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Department of Agricultural SciencesFaculty of Agriculture and Forestry

andDoctoral Program in Sustainable Use of Renewable Natural Resources (AGFOREE)

University of Helsinki

Molecular variability, genetic relatedness and a novelopen reading frame (pispo) of sweet potato-infecting

potyviruses

Milton Untiveros Lazaro

ACADEMIC DISSERTATION

To be presented, with the permission of the Faculty of Agriculture and Forestry ofthe University of Helsinki, for public criticism in lecture room B2, B-building,

Latokartanonkaari 9, Viikki, on 15th June 2015, at 12 noon.

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Supervisors: Docent Dr. Jan F. KreuzeDepartment of Agricultural SciencesUniversity of Helsinki, FinlandScience Leader in Virology, Bacteriology and DiagnosisInternational Potato Center (CIP)

Professor Dr. Jari P. T. ValkonenDepartment of Agricultural SciencesUniversity of Helsinki, Finland

Reviewers: Docent Dr. Minna PoranenDepartment of BiosciencesUniversity of Helsinki, Finland

Professor Dr. Anders KvarnhedenDepartment of Plant Biology and Forest GeneticsThe Swedish University of Agricultural Sciences (SLU),Sweden

Opponent: Professor Dr. John CarrDepartment of Plant SciencesUniversity of Cambridge, UK

Custos: Professor Dr. Jari P. T. ValkonenDepartment of Agricultural SciencesUniversity of Helsinki, Finland

Cover: Nicothiana benthamiana 16c leaf under ultravioletlight in a RNA silencing assay.

ISSN 2342-5423 (Print), 2342-5431 (Online)ISBN 978-951-51-1249-1 (paperback)ISBN 978-951-51-1250-7 (PDF)http://ethesis.helsinki.fiHansaprintHelsinki 2015

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Life has only one orientation: 5’---->3’

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CONTENTS

CONTENTS 4

LIST OF ABBREVIATIONS 6

LIST OF ORIGINAL PUBLICATIONS INCLUDED IN THE THESIS 9

THESIS AT A GLANCE 10

ABSTRACT 11

1. INTRODUCTION 13

1.1 Potyviruses 131.1.1 Genomic structure 131.1.2 Protein functions 141.1.3 Recombination as a means of evolution 151.1.4 Taxonomy 16

1.2 Sweet potato viruses 171.2.1 Sweet potato feathery mottle virus (SPFMV) 181.2.2 Other potyvirus infecting sweet potato 191.2.3 Sweet potato virus disease and other synergisms 20

1.3 RNA interference (RNAi) 211.3.1 Overview 211.3.2 Spread of RNAi in plants 231.3.3 Viral suppressors of RNAi 241.3.4 RNAi suppressors and their role in viral synergism 25

2. AIM OF THE STUDY 26

3. MATERIALS AND METHODS 27

4. RESULTS AND DISCUSSION 28

4.1 Relatedness and geographical distribution of potyviruses infecting 28sweet potato

4.1.1 Re-classification of SPFMV strains 28

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4.1.2 New insights on the distribution of potyviruses infecting 28 sweet potato

4.1.3 The “SPFMV-group” of potyviruses 29

4.2 New genomic organization in the “SPFMV-group” of potyviruses 304.2.1 Intra- and inter-specific recombination events 304.2.2 The P1-N domain 314.2.3 A hyper variable region in P1 324.2.4 A new overlapping open reading frame of P1 encodes PISPO 32

4.3 RNAi suppressors in SPFMV 344.3.1 Novel role of P1 region of SPFMV among potyviruses 344.3.2 Suppression mediated by putative Ago-binding motifs 354.3.3 The role of viral HC-Pro protein in RNAi 35

4.4 An evolutionary model for sweet potato-infecting potyviruses 36

5. CONCLUSIONS AND PERSPECTIVES 38

6. SUPPLEMENTARY MATERIAL 41

ACKNOWLEDGEMENTS 46

REFERENCES 48

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LIST OF ABBREVIATIONS

2b 2b protein3’-end 3’ genomic region3’-proximal amino terminus5’-end 5’ genomic region5’-proximal carboxyl terminus6K1 6-kDa protein 16K2 6-kDa protein 2aa amino acidAGO argonaute proteinBYMV bean yellow mosaic virusCI cylindrical inclusionCMV cucumber mosaic virusCIP International Potato CenterCP coat proteindai days after infiltrationDAS ELISA double antibody sandwich enzyme-linked

immunosorbent assayDCL dicer-like proteinDCL4 dicer-like protein 4DNA deoxyribonucleic acidDRB4 double-stranded RNA binding protein 4eIF4E eukaryotic initiation factor 4EEA East AfricaGUS β-glucuronidaseGFP green florescen proteinGW motif Glycine/Trytophan Ago-binding motifsHC-Pro helper component proteinaseHEN1 Hua enhancer 1HMW RNA high molecular weight RNAhpRNA hairpin RNAkb kilobasekDa kilodaltonLMW RNA low molecular weight RNAMAb monoclonal antibodymRNA messenger RNANIa nuclear inclusion protein aNIb nuclear inclusion protein bnt nucleotideORF open reading frame

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PAb polyclonal antibodyPCR polymerase chain reactionpipo Pretty Interesting Potyviridae ORFpispo Pretty Interesting Sweet potato Potyvirus ORFPTGS post-transcriptional gene silencingPVA potato virus APVX potato virus XPVY potato virus YP0 protein 0P1 protein 1P1-N amino terminus of P1P1N P1-N terminus including the hypervariable regionP1N-PISPO P1N-termius PISPO fusion proteinP1-pro proteinase domain at P1P3 protein 3P3N-PIPO P3 N-terminus PIPO fusion proteinRdRp RNA-dependent RNA polymeraseRdRp6 RNA-dependent RNA polymerase 6RISC RNA-induced silencing complexRNA ribonucleic acidRNAse 3 endoribonuclease type IIIRNAi RNA interferenceRSS RNA silencing suppressionsRNA small RNAsiRNA small interfering RNAssRNA single-stranded RNASCMV sugar cane mosaic virusSGS3 suppressor-of-gene silencing 3SPCFV sweet potato chlorotic fleck virusSPCSV sweet potato chlorotic stunt virusSPFMV sweet potato feathery mottle virusSPLV sweet potato latent virusSPMSV sweet potato mild speckling virusSPV2 sweet potato virus 2SPVC sweet potato virus CSPVD sweet potato virus diseaseSPVG sweet potato virus GTAV cauliflower mosaic virus transactivator proteinVIGS viral-induced gene silencingTCV turnip crinkle mosaic virusTGB triple gene block proteinTGS transcriptional gene silencing

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TMV tobacco mosaic virusToMV tomato mosaic virusTuMV turnip mosaic virusUTR untranslated regionVPg viral genome-linked proteinYFP yellow florescent proteinZYMV zucchini yellow mosaic virus(+) ssRNA positive-sense single-stranded ribonucleic acid(-) ssRNA negative-sense single-stranded ribonucleic acid

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LIST OF ORIGINAL PUBLICATIONS INCLUDED IN THE THESIS

This work is based on the following publications and manuscripts which arereferred to in the text by their Roman numerals.

I Milton Untiveros, Segundo Fuentes and Jan Kreuze. 2008. Molecular variabilityof sweet potato feathery mottle virus and other potyviruses infecting sweetpotato in Peru. Arch Virol (2008) 153:473–483

II Milton Untiveros, Dora Quispe and Jan Kreuze. 2010. Analysis of completegenomic sequences of isolates of the Sweet potato feathery mottle virus strainsC and EA: molecular evidence for two distinct potyvirus species and two P1protein domains. Arch Virol (2010) 155:2059–2063

III Milton Untiveros, Allan Olspert, Katrin Artola, Andrew E. Firth, Jan F. Kreuze, andJari P. T. Valkonen. 2015. An overlapping ORF in sweet potato potyviruses isexpressed via transcriptional slippage and suppresses RNA silencing. Submitted

The published papers I and II are reproduced with permission from the publisher.

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THESIS AT A GLANCEI Molecular variability of Sweet potato feathery mottle virus and other potyviruses infecting sweet

potato in Peru.Objectives Identification of sweet potato-infecting potyviruses in Peru.Methods Field sampling, phylogenetic reconstructions, analysis and mapping of

recombination events.Ilustration

Main findings Isolates of SPFMV strain EA, SPVG and SPV2 first reported in South America.Detection of recombination events in potyviruses infecting sweet potato.Identification of the “SPFMV-group” of potyviruses.

III An overlapping ORF in sweet potato potyviruses is expressed via transcriptional slippage andsuppresses RNA silencing.

Objectives Study of the structure at the 5’-end of the SPFMV genome and its role in RNAisuppression.

Methods Computational analyses for detection of ORFs, siRNA assembling,Agrobacterium-mediated infiltration assays, Western blot, HMW/LMW RNAanalyses.

Ilustration

Main findings Pispo is a novel ORF limited to the members of the “SPFMV-group” and occursthrough transcriptional frameshifting. The predicted novel protein P1N-PISPOand P1 of SPFMV block RNAi at different levels.

II Analysis of complete genomic sequences of isolates of the Sweet potato feathery mottle virusstrains C and EA: molecular evidence for two distinct potyvirus species and two P1 protein domains.

Objectives Sequencing and analysis of the complete genome of strain C of SPFMV.Methods Viral genome sequencing, analysis of nucleotide and amino acid identity

indices, analysis and mapping of recombination events.Ilustration

Main findings Identification of Sweet potato virus C, a new potyvirus in sweet potato.Identification of P1-N domain in the genome of ‘SPFMV-group’ of potyviruses.

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ABSTRACT

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) infects sweetpotato wherever it is cultivated. In single infections, SPFMV often causes only mildsymptoms a situation that changes dramatically when it co-infects the plants withSweet potato chlorotic stunt virus (SPCSV, genus Crinivirus). Co-infection generatesthe sweet potato virus disease (SPVD), the most devastating viral disease of sweetpotato that can cause total loss of yield. Previous studies have described RNase3 asthe SPCSV protein responsible for the occurrence of SPVD. However, attempts todevelop resistance to SPVD based on this knowledge have failed, suggestingpossible SPFMV determinants involved in the development of SPVD. This studyaimed to contribute to the molecular understanding of SPFMV, in particularregarding its phylogenetic relationships, genomic structure, and ability to suppressRNA interference (RNAi).

Deployment of adequate control measures requires proper characterizationof the pathogen. Phylogenetic relationship of SPFMV isolates and closely relatedpotyviruses was ambiguous due to misidentification of viruses often found in mixedinfections, and lack of specific methods of detection. In this study, we sequencedthe complete genome of SPFMV strain C and found phylogenetic evidence tosupport its reclassification as a different species, Sweet potato virus C (SPVC).Furthermore, we demonstrated that Sweet potato feathery mottle virus and Sweetpotato virus C together with Sweet potato virus G (SPVG) and Sweet potato virus 2(SPV2) were found to cluster into one unique monophyletic subgroup among thepotyviruses, the so-called “SPFMV-group”.

Members of the “SPFMV-group” contain some unique genomic features.Based on pairwise comparisons of partial and complete genome sequences wefound recombinant isolates in SPFMV and SPVC. We also identified, novel viraldeterminants characteristic for the “SPFMV-group” of potyviruses, mainly in the P1cistron, a region known for its high variability among potyviruses. An additionaldomain at the N terminus of P1 (P1-N) which is not found in other potyvirus, but isfound in sweet potato mild mottle virus (genus Ipomovirus) is invariably found inmembers of the “SPFMV-group”. The P1-N domain is followed by a hypervariableregion which contains specific hallmarks for each member of the “SPFMV-group”,and is becoming a promising region for their rapid detection and characterization.However, perhaps the most remarkable finding was the identification of an extraopen reading frame (ORF) overlapping the C-terminal part of P1, and which wasdesignated as pispo (Pretty Interesting Sweet potato Potyvirus ORF). pispo istranslated as a result of a transcriptional slippage mechanism occurring in themembers of the “SPFMV-group” of potyviruses and results in a novel protein P1N-PISPO.

