Potato mop top virus, a soilborne virus transmitted by Spongospora subterranea: distribution, problems and possible solutions

Transcription

1 Potato mop top virus, a soilborne virus transmitted by Spongospora subterranea: distribution, problems and possible solutions Jari Valkonen Department of Agricultural Sciences, University of Helsinki FINLAND

2 CONTENTS 1.Etiology and symptoms 2.Geographical distribtion 3.Diagnostics 4.Resistance

3 1. ETIOLOGY and SYMPTOMS

4 Photos taken by Hanne Grethe Kirk in Norway mop-top symptoms Oak leaf pattern (chlorosis) in leaves Foliar symptoms caused by PMTV are rear in Scandinavia, except in Norway. Foliar symptoms occur in Scotland.

5 Potato mop-top virus (PMTV) Infects roots and tubers, but only rarely the green parts of the plant Causes necrotic rings and arcs inside tubers and on tuber surface

6 Other causal agents of spraing symptoms PMTV Tobacco rattle virus (TRV) (nematode-transmitted) TRV Potato virus Y (PVY) (aphid-transmitted)

7 Saturna is a PMTV-sensitive cultivar, but resistant to TRV Therefore, the spraing symptoms observed in Saturna are likely caused by PMTV.

8 Symptoms in tubers

9 Immunostaining with antibodies to PMTV coat protein Infected Healthy (necrotic arcs pointed out by arrows) Germundsson et al. (2002) J. Gen. Virol. 83:

10 Detection of PMTV based on symptoms can be highly unreliable! Latvala-Kilby et al. 2009, Phytopathology 99:

11

12 Potato mop-top virus (PMTV) Transmitted by zoospores, and retained infective in the resting spores, of the soilborne microbe Spongospora subterranea PMTV-infected small root PMTV virions carried inside zoospores Sporangia of S. subterranea (AAB Descr. no. 389) Resting spores in soil maintain PMTV >10 years

13 NATURAL RESERVOIRS OF PMTV Black nightshade (Solanum nigrum) (Photo: Steen Lykke Nielsen) PMTV was detected in the roots of 19 % (n = 222) of the black nightshade plants growing at edges of potato fields in Denmark

14 EFFICIENT SAMPLING for SURVEYING FIELDS for PMTV Sporangia of S.s. are concentrated in the soil on tubers Bait plant roots tested for PMTV Sampling of soil falling under the potato grading machine

15 2. GEOGRAPHICAL DISTRIBUTION

16 Distribution of Potato mop-top virus as known in 2005 E.L. Calvert & B.D. Harrison 1966 R.A.C. Jones, J.I. Cooper 1960s s s 2002 South America Hinostrava & French 1972; Salazar & Jones 1975

17 Survey of PMTV in the Baltic Sea region Enhanced control of Potato mop-top virus in the Nordic and Baltic Sea region GOALS: Defined knowledge on geographic distribution of PMTV - Study on genetic variability (all countries where PMTV is found) Improved diagnostic methods for PMTV Improved resistance to PMTV in potatoes

18 Enhanced control of Potato mop-top virus in the Nordic and Baltic Sea region MEANS: i) Utilisation of the latest biotechnological methods and molecular tools ii) Research training iii) Platform for institutional collaboration within the Baltic Sea region (universities, research institutes and private enterprises) Annual project meetings

19 Funding arrangements NKJ AB-RMS Agrobiotechnology focused on root-microbe systems ScanBalt Roles of donors: NKJ research activities ( ) ScanBalt workshops and training ( ) NOVA education of doctoral students ( )

20 MOP-TOP: Network of research institutions FINLAND - University of Helsinki (Co-ordinator)* - AgriFood Research Finland SWEDEN Swedish University of Agric. Sciences (SLU) NORWAY - Norwegian Crop Res. Institute - Norwegian Food Safety Authority DENMARK - Danish Institute of Agricultural Sciences* - Danish Potato Breeding Foundation GERMANY University of Hamburg* University of Rostock* RUSSIA - All-Russia Res. Institute for Agric. Microbiology* -State Research Concern North-Western Research Insitute of Agriculture -All-Russia Research Institute for Plant Protection ESTONIA -Estonian Agricultural University* -Estonian Plant Production Inspectorate -Agricultural Research Centre -Tallinn University of Technology LATVIA -Latvia University of Agriculture* -Latvian State Plant Protection Service LITHUANIA -Ministry of Agriculture, State Seed and Grain Service* -Institute of Botany -State Plant Protection Service POLAND -Plant Breeding and Acclimatization Institute (IHAR)* -Plant Protection Institute (IOR)

