Functional genomics of Cherry leaf roll virus (CLRV) and molecular analyses of Finnish virus variants
Zusammenfassung der Projektergebnisse
Cherry leaf roll virus (CLRV) belongs to subgroup C of the genus nepovirus within the family Secoviridae of the order Picornavirales. It has a bipartite, positively orientated single-stranded (ss+) RNA genome. In the first project (van Bargen), we could successfully characterize the full length sequence of RNA1 and RNA 2 in silico. The overall genome structure resembles that of other subgroup C nepoviruses such as the representative member Tomato ringspot virus (ToRSV) regarding RNA1, while RNA2 displays several differences. By heterologous expression and purification, the proposed coding region of the proteinase (Pro) on RNA1 could be experimentally verified, and the proteolytic functionality of the VPg-Pro and Pro could be demonstrated by activity assays in vitro. Cleavage sites were predicted computationally. The proteolytic processing for two processing sites of RNA2-encoded polyprotein P2 was proven experimentally, revealing the mature movement protein (MP) and coat protein (CP), verifying their proposed size and localization on the genome. Cleavage by the RNA1-encoded proteinase (VPg-Pro, Pro) between the proposed X3 and X4 proteins encoded N terminally by RNA2 could not be proven to occur in in vitro activity assay. Even by use of the in vitro translation complete X3/X4 coding region as substrate no processing site of the CLRV-E395 proteinase could be identified. Therefore, further studies are advisable to investigate, whether additional factors may be required to assist the CLRV-proteinase in the processing of the N- terminal region of P2 in trans. We could further demonstrate that certain CLRV-variants exhibit putative alternative start codons capable of serving as translation initiation sites in vitro. CLRV RNA2 displays notable differences compared to related subgroup C nepoviruses, especially concerning the 5´ terminal region. The predicted N-terminal proteins do not share similarities to the corresponding regions of related viruses such as ToRSV. Especially the region encoding the putative X4 protein shows differences leading to size and amino acid sequence variation of different CLRV isolates. This indicates that this genomic region is characteristic for certain strains of the virus derived from cherry or birch denominated as group A variants. A viral epidemic associated with CLRV has emerged in Betula species in Fennoscandia exhibiting quick and effective spread during the last 15 years. The “birch leafroll disease” could be linked with a CLRV infection, impairs several important birch species such as B. pendula and B. pubescens and it was shown that atypical CLRV-variants may contribute to the observed disease. In project Büttner, we investigated these CLRV-variants and compared them to characteristic CLRV-isolates derived from diseased B. pendula in Germany by transmission experiments through grafting. It could be demonstrated that the CLRV variants from Finnish accessions were not transmittable to herbaceous hosts but induced typical symptoms of the “leaf roll disease” in grafted B. pubescens rootstocks. Further, a population genetics approach was chosen to characterize the virus diversity and the sources of genetic variation aiming to investigate the epidemiology of the pathogen. In a CLRV population from Rovaniemi urban parks and a population that occurred after infecting young Betula seedlings with scions from the original Finnish trees the genetic diversity is found to be remarkably high and infections by several CLRV variants from different phylogenetic groups were detected in several individual trees. The estimated genetic variability is high and the CLRV haplotypes detected exhibit clear clustering and belong to different phylogenetic groups. The structure of the viral population revealed a pathogen with high evolutionary potential assumed to carry on its effective spread in Finland. In the frame of this project additional evidence accumulated from a isolated stand in Corsica, that CLRV population diversity in birches is higher than previously anticipated. By comparative NGS analyses in combination with RT-PCR it could be shown that additional viruses were present in leaf roll diseased birches (B. pubescens and B. pendula). However, contribution of the identified novel DNA, ssRNA and dsRNA viruses related to Badna-, Carla-, Endorna- and Totiviruses to the observed symptoms leading to decline of birches over time needs to be investigated in more detail in the future.
Projektbezogene Publikationen (Auswahl)
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(2012): Complete nucleotide sequence of Cherry leaf roll virus (CLRV), a subgroup C nepovirus. Virus Research 163, 678-683
von Bargen, S., Langer, J., Robel, J., Rumbou, A., Büttner, C.
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2012. Genome organization of Cherry leaf roll virus and analyses of functions of virus-encoded proteins. 22nd International Conference on Viruses and Other Graft Transmissible Diseases of Fruit Crops (ICVF) 3.-8.6.2012 in Rome, Italy. Petria 22, 303
von Bargen S, Dierker L, Rott M, Langer J, Büttner C
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(2013): Transmission of Cherry leaf roll virus (CLRV) variants from German and Finnish birches by grafting. In: Schneider C, Leifert C, Feldmann F (Eds), Endophytes for plant protection: the state of the art, pp. 326-327. Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany
Breuhahn M, von Bargen, S., Jalkanen, R., Büttner, C.