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Plants rely on RNAi, an antiviral defense machinery, to destroy viralribonucleic acid (RNA) molecules recognized inside cells, and spread the alert signalto neighboring cells. To evade RNAi, viruses encode proteins termed suppressors.Our analyses revealed that both P1 and P1N-PISPO contain RNAi suppressor (RSS)activity. Hence, SPFMV utilizes novel suppressors expressed from the same P1region in different reading frames. The protein P1N-PISPO suppresses cell-to-cellmovement of silencing, probably by blocking the spread of signaling to neighboringcells. In contrast, P1 protein suppresses silencing only locally. In both cases, aconserved Glycine/Tryptophan (GW) motif located in the P1N part of P1 plays acrucial role in the RSS activity. On the other hand, HC-Pro, a widely known RSSprotein of potyviruses was not able to suppress silencing in SPFMV. This particulararrangement, where the RSS activity resides on P1 region and not in HC-Pro, is notreported in potyviruses, but is known, e.g., in the members of the genus Ipomovirusof the Potyviridae family. Taxonomic, structural and functional findings of this studywill contribute greatly to the understanding of the evolution of SPFMV, andcharacterization of diseases it causes.

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1. INTRODUCTION

1.1 Potyviruses

The Potyvirus genus is the largest of the six genera in the Potyviridae familycontaining approximately 90% of its species. The other genera of Potyviridaeinclude Ipomovirus, Macluravirus, Rymovirus, Tritimovirus and Bymovirus.Potyviruses represent almost 30% of all plant viruses infecting angiosperms andmany of them are damaging crop pathogens (Adams et al., 2012). They aretransmitted by aphids, mainly species of Aphidinae, in a non-persistent manner.Virus particles attach to the stylet of the aphid during a short acquisition period andmust be transported to a new host soon (minutes to hours) as they remaininfective. Virions are flexuous filaments of 680-900 nm in length and 11-15 nm inwidth (Urcuqui-Inchima et al., 2001).

1.1.1 Genome structure

The potyviral genome consists of a monopartite single-stranded positive-sense RNA [(+)ssRNA] molecule of around 10 kb. The genome encodes a largepolyprotein of 340-370 kDa in a monocistronic way (Urcuqui-Inchima et al., 2001).The coding region is preceded by a 5’-end non-translated region covalently linkedwith a single molecule of the genome-linked viral protein (VPg) (Carrington andFreed, 1990). The 3’-end contains a polyadenosine (polyA) tail (Carrington andFreed, 1990). The polyprotein is autocatalytically processed to 10 mature proteins,including P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIaPro, NIb, and CP (Figure 1).Additionally, Chung et al. (2008) have reported the occurrence of a small secondORF named pipo (pretty interesting potyvirus ORF) in frame +2 relative to thepolyprotein-encoding ORF and overlapping with the P3 cistron (Chung et al., 2008).PIPO was found to be translated in the transframe manner P3N-PIPO, though themechanism of expression remains unknown.

Figure 1. Schematic presentation of the potyvirus genome. A polyprotein is produced and processedinto 10 mature proteins by three viral proteinases (see main text). A second ORF, pipo, overlappingthe P3 cistron produces a fusion protein P3N-PIPO. VPg is linked covalently to the 5’-end, while a Poly(A) tail is at the 3’-end of the viral RNA.

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1.1.2 Protein functions

Most potyviral proteins are multifunctional, and their roles in the infectioncycle have been determined in studies on several potyviruses. Frequently, data for aprotein of one potyvirus has been applied to other potyvirus species due to thehigh similarity of the genome structure in the genus. Most proteins are importantfor virus amplification and involved in symptom development (Kekarainen et al.,2002). P1 is the first protein produced from the N-terminus of the polyprotein andis the most variable among potyviruses in terms of sequence and structure. Itcontains a protease domain in its C-terminal part cleaving P1 from the polyprotein(Verchot et al., 1991). Additional functions include binding to ss- and ds-RNA(Soumounou and Laliberté, 1994), involvement in genome amplification (Verchotand Carrington, 1995), acting as an auxiliary factor in the RNAi suppression (RSS)activity of the viral helper component proteinase (HC-Pro) (Brigneti et al., 1998;Rajamäki et al., 2005), and probably in host adaptation (Salvador et al., 2008; Shi etal., 2007).

The next protein, HC-Pro, also cleaves itself from the rest of the polyproteinand is one of the most extensively studied potyviral proteins. It contains threefunctional domains: the N-proximal, central and C-proximal domain (Plisson et al.,2003). They are involved independently or jointly in at least five different functions:proteolytic cleavage of the polyprotein (C-proximal) (Carrington and Herndon,1992); aphid-mediated virus transmission (N-proximal and central domain) (Blancet al., 1998; Peng et al., 1998); RNA amplification (central domain) (Kasschau et al.,1997), systemic movement (central domain) (Cronin et al., 1995), and suppressionof RNAi (N-, and C- proximal, and central domain) (Torres-Barceló et al., 2008). Ithas been suggested that the contribution of HC-Pro to genome amplification andlong-distance movement is a consequence of its RSS activity (Kasschau andCarrington, 2001).

The protein P3 contains two hydrophobic domains located at the N- and C-termini (Eiamtanasate et al., 2007). Several studies have suggested its involvementin virus replication (Merits et al., 1999), pathogenicity (Chu et al., 1997), systemicinfection, and movement (Cui et al., 2010). The overlapping protein P3N-PIPOparticipates in the viral cell-to-cell movement with CI (cylindrical or cytoplasmicinclusion protein) that is associated with plasmodesmata (Wei et al., 2010). Thefollowing protein CI aggregates as laminate cytoplasmatic inclusion bodies(pinwheel-shaped) (Edwardson, 1992) and participates in cell-to-cell movement ofthe virus (Carrington et al., 1998) and virus replication (Fernández et al., 1997).

Functions of 6K1 are less known whereas 6K2 is an hydrophobic membraneprotein (Restrepo-Hartwig and Carrington, 1994) that anchors the viral replicationcomplex to the endoplasmatic reticulum playing a crucial role in virus replication(Schaad et al., 1997). 6K2 is also involved in viral long-distance movement andsymptom induction (Spetz and Valkonen, 2004).

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The subsequent NIa (nuclear inclusion protein a) protein possesses twodomains: VPg and NIa-Pro (Murphy et al., 1990). VPg is linked covalently to the 5’-end of the viral RNA via a tyrosine residue (Murphy et al., 1991). VPg interactsphysically with many viral and plant proteins, possibly due to its structural flexibilityand intrinsically disordered nature, and it shows functional diversity (Rantalainen etal., 2011). Depending on the kind of protein interactions, it is found in differentcellular compartments. The VPg–NIa-pro protein is found exclusively in thenucleolus, whereas the 6K2–VPg–NIa-pro complex is found within vesicularstructures derived from the endoplasmic reticulum (Jiang and Laliberté, 2011). VPgplays an important role in accumulation and loading of potyvirus into the phloemfor systemic infection (Rajamäki and Valkonen, 2002). In addition, VPg binds thehost eukaryotic initiation factor 4A (eIF4E) and the viral CI to form the translationinitiation complex (Robaglia and Caranta, 2006; Tavert-Roudet et al., 2012). VPg hasbeen observed to play a role in RSS (Rajamäki and Valkonen, 2009). The proteasedomain NIa-Pro cleaves most of the proteins from the precursor polyprotein(Carrington and Dougherty, 1987).

The NIb (nuclear inclusion protein b) is the RNA-dependent RNA polymerase(RdRp) involved in replication of the viral RNA (Hong and Hunt, 1996). The coatprotein (CP) consists of the N-, C- proximal and central domain. The central domainof CP binds to viral RNA and plays an essential role in virus assembly and cell-to-cellmovement (Rojas et al., 1997). The N- and C-proximal domains are exposed on thevirion surface and may be involved in systemic movement of the virus (Andersenand Johansen, 1998; Dolja et al., 1995). The highly conserved ‘DAG’ amino acidmotif at the N-terminus of CP is essential for aphid transmissibility (Atreya et al.,1995) and the surrounding residues may modulate the efficiency of transmission(Lopez-Moya et al., 1999).

1.1.3 Recombination as a means of virus evolution

Recombination is considered one of the main driving forces in virus variabilityand thus in virus evolution, the others being mutation and re-assortment (forviruses with a segmented genome) (Roossinck, 1997). Of the existing models forRNA recombination, the most widely accepted in RNA viruses is the ‘copy choice’model (Cooper et al., 1974). In this model, RdRp shifts from a viral RNA molecule(the donor template) to another (the acceptor template) during synthesis whileremaining bound to the nascent nucleic acid chain, thus generating an RNAmolecule with mixed parentage (Cooper et al., 1974; Kim and Kao, 2001). Severalfactors can influence this switching, including the extent of local sequence identitybetween the RNA templates, the kinetics of transcription, and secondary structuresin the RNA (Baird et al., 2006). The molecular mechanism of switching by the RdRpremains unclear, despite of the abundance of proposed models, and may involvepresence of signal regions on the RNAs that pause and terminate the synthesis of

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RNAs on the donor RNA and resume from the new acceptor template. Theseregions that promote RdRp pausing or termination constitute recombinationhotspots (Kim and Kao, 2001). From an evolutionary point of view, it has beenproposed that high recombination rates (rg = 3,427x10-5 recombination event pernucleotide site per generation) may be a consequence of selection for fastreplication at the cost of low fidelity (Tromas et al., 2014).

However, despite the high recombination rate predicted in plant viruses, onlya small fraction of the new resulting variants are functional and emerge in thepopulation, possibly due to strong selection pressure. When successful, the newvariant is frequently associated with changes in host range, increases in virulence,or the evasion of host defense (Moreno et al., 2004; Ogawa et al., 2008; Ohshima etal., 2002). Potyviruses are one of the plant virus genera with the highest frequencyof recombinant isolates described (Chare and Holmes, 2006; Revers et al., 1996).Several reports have highlighted the importance of recombination in shaping of thegenetic structure of potyvirus populations (Desbiez and Lecoq, 2008; Farzadfar etal., 2009; Karasev et al., 2011; Kwon et al., 2005; Mangrauthia et al., 2008; Morenoet al., 2004; Ohshima et al., 2002; Seo et al., 2009; Visser et al., 2012; Wylie andJones, 2009). Natural intra-specific recombinants seems to occur much more oftenthan inter-specific events (Chare and Holmes, 2006; Revers et al., 1996). This hasled to the identification for diverse hotspot recombination sites in almost everyregion of the genome of distinct potyvirus species, the most common site of hostspots being the P1 region (Valli et al., 2006) and the CI-6K2-VPg region of thepolyprotein (Desbiez and Lecoq, 2008; Ohshima et al., 2007).

1.1.4 Taxonomy

Deciphering the molecular variability of viruses is necessary for theunderstanding of their molecular evolutionary history in relation to their virulence,geographical distribution, and emergence of new epidemics. Methods to measuremolecular variability and phylogenetic distances of potyviruses have developedalong the new technological tools that have become available. Initially, theclassification of species and strains was limited to comparison of traits such as hostrange and symptomatology, serology, and amino acid composition of CP (Barnett,1992; Moghal and Francki, 1976; Randles et al., 1980; Shukla and Ward, 1989). Thedevelopment of DNA sequencing and the availability of CP sequences, and then fullgenome sequences of viruses, and development of bioinformatics tools forprocessing sequencing data have made continuous contributions for theclarification of the taxonomy potyviruses (Adams et al., 2005; Gibbs and Ohshima,2010). Comparison of sequences is therefore considered currently the mostimportant tool to measure variability in the different cistrons of the genome ofpotyviruses and to distinguish closely-related virus species and strains of the samespecies (Adams et al., 2005; Ward et al., 1992). Accordingly, based on pairwise

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comparison of 187 complete genome sequences of members of the Potyviridaefamily, Adams et al. (2005) found that genetic conservation in potyviruses genomesvaries within regions. The 3´UTR region is among the most conserved regionsamong strains of the same species, (Frenkel et al., 1989; Uyeda, 1992) while the NIbreplicase is relatively conserved in all species (Domier et al., 1987; Hong and Hunt,1996). On the other hand, the P1, P3 and the N-terminus of CP show the highestvariability (Adams et al., 2005; Valli et al., 2007). In addition, Adams et al. (2005),established that the cut off value of demarcation for potyviruses species is 82% and76-77% identity at the amino acid and nucleotide sequence level of the CP,respectively. Based on these criteria, reclassification of a strain to a new potyvirusspecies, or classifying a previously defined species as a strain of a an establishedspecies is possible (Fauquet et al., 2005; King et al., 2012). Species demarcationthresholds have also allowed identifying several subgroups within the Potyvirusgenus. Thus, based on features of the CP, and confirmed later by amino acidsequence comparison, the existence of three well supported subgroups ofpotyviruses have been established and are denoted as the “SCMV”, “BCMV” and“PVY” subgroups predominantly infecting gramineous, leguminous, andsolanaceous plants, respectively (Barnett, 1992; Sáiz et al., 1994; Shukla et al.,1998; Spetz et al., 2003).

1.2 Sweet potato viruses

Sweet potato (Ipomoea batatas Lam.) is a dicotyledonous, perennial plantbelonging to the genus Ipomoea, family Convolvulaceae. The tuberous roots of theplant are edible. It is the only member of the genus with economic importance(Onwueme and Charles, 1994). With more than 105 million tons of sweet potatoesproduced in 2013, sweet potato is the 6th most important food crop globally afterrice, wheat, potato, maize and cassava (FAO, 2013). Most (95%) of the production isin developing countries: in China and countries of East Asia, Africa and theCaribbean. Due to its robustness, need for only low agronomical input, highnutritional value, and an increasing demand for food, sweet potato is an ideal cropin subsistence economies (Wolfe, 1992). This is particular relevant in sub-SaharanAfrican countries where sweet potato is cultivated in 1.8 million hectares with anestimated production of 11.3 million tons (FAO, 2013) mostly by low-incomefarmers. Hence, improvement of food security in such regions requires to study thenegative impact of pests and pathogens affecting sweet potato (Karyeija et al.,1998).

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1.2.1 Sweet potato feathery mottle virus, SPFMV

SPFMV belongs to the Potyvirus genus and can be found in all sweet potatocultivating regions (Karyeija et al., 2000; Loebenstein and Thottappilly, 2004; Moyerand Salazar, 1989; Rännäli et al., 2009). It was first reported in East Africancountries and named Sweet potato virus A (Sheffield, 1957). Despite the earlyawareness of the existence of the virus, very little was done on its study worldwideuntil 1990’s when severe outbreaks of this virus related to the occurrence ofsynergetic diseases in sweet potato in Africa lead to studies in several researchcenters (Karyeija et al., 1998). SPFMV particles are 810-865 nm long and thegenome is 10820-10996 nucleotides (Kreuze et al., 2009; Sakai et al., 1997) makingSPFMV one of the largest viruses in the genus Potyvirus. The size of CP is 38 kDa(Sakai et al., 1997). Based on nucleotide and amino acid sequence comparisons,SPFMV isolates have been grouped to four strains: EA including East Africanisolates, and russet crack (RC), ordinary (O) and common (C) strains distributedworldwide (Kreuze et al., 2000). SPFMV is able to infect a great number of hosts inthe family Convolvulaceae (Loebenstein and Thottappilly, 2004). Several wild plantshave been found to act as reservoirs and play an important role of in shaping thevirus population structure in Africa (Tugume et al., 2010a). SPFMV is transmitted ina non-persistant, non-circulative manner by aphids (Myzus persicae, Aphis gossypiiand Aphis cracivora) which do not colonize sweet potato plants (Aritua et al., 1998).A recent study demonstrated that Aphis gossypii, but not Myzus persicae, couldtransmit SPFMV RC from naturally mixed-infected (SPFMV+SPVG) sweet potatoplants, suggesting that differences in vector specificity may affect the spread ofdifferent strains (Wosula et al., 2012). In single infections, SPFMV may or may notinduce mild symptoms, depending on several factors, such as strain, the sweetpotato cultivar and the environmental conditions. Symptoms occur mostly on oldleaves and include vein clearing, chlorotic spots and purple rings. Only members ofthe RC strain can cause damage on roots of some sweet potato varieties (Clark andMoyer, 1988). Similarly, titers of SPFMV are low in single infections (Karyeija et al.,2000), which in turn makes it difficult to detect the virus by conventional methods,such as immunosorbent electron microscopy (ISEM), double antibody sandwichenzyme-linked immunosorbent assay (DAS-ELISA) or nitrocellulose membraneenzyme-linked immunosorbent assay (NCM-ELISA) (Abad et al., 1992; Moyer et al.,1980). Therefore, the use of molecular assays such as nucleic acid spothybridization (NASH), polymerase chain reaction (PCR), and quantitative PCR (qPCR)are needed for accurate detection (Colinet et al., 1994b; Kokkinos and Clark,2006b). Infection with SPFMV alone in sweet potato does not usually reduce theyield significantly in infected crops of East African landrace varieties that are ratherresistant to SPFMV, however, this changes dramatically when SPFMV coinfectsplants with the heterologous virus Sweet potato chlorotic stunt virus (SPCSV)(genus Crinivirus, family Closteroviridae)

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1.2.2 Other potyviruses infecting sweet potato

Knowledge about other potyviruses infecting sweet potato is limited,although most of them are widely distributed. Sweet potato virus G (SPVG) is thesecond most widely distributed potyvirus after SPFMV in terms of prevalence andwas first reported to occur in China (Colinet et al., 1994a). Due to the limitedspecificity of polyclonal antibodies, detection of SPVG was often masked by cross-reaction with SPFMV antibodies. Currently SPVG is found in USA, China, Egypt,Ethiopia, South Africa, and New Zealand (Rännäli et al., 2008; Wosula et al., 2013).SPVG causes mild symptoms in sweet potato, and less severe symptoms than inSPFMV in I. setosa, which is a wild species often used as a virus indicator andmaintenance host for sweet potato viruses.

Sweet potato virus 2 (SPV2) is the formal species name proposed for severalisolates that were initially reported as new viruses: Sweet potato Y and Ipomoeavein mosaic virus. SPV2 was first reported in diseased plants from Taiwan (Rosseland Thottappilly, 1988); however, no specific antibodies were available until thefirst decade of this millennium and detection of SPV2 was often masked by cross-reaction with SPFMV antibodies. Currently, SPV2 occurs in most sweet potatoproduction areas (Ateka et al., 2007; Souto et al., 2003; Tairo et al., 2006). The virusrelies on aphid transmission (Ateka et al., 2007). Similarly to SPFMV and SPVG, SPV2causes mild symptoms in sweet potato leaves, including leaf mottle, vein yellowingand/or ringspots (Ateka et al., 2007), and there is no accurate estimation of theimpact of this virus in yield reduction. Mixed infections of SPFMV with SPVG/SPV2are not unusual, and they often generate symptoms which are not stronger than insingle virus infections (Wosula et al., 2013). Currently, SPVG and SPV2 can bespecifically detected by polyclonal antibodies and a great number of moleculartechniques, such as PCR and one step qPCR have been developed lately (Li et al.,2012b). The host ranges of SPVG and SPV2 are mostly limited to plants in theConvolvulacea family. SPVG is not able to infect Nicotiana benthamiana orChenopodium quinoa (Souto et al., 2003).

Sweet potato latent virus (SPLV) was first detected in diseased plants inTaiwan (Liao et al., 1979). SPLV is widely distributed in China (Chen et al., 2008), butrarely found in other regions where sweet potato is cultivated. Based on CPsequence comparisons, SPLV isolates can be grouped to two strains, CH (China) andT (Taiwan), with the latter group lacking the DAG motif, a potyvirus signature in CPrequired for aphid transmission (Colinet et al., 1997). Despite this fact, nosuccessful experimental transmission of any strain of SPLV by aphids has beenaccomplished yet. Similarly to SPFMV, SPVG and SPV2, SPLV does not cause severesymptoms in sweet potato (Clark and Moyer, 1988). The host range for isolates ofSPLV isolates expands beyond that of the family Convolvulaceae, and the virusinduces symptoms such as vein banding and leaf mosaic in members of the family

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Solanaceae, and local lesions in members of the family Chenopodiaceae (Clark andMoyer, 1988).

Sweet potato mild speckling virus (SPMSV) was first detected in SouthAmerica (Alvarez et al., 1997). SPMSV is transmitted by aphids, despite the fact thatit does not react positively to PTY1 monoclonal antibodies detecting aphid-transmission epitopes in CP (Di Feo et al., 2000). Similar to SPLV, SPMSV isolatesinfect species of the family Solanaceae and Chenopodiaceae and remain latent insweet potato. However, during the late 1990’s SPMSV was associated with a severedisease named “batata crespa” with a strong negative impact in Argentinian sweetpotato crops (Di Feo et al., 2000). Further characterization of this disease revealedthat it was caused by synergetic action of SPMSV, SPFMV and SPCSV (Di Feo et al.,2000).

1.2.3 Sweet potato virus disease and other viral synergisms

A viral synergism takes place when symptoms are exacerbated as aconsequence of mixed infection of two or more viruses (Roossinck, 2005).Occurrence of synergisms is evidenced by increase in virus titers (Vance, 1991),facilitation of viral movement (Hacker and Fowler, 2000), drastic increase ofsymptoms severity (Scheets, 1998) and enhancement of virus transmission byvectors (Azzam and Chancellor, 2002). Some viral diseases of economic importancein crop plants are generated by viral synergism (Hibino et al., 1978; Pio-Ribeiro etal., 1978). Sweet potato virus disease (SPVD) is the most devastating viral diseaseoccurring in sweet potato and arises from the co-infection of SPFMV and SPCSV(Gibson et al., 1998; Karyeija et al., 2000). SPVD is characterized by extremelysevere symptoms in diseased plants, including leaf distortion, leaf crinkling, foliarlaminar reduction, curling, general chlorosis, and stunting, and leads to a significantyield reduction, usually above 80% (Gutierrez et al., 2003). These severe symptomsare associated with a 600-fold increase of SPFMV titers, as compared with singleinfection with SPFMV (Karyeija et al., 2000). Similar type of synergism has beenobserved also between SPCSV and other potyviruses, such as SPLV, SPVG, SPV2,SPMSV (Di Feo et al., 2000; Kokkinos and Clark, 2006a; Untiveros et al., 2007) andthe ipomovirus Sweet potato mild mottle virus (SPMMV) (Mukasa et al., 2006;Untiveros et al., 2007) under natural or experimental conditions. Nevertheless, theseverity of symptoms and relative enhancement of the potyvirus titers variesamong virus species. Untiveros et al. (2007) reported symptoms of different degreeof severity caused by coinfection of SPCSV and SPFMV (most severe), SPMMV,SPMSV and SPLV (less severe) (Figure 2) (Untiveros et al., 2007). Similarly, Kokkinoset al. (2006), reported significant differences in the enhancement of titers ofdifferent strains of SPFMV, SPVG and SPV2 relative to single infections. Intriguingly,they observed that increase of potyvirus titers was not necessarily related toincrease of the severity of symptoms (Kokkinos and Clark, 2006b).

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Figure 2. Symptoms induced by synergisms resulting from mixed infections of SPCSV and (a)SPFMV (SPVD), (b) SPMSV or (c) SPLV. Modified from Untiveros et al. (2007).