21 MOP-TOP: Private sector Seed producers Pohjoisen Kantaperuna Ltd. Finland, Sweden, Russia Food industry Breeders Graminor (BASF, Svalöf Weibull AB) Norway, Germany, Sweden (Kesko) DANESPO Denmark Chips AB Finland, Sweden, Norway, Denmark, Lithuania, Latvia, Estonia, Russia Kraft Foods Estrella/Maarud Agro Group Norway, Sweden, Finland, Denmark, Estonia A/S Latfood Latvia Agrochemical industry Kemira Grow-How Finland, Denmark Estonia, Lithuania, Poland

22 Occurrence of PMTV, as known at the end of 2008.

23 Distribution of PMTV in Denmark Red: PMTV detected. Yellow: PMTV not detected Data: Steen Lykke Nielsen

24 Final project meeting in Helsinki, February 17-18, 2009 Santala et al Annals of Applied Biology 157:

25 Occurrence of PMTV as known in 2016 Europe since 1960s USA & Canada 2004 Japan since 1980s China 2013 Pakistan 2013 Costa Rica 2008 Colombia 2013* Chile 2016 *a new pomo-like virus infecting potatos

26 3. DIAGNOSTICS

27 Serological detection (ELISA) of PMTV in potato Would it be possible to test PMTV in sprouts rather than the tuber? Sprouts grown in dark

28 Sprout sap interferes less than tuber sap in ELISA Detection with PMTV CP-specific monoclonal antibodies Latvala-Kilby et al. 2009, Phytopathology 99:

29 Tuber Sprout Detection threshold Latvala-Kilby et al. 2009, Phytopathology 99:

30 Tuber Sprout Detection threshold Latvala-Kilby et al. 2009, Phytopathology 99:

31 Dilution of sprout sap / Pooling of samples Latvala-Kilby et al. 2009, Phytopathology 99:

32 Detection by PCR - Genetic variability of PMTV? Main functions of proteins encoded by the three viral (+)ssrnas Multiplication RNA 1 MetT Hel Pol -148K - RT 206K - Val kb Vector transmission RNA 2 -CP - RT 91K - 20K Val kb Movement TGB RNA 3 51K 21K 13K 8K Val kb 1.0 kb 2.0 kb 3.0 kb 4.0 kb 5.0 kb 6.0 kb Savenkov et al Sandgren et al. 2001

33 Phylogenetic analysis of CP and RT domain (RNA 2) RNA2 CP RNA2 RT BBNV PMTV-S BBNV Latvala-Kilby et al Phytopathology 99:

34 Beuch et al Arch Virol (2015) 160: RNA2 CP RNA3 8K

35 Hu et al Can. J. Plant Pathol. 38:

36 TGBp1 (RNA1) CP (RNA2) TGBp1 (RNA3) Gil et al Arch Virol 161:

37 SUMMARY/Genetic variability: Two types (genetic lineages) of PMTV RNA2 and RNA3: their all four different combinations have been detected in tubers but biological differences, if any, remain to be shown. Within each of the three PMTV RNAs genetic variability is limited. Two newly found PMTV-like viruses infect potatoes in Colombia

38 SUMMARY (1) 1.Foliar symptoms caused by PMTV are sporadic and irregularly observed. 2.Symptomless infections in tubers are very common, both in the field and in storage. Virus-specific testing of tubers for PMTV is necessary (like with other potato viruses): DAS-ELISA (MAbs), RT-PCR or IC-RT-PCR. 3.Soil testing for PMTV can be enhanced by collecting soil under potato grading machines and testing samples using bait plants bait plant roots will be tested for PMTV. 4.Viruliferous sporangia in the soil on seed tubers may be the most important means by which PMTV spreads to new areas. Transmission of PMTV from the seed tuber to progeny tubers via stolons seems less efficient than S.s.-mediated transmission by zoospores. 5. It will be an important goal to prevent spread of PMTV to the PMTV-free areas.

