associated with the mild pathogenicity exhibited by some PS subisolates. Site-directed mutagenesis studies demonstrated that both substitutions, W29R and V139E, but especially W29R, resulted in lower levels of virus accumulation and symptom severity in a woody host, Prunus persica. Furthermore, when W29R and V139E mutations were expressed concomitantly, PPV infectivity was completely abolished in this host. In contrast, the V139E substitution, but not W29R, was found to be responsible for symptom attenuation in herbaceous hosts. Deep sequencing analysis demonstrated that the W29R and V139E heterogeneities already existed in the original PPV-PS isolate before its segregation in different subisolates by local lesion cloning. These results highlight the potential complexity of potyviral populations and the relevance of the P1 protein of potyviruses in pathogenesis and viral adaptation to the host. © 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.This work was supported by grants BIO2010-18541 from the Spanish Ministerio de Educación y Ciencia (MEC), SAL/0185/2006 from Comunidad de Madrid and KBBE-204429 from the European UnionPeer Reviewe