1.3 RNA interference (RNAi)

1.3.1 Overview

RNA interference (RNAi), also known as RNA silencing, refers to cellularmechanisms regulating gene expression in eukaryotic organisms at transcriptionalor post-transcriptional level (Transcriptional gene silencing (TGS) and Post-transcriptional gene silencing (PTGS), respectively) (Pumplin and Voinnet, 2013).RNAi can also defend cells against virus infection (Figure 3) (Ding and Voinnet,2007). In plants, RNAi consists of an initiation phase, effector phase andamplification phase (Csorba et al., 2015). Initiation of silencing requires thepresence of double-stranded RNA (dsRNA) which may be generated in several ways:dsRNA viral genomes, replicative forms of an RNA virus genome, from secondarystructures of the transcripts of DNA viruses (Voinnet, 2005), hairpin RNA (hpRNA)(Baulcombe, 1996), or antisense RNA expressed from vector constructs used totarget plant genes (Waterhouse et al., 1998). RNAi induced by overexpression ofgene transcripts (sense-mediated silencing) in plants requires the activity of plantRNA-dependent RNA polymerase (e.g., RdRp6) for conversion of ssRNA to dsRNA(Béclin et al., 2002; Dalmay et al., 2000). During the initiation phase, dsRNAs arepromptly cleaved into small interfering RNAs (siRNAs) of 21 to 24 nucleotides (nt) inlength, by an orchestrated action of dicer-like endoribonucleases (Class 3 RNase IIIenzymes), of which DCL4 is the most important in antiviral RNAi in plants, reviewedin (Bologna and Voinnet, 2014). Thereafter, the siRNAs are stabilized at their 3' endby the HUA Enhancer 1 (HEN1)-dependent methylation (Vogler et al., 2007). Theeffector phase is initiated with the incorporation of the stabilized double strandedsiRNAs into the RNA-induced silencing complex (RISC) containing an RNaseH-likeargonaute (AGO) enzyme. Subsequently, one strand of the siRNA is removed, thecomplementary strand is used to guide AGO to a homologous RNA molecule to slice

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it (Pumplin and Voinnet, 2013). In plants, a consequence of the effector step is theamplification of silencing response involving RdRps (Wassenegger and Krczal, 2006).RdRps generate further substrates for DCL processing, playing a key role in theproduction of secondary siRNAs and further amplification of silencing. Amplificationof RNAi has been implicated in the spread of an RNAi signal, which is a non-cell-autonomous event (Kalantidis et al., 2008).

Figure 3. A schematic model of antiviral RNAi in plants and the diverse RSS strategies that interferewith the pathway. Initiation phase, effector phase and the amplification phase as well as systemicsilencing are pointed in boxes. The diverse viral suppressors and their mode of action to inhibit PTGSand/or TGS pointed in black and red, respectively. For further details, see main text. Modified fromCsorba et al. (2015).

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1.3.2 Spread of RNAi in plants

Regardless of the source of induction of silencing, spreading of silencing willalways occur at some level, to a few cells around the silencing source or to thewhole plant. Therefore it is important to point out the variations between thedifferent phases of silencing, including short range local silencing, extensive localsilencing spread and systemic silencing (Kalantidis et al., 2008). Phenotypicdifferences of the three phases of silencing can be observed in Figure 4 for a gfptransgene in N. benthamiana.

Figure 4. Phenotypes of RNAi of a gfp transgene in N. benthamiana under visualization ofUV-light: (a) induced local silencing, (b) extensive local silencing, and (c) systemic silencing.Leaves (a) and (b) are those in which silencing was induced by agro-infiltration, whereas (c)is an upper non-treated leaf.

The short range spread of local silencing is limited to a distance of 10-15 cellsand occurs without amplification of the silencing signal (Himber et al., 2003). It isoften evidenced when endogenous genes are targeted by RNAi (typically inducedby a dsRNA or an appropriately modified virus). As there is no evidence of action ofRdRps in this phase of silencing, it has been thought to depend on the spreading ofthe original silencing signal produced in the cells expressing the silencing inducer(Kalantidis et al., 2008). Even though there is some evidence that DCL4 (Dunoyer etal., 2005; Smith et al., 2007) and other plant proteins participate in cell-to-cellsignaling of RNAi (Molnar et al., 2011), and that the hypothetical signal may betransported through the plasmodesmata (Kalantidis et al., 2006), the precise signaland mechanism of its movement remain unclear. On the other hand, extensive localspread of silencing occurs when cell-to-cell silencing extends the 10-15 cell widelayer, which requires a silencing amplification mechanism (Schwach et al., 2005).Accordingly, different reports have pointed out the participation of RdRp6 andDCL4, and production of 21-nt small RNAs (sRNAs) in the amplification of thesilencing signal (Bleys et al., 2006; Molnar et al., 2011). So far, extensive localspread has been found operating against transgenes transcripts only (Smith et al.,

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2007). Finally, systemic silencing is believed to be mediated by a silencing signaltraveling along the vascular system and inducing silencing in sink leaves (Voinnetand Baulcombe, 1997) where the silencing signal moves further as local movementto the rest of the leave. The nature of the systemic silencing signal(s) remainsuncertain (Mlotshwa et al., 2002), but it is widely accepted that it may be a type ofRNA (Jorgensen et al., 1998; Yoo et al., 2004). Through the study of viral silencingsuppressors it has become clear that systemic silencing plays a key role in the plantsilencing response in tissues not yet reached by viral infection (Schwach et al.,2005).

1.3.3 Viral suppressors of RNAi

Not long after the discovery that RNAi protects plants against viral infection,it became clear that viruses are able to counter-act and suppress the silencingmechanism. Several previously characterized viral pathogenicity determinants werefound to possess RNA silencing suppression (RSS) activity. The potyviral HC-Pro andthe 2b protein of cucumber mosaic virus (CMV) were among the first RSS proteinsreported (Anandalakshmi et al., 1998; Brigneti et al., 1998; Kasschau andCarrington, 1998). Since then, RSS proteins have been found and characterized fromalmost all plant virus families (reviewed by Csorba et al., 2015)

There is an extraordinary diversity in sequence and domain structure ofviral RSS proteins indicating that they have evolved independently as an example ofconvergent evolution. The viral RSS proteins use diverse mechanisms to blockvirtually any step of the RNAi pathway. A summary of the modes of action isprovided in (Figure 3). RSS proteins may block the initiation phase of antiviralsilencing by sequestration of dsRNA/siRNA, destabilization of host proteins involvedin RISC assembly or inhibition of Dicer or its co-factor DRB4. Several RSS proteinshave been reported to bind/sequester siRNAs (Lakatos et al., 2006). Among them,the best characterized is the tombusviral P19 protein that selects and sequesterssiRNA duplexes in a sequence-independent manner (Silhavy et al., 2002). On theother hand, 2b and other suppressors bind dsRNA in a size-independent manner(Deleris et al., 2006; Goto et al., 2007; Mérai et al., 2006). RNAi suppressors alsodeteriorate the production of siRNAs, as exemplified by the transactivator protein(TAV; also known as P6) of cauliflower mosaic virus (CaMV, a DNA virus). TAVinteracts directly with the DCL4 cofactor DRB4 (Haas et al., 2008). Suppression ofthe effector stage is achieved by inactivation, competition or compromising theintegrity of host effectors proteins. Binding the key AGO1 protein, through GW/WGAgo-binding motifs (GW motifs), and thus preventing RISC assembly, is a commonstrategy for several RNAi suppressors (Giner et al., 2010; Jin and Zhu, 2010; Pérez-Cañamás and Hernández, 2014). The specific binding alters AGO1 loading withsiRNAs into the RISC complex as demonstrated in P38 transgenic plants (Schott etal., 2012). P0 protein is encoded by poleroviruses (family Luteoviridae) and leads to

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ubiquitylation and further disintegration by autophagy of AGO1 (Baumberger et al.,2007). P0 actually hijacks a normal cytological process whereby unloaded AGO1and GW‑rich proteins undergo selective autophagy in healthy plants as a regulatoryprocess which is also found in human cells and Caenorhabditis elegans (Gibbings etal., 2012). P25 protein, encoded by potato virus X (PVX, genus Potexvirus), targetsAGO1 and leads to its destruction through the activity of the 26S proteasome (Chiuet al., 2010).

A number of RNAi suppressors interfere with signaling of RNAi at differentstages. The tombusviral P19, geminiviral AC2 and the potyviral HC-Pro are powerfulsuppressors interfering with the short-distance movement of gfp silencing (Himberet al., 2003). Others block silencing amplification and systemic signal movement todistant tissues to facilitate the virus replication and spread. This is effectivelyachieved by RdRp6-based activity suppression. The rice yellow stunt virus (RYSV,genus Nucleorhabdovirus) protein P6 is able to suppresses systemic silencing bydirectly interaction with RDR6 and blocking secondary siRNA synthesis (Guo et al.,2013). The V2 protein of tomato yellow leaf curl virus (TYLCV, genus Begomovirus)and the potexviral triple gene box protein 1 (TGBp1) directly interacts with SGS3,the cofactor of RdRp6, to block silencing amplification (Glick et al., 2008; Okano etal., 2014). On the other hand, βC1 suppressor of tomato yellow leaf curl China virus(TYLCCNV, genus Begomovirus) DNA satellite, HC-Pro and TAV2b of Sugarcanemosaic virus (SCMV, genus Potyvirus) and Pns10 of Rice dwarf phytoreovirus (RDV,genus Phytoreovirus) down regulate rdrp6 mRNA to repress RdRp6 expression andsecondary siRNA production (Li et al., 2014; Ren et al., 2010; Zhang et al., 2008).

1.3.4 RNAi suppressors and their role in viral synergism

Several studies have demonstrated the role of viral RSS proteins in viralsynergisms. Using different experimental approaches, HC-Pro has been reported asthe responsible protein for titer enhancement of heterologous viruses such as PVX,CMV and tobacco mosaic virus (TMV), and potato leaf roll virus (PLRV) (Brigneti etal., 1998; Pruss et al., 1997; Savenkov and Valkonen, 2001). Similarly, Siddiqui et al.,(2011) used transgenic tobacco plants transformed with the cucumoviral protein 2bto demonstrate that this protein mediates the synergism between CMV and thenon-related TMV (Siddiqui et al., 2011). Furthermore, the molecular mechanism ofSPVD was unraveled when transgenic plants expressing the RNase3 protein ofSPCSV displayed SPVD-like symptoms following infection with SPFMV (Cuellar et al.,2009). The presence of RNase3 induced synergisms also with other non-relatedviruses, such as CMV, sweet potato chlorotic fleck virus (SPCFV, genus Carlavirus)and, recently discovered cavemoviruses, family Caulimoviridae (Cuellar et al., 2011;Cuellar et al., 2009).

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2. AIMS OF THE STUDY

a) To elucidate the phylogenetic relationships among strains of SPFMV andrelated potyviruses.

b) To resolve the genome structure at the 5’-end of SPFMV and proteinexpression from this part of the viral genome

c) To determine the molecular roles of the 5’-proximal region of SPFMVgenome in RNAi suppression.

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3. MATERIALS AND METHODS

A detailed description of the materials and methods used in this study are in theoriginal publications (I-III). For a summary the methods see Table1.

Table 1. Summary of methods used in this study.

Methods Publications

Agrobacterium-mediated infiltration assays III

Analysis and mapping of recombination events in SPFMV I, II

Analysis of nucleotide diversity indices I, II

Computational analysis for detection of ORFs III

Designing and engineering of binary vector III

Expression of recombinant proteins from Escherichia coli III

Field surveys and sampling I

Introduction of mutations III

Molecular characterization of SPFMV I, II

Molecular cloning of viral genes III

Molecular phylogenetic analysis of sequence data I, II

Northern blotting III

PCR and RT-PCR I, II, III

Phylogenetic reconstructions I, II

Primer design I, II, III

Protein extraction from plant tissue III

RNA extraction from plant tissue I, II, III

Sequencing of SPVG and SPV2 by Assembly of siRNA III

siRNA assembly III

Western blotting III

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4. RESULTS AND DISCUSSION

4.1 Relatedness and geographical distribution of potyviruses infecting sweetpotato

4.1.1 Re-classification of SPFMV strains

Re-classification of the SPFMV strain C as a new viral species was proposedby Tairo et al, 2005 (Tairo et al., 2005). In our first study based on pairwisecomparisons of CP sequences of SPFMV isolates (Figure 2, I), we observed thatnucleotide (76-77%) and amino acid (82%) identity values for strain C whencompared to other strains were at or under of values accepted for demarcation of anew species of potyvirus (Adams et al., 2005). The aforementioned cut off valuesseemed to prevent reclassification when studies were limited to the CP. Completegenome sequencing of isolate C1 (Strain C), however, provided new and strongevidence to justify the reclassification of this strain as a new virus, Sweet potatovirus C (SPVC) (II). Analysis of pairwise comparisons of nucleotide and amino acididentity values resulted in 71% (complete genome) and 77.6% (polyprotein) (II),both clearly below the recommended potyvirus species demarcation limit of 76 and83%, respectively (Adams et al., 2005). Similar results were found when individualgenomic regions such as P1, HC-pro, P3, and CI were analyzed, whereas NIa, NIband CP are either at or only slightly above the recommended threshold (II).Reclassification of SPVC as new species was later supported by molecular analysisof complete genome sequences of additional SPVC isolates (Yamasaki et al., 2010),and phylogenetic reconstructions of SPVC isolates and other related potyvirusreported in recent surveys worldwide (Qin et al., 2013).

4.1.2 New insights on the distribution of potyviruses infecting sweet potato

Understanding the molecular variation and geographical distribution ofviruses is essential for the establishment of evidence-based control strategies. Earlyreports indicated that SPFMV strains RC and O, as well as SPVC isolates wereglobally distributed, while EA remained confined to East African countries (Tairo etal., 2005). As part of our results, we reported four EA isolates in America thatclustered together with two other isolates reported outside of Africa (Figure 3B, I).Later studies have verified the existence of more EA isolates outside of Africa, in theEaster Islands, and recently in China (Qin et al., 2013; Rännäli et al., 2009). Withmore and more surveys and quicker and more accurate methods of diagnosis, a firstpicture can be drawn regarding the current distribution of the three strains ofSPFMV: EA strain appears not to be restricted to East Africa, the strain O isprevalent in China and Asia, and RC has been found worldwide with the exceptionof Africa (Rännäli et al., 2009; Tugume et al., 2010a). An evolutionary model for EA

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isolates was suggested, with East-Africa as the center of origin and dispersion ofthis strain (Tugume et al., 2010a), however, with the discovery of more EA isolatesoutside of Africa, this model could be challenged. On the other hand SPVC seems tobe present worldwide with no geographic preference for a specific region (I)(Rännäli et al., 2009; Tairo et al., 2006). So far, there has not been any reportsuggesting classification of SPVC isolates into groups or strains, although our resultsindicate that Peruvian SPVC isolates may belong to an unique clade (Figure 2, I).

Based on CP nucleotide sequences analysis, this study also reportedexistence of isolates of SPVG and SPV2 for the first time in South America. Theisolate Hua2 showed a more distant relationship to other SPVG isolates (Figure 4, I).However, recent studies on SPVG geographical distribution and phylogeneticreconstructions based on CP comparisons have clustered Hua2 together with aTaiwanese isolate Tw2, as part of a separate taxonomic clade (Qin et al., 2013). Asimilar situation can be found for SPV2 isolate Hua4 that clustered with theAustralian isolate Thomas as a unique clade of this virus (Figure 4, I). Curiously,SPVG seems to be consistently more prevalent than SPV2 wherever both viruses arefound despite the fact that the latter has a wider host range (Ateka et al., 2007).Some clues to explain the differences in prevalence of potyviruses infecting sweetpotato can be obtained from Wosula et al, 2013 who indicated that multipleinfections, hosts, aphid species and titer of virus affect the rate of transmission ofthese viruses (Wosula et al., 2013).

4.1.3 The “SPFMV-group” of potyviruses

The potyvirus genus encompasses more than 150 species and the number isincreasing every year (King et al., 2012). Our phylogenetic analysis based onpairwise comparison of CP nucleotide sequences of various isolates of SPFMV,SPVC, SPVG and SPV2 used in (I) together with those from a significant number ofother potyviruses revealed that most of the potyvirus infecting sweet potato form aseparate well supported phylogenetic subgroup within the potyvirus genus (Fig 4, I)and was denoted as the ’SPFMV-group’. A particular characteristic of members ofthis group is the restriction of their host range mainly within the Convolvulaceaefamily suggesting, as in the case of the previous subgroups, a significant role forvirus-host co-evolution in potyvirus speciation (Sáiz et al., 1994; Spetz et al., 2003).Analysis based on CP nucleotide sequences excluded SPLV and SPMSV from the“SPFMV-group” in agreement with the fact that they can readily infect species ofthe Solanaceae and Chenopodiaceae families in contrast to viruses of the SPFMV-group, of which only some can infect few species in these families. This initialpicture could, however, change when complete genome sequences are analyzed.Indeed, phylogenetic reconstruction based on 127 potyviral complete genomesequences including those from our own data (I, III) and recently sequencedgenome sequences of SPVG, SPV2, SPVC and SPLV (Li et al., 2012a; Wang et al.,

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2013; Yamasaki et al., 2010) clustered the ‘SPFMV-group’ and SPLV in a largermonophyletic group as compared to other potyviruses (Supplementary Figure 1).Hence, in order to avoid confusing use of terms, we propose to retain the name“SPFMV-group” for the more closely related SPFMV, SPVC, SPVG, and SPV2 (basedon evidence discussed later) from the clade encompassing all potyviruses infectingsweet potato. The fact that SPLV is distantly related to the SPFMV-group, is inagreement with the extent of their host ranges and suggests that they all may sharea common ancestor. SPFMV and SPVG infect only species in the Convolvulaceaefamily and are unable to infect Nicotiana benthamiana or Chenopodium quinoa(Clark and Moyer, 1988; Souto et al., 2003). On the other hand, SPLV is able toinfect different members of Solanacea and Chenopodiacea families, and SPV2 wasreported to infect Nicotiana benthamiana, Datura stramonium and generate locallesions in Chenopodium amaranthicolor, but not other members of those plantfamilies (Ateka et al., 2007).

4.2 New genomic organization in the “SPFMV-group” of potyviruses

4.2.1 Intra- and interspecific recombination events

Visual inspection, bioinformatic analysis and phylogenetic reconstructionsrevealed the occurrence of recombinants among members of the “SPFMV-group”.Analysis of the C-Term NIb-CP-3’UTR (3’region) (I) or complete genome sequences(II) showed that five SPFMV isolates of RC, O and EA strains, and two SPVC isolatesresulted from intra- or inter- specific recombination events, respectively. Twobreakpoints were detected in the 3’– end region of SPFMV (Figure 3a, I). The first,located at position 9,597 nt (relative to isolate S) within the 3’-terminal part of theNIb gene, was shared by isolates SPFMV Eg-9 and SPFMV Eg-1. Phylogeneticreconstruction of adjacent conflicting regions confirmed that both isolates resultedfrom intra-specific recombination of EA and RC parentals (Figure 3e, I). The secondbreakpoint was located at position 10,500 nt within the 3’-terminal region of the CPand was shared by isolates SPVC-YV and SPVC –C (Figure 3d and 3e, I). Phylogeneticreconstructions revealed a rare case of a potyvirus inter-specific recombinationevent between SPVC and SPFMV isolates (Figure 3b and 3c, I). In this study, we alsoidentified a double recombinant (Figure 3, II). SPFMV 10-O contains twobreakpoints located within the P1 cistron region at position 1,135 nt and the C-terminus of the NIa-pro domain, position 7,193 nt. Comparative analysis fromadjacent regions revealed a conflict between RC and EA sequence, and EA and Osequences, respectively (Figure 3, II). The recombination breakpoints reported herehave been found to occur in other potyviruses. Numerous recombination eventshave been identified to occur in the P1 region (Valli et al., 2007). Similarly,

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‘hotspots’ for recombination have been identified in the 3’-proximal part of the NIbin bean yellows mosaic virus (BYMV) (Wylie and Jones, 2009), and sugarcanemosaic virus (SMV) (Padhi and Ramu, 2011), while the 6K2-VPg-NIaPro region is arecombination ‘hotspot’ relevant in shaping the population of turnip mosaic virus(TuMV) isolates (Ohshima et al., 2007). A later study also confirmed the same 6K2-VPg-NIaPro region as a ‘hotspot’ region among SPFMV isolates (Tugume et al.,2010a). Despite the restricted number and size of sequences analyzed in I and II, EAisolates were observed to more frequently recombine than other strains. Theseresults are consistent with Tugume et al., 2010a who found abundant evidence ofintra-specific recombination within SPFMV EA strain (62% of isolates) as comparedto SPVC (19% isolates) while no evidence for inter-specific recombination betweenisolates of SPVC and SPFMV EA was found (Tugume et al., 2010a). Diverse rates ofinter- and intra-specific recombination in potyviruses infecting sweet potato in acontext where frequent multiple co-infections are reported, suggest that certainlevel of sequence homology is needed for recombination as reported for otherpotyviruses (Chare and Holmes, 2006). In fact, the occurrence of recombinationbetween SPFMV and SPVC isolates around the 3’UTR region may be due to the highnucleotide identity they share at this region (84-87%) which would be favored bythe “copy choice” model of RNA recombination (Cooper et al., 1974) .

4.2.2 The P1-N domain

The P1 cistron of potyviruses is considered the most variable region in termsof sequence identity and length (Adams et al., 2005; Valli et al., 2007). Our aminoacid sequence comparison of the first 337 residues in the P1 region of SPFMV andSPVC isolates revealed consistent presence of a P1N-terminal extension (which wedesignated P1-N) that had been previously noted in SPFMV-S (Figure 1,III). Noputative protease cleavage site could be identified around the demarcation areasbetween P1-N and the P1 region that is common with other potyviruses (which wedesignated P1-pro) revealing no evidence of physical separation of these domains.Later reports have established the existence of the P1-N in SPVG, SPV2 (Li et al.,2012a; Pardina et al., 2012), but not in SPLV (Figure 1, III) and confirmed that thisdomain is restricted to the members of the “SPFMV-group”. Phylogeneticreconstruction using P1-N nucleotide sequences confirmed a relationship of the P1-N of the “SPFMV-group” potyviruses with the corresponding region of SPMMV tothe exclusion of other poty- and ipomoviruses (Figure 2, II). Intriguingly, isolates ofstrain O were grouped together with isolates of strain EA and RC (Figure 2, II). Theseresults were confirmed in a new phylogenetic analysis including other members ofthe “SPFMV-group” (Supplementary Figure 2). Absence of a strain O lineage whenanalyzing P1-N is not congruent with the analysis of complete genome sequenceswhich shows a clear demarcation of strains including the O lineage. It may bepossible that recombination is not an uncommon phenomenon in the 5’-end region

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of SPFMV. In fact we identified one recombination breakpoint in P1-N (Figure 3, II).Recombination in P1-N between strains (II) and genera (Valli et al., 2007) suggeststhat P1-N may represent a functional domain that is somewhat independent of theP1-Pro domain. The N-terminus of P1 has also been proposed to be important forhost specificity in potyviruses (Salvador et al., 2008), a hypothesis that fits with therange of host described for members of the ‘SPFMV-group’. Therefore, furtherevolutionary and functional investigations of the P1-N are merited.

4.2.3 A hyper variable region in P1

Sequence comparison of the P1 region of isolates of SPFMV, strains RC andEA, and SPVC revealed a highly variable region between nucleotide positions 750and 1,250 nt (isolate S) (Fig. 1b, II). Specific insertions/deletions were observed forstrains of SPFMV and for SPVC isolates. Thus, amongst other minor gaps, a non-homologous insertion of 25 and 28 aminoacids is observed for EA and SPVCisolates, respectively as compared to RC isolates. Remarkably, EA isolates of EastAfrica and from non-East African regions shared this particular signature with highhomology (Figure 1b, II) with only one exception: a larger 60-amino acid insertionfound in SPFMV-Piu3 appeared to be a characteristic of that particular isolate. Amore complete analysis including other members of the “SPFMV – group” (Figure5) placed this hypervariable region at position 286 aa residue (according to isolateS). Despite the variability, this region allows a good discrimination among membersof the ’SPFMV’-group. Variable regions are useful for divergence studies anddevelopment of methods of diagnosis for SPFMV (Kreuze et al., 2000; Li et al.,2012b). Therefore, additional sequences in this region may allow the design ofspecific primers for each strain/virus for a rapid and accurate diagnosis of “SPFMV”-group species.

4.2.4 A new overlapping open reading frame of P1 encodes PISPO

Bioinformatic analysis of the complete genome of 31 full-length ‘SPFMV-group’virus isolates revealed the conserved presence of a long +2-frame ORF, namedpispo (Pretty Interesting Sweet potato Potyvirus ORF) overlapping the P1-pro regionof the polyprotein ORF (Figure 1A, III). However, differently from pipo, thatoverlaps the P3 region of all members across the Potyviridae family, pispo is wasonly found in the SPFMV-group members, and its discovery reveals a new categoryof genome structure variability occurring in the P1 region of potyviruses. SlipperyG2A6 sequences at the 5'-end of pispo and pipo (Chung et al., 2008) suggested thatboth ORFs are expressed by the same frameshifting mechanism (Figure 3, Table 1,III). Indeed, analysis of high-throughput sRNA sequencing data obtained fromSPFMV-infected sweet potato plants, and SPV2- and SPVG-infected I. setosarevealed that a significant fraction of sRNA reads contained an additional 'A'

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inserted within the pispo G2A6 sequence (Figure 3, Table 2, III). Presence of G2A6

was confirmed by targeted sequencing of this region in SPFMV-Ruk73 from infectedsweet potato and I. nil plants, revealing a frequency of ‘A’ insertions of 4.98% and5.45% of reads, respectively (Figure 4, Table 2, III). Similarly, 'A' insertions attargeted sequencing of the pipo G2A6 site in SPFMV-Ruk73 were also observed witha frequency of 0.9 and 1.03% from infected sweet potato and I. nil plants,respectively (Figure 4, Table 2, III). Transcriptional slippage occurs in (-) ssRNAviruses including subfamily Paramyxovirinae and members of the genus Ebolavirus(Larsen et al., 2000; Penno et al., 2005; Volchkov et al., 1995), but so far pipo andpispo are the first examples of the utilization of transcriptional slippage for geneexpression in positive-sense RNA viruses.

Figure 5. Partial amino acid alignment of the hypervariable region within the P1 cistron ofmembers of the “SPFMV-group” of potyviruses. The presence of “indels” of specific size foreach member or strain is distinguished. Complete list with the Genebank codes available inSupplementary Table 2

34

Similarly to P3N-PIPO, a transframe protein P1N-PISPO is expected to occuras a consequence of transcriptional frameshifting (Chung et al., 2008). In case ofisolate Ruk 73, P1N-PISPO has a predicted mass of 75.7 kDa, while the native P1 hasa predicted mass of 77.1 kDa (Figure 2C, III). Contrary to PIPO, the predicted aminoacid sequences are highly variable among PISPOs. Even more, PISPO is morevariable than its overlapping region, in contrast to PIPO (Table S1, III) (Chung et al.,2008). Accordingly, evolutionary tests revealed more plasticity for P1N-PISPO thanfor P3-PIPO deduced from lower conservation at polyprotein-frame synonymoussites in pispo than in pipo overlapping regions (Figure 1C, III). This difference mayreflect the functional importance of each corresponding transframe protein P3N-PIPO and P1N-PISPO. Thus, while P3N-PIPO is an essential protein involved in viralmovement, and appears to be of ancient origin as indicated by its conservationthroughout the Potyviridae family, P1N-PISPO seems to be a more recentevolutionary development and may therefore not yet have acquired a critical role,allowing more sequence plasticity.

4.3 RNAi suppressors in SPFMV

4.3.1 Novel role of P1 region of SPFMV among potyviruses

P1 was reported to have a supporting role in enhancement of HC-Pro RSSactivity in potato virus Y (PVY) (Tena Fernández et al., 2013). In this study, wepresent evidence that the P1 region SPFMV encodes two RSS proteins. Presence oftwo and four GW motifs in P1 and P1N-PISPO, respectively (Figure 5, III) led us toasses tentative RSS activity of these proteins using standard silencing assays. Weincluded SPLV P1 to our test as pispo was not found in this virus. Our data at 4 dayspost-infiltration showed that tissues co-infiltrated for expression of GFP and P1 orP1N-PISPO showed brighter GFP fluorescence than those infiltrated for expressionof SPLV P1 or a GUS control (Figure 7A, III). The phenotypic observation correlatedwith protein and RNA analysis (Figure 7 B and C, III), indicating that SPFMV P1 andP1N-PISPO, but not SPLV P1, have RSS activity. This result contrasts previousfindings of Szabó et al (2012) who did not find evidence of RSS activity for P1 ofisolate SPFMV Nig (JQ742091) (Szabó et al., 2012). Isolates Ruk 73 and Nig sharemore than 92% amino acid identity and contain the same number of Ago-bindingmotifs, and thus the contrasting results may be due to the use of differentexperimental design. SPFMV Ruk 73 P1 and P1N-PISPO have different silencingactivities. While a red halo was observed to surround the leaf tissue infiltrated forexpression of GFP+P1 at 4 d.p.i, (being clearer at 6 d.p.i), a clear hallmark of short-distance movement of the silencing signal, no clear halo development wasobserved around the leaf tissue infiltrated with GFP+P1N-PISPO (Figure 7C, III). Thisphenotypic difference indicates that P1 suppresses silencing at local or intracellular

35

level as reported for other RSS proteins (Baumberger et al., 2007; Giner et al.,2010), while P1N-PISPO is additionally able to inhibit the spread of silencing tosurrounding cells as indicated for HC-Pro, P19 and other suppressors (Reviewed inLakatos et al., 2006). We also verified that the phenotype observed for P1 is due tothe sole action of this protein since a mutant version P1ΔPISPO, designed toprevent possible expression of P1N-PISPO from the construct via transcriptionalframeshifting, behaved similarly to P1, and developed a red halo (Figure 8A, III).Thus, SPFMV is included in a group of viruses using different RSS proteins to inhibitRNAi silencing in alternative ways. For instance, the carlavirus potato virus M (PVM)utilizes a cysteine-rich protein (CRP) to suppress both local and systemic silencing,whereas the triple gene block protein 1 (TGBp1) inhibits only systemic silencing(Senshu et al., 2011), the closterovirus citrus tristeza virus (CTV) suppresses localsilencing through the P20 and p23 proteins, and intercellular silencing is inhibitedby CP and P20 (Lu et al., 2004). Finally, the absence of intrinsic RSS activity of SPLVP1 is similar to that found in other potyviruses and most likely is related to theabsence of P1-N.

4.3.2 Suppression mediated by putative Ago-binding motifs

Elimination of the common GW motif in residue 25 aa resulted in theelimination of RSS activity of P1 and P1N-PISPO (Figure 8B, III), while mutations ofthree GW motifs in PISPO did not affect its RSS activity (Figure 8A, III). These resultsresemble those found for SPMMV P1, in which silencing activity relies on three Ago-binding GW motifs found in the P1-N, but mutation of two of them is sufficient toeliminate RSS activity. Both SPMMV and SPFMV P1 operate only at a single-cell orlocal level as shown by the formation of the red halo (Giner et al., 2010). FunctionalAgo-binding motifs at the N-terminal region of different RSS proteins may indicatethe use of a mimicry to compete for and inhibit host AGOs as a general adaptivestrategy used by plant viruses to counteract silencing in hosts (Reviewed by Jin andZhu, 2010). Structural and functional similarities between SPMMV and SPFMV P1sreinforce the hypothesis of a common evolutionary origin (Valli et al., 2007).

4.3.3 The role of viral HC-Pro protein in RNAi

In this study, we evaluated the RSS activity of SPFMV HC-Pro using standardsilencing assays. Our results at 4 d.p.i. did not show significant difference in GFPintensity of the infiltrated patch with HC-Pro when compared to the control vectorexpressing GUS (Figure 7A, III), and a red halo developed around the leaf spotsinfiltrated for expression as illustrated at 8 d.p.i (Figure 7E, III). Accordingly, mRNAand siRNA analysis were in agreement with phenotypic observations supporting thelack of suppression mediated by SPFMV HC-Pro. This results was unexpected sinceHC-Pro is known to be a RSS protein through the well-conserved FRNK motif

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(Shiboleth et al., 2007), a motif also present in SPFMV Ruk 73. However, we cannotdiscard RSS activity of SPFMV HC-Pro, since we were unable to detect HC-Pro usingantibodies against different tags (c-myC, YFP N-terminal) suggesting expression ofHC-Pro by itself may be difficult. Supporting this, Tena Fernandez et al (2013)reported infiltration essays using only PVY HC-Pro failed to suppress silencing, andthe protein remained undetectable in western blot analysis, while when PVYP1+HC-Pro were infiltrated, the RSS activity of HC-Pro became observable,providing evidence of the importance of P1 in the stabilization of HC-Pro RSSactivity. As long as SPFMV Ruk 73 P1 already contains RSS activity, a constructharboring SPFMV P1+HC-Pro would not have elucidated the role of SPFMV HC-Proin silencing. The particular arrangement where P1 is a suppressor, and HC-Pro hasno role in RNAi has not previously been reported in potyviruses, though it is knownto occur in other genera of the Potyviridae family such as among the members ofthe Ipomovirus, Poacevirus and Tritimovirus genera (Giner et al., 2010; Mbanzibwaet al., 2009; Tatineni et al., 2012; Valli et al., 2006; Young et al., 2012). This commonorganization in SPFMV and SPMMV, both infecting hosts in the Convolvulaceaefamily, is particularly interesting and supports the biological importance of the P1-Ndomain in the evolution of these viruses.

4.4 An evolutionary model for sweet potato-infecting potyviruses

The acquisition of SPFMV P1-N domain by a recombination event betweenSPMMV and an ancestor of SPFMV was previously hypothesized (Valli et al., 2007).This study added phylogenetic, structural, and functional findings that highlight theimportant role of P1-N domain on the 5’-proximal region of the genome ofpotyvirus infecting sweet potato, and support and expand the recombinationhypothesis to the origin of the “SPFMV-group”. Based on these findings widerschemes for evolution of potyviruses infecting sweet potato can be hypothesized(Figure 6). In such schemes, an original ancestral population of potyviruses was ableto infect several plant families including Convolvulaceaeas as found in SPLV. One ofthese ancestors hereafter named the ancestor of the “SPFMV-group” acquired theP1-N domain through one or more recombination events with SPMMV possibly in awild Ipomoea host, as mixed infections occur often (Tugume et al., 2010b). Thismodel is depicted in path A (Figure 6). Detection of recombination breakpointsaround 1200-1600 nt in isolates of SPMMV (Tugume et al., 2010b; Valli et al., 2007)and SPFMV (this study) support the occurrence of such recombination events.However, this does not exclude the possibility that ancestors of “SPFMV-group”could have obtained the P1-N domain from an as yet unknown source, andsubsequently been the donor of P1-N for SPMMV, or that both viruses acquired P1-N independently, generating alternative paths B and C (Figure 6). The acquisition ofP1-N domain could have directly or indirectly instigated two critical outcomes that

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enabled the particular evolution of the “SPFMV-group”: loss of motifs needed toinfect other plant families, limiting them to the Convolvulaceae family, andintroduction of an additional RNA slippery context (G2A6) recognized by an RNApolymerase already familiarized with the synthesis of the much older P1N-PIPO.Thus, the acquisition of P1-N, and new biological properties encoded by this regionmay have influenced host preferences as well as RSS activity of the “SPFMV-group”in a similar way as in SPMMV where P1 has the main role in RSS activity instead ofHC-Pro. The effects of high diversification of P1-N and PISPO in the “SPFMV group”remain unclear. Functions of P1N-PISPOs may differ due to the high degree ofsequence variability and pI values [ranging from 5.7 (SPV2) to 9.34 (SPFMV-S)].Here, we provide evidence that PISPO transcriptional RNA slippage occurs inmembers of this group, and that P1 and P1N-PISPO are RSS proteins, but thefunctional characterization of PISPO from other viruses and strains requires furtherexperimental data.

Figure 6. Schematic representation of an evolutionary model of sweet potato-infectingpotyviruses including three alternative paths (A, B and C). (SP potyvirus, Sweet potato-infecting potyvirus; anc. SPMMV, ancestor of SPMMV; anc. “SPFMV-GROUP”, ancestor ofthe “SPFMV-group”)

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5. CONCLUSIONS AND PERSPECTIVES

This study has contributed to the understanding of the molecular biology ofpotyvirus infecting sweet potato. A main result was sequencing and furtheridentification of Sweet potato virus C as a separate virus species in sweet potato.This study also contributed with new insights into the distribution of potyviruses insweet potato. The presence of SPFMV EA isolates in fields in Peru and clustering ina single clade was an unexpected finding since EA isolates were considered mostlyrestricted to East Africa previous to that. Later detection of isolates in Oceania andthen in China indicate that EA isolates are more widespread than initially thought.More surveys and sequence data are needed to clarify the evolution of the EAstrain. Similarly, isolates of SPVG and SPV2 were detected for the first time in SouthAmerica. Specific antibodies are now available for SPVG and SPV2 contributing tothe study of their distribution globally. Specific antibodies are not yet available forSPVC. As many complete genomic sequences are currently accessible for SPVC, thedesign and production of specific antibodies should be a worthwhile target to moreeasily determine its geographical distribution and prevalence in sweet potatocultivating areas. Taken together these results are relevant for further studies onthe epidemiology of these potyviruses and deployment of more effective strategiesof disease control.

As a result of phylogenetic reconstructions based on the CP and completegenome sequences, this study identified a new subgroup of potyviruses named the“SPFMV-group” encompassing the four related viruses SPFMV, SPV2, SPVG andSPVC. This group together with SPLV belongs to a broader clade that we suggest tobe named as “Sweet potato-infecting potyviruses”. This study detected theoccurrence of recombination events in ”SPFMV-group” potyviruses. Recombinationbreakpoints were observed in different regions including P1, NIa/b, 5’-proximal partof NIb, and the 3’UTR. Results of this study evidenced that intra-specificrecombination events were more common than inter-specific events and wasconfirmed by later reports (Tugume et al., 2010a). Phylogenetic and recombinationanalysis based on the P1-N region revealed that recombination may be playing aparticularly important role on the molecular evolution of strain O of SPFMV sinceno O specific P1 region could be identified and instead the P1 region of O strainscorresponded to either EA or RC. To elucidate the relationship among SPFMVstrains O and other strains, additional complete genome sequences of strain Oisolates, specially from areas with high prevalence of this strain, and furtherrecombination analysis are required.

Different from SPLV, members of the ‘SPFMV-group’ contain unique genomicfeatures that were identified and/or characterized in this study. The first was theidentification of P1-N, a conserved domain which shares amino acid identity withthe homologous P1-N region of SPMMV. Indeed, due to the high homology, this

39

domain was proposed to be the result of a recombination event between SPMMVand SPFMV. Our data expands this suggestion by directing it’s occurrence to anearlier evolutionary time, as it could only have occurred with a common ancestor ofthe ‘SPFMV-group’, although there is currently no certain way to ascertain whichvirus acquired the P1-N domain first, or from where. P1-N is followed by anhypervariable region that, however, seems to be conserved only within each specieand even strains of the ’SPFMV-group’, and thus has potential for design of rapidand accurate diagnostics of virus strains. Possibly, the most remarkable genomicproperty this study identified was the discovery through bioinformatics of pispo, anew ORF overlapping the P1-pro region of all members of the ‘SPFMV-group’. Pisporesults from a transcriptional slippage occurring after a slippery G2A6 sequences,similarly to pipo. Pispo, however, seems to be a more recent event than pipo whichis inferred from the restricted presence in only SPFMV-group viruses and it’spredicted genetic plasticity. Pispo is expected to be translated as a transframeprotein P1N-PISPO, in a similar manner as pipo and P3N-PIPO. Although our effortsto detect this predicted protein in natural SPFMV infections from either I. nil or I.batatas plants were not successful, future efforts should focus to achieve this. P1N-PISPO is possibly present at very low amounts and may be expressed during specifictime points in the viral infection cycle, and therefore conventional detection test,such as western blot, may have failed. The use of more sophisticated, antibodyindependent, techniques such as liquid chromatography-tandem massspectrometry (LC-MSMS) and/or development of a SPFMV infectious clone mayprovide alternative strategies to detect P1N-PISPO in infected plants.

This study found that P1N-PISPO is a RSS which can efficiently eliminate thespread of the short distance silencing signal. Furthermore, this work showedevidence that the SPFMV P1 is also an RSS, inhibiting silencing only at local level. Inboth cases, the suppressor activity is related to the presence of a WG motif in P1-Nand could possibly be related to interaction with host Ago proteins as reported inother RSS including P1-N of SPMMV. More experimental data are necessary toelucidate the functional characteristics of P1 and P1N-PISPO. Co-inmunoprecipitation tests could verify the interaction between the host Agoprotein with P1 and P1N-PISPO. Similarly, the role of extra WG motifs in P1N-PISPOand P1 in the RSS activity or other functions in viral cycle requires further studies.This study did not elucidate if PISPO may suppress the signal without P1-N or if P1-pro may be unnecessary for P1 RSS activity. Therefore, additional experimentswhere entire domains P1-N, PISPO and P1-pro can be evaluated independently orin combinations are worthwhile.

Our study did not find RSS activity in SPFMV HC-Pro and SPLV P1. However,we cannot discard that HC-Pro has RSS activity. In potyviruses, P1 is known tostabilize HC-Pro and thus to enhance HC-Pro RSS activity. In this study, however, inorder to evaluate the role of P1and HC-Pro separately, we used a binary vectorexpressing SPFMV HC-Pro alone. This may explain the negative outcomes when

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trying to detect HC-Pro from infiltrated leaves. Perhaps, the best way to evaluateSPFMV HC-Pro RSS activity is by stabilizing it with a P1 containing non-functionalWG motif, and thus unable to suppress RNAi. On the other hand, elucidation of RSSactivity of SPLV HC-Pro could be achieved by P1 stabilization since this protein doesnot have RSS activity. Elucidation of SPLV HC-Pro RSS activity may shed more lighton the functional genomics of potyviruses infecting sweet potato and contribute tothe understanding of the possible opposite of roles of P1 and HC-Pro for RSSactivity in ‘SPFMV-group’ and other potyviruses.

Differences not only in PISPO, but other domains in P1, may affect theperformances of distinct potyviruses when infecting sweet potato, in particularduring SPCSV synergisms. Cuellar et al. (2009) established that SPCSV-RNase3protein mediates these synergisms through the suppression of the antiviral defensein sweet potato plants, allowing potyviruses to enhance titers (Cuellar et al., 2009).Nevertheless, symptoms severity, even among the “SPFMV-potyviruses” varygreatly (Gutierrez et al., 2003; Untiveros et al., 2007) which is not necessarilyrelated to the enhancement of replication of viruses (Kokkinos and Clark, 2006a).This suggests that additional viral determinants may be required for thedevelopment of unusually severe diseases in sweet potato plants. Under such ascenario, inclusion of P1 and P1N-PISPO in the equation, through complementaryactions of RNAi-suppressor proteins encoded by SPCSV and potyviruses as theultimate cause of major failure of antiviral defense and the subsequentdevelopment of diseases, may be worthwhile to assess.

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6. SUPPLEMENTARY MATERIAL

Supplementary Figure 1. Phylogenetic tree reconstructed based on completegenome nucleotide sequences of 127 potyviruses. The ‘SPFMV-group’ and ‘Sweetpotato-infecting potyviruses’ are located inside blue and red boxes, respectively.The scale bar indicates Kimura nucleotide distance. List of Genebank codes isavailable in Supplementary Table 1. Figure was electronically modified for a bettervisualization of the aforementioned groups.

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Supplementary Table 1. List of virus isolates and species of the Potyviridae familyused in Supplementary Figure 1 (recognized species are in italics, according to ICTVMaster Species List 2014 v3,http://talk.ictvonline.org/files/ictv_documents/m/msl/5208.aspx)

Species, isolate Accession No Species, isolate Accession No

Agropyron mosaic virus NC 005903.1 Leek yellow stripe virus NC 004011.1Algerian watermelon mosaic virus NC 010736.1 Lettuce mosaic virus NC 003605.1Apium virus Y NC 014905.1 Lily mottle virus NC 005288.1Arracacha mottle virus NC 018176.1 Lupine mosaic virus NC 014898.1Asparagus virus 1 NC 025821.1 Maize dwarf mosaic virus NC 003377.1

Banana bract mosaic virus NC 009745.1 Moroccan watermelonmosaic virus

NC 009995.1

Basella rugose mosaic virus NC 009741.1 Narcissus degeneration virus NC 008824.1Bean common mosaic necrosisvirus

NC 004047.1 Narcissus late season yellowsvirus

NC 023628.1

Bean common mosaic virus NC 003397.1 Narcissus yellow stripe virus NC 011541.1Bean yellow mosaic virus NC 003492.1 Onion yellow dwarf virus NC 005029.1Beet mosaic virus NC 005304.1 Ornithogalum mosaic virus NC 019409.1Bidens mosaic virus NC 023014.1 Panax virus Y NC 014252.1

Bidens mottle virus NC 014325.1 Papaya leaf-distortion mosaicpotyvirus

NC 005028.1

Blue squill virus A NC 019415.1 Papaya ring spot virus NC 001785.1Brugmansia suaveolens mottlevirus

NC 014536.1 Passion fruit woodiness virus NC 014790.1

Calla lily latent virus NC 021196.1 Pea seed-borne mosaic virus NC 001671.1Canna Yellow Streak Virus NC 013261.1 Peanut mottle virus NC 002600.1Carrot thin leaf virus NC 025254.1 Pennisetum mosaic virus NC 007147.1Cassava brown streak virus. NC 012698.2 Pepper mottle virus NC 001517.1Chilli ringspot virus NC 016044.1 Pepper severe mosaic virus NC 008393.1Chilli veinal mottle virus NC 005778.1 Pepper veinal mottle virus NC 011918.1Clover yellow vein virus NC 003536.1 Pepper yellow mosaic virus NC 014327.1Cocksfoot streak virus NC 003742.1 Peru tomato mosaic virus NC 004573.1Colombian datura virus NC 020072.1 Plum pox virus NC 001445.1Cowpea aphid-borne mosaicvirus NC 004013.1 Pokeweed mosaic virus NC 018872.2Cyrtanthus elatus virus A NC 017977.1 Potato virus A NC 004039.1Daphne mosaic virus NC 008028.1 Potato virus V NC 004010.1Dasheen mosaic virus NC 003537.1 Potato virus Y NC 001616.1Donkey orchid virus A NC 021197.1 Scallion mosaic virus NC 003399.1East Asian Passiflora virus NC 007728.1 Shallot yellow stripe virus NC 007433.1Freesia mosaic virus NC 014064.1 Sorghum mosaic virus NC 004035.1Fritillary virus Y NC 010954.1 Soybean mosaic virus NC 002634.1Habenaria mosaic virus NC 021786.1 Sugarcane mosaic virus NC 003398.1

Hippeastrum mosaic virus NC 017967.1 Sunflower chlorotic mottlevirus

NC 014038.1

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Continuation

Hordeum mosaic virus NC 005904.1 Sweet potato feathery mottlevirus CW137

KP115608.1

Iranian johanson grass mosaicvirus

NC 018833.1 Sweet potato feathery mottlevirus GJ122

KP115609.1

Japanese yam mosaic virus NC 000947.1 Sweet potato feathery mottlevirus IS90

KP115610.1

Johnsongrass mosaic virus NC 003606.1 Sweet potato feathery mottlevirus O-Arg

KF386013.1

Konjac mosaic virus NC 007913.1 Sweet potato feathery mottlevirus Piu3

FJ155666.1

Sweet potato feathery mottlevirus RC-Arg

KF386014.1 Sweet potato virus G WT325 KF790759.1

Sweet potato feathery mottlevirus 10-O

AB439206.1 Sweet potato virus G Z01001 JN613806.1

Sweet potato feathery mottlevirus Bungo

AB509453.1 Sweet potato virus G HG167 KM014814.1

Sweet potato feathery mottlevirus O

AB465608.1 Tamarillo leaf malformation virusA

NC 026615.1

Sweet potato feathery mottlevirus Ruk73

KP729265 Telosma mosaic virus NC 009742.1

Sweet potato feathery mottlevirus S

D86371.1 Thunberg fritillary virus NC 007180.1

Sweet potato latent virus NC 020896.1 Tobacco etch virus NC 001555.1Sweet potato virus 2 CW142 KP115615.1 Tobacco vein banding mosaic

virusNC 009994.1

Sweet potato virus 2 GJ118 KP115616.1 Tobacco vein mottling virus NC 001768.1Sweet potato virus 2 GWB-2 JN613807.1 Tomato necrotic stunt virus NC 017824.1Sweet potato virus 2 HN77 KP115617.1 Turnip mosaic virus NC 002509.2Sweet potato virus 2 SC6 KP115618.1 Vanilla distortion mosaic virus NC 025250.1Sweet potato virus 2 SCN20 KP115619.1 Watermelon mosaic virus NC 006262.1Sweet potato virus C C1 GU207957.1 Verbena virus Y NC 010735.1Sweet potato virus C Arg KF386015.1 Wild potato mosaic virus NC 004426.1Sweet potato virus C CW135 KP115620.1 Wild tomato mosaic virus NC 009744.1Sweet potato virus C HN52 KP115621.1 Wisteria vein mosaic virus NC 007216.1Sweet potato virus C IL JX489166.1 Yambean mosaic virus NC 016441.1Sweet potato virus C UN202 KP115622.1 Yam mild mosaic virus NC 019412.1Sweet potato virus G GWB-G JN613805.1 Yam mosaic virus NC 004752.1Sweet potato virus G IS103 KM014815.1 Zantedeschia mild mosaic virus NC 011560.1Sweet potato virus G Jesus Maria JQ824374.1 Zucchini tigre mosaic virus NC 023175.1Sweet potato virus G SC11 KP115623.1 Zucchini yellow mosaic virus NC 003224.1

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Supplementary Figure 2. Phylogenetic relationships among isolates of “SPFMV-group” and SPMMV, based on the alignment of the nucleotide sequence of the first~1.3kb of the 5’-end region of the complete genome. List of Genebank codesavailable in Supplementary Table 2.

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Supplementary Table 2. List of sweet potato-infecting potyvirus isolates used inFigure 5 and Supplementary Figure 2.

Species, isolate Accesion No Species, isolate Accesion No

Sweet potato feathery mottle virus 46b AY523546S1 Sweet potato featherymottle virus S D86371.1

Sweet potato feathery mottle virus Ch2 GU207951.1 Sweet potato virus 2 CW142 KP115615.1

Sweet potato feathery mottle virus CW137 KP115608.1 Sweet potato virus 2 GJ118 KP115616.1

Sweet potato feathery mottle virus GJ122 KP115609.1 Sweet potato virus 2 GWB-2 JN613807.1

Sweet potato feathery mottle virus IS90 KP115610.1 Sweet potato virus 2 HN77 KP115617.1Sweet potato feathery mottle virus

KmtMil GU207956.1 Sweet potato virus 2 SC6 KP115618.1

Sweet potato feathery mottle virus O-Arg KF386013.1 Sweet potato virus 2 SCN20 KP115619.1

Sweet potato feathery mottle virus Piu3 FJ155666.1 Sweet potato virus C 19-T AB509463.1

Sweet potato feathery mottle virus RC-Arg KF386014.1 Sweet potato virus C Arg KF386015.1

Sweet potato feathery mottle virus 10-O AB439206.1 Sweet potato virus C CW135 KP115620.1

Sweet potato feathery mottle virus 115/1s Y523538S1 Sweet potato virus C HN52 KP115621.1

Sweet potato feathery mottle virus B5 GU207949.1 Sweet potato virus C IL JX489166.1

Sweet potato feathery mottle virus Bungo AB509453.1 Sweet potato virus C UN202 KP115622.1

Sweet potato feathery mottle virus Fio GU207952.1 Sweet potato virus C 11-T AB509461.1

Sweet potato feathery mottle virus 19-S AB509457.1 Sweet potato virus C C1 GU207957.1

Sweet potato feathery mottle virus 2-S AB509455.1 Sweet potato virus C C18 GU207950.1Sweet potato feathery mottle virus 5-O AB509459.1 Sweet potato virus G GWB-G JN613805.1

Sweet potato feathery mottle virus M2-41 GU207953.1 Sweet potato virus G IS103 KM014815.1Sweet potato feathery mottle virus

Nigeria JQ742091.1 Sweet potato virus G JesusMaria JQ824374.1

Sweet potato feathery mottle virus O AB465608.1 Sweet potato virus G SC11 KP115623.1

Sweet potato feathery mottle virus R5 GU207954.1 Sweet potato virus G WT325 KF790759.1

Sweet potato feathery mottle virus R6 GU207955.1 Sweet potato virus G Z01001 JN613806.1

Sweet potato feathery mottle virus Ruk73 KP729265 Sweet potato virus G HG167 M014814.1

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ACKNOWLEDGEMENTS

The present study was carried out in two stages, and therefore feelings ofthankfulness are divided. The first stage was done a while ago at the Applied Biotechnology Laboratory atCIP (Peru) under supervision of Dr Jan Kreuze. We did a great job in old good daysJan! Thanks for the many opportunities you have given to me to develop my career,for your support, advices and friendshipJ. The final stage was carried out in thelast three years at the Department of Agricultural Sciences, at the University ofHelsinki under supervision of Professor Jari Valkonen. My deepest gratitude to Jari.Thanks for building up the “pispo team” and giving me the opportunity to work onthe most fascinating part of this project. Your guidance and advices have beeninvaluable along the realization of this work. I have enjoyed most of our meetingsfrom where I have learned not only tons of knowledge concerning plant diseases,but also from the hundreds of funny anecdotes you shared with meJ. I gratefully acknowledge institutions and colleagues who were directly involvedin the development of this project. Thanks to CIP and the Academy of Finland(grants # 1253126 and #1276136 to Prof. Jari Valkonen) for providing the financialsupport for this study, and to the Doctoral Program in Sustainable Use ofRenewable Natural Resources (AGFOREE) for funding my academic trips. I deeplyappreciate the efforts of my co-authors in the different papers included in thisthesis, starting with Segundo Fuentes and Dora Quispe from CIP. Thanks also to Dr.Andrew Firth, and Dr. Allan Olspert from Cambridge University for thebioinformatics support, and intense brainstorming along these last years. I extendmy appreciation to Katrin Artola who did a great job on the “pispo project” prior tomy arriving to Finland. Here, I would also like to include my pre-examiners MinnaPoranen and Anders Kvarnheden for their extraordinary job in such a tighttimeframe J. I would also like to express my gratitude to Dr. Marc Ghislain fromCIP who provided economical support at very critical stages of this project, and DrSalazar who was my first mentor in the world of plant viruses. I gladly extend my appreciation to CIP staff. Thanks for having been a constantsource of support to face challenges in my career and sharing so many“Olimpapas”J. I am thankful to my ex-colleagues Kathy, Jose Carlos, Sandra,Fernando, Dina, Heydi, Ana, Betty, Karina, Rocio, Jorge T, and in special to theformer members of the dear “Jan’s group”: Lorena, Ilanit, Ada, Elizabeth, Wilbert,Bruno and Gabriela. The technical staff are part of my deepest gratitude tooincluding Jaime, Marco, Hugito, La señora Catita, Martin, Totis, Orejitas, Gallito,Luciano P, and Luciano F. Special thanks to Wilmer Cuellar and Carl Spetz for thosechats (and beers) when needed most! Here in Helsinki time flied!, and luckily I came up to know many colleagues andfriends with whom I shared productive brainstorming, funny moments, many

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lunches and coffees, and also our disappointments when experiments failed L.Thanks to former PhD candidates from KPAT to whose Doctoral defenses Ieventually assisted. I am grateful to Tian; you always had time to reply my millionsof questions regarding viruses. Thanks to Tuuli, Mikko and Marjo for giving cluesabout the many silly questions I made, and showing that it is not 100% true thatFinns do not like chatting with strangers when they first meet one J. Thanks toIsabel for her secret protein extraction buffers, to Sascha for introducing me somany aphids, and to Minna for the endless list of advices in molecular biology.Thanks to Eva M. for being so helpful in my first days in Finland!. Thanks also to the“next generation” of PhD candidates for the company, thai cubes and now millionquestions they made. Thanks to Delfia, Veru, Yussuf and Linping. I would like toextend my gratitude to other current PhD candidates and M Sc students of the lab:to Mirko (still cannot believe you are from Chile), Marcelina, Tanja, Enni!, Jinhui,Amigo Hany, Bahram&Iman, Sussana, Monica, SuperChen!, Tomasso, Eva T., Ashok,Aurora, Lanman&Ying, Ana S, Paulina, and in special to Päivi (for the rides to andfrom work!) Life in Finland has been much easier having “latin” culture and good Finns andother foreigner as friends around me all the time. Immense gratitude is extended tomy dear dancing mates of Askelten Palo. Thanks for those amazing presentationsand having shared a little bit of our culture with Finland. Many thanks to Mi presiNieves, Isabel, Janeth and Telma (Charlie Angels), to my young dance partnerCristina!, to Rita and Daniel for the hundreds of invitations to spend time together,and to the “fantastic andean heroes”: Elias and Alex. Thanks also to Suvi, Riikka,Maiju, Kaissa, Karoliina, Tiina, Henrika and Marjaana for the Finnish culture lessonsJ. I also enjoyed so much friendship and company of Susana, Cecilia, Cynthia,Melissa (y su mama), Juha, Osquitar, Paisano Camilo, Zubair, Naseer, Rodrigo andHarold. Last but not least, I am deeply thankful to my family. We have missed so muchtime together for the last three years. Thanks to my parents Monica and Demetriofor the huge support they provided and endless Skype chats. I apologize for nothaving been there with you last Christmas and missed other great moments duringthese years. To my little brave brother Erick, you are the best man ever!, you knowhow much I admire you, thanks for taking care of our parents during my absence.Thanks to my fearless sister Monica and her Micho, I enjoyed so much having youhere in Helsinki for that day!, and I am looking forward to become an uncle soon J.

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