39 SUMMARY (2) 6. All potato cultivars are susceptible to PMTV but their tolerance varies, which is observed as differences in the proportion of infected tubers developing symptoms. Symptom expression varies from year to year and is unpredictable. 7. True resistance to PMTV in potato cultivars will be the only sustainable solution to problems caused by the spraing disease. Potato germplasm should be screened for resistance and suitable accessions included in breeding programmes. Genetic engineering of cultivars for PMTV resistance could be used, too. 8. The limited genetic variability of PMTV implies that resistance breeding should provide an efficient means to control PMTV.

40 SUMMARY (2) 6. All potato cultivars are susceptible to PMTV but their tolerance varies, which is observed as differences in the proportion of infected tubers developing symptoms. Symptom expression varies from year to year and is unpredictable. 7. True resistance to PMTV in potato cultivars will be the only sustainable solution to problems caused by the spraing disease. Potato germplasm should be screened for resistance and suitable accessions included in breeding programmes. Genetic engineering of cultivars for PMTV resistance could be used, too. 8. The limited genetic variability of PMTV implies that resistance breeding should provide an efficient means to control PMTV. COULD WE MANIPULATE PLANT FACTORS (GENES) REQUIRED BY PMTV FOR EFFICIENT INFECTION OF POTATO PLANTS, CONVERT SUSCEPTIBILITY FACTORS TO RESISTANCE FACTORS?

41 Coordinated functions of TGB proteins RNA 2 p1 TGB p2 p3 8K Val GFP GFP-p1 GFP-p1 + p2 + p3 10 µm 10 µm 20 µm Model: TGBp3 and TGBp2 of PMTV mediate the transport of a TGBp1- containing ribonucleoprotein complex to and through the plasmodesmata, whereas TGBp2 and TGBp3 are not transported to the adjacent cell. (Zamyatnin et al. 2004, MPMI 17: )

42 TGB proteins must be expressed at suitable relative molarities for viral movement to occur. TGBp3 should be expressed at 10-fold lower levels than TGBp2, and 100-fold lower levels than TGBp1 (Tilsner et al. 2010). Careful regulation of expression levels seems pivotal. Could we interfere with the regulation of viral functions, e.g., by affecting plant factors possibly needed in the regulation? Tool for the study: infectious clone of PMTV Infectious PMTV was generated using transcripts of the full-length cdna clones of RNA1, RNA2 and RNA3. Savenkov et al. (2003) J. Gen. Virol. 84,

43 Protein phosphorylation is a typical mechanism to regulate functions Is TGBp3 phosphorylated? The putative phosphotyrosine sites in PMTV TGBp3 were predicted using NetPhos 2 and Scansite. p3 detected with anti-py antibodies in a PMTV-infected plant Infectious clone of PMTV was engineered to express Myc-tagged TGBp3 Tyrosine-phosphorylated higher molecular weight host proteins were also detected.

44 Multiplication and/or cell-to-cell movement of PMTV is inhibited, when tyrosine residues of TGBp3 are substituted for alanine A. Detection of PMTV in inoculated (I) leaves (cell-to-cell movement) and upper leaves (S) following long-distance movement, by RT-PCR shows that both functions are affected in all mutant viruses. Days postinoculation (P.S. No infectivity at all was observed with virus mutants with Tyr120 substituted for alanine.) B. Confocal microscopy of N. benthamiana leaves inoculated with PMTV that express GFP fused to TGBp1. - GFP-PMTV expresses wild-type TGBp3 (large number of cells infected by cell-to-cell movement). - GFP-PMTV21K 87 89A : only single cells infected, no cell-to-cell movement. (GFP-PMTV21K 120A : no infection detected) 100 µm 100 µm Samuilova et al. 2013, J. Virol. 87:

45 Future prospects: Proteins of many mammalian viruses are phosphorylated by tyrosine kinases, and kinase inhibitors can increase survival of the host by reducing viral load and dissemination of the virus to distal tissues. It is conceivable that inhibition of tyrosine kinases required for phosphorylation of viral proteins could have a role to play in combating viral infections also in plants.