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2013. Genome organization of Cherry leaf roll virus and comparative analyses of RNA2-encoded proteins. In: Schneider C, Leifert C, Feldmann F (Eds), Endophytes for plant protection: the state of the art, pp. 307- 308. Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany
von Bargen S, Langer J, Büttner C
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2013. Heterologous expression of the viral proteinase of Cherry leaf roll virus (CLRV). In: Schneider C, Leifert C, Feldmann F (Eds), Endophytes for plant protection: the state of the art, pp. 311-312. Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany
Rott M, Büttner C, von Bargen S
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2013. Heterologous expression of the viral proteinase of Cherry leaf roll virus (CLRV). In: Schneider C, Leifert C, Feldmann F (Eds), Endophytes for plant protection: the state of the art, pp. 311-312. Deutsche Phytomedizinische Gesellschaft, Braunschweig, Germany
Rott M, Büttner C, von Bargen S
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(2014): chapter 15: Molecular methods for detection of plant pathogens in irrigation water. In: Biology, detection and management of plant pathogens in irrigation water (Eds. Hong, C., Moorman, G. W., Wohanka, W., Büttner, C.). APS Press, St. Paul, USA, 161-174
von Bargen, S.
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2014. Cherry leaf roll virus in Betula sp. in Finland: what do we know about its population diversity? 59. Deutsche Pflanzenschutztagung, 23.-26.9. in Freiburg, Germany. Julius-Kühn Archiv 447, p. 516-517
Rumbou A, von Bargen S, Rott M, Jalkanen R, Büttner C
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2014. Funktionelle Charakterisierung der viralen Proteinase des Cherry leaf roll virus (CLRV). 59. Deutsche Pflanzenschutztagung, 23.-26.9. in Freiburg, Germany. Julius-Kühn Archiv 447, p. 286
Rott M, Büttner C, von Bargen S
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2015. Current impact and future directions of high throughput sequencing in plant virus diagnostics: the drivers of COST Action 1407. 18th International Plant Protection Congress, 24.-27.8.2015 in Berlin, Germany. Book of abstracts, p. 594-595
Wetzel T, Büttner C, von Bargen S, Rumbou A, Olmos A, Boonham N, Candresse T, Felix R, Font I, Glasa M, Jalkanen R, Kominek P, Laimer M, Malinowski T, Maliogka V, Minafra A, Ortega Parra N, Poliverari A, Ravnikar M, Safarova D, Vandervlugt R, Varveri C, Witzell J, Zagrai I, Massart S
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2015. Establishment of an in-vitro assay for functional characterization of the viral proteinase and processing of RNA2-encoded polyprotein P2 of Cherry leaf roll virus (CLRV). 18th International Plant Protection Congress, 24.-27.8.2015 in Berlin, Germany. Book of abstracts, p.12-13
Rott M, Büttner C, von Bargen S
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2015. The `birch-leafroll disease´ emerging in forests and urban parks in Fennoscandia - Viral agents associated with the disease. 18th International Plant Protection Congress, 24.-27.8.2015 in Berlin, Germany. Book of abstracts, p. 46
Rumbou A, von Bargen S, Jalkanen R, Büttner C
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2015. Translation initiation studies of the polyproteins encoded by RNA1 and RNA2 of Cherry leaf roll virus. 18th International Plant Protection Congress, 24.-27.8.2015 in Berlin, Germany. Book of abstracts, p. 350
Breuhahn M, von Bargen S, Rott M, Langer J, Büttner C
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2015. Virus discovery using NGS in trees from urban/forest ecosystems. 1st conference of the COST action FA1407 DIVAS, 16- 18.11. in Ljubljana, Slowenia
Rumbou A, von Bargen S, Büttner C
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(2016): Comparison of diagnostic techniques for the detection and differentiation of Cherry leaf roll virus strains for quarantine purposes. Journal of Virological Methods. Volume 234, 142–151
Lebas, B.S.M., Veerakone, S., Liefting L.W., Tang, J., Perez-Egusquiza, Z., von Bargen, S., Ward., L.
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(2016): High genetic diversity at the inter-/intra host level of Cherry leaf roll virus population associated with the “birch leaf-roll disease” in Fennoscandia. Scandinavian Journal of Forest Research 31, 546-560
Rumbou, A., von Bargen, S., Demiral R, Langer, J., Rott, M., Jalkanen, R., Büttner, C.
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(2016): High genetic variation in a small population of Cherry leaf roll virus in Betula sp. of montane origin in Corsica. Forest Pathology
Langer, J., Rumbou, A., Fauter, A., von Bargen, S., Büttner, C.
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2016: Viruses associated with declining deciduous tree species. International Advances in Plant Virology in conjunction with COST Action FA1407, 7.-9. Sep., Greenwich, UK
von Bargen S, Bandte M, Rumbou A, Wetzel T, Candresse T, Marais-Colombel A, Faure C, Landgraf M, Langer J, Roßbach J, Rott M, Büttner C
