Modes of transmission of the Cherry leaf roll virus: genetic basis of seed transmissibility and investigation of possible arthropod vectors

• Antragstellerin: Professorin Dr. Carmen Büttner
• Fachliche Zuordnung: Pflanzenzüchtung, Pflanzenpathologie
• Förderung: Förderung von 2010 bis 2014
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 158907904

• Erstellungsjahr: 2014

Zusammenfassung der Projektergebnisse

We investigated protein-interactions involved in cell-to-cell and systemic movement of Cherry leaf roll virus (CLRV), a globally distributed plant virus with a wide host range especially in woody plant species which is transmitted in nature mainly via seed and pollen. The first full length sequence of a rhubarb-isolate of CLRV was determined throughout this project and the coding regions of the putative movement protein (MP) and coat protein (CP) of the virus were identified on RNA2 of the bipartite ss(+)RNA virus. Both viral proteins are essential factors contributing to tubule-guided cell-to-cell movement and systemic infection of plant tissue of CLRV particles. It was possible to raise specific antibodies against the CP as well as the MP enabling first localization studies of CLRV by tissue print. The virus was detectable in male as well as in female birch catkins in different developmental stages. It was most prevalent in seed producing female catkins in June supporting previous findings that the virus invades Betula spp. gametophytes as a prerequisite for effective transmission by seed. The CLRV-content in male pollen producing catkins was lower as estimated by tissue print. Yet we established a sensitive IC-RT-PCR for the detection of CLRV even in low amounts of pollen. Comparative sequence analyses of CLRV-MP and CLRV-CP including 13 different isolates representing different main phylogenetic/serological groups originating from various countries and host plants revealed that the CP is more conserved than the putative viral transport protein (MP). We applied the yeast-two hybrid (YTH) system to study these viral proteins responsible for systemic invasion of the plant by CLRV in more detail. Dimerization of the MP and the CP could be demonstrated as well as the specific binding of the CLRV-MP to the corresponding CP of the isolate. Heterodimerization of both virus proteins was confirmed by protein overlay blot employing the polyclonal CLRV- CP specific antibody developed in this study. On the contrary while MPs derived from different virus strains showed dimerization, no protein-protein interaction was found between MP and CP originating from different isolates. Together with results obtained by application of deletion constructs of the CLRV-MP in YTH system, functional domains were identified facilitating dimerization of the MP and specific recognition of the CP. The YTH assay was also employed to determine host proteins interacting with the viral proteins involved in virus transport, thus likely contributing to systemic infection of the model plant Arabidopsis thaliana. A single host factor denominated At-4/1 was identified throughout screening of a cDNA-library applying the full length CP as well as the putative MP of the rhubarb isolate of CLRV. At-4/1 showed specific binding to the putative CLRV- MP but did not interact with the viral CP. It has been demonstrated previously that the At- 4/1 protein is involved in cell-to-cell movement of Tomato spotted wilt virus by binding specifically to the MP of the virus. Also its orthologs from Nicotiana species have been shown to affect the systemic spread of Potato spindle tuber viroid indicating towards a conserved and fundamental mechanism of transport of pathogens within the plant. Results and developed tools within this project provide fundamentals for more detailed localization studies of the viral proteins associated with systemic virus transport in the host tissue. Follow up studies are required though, to corroborate the hypothesis that the interaction identified between the CLRV-MP and the plant factor At-4/1 is associated with fundamental transport processes of plant viruses employing the tubule-guided cell-to-cell movement for intercellular dispersal. Initial studies regarding alternative routes of CLRV dissemination by arthropod vectors were not further pursued throughout this project, but are currently being addressed in an independent DFG-project “Modes of vector transmission of Cherry leaf roll virus (CLRV) – molecular basis and potential arthropod vector species”.

Projektbezogene Publikationen (Auswahl)

• 2011. Analyse des Artenspektrums der Ordnung Hemiptera an Betula pendula (Roth.) und Nachweis von Cherry leaf roll virus (CLRV) in potentiellen Vektoren. In: Dujesiefken, D. (Ed.), Jahrbuch der Baumpflege, Haymarket Media, Braunschweig, 215-221
  Bandte M, Schuster AK, von Bargen S, Büttner C
  
• 2011. Chapter 24: Cherry leaf roll virus. In: Virus and Virus-Like Diseases of Pome and Stone Fruits (Eds. Hadidi A, Barba M, Candresse T, Jelkmann W). APS PRESS, St. Paul, USA, 119-125
  Büttner C, von Bargen S, Bandte M, Myrta A
  
• 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
  
• 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 Rom, Italy. Petria 22, 303
  von Bargen S, Dierker L, Rott M, Langer J, Büttner C
  
• 2012. Identifizierung von Protein-Protein- Interaktionen im Wirt-Pathogen-System Arabidopsis thaliana/Cherry leaf roll virus. 58. Deutsche Pflanzenschutztagung 10.-14.9. in Braunschweig. Abstract erschienen in: Julius-Kühn Archiv 438, p. 225
  Dierker L, von Bargen S, Büttner C
  
• 2013. chapter 3: Forest diseases caused by viruses. In: Infectious forest diseases. Gonthier P, Nicolotti G (eds), CABI, 50-75
  Büttner C, von Bargen S, Bandte M, Mühlbach HP
  
• 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
  von Bargen S, Langer J, Büttner C
  
• 2013. Identification of protein-protein-Interactions in the host-pathogen-system Arabidopsis thaliana/Cherry leaf roll virus (CLRV). In: Schneider C, Leifert C, Feldmann F (Eds), Endophytes for plant protection: the state of the art, pp. 309-310. Deutsche Phytomedizinische Gesellschaft, Braunschweig
  Dierker L, von Bargen S, Büttner C
  
• 2013. Lokalisation des Cherry leaf roll virus (CLRV) in Blütenständen der Hänge-Birke (Betula pendula) 44. Tagung des DPG-Arbeitskreises „Virologie der Pflanzen“, 14.-15. Oktober 2013 in Braunschweig, Germany
  Dierker L, von Bargen S, Büttner C
  
• 2014: Charakterisierung der Transportproteinkodierenden Region des Cherry leaf roll virus (CLRV). 59. Deutsche Pflanzenschutztagung, 23.-26.9. in Freiburg. Julius-Kühn Archiv 447, p. 287
  Dierker L, von Bargen S, Büttner C
  
• 2014: Identification of protein interactions of the putative movement protein of Cherry leaf roll virus (CLRV). The 5th Asian conference on plant pathology, 3.-6. November in Chiang Mai, Thailand. Book of abstracts, p. 20
  Dierker L, von Bargen S, Büttner C
  
• 2015. Interaction studies of the Cherry leaf roll virus (CLRV)-encoded proteins involved in intercellular VIRUS MOVEMENT IN HOST PLANTS. ISHS Acta Horticulturae 1099: II International Symposium on Horticulture in Europe, 681-686
  Dierker L, von Bargen S, Büttner C