46 Thank you! The Viikki Life Science Campus of University of Helsinki, Finland

47 Savenkov, E.I., Sandgren, M. & Valkonen, J.P.T Complete sequence of RNA 1 and presence of trna-like structures in all RNAs of Potato mop-top pomovirus. Journal of General Virology 80: Sandgren, M., Savenkov, E.I. & Valkonen, J.P.T The readthrough region of Potato mop-top virus (PMTV) coat protein encoding RNA, the second largest RNA of PMTV genome, undergoes structural changes in naturally infected and experimentally inoculated plants. Archives of Virology 146: Germundsson, A., Sandgren, M., Barker, H., Savenkov, E.I. & Valkonen, J.P.T Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus. Journal of General Virology 83: Sandgren, M., Plaisted, R.L., Watanabe, K.N., Olsson, S. & Valkonen, J.P.T Evaluation of some North and South American potato breeding lines for resistance to Potato mop-top virus in Sweden. American Journal of Potato Research 79: Savenkov, E.I., Germundsson, A., Zamyatnin, A.A. Jr., Sandgren, M. & Valkonen, J.P.T Potato mop-top virus coat protein-encoding RNA and the gene for cystein-rich protein are dispensable for systemic virus movement in Nicotiana benthamiana. Journal of General Virology 84: Zamyatnin, A.A., Solovyev, A.G., Savenkov, E.I., Germundsson, A., Sandgren, M., Valkonen, J.P.T. & Morozov, S.Yu Transient coexpression of individual genes encoded by the triple gene block (TGB) of Potato mop-top virus reveals requirements for TGBp1 trafficking. Molecular Plant-Microbe Interactions 17: Lukhovitskaya, N.I., Yelina, N.E., Zamyatnin Jr. A.A., Schepetilnikov, M.V., Solovyev, A.G., Sandgren, M., Morozov, S.Yu., Valkonen, J.P.T. & Savenkov, E.I Expression, localization and effects on virulence of the cysteine-rich 8-kDa protein of Potato mop-top virus. Journal of General Virology 86: Latvala-Kilby, S., Aura, J.M., Pupola, N., Hannukkala, A. & Valkonen, J.P.T Detection of Potato mop-top virus in potato tubers and sprouts: combinations of RNA2 and RNA3 variants and incidence of symptomless infections. Phytopathology 99: Santala, J., Samuilova, O., Hannukkala, A., Latvala, S., Kortemaa, H., Beuch, U., Kvarnheden, A., Persson, P., Topp, K., Ørstad, K., Spetz, C., Nielsen, S.L., Kirk, H.G., Uth, J.G., Budziszewska, M., Wieczorek, P., Obrepalska-Steplowska, A., Pospieszny, H., Kryszczuk, A., Sztangret-Wisniewska, J., Yin, Z., Chrzanowska, M., Zimnoch-Guzowska, E., Jackeviciene, E., Taluntytė, L., Pūpola, N., Mihailova, J., Lielmane, I., Järvekülg, L., Kotkas, K., Rogozina, E., Sozonov, A., Tikhonovich, I., Horn, P., Broer, I., Kuusiene, S., Staniulis, J., Adam, G. & Valkonen, J.P.T Detection, distribution and control of Potato moptop virus, a soil-borne virus, in northern Europe. Annals of Applied Biology 157: Samuilova, O., Santala, J. & Valkonen, J.P.T Tyrosine phosphorylation of the triple gene block protein 3 regulates cell-to-cell movement and protein interactions of Potato mop top virus. Journal of Virology 87: Beuch, U., Berlin, S., Åkerblom, J., Nicolaisen, M., Nielsen, S.L., Crosslin, J.M., Hamm, P.B., Santala, J., Valkonen, J.P.T. & Kvarnheden, A Diversity and evolution of Potato mop-top virus. Archives of Virology 160: