Poster Session Anelloviridae Epidemiology Virus Family: Anelloviridae

Category: Epidemiology/Transmission

Title: Genogroup distribution and viral load of Torquetenovirus in fecal samples

Authors: C. A. PINHO-NASCIMENTO1, J. P. G. LEITE 2, C. NIEL1, L. DINIZ- MENDES1

Address: 1Laboratório de Virología Molecular; 2Laboratório de Virología Comparada e Ambiental, Instituto Oswaldo Cruz, Fiocruz, Avenida Brasil 4365, 21040-360 Rio de Janeiro, RJ, Brasil. E-mail:[email protected]

Abstract: Torquetenovirus (TTV, floating genus Anellovirus) is a non enveloped DNA virus with circular, single-stranded genome with size of 3.8 kb. TTV is highly prevalent in populations from around the world. TTV isolates have been classified into five main phylogenetic groups (1-5) showing a large genetic distance between them. The presence of TTV has been detected in feces. However, whether all five TTV genogroups are excreted in feces and the frequency of these events are presently unknown. The presence of TTV DNA was assessed in feces from 135 Brazilian (0-90 years old) patients with gastroenteritis by using three PCR methods, including real-time PCR. One hundred and twenty one (91.1%) samples were positive with at least one method. Using a genogroup-specific assay, it was shown that all genogroups were present. Thirty-seven (27.4%), 27 (20.0%), 57 (42.2%), 29 (21.5%) and 33 (24.4%) fecal samples contained TTV isolates belonging to genogroups 1 to 5, respectively. Coinfections with two, three, four and five TTV genogroups were found in 23 (17.0%), 15 (11.1%), 7 (5.2%) and 7 (5.2%) fecal samples, respectively. Thus, 52 (38.5%) samples contained more than one TTV genogroup. Viral loads ranged from 2.6 to 6.5 log genome equivalents per gram of feces. However, only moderate variations of viral load were noted depending on genogroup and number of coinfecting TTV genogroups. These results are the first to show high prevalence and the diversity of TTV isolates in feces.

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Poster Session Anelloviridae Replication Virus Family: Anelloviridae

Category: Replication/Gene expression

Title: Transcription of TTV-HD types during in vitro virus replication

Authors: JIAN-WEI FEI, ROMANA KIMMEL AND ETHEL-MICHELE DE VILLIERS

Address: Division for the Characterization of Tumorviruses, German Cancer Research Centre, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany

Abstract: Transcription of single TTV types has previously been reported. We replicated full-length genomes and propagated virus of 12 isolates of 7 TTV-HD types in vitro. Transcripts from all cultures were analyzed using two different approaches. Single- as well as double stranded cDNA served as template for 5’- and 3’-RACE–PCR. We report a large range of transcripts resulting from varying splicing events. Our data indicate possibilities of additional proteins being formed during the replication of TT viruses. These data will facilitate future attempts to associate TTV infection with the pathogenesis of human disease.

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Poster Session Anelloviridae Replication Virus Family: Anelloviridae

Category: Replication/Gene expression

Title: Stimulation of TT replication by EBV in cells of lymphatic origin.

Authors: SILVIA S. BORKOSKY, CORINNA WHITLEY, ANNETTE KOPP- SCHNEIDER, HARALD ZUR HAUSEN AND ETHEL-MICHELE DE VILLIERS

Address: Division for the Characterization of Tumorviruses, German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany.

Abstract: Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate. Torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication of two TTV-HD isolates obtained from multiple sclerosis brain tissue in a series of EBV- positive and -negative hematopoietic cell lines. Our results demonstrate the replication of both transfected TTV genomes in all the evaluated cell lines. Quantitative amplification indicates an enhanced TTV replication in the EBV- positive cell lines in comparison to the EBV-negative Burkitt’s lymphoma cell line BJAB, suggesting a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis.

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Poster Session Anelloviridae Other Virus Family: Anelloviridae

Category: Other

Title: Assessment of human torque teno virus removal from two wastewater treatment plants

Authors: L. DINIZ-MENDES1, C.A. PINHO-NASCIMENTO1, L.R. BERGSTEN- TORRALBA1, V.S. DE PAULA3, M.P. MIAGOSTOVICH2 AND C. NIEL1

Address: 1Laboratório de Virologia Molecular; 2Laboratório de Virologia Comparada e Ambiental; 3Laboratório de Desenvolvimento Tecnológico em Virologia, Instituto Oswaldo Cruz, FIOCRUZ – Avenida Brasil, 4365 – CEP21040-360 Rio de Janeiro, RJ, Brasil. E-mail: [email protected]

Abstract: Sewage is one of the primary sources of human viruses in environment. It is thus important to investigate the effectiveness of viral removal in sewage plants. Human torque teno virus (TTV) has been suggested as a possible indicator of viral contamination in environment, due to its long-term excretion in faeces, high persistence and elevated concentrations in sewage and polluted aquatic environments. The ability of two sewage treatment plants (STP1 and STP2) to remove TTV from effluents was assessed by real time PCR, and compared to removal profile of other common viruses found in wastewaters, namely enteric adenovirus (AdV) and hepatitis A virus (HAV). In STP1, the presence of viruses was evaluated monthly, for one year, in raw and treated sewage. In STP2, samples from raw sewage, as well as after primary and secondary treatment, were assessed over 20 collections which were done twice a week. Viruses were concentrated 1000-fold from two liters of water by adsorption-elution to a negatively charged membrane. The loads of both TTV and AdV reached about 4.3 log copies per 100 mL of raw sewage in STP1, and the treatment used in this plant removed approximately 68% of the input amount of these viruses. Furthermore, 4.6 log copies of TTV and AdV, and 2.7 log copies of HAV were detected per 100 mL of raw sewage in STP2. This plant was able to eliminate about 98% of the input amount of both TTV and AdV, and 82% of HAV content. STP1 effluents contained about 3.7 log copies of TTV and AdV. Viral amounts in STP2 effluents were 2.6 log copies of both TTV and AdV and 2 log copies of HAV per 100 mL. We conclude that, despite an appreciable reduction of viral loads, notable amounts were still discharged in the environment after treatment. These results reinforce the necessity of surveillance for viruses in sewage and the establishment of a viral indicator for water quality.

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Poster Session Circoviridae G enetic Diversity/Evolution Virus Family: Circoviridae

Category: Genetic Diversity/Evolution

Title: Origin and evolution of porcine circovirus type 2 in Cuba

Authors: Lester Josué Pérez, Heidy Díaz de Arce, Martí Cortey, Patricia Domínguez, Maria I. Percedo, Carmen Laura Perera, Joan Tarradas, Maria T. Frías, Joaquim Segalés, LLilianne Ganges, José Ignacio Núñez.

Address: Centro Nacional de Sanidad Agropecuria (CENSA), La Habana, Cuba

Abstract: Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour- Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and experimental data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12 × 10−3 and 6.57 × 10−3 substitutions/site/year, which are closer to those reported for RNA viruses.

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Poster Session Circoviridae G enetic Diversity/Evolution Virus Family: Circoviridae

Category: Genetic Diversity/Evolution

Title: Discovery of a novel single-stranded DNA virus in dragonflies

Authors: K. Rosario1, M. Marinov2, D. Stainton2, S. Kraberger2, D. Martin3, M. Breitbart1, and A. Varsani2

Address: 1College of Marine Science, University of South Florida, St. Petersburg, FL, USA 2School of Biological Sciences, University of Canterbury, Chirstchurch, New Zealand 3Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Rondebosch, South Africa

Abstract: To date, no circular single-stranded DNA (ssDNA) viruses infecting insects have been described. Here we report a new species of ssDNA virus isolated from dragonflies (Libellulidae family) using a viral metagenomic approach. The novel virus was identified in three dragonfly species (Pantala flavescens, Tholymis tillarga, and Diplacodes bipunctata) collected in the South Pacific Islands of Tongatapu and Vava’u, Kingdom of Tonga. Initially viral particles were purified from the homogenates of the abdomens of three P. flavescens individuals using filtration. Nucleic acids were extracted from the purified viral fraction and amplified using multiple displacement amplification (MDA). MDA products were fragmented and further amplified using a Whole Genome Amplification (WGA) kit. Cloning and sequencing of WGA products revealed several sequences with significant similarities to members of the recently described Cyclovirus genus within the Circoviridae family. PCR primers were designed to recover unit length full genomes of the dragonfly cycloviruses through inverted PCR. In total, thirty full genomes were recovered, cloned and sequenced from the three dragonfly species. These cyclovirus genomes share greater than 95% nucleotide identity indicating the presence of a single strain in the Libellulidae family members screened. The new Drangonfly cycloviruses (DFCyV) share approximately 63% full genome nucleotide identity to a cyclovirus genome identified in human stool (69% within the Rep region). DFCyV have greater nucleotide identity to other recently characterized cycloviruses (48.3 - 63%) than to circoviruses (41 - 46.9%). The DFCyV genomes encode putative replication (Rep) and capsid (CP) proteins arranged in an opposite orientation and contain a stem loop structure and nonanucleotide motif. Recombination analysis of the DFCyV genomes revealed two recombination events, one within the CP and another spanning the Rep, CP, and intergenic region. To our knowledge this is the first report of a circular ssDNA virus infecting insects, which may help elucidate evolutionary links between cycloviruses and circoviruses.

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Poster Session Circoviridae G enetic Diversity/Evolution Virus Family: Circoviridae

Category: Replication/Gene expression

Title: Identification of replication specificity determinants in eukaryotic ssDNA viruses

Authors: A. LONDOÑO, L. RIEGO-RUIZ, G.R. ARGUELLO-ASTORGA

Address: Instituto Potosino de Investigación Científica y Tecnológica, A.C. Camino a la Presa San José 2055, Colonia Lomas 4ta. Sección, San Luis Potosí, S.L.P., México C.P. 78216. E-mail: [email protected]

Abstract: A huge assortment of genetic entities multiply their genomes by the mechanism of rolling circle replication (RCR). These systems encode a replication initiation protein (Rep) that binds DNA in a sequence specific fashion and possesses DNA nicking-closing activity. A large superfamily of Rep proteins share certain amino acid sequences (Motifs I-III) that are arranged in a characteristic way, and are encoded by several lineages of prokaryotic and eukaryotic replicons, including ssDNA viruses of plants and animals, plasmids of red algae and protozoos, and phages and plasmids of bacteria and archeas. We have used a comparative approach based on several heuristic hypotheses to identify, in the Rep proteins from four groups of ssDNA viral systems (circoviruses, nanoviruses, geminiviruses and alphasatellites), the amino acid residues that presumably determine their high- affinity DNA binding specificity. These putative ‘‘specificity determinants’’ (SPDs) cluster in two discrete protein regions, which are adjacent to distinct RCR conserved motifs. Modeling of the tertiary structure of several Rep proteins showed that SPD regions interact to form a small beta-sheet element that seems to be critical for specific DNA-binding. Our findings suggest that SPDs present in the replication proteins from an enormous diversity of RCR systems, including new circovirus-like genomes identified in metagenomic studies, are associated with the same Rep conserved motifs.

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Molecular characterization and phylogenetic relationships of two novel begomoviruses infecting weeds in Brazil

Authors: A.T.M. LIMA1, D.R. BARROS1, F.N. SILVA1, P. ALFENAS-ZERBINI1, C.S. ROCHA1, E.W. KITAJIMA2 & F. M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; 2NAP/MEPA, ESALQ-USP, Piracicaba, SP, 13418- 900, Brazil

Abstract: Begomoviruses (family Geminiviridae) have a circular, single-stranded DNA genome encapsidated in twinned icosahedral particles. In Brazil, a number of begomoviruses infecting weeds and wild hosts have been described, and available evidences suggests that viruses present in these hosts have given rise to the viruses currently found in crop plants. In this study we describe two novel begomoviruses infecting weeds. One Desmodium tortuosum plant (family Fabaceae) with mosaic and yellow spots was collected in the city of Belo Horizonte, MG, in December 2008. Leaves of a very old Malvaviscus arboreus plant (approximately 50 years old; family Malvaceae) showing a bright yellow mosaic were collected at the experimental farm of the Campinas Agronomical Institute (IAC), in Campinas, SP, Brazil, in May 2005. Total DNA was extracted from each sample and viral genomes were amplified by RCA, cloned and sequenced. Sequence analysis (DNA-A) indicated that both viruses corresponded to novel begomoviruses, for which the names Desmodium yellow spot virus (DesYSV) and Malvaviscus yellow mosaic virus (MalYMV) are proposed. Based on phylogenetic analysis, DesYSV clustered with other begomoviruses from Brazil that infect weeds and tomatoes. Although MalYMV has been collected in Brazil, this isolate has closer phylogenetic relationship with begomoviruses from Central and North America. Additionally, MalYMV has an identical nanovirus and alphasatellite nonanucleotide (TAGTATTAC). The M. arboreus plant has been displaying the observed yellow mosaic symptoms at least since the 1960's (as noted by the sixth author), which suggests that MalYMV maybe poorly transmitted (or not transmitted at all) by local whitefly vector populations.

Financial support: National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Begomoviruses infecting weeds in Bolivia

Authors: A.T.M. LIMA1, F.N. SILVA1, C.S. ROCHA1, T.F.S. ANTUNES1, D.R. BARROS1, G.P. ROSALES2, N. ORTUÑO2, A. GANDARILLAS2, R.O. RESENDE3 AND F. M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; 2Fundación PROINPA, Av. Meneces s/n. Km. 4, Zona El Paso, Cochabamba, Bolivia; 3Dep. de Biologia Celular/Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.

Abstract: The genus Begomovirus (family Geminiviridae) includes dicot-infecting, whitefly-transmitted viruses with the genome comprised of one or two molecules of circular, single-stranded DNA. Begomoviruses cause serious damage to crops in several Latin American countries, and are also associated with the wide range of wild and weed hosts. However, information on the occurrence of begomoviruses is lacking in some countries, such as Bolivia. Therefore, the aim of this study was to assess the presence of begomoviruses infecting weeds in Bolivia. Total DNA was extracted from 35 weed samples collected in March of 2009 in tomato, pepper and bean fields in the area known as the "Mesothermal Valleys" (towns of Comarapa, Saipina, San Isidro and Pulquina in the Province of Manuel M. Caballero, Department of Santa Cruz). The RCA-amplified DNA was cleaved with BamH I and Hind III, cloned and completely sequenced. Sequences were compared to those of previously characterized begomovirus species, and the ICTV-established 89% DNA-A identity threshold was used to determine taxonomic placements. Out of nine clones sequenced so far, four obtained from Sida spp. and one from Physalis sp. displayed 95% identity with the recently described begomovirus Sida mosaic Brazil virus (SiMBV). Bean golden mosaic virus (BGMV) was found in four unidentified (most likely leguminous) weeds. These initial results demonstrate the existence of begomoviruses infecting wild hosts in Bolivia, and suggest that there is a low species diversity among weeds. We are currently analyzing the presence of begomoviruses in tomato, pepper and bean samples collected at the same fields.

Financial support: PROSUL-CNPq, National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Malvaceous plants are infected with novel Begomovirus species

Authors: M. M. S. ALMEIDA1,2, L. V. HOFFMANN3, J. C. BARBOSA4, S. A. ARANHA1,2, AND A. K. INOUE-NAGATA1,2

Address: 1Universidade de Brasília, 70910-900 Brasília, Brazil; 2Embrapa Vegetables, BR 060, Km 09, C.P. 218, 70359-970, Brasília, Brazil; 3 Embrapa Algodão, GO 462, Km 12, zona rural, C.P. 179, 75375-000, Santo Antônio de Goiás, Brazil; 4ESALQ/USP – Departamento de Fitopatologia e Nematologia, Piracicaba, Brazil.

Abstract: In Brazil, malvaceous plants are widely spread throughout the country, as weeds or wild species. It is common to see clear mosaic symptoms on these plants, and invariably they are infected by begomoviruses. However, it is rare to observe virus-like symptoms on cultivated malvaceous plants. Here, we carried out a survey on begomovirus occurrence in three malvaceous plants: okra, cotton and hibiscus plants. As expected, only a few infected plants were found. The genome sequence of these begomoviruses (DNA-A and DNA-B, 2646 to 2711 nucleotide- long) was analyzed after cloning of rolling circle amplified products of each DNA sample. Their DNA-A sequences were less than 88% identical to each other, suggesting that distinct begomoviruses were present on these samples. From okra plants, a DNA-A component sequence with 88% nucleotide identity to Sida micrantha mosaic virus was found, suggesting the presence of a new begomovirus species. From this sample, the DNA-B sequence shared 89% nucleotide identity with Sida micrantha mosaic virus too, the best matched sequence. From cotton plants, a DNA-A sequence sharing the maximum nucleotide identity of 81% with Tomato common mosaic virus and a DNA-B sequence sharing 74% identity with Chino del tomate virus were found. Finally, from Hibiscus sp. plants, the DNA-A sequence was 83% identical to Tomato yellow spot virus and the DNA-B 78% to Tomato golden mosaic virus. It was clear that new begomovirus species are occurring on cultivated malvaceous plants in Brazil, and may cause future epidemics in Brazil and surrounding countries. A detailed analysis is being carried on with these sequences, some of them lacking some particular characteristics commonly found on geminiviruses, such as the conserved nonanucleotide motif.

Financial support: CNPq, Embrapa, National Research Institute for Plant-Pest Interactions

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Circoviridae

Category: Genetic diversity / Evolution

Title: Beak and feather disease virus: Global genetic diversity, geographical and host species distribution of isolates Authors: Arvind Varsani1,2, Guy L. Regnard3, Inga I. Hitzeroth3, Robert R. Bragg4, Edward P. Rybicki3,5

Address: 1 School of Biological Sciences, University of Canterbury, Christchurch, 8140, New Zealand, 2 Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa, 3 Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, 7701, South Africa, 4 Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, 9301, South Africa, 5 Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town, 7925, South Africa

Abstract: Psittacine beak and feather disease (PBFD) has a broad host range and is widespread in both wild and captive psittacine populations in Asia, Africa, the Americas, Europe and the Pacific region (mainly New Zealand and Australia). Beak and feather disease virus (BFDV) is the causative agent of PBFD. It belongs to the genus Circovirus, family Circovirdae, and has a ~2 kb single stranded DNA genome coding for two proteins (Rep and CP). In this study we provide support for accurate demarcation of BFDV strains by analysing 66 full genomes present in Genbank, as well as 22 new BFDV sequences isolated from infected psittacines in South Africa, for the frequencies of pairwise identities and maximum likelihood phylogeny prediction. We propose a 94% genome-wide sequence identity as a strain demarcation threshold, with isolates that share >94% genome wide sequence identity belonging to the same strain, and sequences that share >98% being considered subtypes of the strain. Our analysis indicates that current BFDV diversity falls within 14 strains, and that the 5 highly divergent isolates from budgerigars should be considered members of a new species of circovirus (Budgerigar circovirus; BCV) and fall within 3 strains (-A, -B and –C). We determined that the distribution of the BFDV and BCV strains cannot clearly be linked to geography or host species. It seems that the geographic distribution is rather linked to the international trade in exotic birds, and that the viral strains with more than one host are generally located in the same geographical area. Lastly, we examined the BFDV and BCV sequences for evidence of recombination, and determined that recombination has occurred in most BFDV and BCV strains. We established that there were two globally significant recombination hotspots, the first being along the entire intergenic region and the second in C-terminal portion of the CP ORF.

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Molecular characterization of two new strains of Okra yellow mosaic Mexico virus

Authors: Y. CARDENAS-CONEJO, M.CANTU-IRIS, S. AMBRIZ-GRANADOS, AND G. ARGÜELLO-ASTORGA

Address: Instituto Potosino de Investigación Científica y Tecnológica, A.C. Camino a la Presa San José 2055, Colonia Lomas 4ta. Sección, San Luis Potosí, S.L.P., México C.P. 78216. E-mail: [email protected]

Abstract: Sida rhombifolia and Herissantia crispa plants showing yellow mosaic symptoms were collected in Colima, Mexico. Total DNA was isolated from symptomatic plants, and begomovirus universal primers were used for the amplification of viral DNA-A and DNA-B by polymerase chain reaction (PCR). Overlapped PCR products encompassing the complete individual genomic components were cloned and sequenced. Analysis from H. crispa plant samples revealed just a single begomovirus (GenBank Acc.No. GU990614). When compared with sequences from other geminiviruses, its DNA-A displayed a 90% sequence identity with Okra yellow mosaic Mexico virus (OkYMMV) from Mezatepec, Mexico; therefore, it was considered to be a new strain of this virus species. On the other hand, a S. rhombifolia plant, which displayed an exceptionally intense leaf mosaic phenotype, contained two different begomoviruses. The first virus (Acc.No. GU990612) was very similar (97% identity) to OkYMMV-[Herissantia], whereas the second one (Acc.No. GU990613) had a lower nucleotide identity (91%) with that virus and only 88% sequence identity with OkYMMV-[Mezatepec]. Consequently, this second begomovirus in the examined S. rhombifolia plant represents a new strain, OkYMMV-[Sida], which is the third strain identified from this malvaceous-infecting begomovirus species. Surprisingly, two of these OkYMMV strains exhibited iterons (i.e., Rep-binding sites) with a nucleotide sequence GGTACACA that is unique among the New World begomoviruses, while the OkYMMV-[Sida] displayed clearly different iterons (i.e., GGAGTA). This observation suggests a probable replication incompatibility between the latter and the former two strains, that could explain the stable coexistence of two OkYMMV strains in a single S. rhombifolia plant.

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy / Emerging viruses

Title: Genetic diversity and synergistic interaction among newly emerging begomoviruses associated with chilli leaf curl disease in India

Authors: S. Chakraborty, A. K. Singh, R. Vinothkumar, N. Khushwaha, B. Chattopadhyay

Address: Molecular Virology laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110 067.

Leaf curl disease of chilli (ChiLCD) is a major constraint for successful chilli (Capsicum annuum) production in India. We have cloned full-length genomes of DNA-A, DNA-B and satellite DNA-βs of 26 chilli-infecting begomovirus isolates from major 15 chilli growing states of the country. Based on phylogenetic analyses of the DNA-As, we have identified association of ten distinct species of begomoviruses viz., Chilli leaf curl Multan virus (ChiLCV-Mul), Chilli leaf curl India virus (ChiLCV-In), Pepper leaf curl Bangladesh virus (PepLCBDV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl New Delhi virus, Tomato leaf curl Gujarat virus (ToLCGV), Tomato leaf curl Karnataka virus, Croton yellow vein mosaic virus including two new species reported in this study. Out of these, four begomovirus species viz., Chilli leaf curl Multan virus, Chilli leaf curl India virus, Pepper leaf curl Bangladesh virus and Tomato leaf curl Joydebpur virus are the predominant ones associated with ChiLCD throughout the country, without being restricted to any particular geographical area. Presences of betasatellites were ubiquitously observed with the disease. Based on nucleotide sequences, betasatellites were categorized into four distinct species viz., Tomato leaf curl Bangladesh betasatellite, Tomato leaf curl Joydebpur betasatellite, Tobacco leaf curl betasatellite and Croton yellow vein mosaic betasatellite. Association of DNA- B was detected in fewer samples. Mixed infection of genomic components of ChiLCV-Mul & ToLCNDV (in New Delhi), ToLCJoV & PepLCBDV (in Ghazipur), and ToLCJoV & ToLCGV (in Kolkata) were found to be associated with this disease. Synergistic interaction among chilli begomoviruses resulted in occasional breakdown of host resistance of hitherto known resistant cultivars. Molecular diversity of these chilli-infecting begomoviruses and mechanism of synergistic interaction leading to severe leaf curl disease will be presented.

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging virus

Title: Molecular characterization of a novel begomovirus infecting the weed Corchorus hirtus in Brazil

Authors: R. S. FONTENELE1, 2, R. BLAWID1, 2, S. G. RIBEIRO 1

Address: 1Embrapa Recursos Genéticos e Biotecnologia, Pq Estação Biológica, Brasília, Brazil, 2Universidade de Brasília, Instituto de Biologia, Brasília, Brazil.

Abstract: Corchorus spp are indigenous Brazilian weeds. The genus is distributed throughout all regions of the country. Weeds are supposed to be the original hosts and could be reservoirs of begomoviruses. Therefore, it is important to evaluate the presence and identity of the viruses infecting weeds. A sample of Cochorus hirtus L. showing yellow mottle symptoms was collected in the state of Paraiba, Brazil. Following PCR confirmation of begomovirus infection, viral DNA was amplified by rolling circle amplification (RCA) using Phi-29 DNA polymerase and cloned in pBluescript SK+ vector at the Kpn I site. Two clones containing the expected insert of approximately 2.6 kbp were obtained and sequenced. Clone 62k-2 was 2,650 nucleotides-long and corresponded to the DNA-A, possessing the typical bipartite, New World begomovirus ORFs: one in the sense orientation (AV1) and four in the complementary sense (AC1, AC2, AC3 and AC4). Clone 62k-3 was 2,611 nucleotides-long and corresponded to a DNA-B molecule also possessing the typical begomovirus ORFs: BV1 on the virion strand and BC1 on the complementary strand. The common region of these two components comprised 170 nt with 93% identity, suggesting that the two clones correspond to cognate components of the same virus. The DNA-A and DNA-B shared 88% and 87% nucleotide identity, respectively, with Abutilon Brazil virus (FN434438 and FN434439). Considering the ICTV-defined species demarcation threshold of >89% identity for the complete DNA-A sequence, this virus represents a new virus species infecting Corchorus hirtus, and the name Corchorus mottle virus (CoMoV) is proposed. Infectivity tests to fulfill Koch's postulates and biological characterization are in progress.

Financial support: Embrapa, National Research Institute for Plant-Pest Interactions; CNPq.

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Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus family: Geminiviridae

Category: Virus taxonomy/Emerging viruses

Title: Characterization of two newly discovered curtoviruses isolated from spinach in south-central Arizona

Authors: Cecilia Hernández-Zepeda1, Gerardo Arguello-Astorga2, and Judith K. Brown1 Address: 1School of Plant Sciences, The University of Arizona, 85721, Tucson, AZ USA; 2Division de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A.C. San Luis Potosí, México Abstract: In 2009, a commercial spinach field in south-central Arizona developed geminivirus-like disease symptoms. Total DNA extracts from spinach plants were used as template for rolling circle amplification (RCA) of the full-length viral genomes. RCA products were digested using a restriction enzyme (endonuclease) that was capable of linearizing the genomes, and cloned to obtain two full-length curtovirus genomes, each of approximately 3 Kbp in size. One curtovirus was cloned using PstI, and upon determining the complete DNA sequence, was found to be 3065 bases in size. Alignment (ClustalV; MegAlign, DNASTAR) with sequences of all curtovirus species available in GenBank indicated that this spinach isolate shared the highest nt sequence identity, at 59%, with Horseradish curly top virus (HrCTV). The next closest relatives were Beet mild curly top virus, Beet severe curly top virus, and Spinach curly top virus, at 50%. This isolate has been tentatively named Spinach severe curly top virus-[AZ] (SSCTV-[AZ]){Genbank No. GU734126}. The genome consists of six open reading frames and contains no discernable AC3 ORF, an arrangement most similar to HrCTV, among other curtoviruses. SSCTV infectious 1.5-mer clones were constructed and used to Agro-inoculate Nicotiana benthamiana seedlings (6-8 leaf stage). Severe leaf distortion symptoms were observed in 4 of 7 plants 7 days post inoculation (dpi), whereas mock-inoculated plants were asymptomatic (0/2). A second curtovirus was cloned using NdeI, and its genome was determined by DNA sequencing to consist of 2860 bases. This isolate has been named Spinach Arizona curly top virus (SACTV). ClustalV alignment of SACTV with other well- studied curtovirus species and SSCTV indicated that it shared its highest nt sequence identity, at 66%, with Beet curly top Iran virus (BCTIV). The SAZCTV genome has three virion-sense and two complementary- sense ORFs, however, neither a C3 nor a C4 ORF could be located on the SACTV genome, indicating that among extant curto-like viruses, its gene organization is most like that of BCTIV from the Middle East, instead of other extant New World curtoviruses. Consistent with this observation is that SACTV contains the same novel nonanucleotide as does BCTIV, which is the sequence: TAAGATT/CC. Phylogenetic analysis placed each Arizona spinach isolate into a separate subclade, with the SSCTV being a sister species to HrCTV, and SACTV as sister species to BCTIV. Preliminary recombination analysis indicated two possible recombinant fragments comprising: (i) the portion of the SSCTV genome containing the intergenic region and Rep, which has probably arisen from interspecific recombination with the HrCTV, and (ii) a small fragment containing iterons identical in sequence to begomoviruses from the Squash leaf curl virus (SLCV) clade.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: TYLCV in Northern Mexico, an emerging pathogen of tomato crops is found in single or mixed infection with native begomoviruses.

Authors: Méndez-Lozano J., Romero-Romero, J.L., Camacho-Beltrán E., Orduño- Vega, W., Gámez-Jiménez, C., Magallanes-Tapia, M.A., and Leyva-López, N.E.

Address: Departamento de Biotecnología Agrícola, CIIDIR Unidad Sinaloa del Instituto Politécnico Nacional. Blvd. Juan de Dios Bátiz Peredes 250, C.P. 81101. Guasave, Sinaloa, México.

Abstract: Viral diseases are an important limiting factor in many crop production systems. The trade of plant materials enhances the risk of introducing new viruses into a different agriculture area. In addition, plant virus interaction with natives virus and their vectors can contribute to a successful spread of a newly introduced virus in areas that were previously not present. Tomato yellow leaf curl virus (TYLCV) is one of the most devastating viral diseases of cultivated tomato (Lycopersicon esculentum) in tropical and subtropical regions worldwide. Tomato is an economically important vegetable crop in Sinaloa, Mexico and became cultivated extensively in the Northern states of in Baja California, Nayarit, Durango and Coahuila. Begomoviruses diseases have been associated to different crops in Sinaloa for the last decades. However, the incidence of begomovirus infections in tomatoes and others Solanaceous crops has severely increased following the introduction of TYLCV in the lately 2005. In this work a survey was performed in tomato crops for the last 5 years in order to monitor the spread and establishment of TYLCV. Infections of TYLCV and others native begomovirus were identify as single or mixed infection in tomato crops.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: New Emerging Viruses

Veni Vidi Vici- The tale of the Cotton leaf curl Burrewala virus in India

Prem Rajagopalan1, Prashanth Katturi1, Amruta S. Naik1, Meera Kurulekar1, Ravi Kankanallu2, Radha Anandalakshmi1, 1-Plant-Virus Interactions Lab, Mahyco Research Center, Post Box no. 76, Jalna - 431203, India, 2- Vegetable Research Center, Mahyco, Bengaluru, India

Cotton leaf curl disease (CLCuD) is the major limitation to cotton production in the Indian subcontinent. We conducted surveys for Cotton leaf curl viruses (CLCuV) incidence in the Northwestern cotton growing belt of India spread over in the states Punjab, Haryana and Rajasthan during the 2009 and 2010 cropping seasons. We have characterized partial and complete genomes of several CLCuV isolates collected during these two years. In addition, to the earlier reported CLCuV from India, we have documented the dominance of the Gossypium hirsutum CLCuD resistance breaking Cotton leaf curl Burewala virus (CLCuBV, Amrao etal , 2010) from many fields. The spread and establishment of the mutant CLCuV in North India, the variation in the genomic sequence, the virus fitness and implications for cotton breeding will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging virus

Title: Molecular characterization of an isolate of bean golden mosaic virus infecting soybean in Argentina.

Authors: P.E RODRÍGUEZ-PARDINA1, B. MÁRQUEZ-MARTÍN2, I.G. LAGUNA1 AND J. NAVAS-CASTILLO2

Address: 1 INTA-IFFIVE, Camino 60 Cuadras Km 5 ½, X5020ICA Córdoba, Argentina. 2 Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), 29750 Algarrobo-Costa, Málaga, Spain.

Abstract: Both whiteflies and begomoviruses are limiting factors for soybean production in Argentina. Three begomoviruses have been found infecting this crop: tomato yellow spot virus, Euphorbia mosaic virus and a novel virus for which the name soybean blistering mosaic virus has been proposed. Specific DNA probes were developed to differentiate these three begomoviruses, and were used to analyze field samples. Some symptomatic plants did not react with either of the probes. One of these samples, collected in Campichuelo (Salta province), was selected for further analysis. Total DNA was extracted by the CTAB method and amplified by RCA using φ29 DNA polymerase. A DNA fragment obtained after digestion with SalI with a size corresponding to a full genome component was cloned into pBluescript II SK. Two clones, S27(1) and S27(4), which differed in restriction enzyme pattern, were fully sequenced. Nucleotide similarity comparisons demonstrated that clone S27(1) had the highest nucleotide identity (96%) with DNA-A of a bean golden mosaic virus (BGMV) isolate from Brazil. Clone S27(4) had 96% nucleotide identity with DNA-B of the same virus isolate. To our knowledge, this is the first report of BGMV infecting soybean in Argentina. The epidemiological importance of this finding is clear due to the fact that, in the Northwest region of Argentina, bean and soybean crops share production areas. Earlier sown soybean could serve as inoculum source for bean crops.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Tomato severe rugose virus and Tomato mild mosaic virus detected in weeds in natural infections in Southeastern Brazil

Authors: F.N. SILVA, C.S. ROCHA, P. ALFENAS-ZERBINI, G.P. CASTILLO- URQUIZA, A.T.M. LIMA & F. M. ZERBINI

Address: Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil

Abstract: Begomoviruses (family Geminiviridae) are currently the most economically important viruses in tomato crops in Brazil. They are present in all major tomato-growing areas of the country, in both fresh market and processing tomatoes. So far, 11 distinct viral species have been detected (plus 5 tentative species), all indigenous to Brazil. Previous results by other groups indicated that weeds may act as a source of novel viruses, as well as begomovirus reservoirs. Viruses originally infecting weeds can be whitefly-transmitted to tomatoes, where they may rapidly adapt, giving rise to novel species. Large-scale sampling of tomato fields in the states of Goiás, Minas Gerais, Rio de Janeiro and São Paulo, the four main tomato producing states of the country, indicates a clear distinction between viruses found in tomatoes and those detected in associated weeds. Here, we report the detection in weeds of two of the most prevalent begomoviruses in tomatoes. Symptomatic weed samples were collected in an experimental tomato field at Universidade Federal de Viçosa, Viçosa, Minas Gerais, in May 2010. Total DNA was extracted from each sample and complete viral genomes were amplified by RCA using phage phi29 DNA polymerase, cloned into plasmid vectors and completely sequenced. Sequences were compared to those of previously characterized begomovirus species, and based in the ICTV-established threshold of 89% identity for the DNA-A, we identified Tomato mild mosaic virus (ToMlMV) infecting Sida urens and Sida sp., and Tomato severe rugose virus (ToSRV) infecting Physalis sp. ToSRV is one of the most prevalent begomoviruses in tomato crops throughout the country. To our knowledge, these viruses had never before been detected in weeds. Our results indicate that Brazilian begomoviruses which are well adapted to tomato crops can reinfect weeds under field conditions. Analysis of a larger number of weed samples should indicate whether they can act as effective begomovirus reservoirs.

Financial support: National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Isolation and characterization of a pepper-infecting curtovirus from Mexico

Authors: M. TAJA, J.A. MAURICIO-CASTILLO, B. BAÑUELOS-HERNANDEZ AND G.R. ARGUELLO-ASTORGA

Address: Instituto Potosino de Investigación Científica y Tecnológica, A.C. Camino a la Presa San José 2055, Colonia Lomas 4ta. Sección, San Luis Potosí, S.L.P., México C.P. 78216. E-mail: [email protected]

Abstract: Geminiviruses in the genus Curtovirus are vectored by the beet leafhopper (Circulifer tenellus) and infect a wide range of dicotyledonous plants. Most of the curtoviruses known until now have been isolated and characterized in the United States; however, none of the curtoviruses isolated in Mexico have been previously characterized at the molecular level. To search for curtoviruses in the dry lands of North-Central Mexico, where the viral vector is broadly distributed, we designed two sets of degenerated primers that made feasible the amplification by PCR of the complete genomic DNA of those viruses. Pepper plants displaying upwardly rolled leaves, yellowing and stunted growth were collected during the summer of 2006 in commercial fields of San Luis Potosi, Mexico. Several curtovirus-positive samples were found, and PCR-RFLP analyses showed that a single type of virus was present in all of them. The complete genome from this curtovirus was sequenced (GenBank No. EU193175). Full-length genome comparisons revealed that the isolate was a new strain of Beet mild curly top virus (BMCTV) that shared 88.1% nucleotide sequence identity with its closest relative BMCTV-[Worland]. Moreover, to determine experimentally the host range of BMCTV-[Mexico], the multimeric construct pBMCTV-[MX]1.2 was generated. This infectious clone was inoculated by either microparticle bombardment or mechanical transmission into Nicotiana benthamiana, Serrano pepper and chard seedlings. Infected plants of all the species developed disease symptoms. The known curtoviruses are geographically restricted to the North Hemisphere, and BMCTV- [Mexico] is the member of this viral group which has been isolated closer to the earth tropical belt.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/ Emerging viruses

Title: Identification of a Begomovirus weed (Allosidastrum cf pyramidatum (Lav.) Kropov) associated with the tomato crop in Colombia

Authors: J. C. VACA-VACA AND K. LOPEZ-LOPEZ.

Address: Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia. Palmira, Valle del Cauca, Colombia.

Abstract: Native species, acting as reservoirs can play an important role in the emergence of plant virus epidemics. However, studies to understand the genetic structure and dynamics of Begomovirus populations in wild reservoirs and possible effects on epidemics of cultivated species are scarce and less detailed. Since 2008 we are conducting a survey in order to detect and identify Begomovirus which are affecting tomato crops in Colombia. During this survey we found a perennial weed closer to tomato crops which exhibit bright or diffuse yellow mosaic patterns on leaves that are like those associated with diseases caused by geminivirus. First of all we identify the weed related to family Malvaceae and also it was new botanical specimen never seen or studies before in Colombia. For this reason it was called Allosidastrum cf pyramidatum (Lav.) Kropov. Then pieces of young leaves of this perennial weed showing above symptoms were recollected and total DNA was extracted. Begomovirus infection in these plants was confirmed by PCR using pairs of primers: PAL1v1978/PAR1c496 and PBL1v2040/PCRc1, which direct the amplification of regions approximately 1.2 kb DNA-A kb and 0.6kb DNA-B, respectively. These fragments were cloned and sequenced. Partial bioinformatics sequence analysis confirmed that those belongs bipartite Begomovirus (Geminivirus Family). When CP nucleotide sequence clone was submitted to a blastn search identity was closer to 86% identical with Bean dwarf mosaic virus. According to the analysis of the sequence of the cloned fragment of the geminivirus that causes bright or diffuse yellow mosaic patterns on leaves of Allosidastrum cf pyramidatum (Lav.) Kropov this could eventually be a different, previously unreported virus.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/ Emerging viruses

Title: First Identification of a Begomovirus infecting yellow passion fruit in Colombia.

Authors: J. C. VACA-VACA, J. F. BETANCUR-PEREZ, AND K. LOPEZ-LOPEZ.

Address: Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia. Palmira, Valle del Cauca, Colombia.

Abstract: The Yellow Passion fruit (Passiflora edulis var. flavicarpa) is one of the most important tropical fruit consumed word-wide. Colombia is the third largest producer of passion fruit in the world. Around seven species of virus are recorded as causing disease on yellow passion fruit. In Colombia just virus which belongs to Tymovirus and Potyvirus family has been identified affected this fruit. Since February 2010 a high incidence of plant exhibiting intense yellow mosaic symptoms of leaves, drastic reduction of the leaf lamina and fruit deformation has been observed in a fruit crops located on La Union (Valle de Cauca-Colombia). Pieces of young leaves showing above symptoms were recollected and total DNA was extracted. Begomovirus infection in these plants was confirmed by PCR using pairs of primers: PAL1v1978/PAR1c496 and PBL1v2040/PCRc1, which direct the amplification of regions approximately 1.2 kb DNA-A kb and 0.6kb DNA-B, respectively. These fragments were sequenced, and partial bioinformatics sequence analysis confirmed that those belongs Begomovirus genus (Geminivirus Family) but these virus are not related to Begomovirus previously identified in Brazil founded affecting yellow passion fruit up there. To our knowing this is the first report of a bipartite Begomovirus which are affecting the passion fruit crop in Colombia. Further research has been done in order to get the complete genome in order to get its identity.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy / Emerging viruses

Title: Divergent monopartitite geminiviruses: Eragrostis curvula streak virus and Turnip curly top virus

Authors: Arvind Varsani1,2, Dionne N Shepherd3, Kyle Dent 2,3, Aderito L Monjane3, Edward P Rybicki 3,4, Jahangir Heydarnejad5, Fakhrosadat Khosrowfar6, Hossain Massumi5, Rob W. Briddon7, Darren P Martin4

Address: 1School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand; 2Electron Microscope Unit, University of Cape Town, Rondebosch, Cape Town, , South Africa; 3Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa; 4Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town, South Africa; 5Department of Plant Protection, College of Agriculture, Shahid Bahonar University of Kerman, Kerman, Iran; 6Plant Protection Management, Fars Province, Shiraz, Iran; 7National Institute for Biotechnology and Genetic Engineering, Jhang Road, P.O. Box 577, Faisalabad, Pakistan

Abstract: We have characterised two new highly divergent geminivirus species, Eragrostis curvula streak virus (ECSV), found infecting a hardy perennial South African wild grass and Turnip curly top virus (TCTV), found infecting turnip crops in Fars province of Iran.

The ECSV genome is predicted to express a replication associated protein (Rep) from an unspliced complementary strand transcript that is most similar to those of begomoviruses,curtoviruses and topocuviruses. Similarly, while ECSV has the same unusual TAAGATTCC virion strand replication origin nonanucleotide found in another recently described divergent geminivirus, Beet curly top Iran virus (BCTIV), the rest of the transcription and replication origin is structurally more similar to those found in begomoviruses and curtoviruses than it is to those found in BCTIV and mastreviruses. ECSV also has what might be a homologue of the begomovirus transcription activator protein gene, a mastrevirus-like coat protein gene and two intergenic regions. Although it superficially resembles a chimaera of geminiviruses from different genera, the ECSV genome is not obviously recombinant, implying that the features it shares with other geminiviruses are those that were probably present within the last common ancestor of these viruses.

In the case of TCTV, its genome arrangement in the complementary-sense is similar to that of curtoviruses (consisting of four overlapping genes) but it has only two open reading frames in the virion-sense (the curtoviruses encode three).The complementary-sense genes are homologuess of those found in curtoviruses but, with the exception of the C4 ORF, show little sequence identity to these genes,. In the virion-sense the predicted product of the V2 open reading frame of TCTV shows no significant similarity with any proteins in the databases whereas the coat protein shows low levels of sequence identity to the CPs of curtoviruses.

These findings reveal that ECSV and TCTV each represent new genus-level geminivirus lineages, displaying mixtures of features normally associated with other specific geminivirus genera.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Taxonomy/Emerging viruses

Virus Family: Geminiviridae

Category: Taxonomy/Emerging viruses

Title: Characterization of a new begomovirus infecting Solanum lycopersicum in Venezuela.

Authors: K.A ZAMBRANOa, J. CARRILLO-TRIPPb, O. CARBALLOa, R.F. RIVERA- BUSTAMANTEb, AND E. MARYSa

Address: aCentro de Microbiología y Biología Celular, Instituto Venezolano de Investigaciones Científicas (IVIC), Panamericana Km 11, Caracas, Venezuela. bDepartamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6, Libramiento Norte, P.O. Box 629, C.P. 36500,Irapuato, Guanajuato, México.

Abstract: In Venezuela, an important food crops, tomatoes (Solanum lycopersicum), have been severely affected by begomovirus infection, causing significant yield losses. At least five begomovirus species have been reported in tomato in this country. Virus isolate V10 was obtained from diseased tomato plants showing mild yellowing and curl of leaves symptoms in Aragua state. Full-length DNA-A and DNA-B-like molecule of V10 were cloned and sequenced. The genome organization of V10 was identical to the bipartite genomes of other begomoviruses described from the Americas. Characteristic disease symptoms were reproduced in Solanum lycopersicum and Capsicum annuum plants biolistically inoculated using the cloned viral DNA-A and DNA-B components, confirming disease aetiology. A phylogenetic analysis of DNA-A (AY927277) showed that V10 isolate was most similar to western hemisphere begomoviruses, having the highest sequence identity (86%) with Euphorbia mosaic virus (EuMV) isolates. This is less than the 89% identity in the complete genome that has been defined as the threshold value for the demarcation of species in the genus Begomovirus. The molecular data show that isolate V10 from tomato in Aragua, Venezuela, is a novel Begomovirus species, for which the name Tomato mild yellow leaf curl Aragua virus (TMYLCAV) is proposed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Four suppressors of RNA silencing are encoded by cotton leaf curl Multan begomovirus complex Authors: Imran Amin1*, Khadim Hussain1,2*, Rashid Akbergenov2, Jitender S. Yadav3, Javaria Qazi1, Shahid Mansoor1, Thomas Hohn2, Claude M. Fauquet3 and Rob W. Briddon1

1National Institute for Biotechnology and Genetic Engineering, Jhang Road, P.O. Box 577, Faisalabad, Pakistan. 2Institute of Botany, University of Basel, Switzerland. 3ILTAB/Danforth Plant Science Center, 975 N. Warson Rd, St Louis, MO 63132, USA. *These authors equally contributed Abstract: Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomviruses, particularly in tropical and sub tropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to mainstay of the economy of Pakistan, cotton. RNAi or gene silencing is a natural defense response of plants against invading viruses. In counter-defense viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan betasatellite (CLCuMB), which together cause CLCuD, and the non-essential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However in the presence of the betasatellite gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was for the first time shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real time polymerase chain reaction (qPCR) assay and northern blot analysis, the ability of all genes encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNA interference and the relative strengths of their suppression activity were compared. The analysis showed that the V2, TrAP, C4 and βC1 proteins exhibit suppressor activity, with theV2 showing the strongest activity. In addition, V2, C4 and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses/betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminivirus (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: A reverse genetics approach to discover tomato genes underlying resistance to Tomato yellow leaf curl virus

Authors: Eybishtz Assaf, sade dagan and Czosnek Hanokh (Henryk)

Address: Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Abstract: Plants have evolved sophisticated signaling mechanisms to deal with environmental challenges, abiotic and biotic stresses. Tomato yellow leaf curl virus (TYLCV) is a whitefly-transmitted geminivirus that causes devastating loss of tomato crops worldwide. Domestication of tomato from the wild and selection for high yield led to the loss of many biotic, including TYLCV, and abiotic resistant alleles, which might have been linked with poor fruit quality. Breeding for resistance consisted in reintroducing resistance genes found in wild tomato species such as S. peruvianum, S. chilense and S. habrochaites into the domesticated tomato S. lycopersicum. The genes conferring resistance to TYLCV and the biochemical and molecular events sustaining resistance are unknown. I have used two tomato lines issued from the same breeding program with S. habrochaites as source of resistance: susceptible (S), and resistant (R). I postulated that genes involved in resistance to TYLCV in R plants are more expressed than in S plants, and that silencing them by TRV-VIGS would lead to the collapse of resistance. I have isolated about 70 genes preferentially expressed in the R line after screened cDNA libraries from infected and non- infected R and S plants. From the 20 genes I tested up to now, silencing of 6 genes lead to the collapse of resistance of R plants. I present the results obtained with three genes: a Permease I homologue, the Hexose transporter LeHT1 and a lipocalin-like gene coined SlRSLip. Silencing either one in R tomatoes resulted in typical disease symptoms, increased virus movement, accumulation of virus amounts as in S plants. R plants where LeH1 and SlRSLip have been silenced presented a necrotic reaction along the stem and petioles upon TYLCV infection reminiscent of an apoptotic response: DNA laddering, ROS production, JNK expression. These results confirmed that plant defenses are organized in multiple layers, and demonstrate that Permease I, LeHT1 and SlRSLip are essential for the expression of natural resistance against TYLCV and that their expression correlates with inhibition of virus replication and movement.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Interaction of the movement protein with a begomovirus-induced cell wall protein.

Authors: K.M. BALMANT1,2, M.C.T. LEITE2, C.M. CARVALHO1,3, AND E.P.B. FONTES1,2

Address: 1INCT em Interações Planta-Praga/Fapemig/CNPq/MCT 2Departamento de Bioquímica e Biologia Molecular/BIOAGRO 3Departamento de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brasil, 36570-000

The movement protein (MP) of begomovorus facilitates cell-to-cell as well as long- distance transport of viral DNA within plants, besides influencing viral pathogenicity. However, functional details of this process remain to be determined. Since MP influences viral pathogenicity, it is important to find putative host factors that interact with MP. To identify such host factors, we screened Arabidopsis thaliana proteins by the yeast two-hybrid system using MP as a bait and a cDNA library prepared from mRNA of infected Arabidopsis thaliana leaves. The yeast strain AH109, previously transformed with pBD-MP, was co-transformed with a cDNA library from infected Arabidopsis thaliana cloned into pEXP-AD502 vector. Potential cDNAs encoding MP-interacting proteins were first selected on selective medium lacking Leu and Trp to select for the presence of both bait expression plasmid and cDNA library plasmid and on medium lacking His and supplemented with 10mM 3AT to select for interactions between the bait BD-MP and infected Arabidopsis cDNA library-encoded proteins. The positive clones were further confirmed by assaying β-gal expression on X-gal indicator plates. Among the two isolated cDNAs that displayed His prototrophy and β-galactosidase activity, we selected ATEXL3 (Exordium like 3), a cell wall protein, for future analyses. We are currently confirming the in vitro and in vivo interactions between MP and ATEXL3 and assessing the biological significance of these interactions.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Identification of cellular targets for NIG (NSP-interacting GTPase) involved in nucleocytoplasmic transport of proteins.

Authors: J.P.B. MACHADO1,2, I.P. CALIL1,2, C.M. CARVALHO2,3, AND E.P.B. FONTES1, 2

Address: 1Departamento de Bioquímica e Biologia Molecular/BIOAGRO 2INCT em Interações Planta-Praga/CNPq/Fapemig/MCT 3Departamento de Fitopatologia/BIOAGRO Universidade Federal de Viçosa, Viçosa - MG, Brasil, 36570-000

Abstract: The geminivirus nuclear shuttle protein (NSP) facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts along with the movement protein (MP) to translocate the vrial DNA to adjacent cells. However, the mechanism by which NSP mediates the nucleocytoplasmic movement of the viral DNA is unknown. Recently, we identified a GTPase, designated NIG (NSP- interacting GTPase), which displays biochemical and structural properties consistent with a role in the nucleocytoplasmic transport of viral DNA. NIG may act as a cellular cofactor for NSP function. To assess the potential role of NIG in general cellular nucleocytoplasmic transport of protein complexes, we performed yeast two-hybrid screens with a Pro-rich domain of NIG as bait. The yeast strain AH109, previously transformed with pBD-Pro-Rich, was co-transformed with a cDNA library from Arabidopsis thaliana cloned into pEXP-AD502 vector. Double transformants were plated on medium lacking Leu and Trp to select for the presence of both bait expression plasmid and cDNA library plasmid and on medium lacking His and supplemented with 10mM 3AT to select for interactions between the bait BD-Pro-Rich and Arabidopsis cDNA library-encoded proteins. Among five isolated cDNAs that displayed His prototrophy and β-galactosidase activity, CSN5A and At2g41020 were selected for further analyses. CSN5A, COP9 SIGNALOSOME 5A, is also annotated as AJH1 that encodes a protein similar to JAB1, a specific mammalian coactivator of AP-1 transcription. Here we showed that a CSN5A-GFP fusion is fractionated between the nucleus and the cytoplasm, whereas the At2g41020-encoded protein was found to be located predominantly in the nucleus. We have previously demonstrated that NIG is located in the cytosol. However, co-expression of NIG-YFP with either CSN5A-GFP or At2g41020-GFP promoted its relocation to the nucleus. These results implicate both NIG cellular partners as potential targets for the elucidation of the NIG role in the nucleocytoplasmic transport of protein complexes in plant cells. We are currently confirming the in vivo interactions between NIG and CSN5A or At2g41020- encoded protein and assessing the biological significance of these interactions.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: APC7, a component of the Anaphase Promoting Complex, in begomovirus replication

Authors: F.P. BRUCKNER1, F.N. SILVA2, T.B. CARVALHO3, P.C.G. FERREIRA3, F.M. ZERBINI2, E.P.B. FONTES4 AND P. ALFENAS-ZERBINI1

Address: 1Dep. de Microbiologia/BIOAGRO, Universidade Federal de Viçosa, 36570-000, Viçosa, MG, Brazil; 2Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa; 3Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil; 4Dep. de Bioquímica e Biologia Molecular/BIOAGRO, Universidade Federal de Viçosa

Abstract: The Anaphase Promoting Complex (APC) is a multi-subunit ubiquitination complex that controls CDK activity by targeting the ubiquitin-dependent proteolysis of S- phase factors including cyclins, kinases and proteins associated with the assembly of the DNA replication complex. During the cell cycle, the APC has an extensive array of functions and a wide range of substrates have been identified. APC plays a key role in maintaining G0/G1 cells in a quiescent state and, similarly to pRb and p53, plays a major role as a check point of entry into the S phase. Geminiviruses are dependent on the host's replication machinery and infect differentiated cells, modulating cell cycle progression in order to create a favorable environment for genome replication. To assess whether APC7, a component of the APC, plays a role during geminivirus infection, transgenic A. thaliana plants overexpressing wild-type APC7 or an APC7 deletion mutant were inoculated with the begomovirus Cabbage leaf curl virus (CaLCuV), and viral load was determined by qRT-PCR. Plants overexpressing APC7 had reduced virus accumulation compared to non- transformed controls (Col-0) at both 14 and 28 days post-inoculation (dpi). Plants expressing only the APC7 C-terminal portion had similar viral accumulation to that observed for Col-0 plants at 14 dpi. However, a decrease in viral accumulation was observed at 28 dpi, equivalent to that observed in plants overexpressing APC7. Viral accumulation was also evaluated in two Arabidopsis T-DNA mutant lines for APC7. The SALK 036724 line has a T-DNA insertion at the 5'NTR of the APC7 gene, and accumulated the same amount of virus compared to wild-type Col-0 plants at either 14 and 28 dpi. For the SALK 109118 line, which has an insertion at the 5'-region of the gene, thus allowing expression of a portion of the N-terminal region of the APC7 protein, a decrease in viral accumulation was observed at 14 dpi, similar to that observed for the plants overexpressing APC7. This effect was no longer observed at 28 dpi, when CaLCuV- infected plants showed a viral load similar to Col-0 plants. These results suggest that APC7 has a negative effect on geminivirus replication, and that the APC7 N-terminal region is involved in this effect. Analyses of transgenic plants overexpressing only the N- terminal portion of ACP7, as well as other APC proteins, are being performed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Differentially expressed proteins in begomovirus-resistant tomato inoculated with Tomato chlorotic mottle virus

Authors: L. S. T. CARMO1, 2, C. LACORTE1, R. S. FONTENELE1, 2, R. O. RESENDE2, L. P. SILVA1, C. BLOCH1, S. G. RIBEIRO1, A. MEHTA1.

Address: 1Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Brasília, Brasil, 2Universidade de Brasília, Departamento de Biologia Celular, Brasília, Brasil Abstract: Tomato chlorotic mottle virus (ToCMoV) is a begomovirus species widely distributed in Brazil. Recently, the tomato introgression line LAM 144 carrying the locus Ty-1 and resistant to a wide range of bipartite begomoviruses was developed. However, the metabolic pathways triggered during viral infection in plants containing this locus are not known. The present work aimed to study the differential proteomic profile of susceptible (Santa Clara) and resistant LAM 144 tomato genotypes inoculated with ToCMoV. The infectious clone of ToCMoV was inoculated in resistant and susceptible genotypes by particle bombardment. At 3 and 12 days after inoculation, leaves were collected and grounded in liquid nitrogen. Protein extraction was performed by adding 750 ul of extraction buffer and the same volume of phenol. Proteins were precipitated with amonium acetate 0,1 M in methanol, washed with acetone 80% and quantified by the Bradford method. Proteome-level changes in response to TocMoV were investigated using two-dimensional electrophoresis. For each genotype, approximately 300 protein spots were reproducibly obtained in Coomassie-stained 2-D gels over a combined pH range of 3-10 NL. The proteomic profiles were very similar for the two genotypes and most proteins were distributed in the pH range of 4-7 and size range of 10-120 kDa. Some differentially expressed protein spots were excised, in gel digested and analyzed by mass spectrometry (MALDI TOF/TOF). Preliminary results identified three proteins including a nucleoside diphosphate kinase (NDPK), an enzyme involved in maintaining cellular levels of nucleoside triphosfate pool and in signal transduction pathways, a plastocyanin, a copper protein involved in photosynthesis and a putative peptidyl-prolyl cis-trans isomerase, an enzyme involved in protein folding. These proteins were up-regulated in the resistant genotype. The analysis of other differentially expressed proteins is in progress.

Financial support: Embrapa/Monsanto, National Research Institute for Plant-Pest Interactions

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Soybean golden mosaic virus: a new soybean-infecting begomovirus in Southeast Brazil

Authors: D. CÔCO1,2, F.M. ZERBINI1,3, P. ALFENAS-ZERNINI4, A. NAGATA1,5, AND E.P.B. FONTES1,2

Address: 1INCT em Interações Planta-Praga, Fapemig/CNPq/MCT 2Departamento de Bioquímica e Biologia Molecular/BIOAGRO, 3Departamento de Microbiologia/BIOAGRO 4Departamento de Microbiologia /BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brasil, 36570-000 5Embrapa Soja, Londrina, Paraná, Brasil

Abstract: Species of the Begomovirus genus (família Geminiviridae) found in Brazil typically have a bipartite genome that consists of two 2.6-2.7Kb genomic components, designated DNA-A and DNA-B. In the last two decades, begomoviruses have emerged as economically relevant pathogens, particularly in tropical and sub-tropical regions. In Brazil, begomoviruses remain as a major constraint for common bean productivity and, more recently, the frequency of begomovirus-associated epedemics has increased considerably with the proliferation of new species remarkably in tomato plantation. However, begomovirus infection in soybean (Glycine max) has been only sporadically reported. Here, we describe a soybean-infecting begomovirus found in the Southeast Brazil that may be considered a new species of the Begomovirus genus, on the basis of DNA-A and DNA-B sequence identities. Soybean leaves displaying typical symptoms of viral infection were harvested in the Jaiba-MG region. Both DNA-A and DNA-B components were amplified from infected leaf DNA with phi29 DNA polymerase, cloned in pBS vector and sequenced. The DNA-A is more closely related to Tomato chlorotic mottle virus-[Brazil] (80% identity), whereas the DNA-B shares up to 72% identity with Bean golden mosaic virus-[Brazil]. The common region of DNA-A and DNA-B are 95% identical and they harbor all the functional domains for virus replication. We tentatively named this new species of Begomovirus as Soybean golden mosaic virus (SoGMV).

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: A broad approach to identify cellular proteins required for Geminivirus infection using virus induced gene silencing.

Authors: T. ROSAS-DÍAZ, R. LOZANO-DURÁN, AND E.R. BEJARANO.

Address: Instituto de Hortofruticultura Subtroplical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigación Científicas (IHSM-UMA-SCIC).

Abstract: Tomato yellow leaf curl Sardinia virus (TYLCSV) is the causal agent of the tomato yellow leaf curl disease, one of the most important threats to tomato crops in many tropical and subtropical regions of the world. Geminiviruses do not encode their own DNA polymerases and rely on the nuclear DNA replication machinery. Strikingly, they replicate in nuclei of mature cells, which are inactive in DNA replication. An early step in geminivirus infection is therefore the induction of host DNA replication enzymes. Nevertheless, we know very little about the cellular factors involved in a successful infection. The identification of host proteins required for viral infection will be an important step towards the understanding of the mechanisms underlying this process.

In our laboratory, transgenic Nicotiana benthamiana plants containing a green fluorescent protein (GFP) expression cassette flanked by two direct repeats of the intergenic region of the Tomato yellow leaf curl Sardinia virus (TYLCSV) have been constructed (2IR plants). When these plants are infected with TYLCSV, an overexpression of the reporter gene is observed. These plants, in combination with VIGS (Virus Induced Gene Silencing) technology, could be used as a powerful tool to easily identify host factors required for the development of normal TYLCSV infection.

We have cloned into VIGS vectors 37 plant candidate genes, selected because may have a role in the infectious process, including those encoding for: (i) proteins that physically interact with geminiviral proteins, (ii) proteins involved in cellular processes potentially required for viral infection, (ii) proteins expressed preferentially in phloem tissues. At the moment, these genes are being assayed in the 2IR transgenic plants. Iidentified host genes for virus infection will be presented and their possible roles will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Systemic acquired resistance (SAR) is involved in the geminivirus resistance observed in an accession of Capsicum chinense Jacq.

Authors: M. A. García-Neria and R. F. Rivera-Bustamante.

Address: Departamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6 Libramiento Norte, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México.

Abstract: Pepper golden mosaic virus (PepGMV) and Pepper huasteco yellow vein virus (PHYVV), members of the Geminiviridae family, are important pathogens of pepper (Capsicum annuum L.) and other solanaceous crops. Nowadays, control of diseases caused by geminiviruses is largely based on a successful management of the vector (Bemisia tabaci Genn.). The generation of geminivirus-resistant varieties is a major objective in breeding programs, therefore the identification and characterization of resistance sources in wild relatives is a priority activity. Accession BG-3821 of Capsicum chinense Jacq. was reported earlier as resistant to mixed infection with PepGMV and PHYVV. In this work, we carried out a general characterization of this resistance trait. Virus replication and movement, two important processes in viral cycle were evaluated. Virus replication was evaluated by real-time PCR in protoplast from resistant and susceptible plants. Our data showed that PepGMV replication in resistant plants is approximately 70% lower when compared with susceptible ones. Viral movement was evaluated using several techniques including grafting, tissue printing and GFP fusions. The results suggested that viral movement in BG-3821 is less efficient in resistant plants when compared with the one observed in susceptible plants. We also evaluated several characteristics commonly associated with systemic acquired resistance (SAR), which is a conserved defensive mechanism. The concentration of salicylic acid (SA) was higher in resistant plants than in susceptible plants, inoculated with PepGMV. Marker genes for SAR were induced after inoculation with PepGMV in resistant leaves. Similarly, we found a higher accumulation of reactive oxygen species (ROS) on resistant leaves as compared with susceptible ones. A model for the mechanism acting in the geminivirus resistance detected in BG-3821 is proposed. Finally, the importance of BG-3821 in geminivirus resistance breeding programs is discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Quantitative trait loci analysis and whole genome sequences as tools to decipher the resistance to Cabbage Leaf Curl Virus in the Pla-1 ecotype of Arabidopsis thaliana.

Authors: O. GUERRA-PERAZA AND D. ROBERTSON

Address: North Carolina State University, Raleigh NC 27695, USA

Abstract: Geminiviruses are emerging pathogens that cause severe damage to subsistence-level crops in many parts of the world. Identification of a geminivirus resistance gene would be a break-through for understanding virus-host interactions and for designing better strategies for crop protection. We screened over 200 Arabidopsis ecotypes and found one ecotype, Pla-1, that was resistant to Cabbage leaf curl virus (CaLCuV), a member of the Begomovirus genus. In addition it also gave resistance to Beet curly top virus (BCTV), a member of the Curtovirus genus of Geminiviridae. On the other hand, inoculation of Pla-1 with the RNA potyvirus turnip mosaic virus (TuMV) or tobacco rattle virus (TRV) did result in infection indicating that the resistance is specific to geminiviruses. Analysis of segregating F2 plants from a Col-0 x Pla-1 cross showed that the resistance was multigenic, recessive, and quantitative, similar to what has been found for geminivirus resistance genes in crop plants such as bean and tomato. Using quantitative trait loci (QTL) analysis of an Col-0xPla-1 F2:3 population, we found several regions in chromosome 1 that are important for CaLCuV resistance. To increase marker density we used Illumina short read sequencing to obtain low coverage of the entire Pla-1 ecotype to be able to identify SNP markers. These SNP markers will be used to determine genes producing major QTLs as a result of CaLCuV infection.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus family: Geminiviridae

Category: Virus-host interactions

Title: Analysis of DNA and small RNA accumulation during the interaction of pepper plants with different strains and chimeric viruses of Pepper golden mosaic virus

Authors: Cecilia Hernandez-Zepeda, Ali M. Idris, Judith K. Brown

Address: School of Plant Sciences, The University of Arizona, 85721, Tucson, AZ. USA

Abstract: Two different strains of the bipartite Pepper golden mosaic virus (PepGMV) [genus, Begomovirus; family, Geminiviridae) cause different symptom phenotypes in pepper plants. The mosaic strain, PepGMV-Mo, causes severe, systemic yellow mosaic symptoms, whereas, the distortion strain, PepGMV-Di, causes leaf distortion symptoms on inoculated leaves subsequent recovery in developing leaves. Previous data indicated that the ‘remission’ phenotype associated with the Di strain was mapped to a defective promoter region of the PepGMV-Di cell-to-cell movement protein (BL1). Chimeric viruses were previously obtained by exchanging the BL1 ORF upstream region (putative promoter) between the Mo and Di strains. Pepper (Capsicum annum var. Annaheim) plants were inoculated with the wild type PepGMV-Mo and PepGMV-Di and the chimeric viruses Mo DNA A + Mo DNAB carrying the Di promoter, and DiDNA A+ Di DNA B carrying the Mo promoter. Plants were maintained in a controlled environment (79-80C; 80% humidity; 12/12 hr light/dark regime) for 21 days. Total DNA was isolated from a pool of five to six leaves comprising the first to sixth leaf above the point of inoculation. Radioactive labeled PepGMV-Mo and -Di DNA-A were used as probe to assess viral accumulation in pooled leaves. Preliminary results indicate that there was no significant difference in viral DNA concentration between the ‘wild type’ symptomatic leaves tissue compared to the analogous ‘recovered’ leaf tissue. Small RNAs of 21-24 nt homologous to the BL1 gene were detected in both symptomatic and recovered leaf tissue inoculated with the wild type PepGMV-Di, PepGMV-Mo and chimeric viruses. The highest concentration of small RNA accumulation was observed in the recovered leaf tissue inoculated with PepGMV-Di or with the chimeric PepGMV-Mo DNA-A component and PepGMV Mo DNA-B component that contained the Di putative promoter region. Thus far, these results support the hypothesis that DNA replication is not directly related to the ‘recovery phenotype’ mechanism and that reduced BL1 expression may be caused by a defective promoter, with one downstream outcome being insufficient amounts of transported viral DNA.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: BCTV and CaLCuV display a different interaction with the cell cycle machinery in Arabidopsis.

Authors: J. T. ASCENCIO-IBAÑEZ, L. SARDO, D. VU, R. COPPERSMITH, G.P. ACOTTO AND L.K. HANLEY-BOWDOIN.

Address: Molecular and Structural Biochemistry Department, 128 Polk Hall, North Carolina State University, Raleigh NC 27606.

Abstract: Cell cycle control is an important process for all living organisms. Geminivirus impinge on the cell cycle network by sequestering the DNA replication machinery of their hosts to induce terminally differentiated cells into replication competent cells. However, observed differences between curtovirus and begomovirus during infection suggest that these two genera may rely on a different subset of cell lineages or cell cycle players to progress their infection. Even though both viruses increase the general ploidy levels in Arabidopsis Col-0, BCTV seems to be impaired to induce the endocycle in cycD3 triple knockout mutants. There is also an apparent discrepancy in which stock of cells are induced in the mutants. While CaLCuV shows a great increase in higher ploidy, mainly from the 4C cell stock, BCTV seems capable of activate cells in 2C. Beside these differences, morphology of infected floral structures also showed divergences. Together, these differences suggest that geminivirus may have a battery of targets to attain infection thus strategies to protect plants against these pathogens may need to be adjusted accordingly.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus family: Geminiviridae

Category: Virus-host interactions

Title: The contribution of an alphasatellite-like molecule to symptom severity when in the presence of a begomovirus-beta satellite complex infecting tomato in Oman

Authors: A.M. Idris1,2, Cecilia Hernández-Zepeda1, J.-K. Zhu2, Judith K. Brown1

Address: 1School of Plant Sciences, The University of Arizona, 85721, Tucson, AZ. USA; 2Plant Stress Genomics and Technology Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

Abstract: Rolling circle amplification was used to amplify, clone, and sequence the genomes of two suspect begomoviruses and satellites from symptomatic tomato plants collected in commercial fields in Oman during 2005. Tomato yellow leaf curl virus-Oman (TYLCV-OM), and an associated betasatellite have been reported previously by our group to be associated with symptomatic tomato plants. From those same samples, we cloned a second, recombinant begomovirus that is sufficiently divergent from other begomoviruses to warrant its’ classification as the distinct species, Tomato leaf curl Oman virus (ToLCOMV). Also cloned from the same sample was an alphasatellite-like molecule that is surprisingly closely related to another alphasatellite (AYVSGA) from Ageratum in Singapore. Infectious clones of ToLCOMV when inoculated to tomato seedlings caused mild leaf curl symptoms in Nicotiana benthamiana and tomato, whereas, TYLCV-OM inoculated N. benthamiana and tomato seedlings developed more severe symptoms than those observed in plants infected by TYLCV-Om. N. benthamiana and tomato seedlings co-inoculated with each helper virus (separately or together) and the betasatellite developed intensified symptoms in both hosts and for both virus(es)-beta satellite combinations. However, co-inoculation of plants with each of the helper viruses (separately), plus the betasatellite and the alphasatellite, resulted in a notably reduced symptom severity in both test plants. This is the first report of the amelioration of disease symptoms by an alphasatellite and the phenotype was observed only in plants when the alphasatellite was present with one or both helper begomoviruses and the associated betasatellite.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Characterization of recovery phenomenon in Tomato leaf curl New Delhi virus : Correlation between viral DNA, transcripts and miRNA levels

Authors: JYOTHSNA. P AND MALATHI V.G. Address: Advanced Centre for Plant Virology, Div, of Plant Pathology, Indian Agricultural Research Institute, New Delhi, INDIA.

Abstract: Tomato leaf curl disease in India is caused both by monopartite and bipartite tomato leaf curl begomoviruses. Association of satellite DNA β was found to be essential in increasing the severity of symptoms in the plants and is also known to enhance the replication of DNA A (Saeed et al 2007). A variant of ToLCNDV was isolated from the samples showing yellow mosaic symptoms in sponge gourd which was identified as a variant of ToLCNDV-Mild (Fauquet et al., 2008) and designated as ToLCNDV-[IN:ND:Luffa:2010] (GenBank Accession No. HM989845 (DNA-A) and HM989846 (DNA-B). Samples showed association of Luffa leaf distortion beta satellite (LuLDB)). Infectivity was achieved by both agro infiltration and agro inoculation. Plants inoculated with DNA-A alone or DNA-A and DNA-B, showed recovery from the symptoms in 15–20 dpi respectively. The expression of the symptom was restored by re-inoculating LuLDB onto the completely recovered or silenced plants. While irrecoverable severe leaf curl symptoms were seen when inoculated along with DNA-β. The extent of PTGS activity of the host and the suppressor activity of the LuLDB was measured by comparing the levels of viral titer at different stages of infection i.e., symptom initiation, severe symptom production, symptom recovery and reversal of recovery (suppression). Viral DNA level was assessed by qRT-PCR using CP region specific primers. There is a three fold decrease in the level of DNA-A accumulation in the presence of satellite DNA-β although the symptom severity is more than DNA-A or DNA-A+DNA-B inoculated plants. There is an interesting observation of generation of subgenomic DNA (900bp) which was confirmed by sequencing. The retained segment spans 5’ half of Rep gene and intergenic region (IR). It is hypothesized that it is the level of subgenomic DNA which must be facilitating the replication of DNA-β which is making possible the interaction of βC1 protein with the host factors involved in PTGS activity and the reversal of recovery (suppression) is taking place. In addition, these changes also showed some correlation with the up / down regulation of host microRNA found in the different developmental stages along with the recovery process. References: 1. Saeed M, Zafar Y, Randles JW, Rezaian MA (2007) A monopartite begomovirus- associated DNA beta satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection. J Gen Virol 88: 2881-2889. 2. Fauquet CM, Briddon RW, Brown JK, Moriones E, Stanley J, Zerbini M, Zhou X (2008) Geminivirus strain demarcation and nomenclature. Arch Virol 153:783–821.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Molecular dissection of Ty-5, a Tomato yellow leaf curl virus resistance locus in the tomato line TY172 derived from Solanum peruvianum

Authors: I. LEVIN, U. KARNIEL, I. ANBINDER, M. REUVENI, S. NAHON, H. SHLOMO, L. CHEN, AND M. LAPIDOT

Address: Institute of Plant Sciences, Agricultural Research Organization, the Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

Abstract: Tomato yellow leaf curl virus (TYLCV) is devastating to tomato (Solanum lycopersicum) crops and development of resistant cultivars is highly effective in controlling the disease. The breeding line TY172, originating from Solanum peruvianum, is highly resistant to TYLCV. To map quantitative trait loci (QTLs) controlling TYLCV resistance in TY172, appropriate segregating populations were analyzed using 69 polymorphic DNA markers spanning the entire tomato genome. Results show that TYLCV resistance in TY172 is controlled by a previously unknown major QTL, originating from the resistant line, and four additional minor QTLs. The major QTL, termed Ty-5, maps to chromosome 4 and accounts for 39.7-to-46.6% of the variation in symptom severity among segregating plants (LOD score: 33-to-35). The minor QTLs, originated either from the resistant or susceptible parents, were mapped to chromosomes 1, 7, 9 and 11, and contributed 12% to the variation in symptom severity in addition to Ty-5. Further analysis of Ty-5, recently completed, indicated that: (1) the minor QTLs we have previously identified are in effect not reproducible; (2) Ty-5 alone can yield highly resistant plants with practically no extra-chromosomal effects, and (3) narrow-sense heritability estimate of the resistance levels does not significantly deviate from 1. These results point to Ty-5 as the sole resistance locus in TY172, thus increasing the likelihood of successful molecular identification of genes controlling TYLCV resistance in this locus.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Vírus Family: Geminiviridae

Category: Vírus-host interactions

Title: The rpL10 family as NIK substrates

Authors: K.V.G. LOPES1,2, A.R. PATARO1,2, F.A. COSTA1,2, A.A. SANTOS1,2, E.P.B. FONTES1,2

Address: 1Departamento de Bioquímica e Biologia Molecular/BIOAGRO, 2 INCT em Interações Planta-Praga/CNPq/Fapemig/MCT, Universidade Federal de Viçosa, Viçosa, MG, Brasil, 36570-000

Abstract: The NSP-interacting kinase (NIK)-mediated defense response has been identified through interactions with the begomovirus nuclear shuttle protein (NSP). Recent progress in elucidating this signaling pathway identified a ribosomal protein L10 as the immediate effector of the pathway. NIK binds to and phosphorylates rpL10 in vitro and in vivo and redirects its localization to the nucleus. rpL10 is represented by a small subfamily in the Arabidopsis genome that includes three genes, rpL10A, rpL10B and rpL10C. Despite the extraordinary conservation of sequence among them, these ribosomal proteins are not functionally analog displaying discrete and distinct functions in development and response to abiotic stresses. Here we have characterized the rpL10 subfamily in the context of the NIK defense signaling. The ribosomal proteins rpL10A, rpL10B and rpL10C were expressed in E. coli as a GST-fusion and purified by affinity. The E.coli-produced proteins were assayed for NIK substrates in phosphorylation assays. Except for the rpL10A that serves as a substrate for NIK, GST-L10B ad GST-L10C failed to be in vitro phosphorylated by the receptor kinase. Inspection of the primary structure of the proteins revealed the presence of a conserved Ser residue at position 104 that has been proposed to be the primary site for NIK phosphorylation. In fact, replacement of Ser-104 with Ala abolished phosphorylation by NIK in vitro and the mutant protein fused to YFP was predominantly localized in the cytoplasm with a low frequency of cells with nuclear rpL10 in the absence (6.8%) and presence (10.9%) of NIK1. In order to identify other possible sites of phosphorylation on rpL10 that could explain the lack of NIK-induced phosphorylation of rpL10B and rpL10C, we have replaced the Ser residues at positions 168, 68 and 137 by Ala but, except for the mutant S168A protein, the other two mutant proteins did not accumulate stably in E. coli. We found that the mutant S168A fused to YFP is mislocalized in agroinfiltrated tobacco leaves as it concentrated predominantly in the nucleus in the absence (48.4% of cells with rpL10 in the nucleus) and presence (41.2%) of NIK1. We are currently detecting the phosphorylation sites on rpL10A by MS-MS analysis and we will discuss the possible involvement of different phosphorylation sites in the subcellular localization of the protein.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Geminiviruses subvert ubiquitination and inhibit jasmonate signalling by altering CSN-mediated de-rubylation of SCF E3 ligase complexes.

Authors: R. LOZANO-DURÁN(1), T. ROSAS-DÍAZ(1), G. GUSMAROLI(2,4), A.P. LUNA (1), RENAU JP (3), X.W. DENG(2) AND E.R. BEJARANO(1)*

Address: (1) Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Dept. Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain. (2) Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven. Connecticut 06520-8104, USA. (3) UMR INRA 1165 - CNRS 8114 - UEVE, 2 rue Gaston Crémieux, CP 5708, 91057 Evry, France. (4) Current address: Department of Science and Mathematics, University of South Carolina Beaufort, Bluffton, SC 29909 USA

Abstract: Viruses have to create a suitable cell environment and elude defence mechanisms, which most likely involve interactions with the host proteins and subsequent interference with or usurpation of the cell machinery. In this work, we describe a novel strategy used by plant DNA viruses (Geminiviruses) to redirect the ubiquitination system by interfering with the activity of the CSN (COP9 signalosome) complex. Geminiviral C2 protein interacts with the plant CSN5 protein, catalytic subunit of the CSN complex, in the nuclei of plant cells. The activity of the CSN over CUL1 seems to be compromised in transgenic Arabidopsis lines expressing C2 and, consequently, several responses regulated by the CUL1- based SCF ubiquitin E3 ligase complexes (including plant responses to jasmonates, auxins, gibberellins, ethylene, ABA) are altered in these lines. Impairment of SCF function is confirmed by the stabilization of YFP-GAI, substrate of SCFSLY1 complex, in the presence of C2. Transcriptomic analysis of C2 transgenic plants highlights the response to jasmonates as the main SCF- dependent hormonal signalling pathway affected in these plants. Since we describe that jasmonate treatment of Arabidopsis plants disrupts geminivirus infection, the suppression of the response to this hormone might be an important requisite to accomplish the infection. Altogether, these data suggest that C2 is affecting the activity of this major class of E3 ligases, most likely through the partial inhibition of the CSN. Taking into consideration that SCFs are key regulators of many cellular processes, the capability of viruses to selectively interfere with or hijack the activity of these complexes might mean a novel and powerful strategy in virus infection.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Functional diversity of gene silencing suppressors from Tomato yellow leaf curl disease viruses.

Authors: A.P. LUNA(1), G. MORILLA(2), O. VOINNET(2)AND E.R. BEJARANO(1)*

Address: (1) Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Dept. Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain. (2) Institut de Biologie Moléculaire des Plantes du Centre National de la Recherche Scientifique, 67084 Strasbourg Cedex, France.

Abstract: TYLCD is caused by a complex of phylogenetically related begomovirus species that produce similar symptoms when infecting tomato plants. In Spain, the first reports of infections by TYLCV-related viruses were in the early 1990s and were associated with the presence of the ES strain of Tomato yellow leaf curl Sardinia virus (TYLCSV) (Noris et al. 1994). Subsequent introductions of isolates of the Israeli severe (IL) and mild (Mld) strains of TYLCV (Morilla et al., 2003; Navas-Castillo et al. 1997, 1999) resulted in novel sources of variation. In fact, only two years after the detection of TYLCV, a novel recombinant variant named Tomato yellow leaf curl Málaga virus (TYLCMalV) emerged as a result of a genetic exchange between isolates of TYLCSV and of the Mld strain of TYLCV (Monci et al. 2002). This recombinant variant was ecologically well adapted and spread within the population (Garcia-Andres et al. 2007)

All plants viruses examined to date encode at least one protein that suppresses the antiviral silencing targeting different steps in the virus infection cycle. The RNA silencing-suppressors promote viral invasiveness by enhancing virus replication in infected cells, and/or by inhibiting local or long distance spread of antiviral silencing. They show different mechanisms of action. In recent years several studies have determined the silencing suppression activity of TrAP, C4 and or V2 from various begomovirus. These analyses have confirmed that geminiviruses can encode more than one suppressor and that similar proteins from different viruses do not have equivalent suppressor activities (Nawaz-Ul-Rehman, Nahid et al. 2010; Sharma et al., 2010; Chowda-Redd et al. 2009; Zrachya, Glick et al. 2007; Gopal et al. 2007). However, since all these results have been obtained using assays in N. benthamiana widely employed in plants for this purpose, no data is yet available on the suppressor activity of these proteins in other plant species and its possible relation with the observed differences in virulence and host range of closely related geminiviruses We have determined the suppressor activity in N. benthamiana of C2, C4 and V2 proteins of four Spanish isolates causing TYLCD that belongs to three different th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions species and we also analyzed the suppressor ability of these proteins in two natural hosts of agronomical importance: tomato and bean to check if the absence or presence of suppressor activity in a determined host could be related with the ability of the virus to infect the plant or the virulence of this infection.

Chowda-Reddy R. V. et al., Virus Res 145, 270 (2009). Garcia-Andres S., et al., Virology 359, 302 (2007). Gopal P. et al., Virus Res 123, 9 (2007). Monci, F. et al., Virology 303, 317 (2002). Navas-Castillo, J. et al., Plant Disease 81, 1461 (1997). Navas-Castillo, J. et al., Plant Disease 83, 29 (1999). Nawaz-Ul-Rehman M. S., et al.,Virology 405, 300 (Sep 30). Morilla G. et al., Plant Disease (2003) Noris, E. et al., Arch. Virol 135, 165 (1994). Sharma, P. et al.,Virus Res 149, 19 (2010). Zrachya A. et al., Virology 358, 159 (2007).

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Genetic silencing of cytoplasmic thioredoxins based on VIGS vector in pepper plants (Capsicum annuum L (cv Anaheim) infected with PepGMV

Authors: Luna-Rivero Marianne1, Villanueva-Alonzo Hernan1, Cecilia Hernández- Zepeda2 y Moreno-Valenzuela Oscar A.1 Address: 1. Unidad de Bioquímica y Bilogía Molecular de Plantas, Centro de Investigación Científica de Yucatán. Calle 43 No. 130, Colonia Chuburná de Hidalgo, Mérida, Yucatán, México. 2. School of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA

Abstract: Thioredoxins (Trxs) are small and ubiquitous proteins involved in disulfide bond reduction, with two close and active redox-active Cys residues in a conserved WCG/PPC motif. In plants, the thioredoxins appear to play a fundamental role in plant tolerance of oxidative stress, avoiding the oxidative damage by supplying reducing power. Furthermore, they could act as regulators scavenging mechanisms, and as components of signaling pathways in the plant antioxidant network. The objective of this work is to characterize the cytosolic Trxs in pepper plants (Capsicum annuum cv Anaheim) and analize their role in the pepper plants- Pepper golden golden mosaic virus (PepGMV) interaction using VIGs vector. Previously it has been reported a thioredoxin h gene that participate in plant pathogen interaction in pepper infected with Xanthomonas axonopodis. Using primers designed on the reported sequences from pepper, we isolated a fragment of 197 pb in C. annuum cv Anaheim, which has 98% identity with the reported thioredoxin h (EF371503). This cloned fragment will be used as probe to analyze the trxs expression in different organs of pepper plants, and will be used to construct two VIGS vectors using the Euphorbia mosaic virus-Yucatan Peninsula and Tobacco rattle virus. Pepper plants will be infected with PepGMV. After the infection, trx h gene will be silenced using the VIGS vectors. To examine the effect of this gene silencing on the oxidative stress, we will analyze the concentration of salicylic acid, hydrogen peroxide, and the activity of the scavenger enzymes catalase, peroxidase, superoxide dismutase in control plants, infected plants and silenced plants.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: “Development of vectors-mediated expression of dsRNA for PTGS activation in Lycopersicum esculentum”

Authors: D. MEDINA-HERNANDEZ1, F. TENLLADO-PERLALO2, C.E. ANGULO- VALADEZ1, AND R.J. HOLGUIN-PEÑA1

Address: 1Biología Molecular de Plantas, CIBNOR, Mar Bermejo No. 195, Col. Playa Palo de Santa Rita Apdo. Postal 128; La Paz, BCS 23090, México. 2Biología Medioambiental, CIB; Ramiro de Maeztu 9, 28040 Madrid. Post-transcriptional gene silencing (PTGS) of plant is proving to be a powerful tool for genetic, developmental, and physiological analyses. Recently, it has been demonstrated the potential of constructs encoding self-complementary `hairpin' RNA (ihpRNA) for efficient gene silencing. Four different dsRNA constructs derived from same viral gene were developed for evaluate viral suppression in tomato infected with Tomato chino de la Paz (ToChLPV) and Pepper golden mosaic virus (PepGMV). . By Gateway Cloning Tecnology, the first step was to insert our gene of interest into a plasmid (pDONOR) containing attP sites to be arecombination reaction between an attB-flanked PCR product using the coat protein region (CP) (AC1048attB2 and AV494attB1), and intergenic region (IR) (PAL1v1978 attB2 and PAR1c496attB1) from ToChLPV and PepGMV, creating an entry clone. Now all entry clones have attL's on both sides of the gene of interest. Those sticky ends arematching up with the sticky ends on a destination vector (pH7GW1WG2(II)), which contains attR restriction sites. This recombination reaction between aclone and a destination vector generated an expression clone with inverted repeat (dsRNA), with an intron as a spacer (ihpRNA). Vector-mediated expressions were cloned in Agrobacterium tumefaciens (strain GV2260). The integration of constructs was verified by protein fluorescents green as control using agroinfiltration in Nicotiana benthamiana plants. The results showed that the fluorescence induced in leaves. To evaluate the capability of dsRNA to interfere with ToChLPV and PepGMV infection a use of a factorial experiment with two levels will be performed. The first level is the use of constructs and second one inoculation methods of constructs (agroinfiltration, biobalistic and mechanical inoculation carbarundum). La experimental infection will be performed by biobalistic using the tandem clones of ToChLPV and PepGMV. Absolute viral load and rate of infection will be measured.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Silencing CchGLP gene increase symptoms severity by geminivirus infections in Capsicum chinense BG-3821

Authors: L. MEJIA-TENIENTE1, I. TORRES-PACHECO1, C.I. MUNOZ- SANCHEZ2, L. GUEVARA-OLVERA2, G. ACOSTA-GARCÍA2, M.M. GONZÁLEZ- CHAVIRA3, R.F. RIVERA BUSTAMANTE AND R.G. GONZALEZ-GUEVARA1

Cerro Universitario Cerro de las Campanas, s/n, Col. Las Campanas. C.P. 76010. Santiago de Querétaro, Querétaro, México. Abstract Pepper Huasteco Yellow Vein Virus (PHYVV) and Pepper Golden Mosaic Virus (PepGMV) are begomoviruses widely distributed in Mexico and mainly affect to solanaceous crops among which highlighting wide varieties of genus Capsicum spp. C. chinense BG-3821, is a pepper accession with resistance to geminivirus infections, and a gene encoding to a putative germin-like protein (CchGLP) is highly induced during infection. CchGLP displays a Mn-SOD activity in vitro.Thus, in order to unravel the role of CchGLP gene in the resistance mechanism in this plant to geminivirus infections, we silence this gene using a PHYVV based-vector. When silenced CchGLP, plants´ susceptibility was increased to both single and mixed infections caused by PHYVV and PepGMV, never reaching severity levels as in susceptible control plants. CchGLP silencing was maintained from 4-6 true leaves stage in plants to flowering stage. On the other hand, the null expression of CchGLP transcripts in RT-PCR analysis of apical and basal leaves of plants locally inoculated with PHYVV based-vector confirmed the systemic silencing of the gene tested. Thus, it is concluded that CchGLP gene of C. chinense BG-3821 is involved (but not the only one) in the resistance mechanism of this accession to geminivirus infections.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Deep sequencing analysis of virus-derived small RNAs in tomato plants infected by Tomato yellow leaf curl Sardinia virus

L. Miozzi, V. Pantaleo, E. Noris, J. Burgyan, G.P. Accotto

Istituto di Virologia Vegetale – C.N.R., Strada delle Cacce 73, 10135 Torino, Italy

Tomato yellow leaf curl Sardinia virus (TYLCSV) is a Begomovirus with a genome consisting of a circular single-stranded DNA molecule ca. 2800 bases in length. Its bidirectional promoter drives the generation of viral RNA transcripts coding for viral proteins required for its entire life cycle in the host plant. The RNA silencing machinery recognises the viral transcripts as foreign RNAs, thus leading on to the production of viral small interfering RNAs (v-siRNAs), likely responsible for v- siRNA-mediated antiviral defense. A cDNA library of small RNAs was generated from tissues of TYLCSV-infected tomato plants and sequenced on Solexa/Illumina sequencing platform. The subset of v-siRNAs was therefore identified; the major size classes of TYLCSV-derived siRNAs were 21 and 22 nt species spanning the entire viral genome but being discontinuously distributed throughout it. The most abundant v-siRNAs are from CP and C4 genes, whereas those from the intergenic region are poorly represented. Moreover, the majority of v-siRNAs are of sense polarity suggesting that they are from folded single-stranded viral transcript, similarly to what observed in the case of other v-siRNAs from positive stranded RNA plant viruses. The possible mechanism/s of v-siRNA biogenesis and their role in antiviral plant response will be also discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Tomato leaf curl virus or Tomato leaf curl New Delhi virus DNA A and Cotton leaf curl disease betasatellite can cause mild transient symptoms in cotton.

Authors: MUHAMMAD SAEED

Address: National Institute for Biotechnology and Genetic Engineering, PO Box 577, Jhang Road, Faisalabad, Pakistan

Abstract: Tomato leaf curl virus (ToLCV) from Australia is a monopartite begomovirus which is naturally associated with a DNA satellite, a vestigial betasatellite. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus that requires both DNA components for systemic infection. Cotton leaf curl disease is caused by a complex consisting of one or more begomoviruses (eight species have been identified so far) associated with a single DNA β satellite named as Cotton leaf curl Multan betasatellite (CLCuMB). Inoculation studies using either ToLCV or ToLCNDV DNA A and CLCuMB caused mild symptoms in cotton plants 18-21 days post inoculation. The mild symptoms caused by either ToLCV and CLCuMB or ToLCNDV DNA A and CLCuMB in cotton plants began to diminish 6 weeks post inoculation and completely disappeared 8-10 weeks post inoculation, raising the possibility that ToLCV and ToLCNDV DNA A may lack some factor(s) essential for persistent systemic infection of cotton.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Altered Gene Expression Profile of Resistant Chilli Genotype Infected With Chilli Leaf Curl Virus

Authors: Nirbhay Kushwaha1, Pranab Sahu2, Manoj Prasad2 and Supriya Chakraborty1

Address: 1Molecular Virology laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi – 110 067. 2National Institute of Plant Genome Research, New Delhi – 110067.

Chilli leaf curl disease caused by begomoviruses is a major constraint for successful cultivation of chilli in the Indian sub-continent. Chilli leaf curl virus (ChiLCV) is a monopartite begomovirus having single stranded circular DNA (~2.7 kb) along with betasatellite (~1.3 kb) which causes leaf curling, stunting of plants, along with no or fewer deformed fruits. Several efforts and strategies have been attempted to control this disease but none of them are found to be effective except use of resistant genotypes. In the present investigation, we have studied differential genetic expression of chilli variety (Punjab lal) resistant to ChiLCV. For this, chilli plants were agroinoculated with infectious cloned DNAs of ChiLCV (Varanasi isolate) (DNA A and DNA β). Total DNA was isolated at 7dpi, 14dpi, 21dpi, 28dpi, 35dpi. Semiquantitative PCR indicated relatively higher level of viral DNA accumulation in resistant genotype till 21 dpi, followed by decrease in viral accumulation. Total RNA was isolated at 21 dpi from inoculated and non inoculated plant and suppression subtractive hybridization (SSH) was carried out to identify gene(s) whose expressions were altered in resistant chilli plant following ChiLCV infection. Selectivity of SSH with sensitive PCR techniques allowed us to detect the low abundance of differentially expressed transcript(s). Some of the differentially expressed genes are categorised as defense related genes, resistant gene (R Gene), cell cycle regulation genes, genes involves in protein degradation, histone proteins, elongation factors 1-alpha, small ubiquitin-like modifier(SUMO) etc. Relative level of differentially expressed transcripts and their putative role in governing resistance in chilli will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Tomato cultivar tolerant to Tomato leaf curl New Delhi virus infection induces virus-specific siRNA accumulation and defense associated host gene expression.

Authors: P. P. SAHU1*, S. CHAKRABORTY2, M. SINGH3, D. CHATTOPADHYAY1, AND M. PRASAD1*

Address: 1National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi- 110067, INDIA 2School of Life Sciences, Jawaharlal Nehru University, New Delhi- 110067, INDIA 3Indian Institute of Vegetable Research, Gandhinagar, Varanasi- 221005, INDIA *E-mail: [email protected]; [email protected]

Abstract: Tomato leaf curl New Delhi virus (ToLCNDV) infection causes significant yield loss in tomato. Availability of conventional tolerance source against this virus is limited in tomato. To understand the molecular mechanism of virus tolerance in tomato, the abundance of viral genomic replicative intermediate molecules and virus-directed short interfering viral RNAs (siRNAs) by host plant in a naturally tolerant cultivar H-88-78-1 and a susceptible cultivar Punjab Chhuhara at different time points after agroinfection were studied. We report here that less abundance of viral replicative intermediate in tolerant cultivar may have a co-relation with a relatively higher accumulation of virus-specific siRNAs. To study defense-related host genes expression in response to ToLCNDV infection, suppression subtractive hybridization technique was used. A library was made from tolerant cultivar H-88- 78-1 between ToLCNDV-inoculated and Agrobacterium mock inoculated plants of this cultivar at 21 day post-inoculation (dpi). A total of 106 non-redundant transcripts were identified and classified into 12 different categories according to their putative functions. By reverse northern analysis, we identified differential expression pattern of 106 transcripts, out of which 34 transcripts were up-regulated (>2.5 fold induction). Of these, 8 transcripts showed more than four-fold induction. Quantitative real time-PCR (qRT-PCR) analysis was carried out to obtain a comparative expression profiling of these 8 transcripts between Punjab Chhuhara and H-88-78-1, upon ToLCNDV infection. The expression of these transcripts showed a significant increase in the tolerant cultivar mostly at 14 dpi and 21 dpi in comparison to that in the susceptible cultivar as analyzed by qRT-PCR. Virus induced gene silencing of some of the ubiquitin proteasomal system components in the tolerant cultivar H-88-78-1 and its implication in the viral resistance are underway.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Natural resistance against Mungbean Yellow Mosaic India Virus correlates with early viral transcript degradation and differential siRNA production in Glycine max.

Authors: RAJIV KUMAR YADAV AND DEBASIS CHATTPOADHYAY

Address: Lab 103, National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi-110067, India

Abstract: Yellow mosaic disease caused by whitefly-transmitted bipartite Geminiviruses is one of the major constraints on productivity of a number of pulse crops. We have cloned the bipartite genome of Mungbean Yellow Mosaic India Virus isolated from infected Soybean. We report here that agroinfection of Soybean seedlings with a single uncut recombinant binary plasmid containing tandem dimers of both DNA A and DNA B resulted in 100% infectivity in susceptible varieties. To understand the mechanism of natural resistance in a Soybean variety, we compared the abundance of the viral RNAs in a resistant and a susceptible variety at the early time points after agroinfection. Whilst the resistant variety displayed synthesis but rapid degradation of the early viral RNAs; the degradation in the susceptible variety was delayed resulting in accumulation of those transcripts later in infection. Accumulation of the late viral transcripts and DNA replication were detectable only in the susceptible variety. This indicates that rapid degradation of the early viral transcripts, possibly through siRNA mechanism, is one of the probable mechanisms of natural resistance against geminivirus. We have also compared siRNA population and their types in both susceptible and resistant soybean verities after infection.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Global transcriptome profile of Arabidopsis thaliana infected with South African cassava mosaic virus

Authors: M.E.C. Rey, E. Pierce and F. van Schalk

Address: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, South Africa, 2050

Abstract: Changes in transcriptome patterns in general pathogen-responsive proteins, and host proteins involved in active cell cycle regulation and host replication, are induced by geminiviruses. Cassava (Manihot esculenta Crantz) is an important food security crop in sub-Saharan Africa and is affected by Cassava mosaic disease (CMD) caused by 7 begomovirus species, including South African cassava mosaic virus (SACMV). In the previous absence of cassava genome sequence data, we chose to examine global changes in gene expression profiles, at 14, 24 and 36dpi, in Arabidopsis ecotype Col-0 infected with SACMV compared to mock-inoculated (agroinfected) plants, under controlled environmental conditions. Viral DNA accumulation for 3 biological replicates (independent RNA) was measured in infectivity assays, and mean values obtained at each time point. In 200ng of total nucleic acid, 10920 SACMV copies were present at 14dpi, 57467 copies at 24dpi, and 62967 copies at 36dpi. Symptom severity thus correlated with an increase in SACMV copy number. Agilent 4 x 44k Arabidopsis gene expression microarray slides were used to establish global profiles of SACMV-infected plants at 14, 24, and 36dpi. Three biological replicates and 1 technical replicate were analyzed per time point using a direct comparison experimental design. An output of 13,9334 differentially expressed genes was obtained with an adjusted p-value statistic at 0.05 after normalization of data. A total of 1660 genes were common across the three time points. The number of genes restricted to a particular time point was shown to be 1714 for 14dpi, 4259 for 24dpi, and 1690 for 36 dpi indicating unique significant genes at each time point. As shown with the geminivirus Cabbage leaf curl virus (CaLCuV), several common transcripts associated with biotic stress, calcium signaling, protein degradation, and SA-induced defense responses were detected in SACMV-challenged Arabidopsis. Similarly to CaLCuV (5365 at 12dpi), SACMV demonstrated 5995 differentially- expressed RNAs at 14dpi, while at 24dpi, the number of transcripts increased to 9857 correlating to fully symptomatic plants and an increase in viral titre. Our study identified differential expression of a set of ion channel & calcium pumps and endomembrane post-golgi vesicular trafficking proteins, as well as PM, cell –wall and PD localized proteins, which provides a putative integrated multi-component model for host factors involved in the movement of SACMV from the nucleus through cytoplasm and plasmodesmata to the next cell. Comparison of differentially expressed core cell cycle genes by SACMV and CaLCuV demonstrated a similar pattern for CYCD 3:2, CYC1 and CYCD4:2 at 14 dpi and 12dpi, respectively, while down regulation of many other core cell cycle genes at 24dpi in SACMV was consistent with the later and less severe induction of disease in Arabidopsis by SACMV compared to CaLCuV. Currently we have identified over 60 differentially expressed Arabidopsis transcript orthologs in cassava, and others are being explored.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Transcriptomic and Proteomic Analysis of Maize Infected with of Maize Streak Virus.

Rizwan Ali Syed1,3, Zac Mcdonald2,3, Dionne N. Shepherd1, Darren P. Martin2, Suhail Rafudeen1, Edward P. Rybicki1

1Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa 2Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, Cape Town, South Africa 3 These authors are co-first authors

Maize streak disease, caused by Maize streak virus (MSV), is peculiar to the African continent where epidemics commonly cause devastating maize crop losses. MSV belongs to the family Geminiviridae and is transmitted by Cicadulina leafhoppers. MSV infection is systemic with the virus moving through the vascular system of the maize leaf, on which symptoms are evident as chlorotic streaks along the leaf veins. As maize cultivation is vital to African food security, extensive breeding programs and, more recently, transgenic programs have been undertaken to protect maize against MSV infection. To aid and direct these programs it is important to gain an understanding of host-pathogen interactions at the molecular and cellular levels.

With recent technological advancements in the field of Systems Biology it is currently possible to look at global changes in the proteome and transcriptome of perturbed systems, such as an MSV-infected maize leaf. The proteomic approach applied here involved the iTRAQ technique (isobaric tags for relative and absolute quantitation) which is based on isobaric tagging of peptides and allows for the simultaneous measurement of protein levels in multiple samples. In the current study, non-infected and MSV-infected maize plants were compared 30 days post agroinoculation using biological and technical replicates in an 8- plex iTRAQ experiment. This revealed that MSV infection of maize results in up– regulation of malate dehydrogenase and proteins associated with photosystem II and the Calvin cycle. Certain other proteins involved in signaling, carbohydrate metabolism, ATP synthesis and glycolysis were down-regulated. The up-regulation of photosystem II components may be compensation for the well-established damage that MSV causes to the photosynthetic machinery of maize chlorenchyma cells.

The MSV transcriptome was also investigated in the context of MSV infections using microarray analysis. Differential expression data will be presented. Investigation of these transcriptome and proteome data sets should further advance our understanding of the molecular interface between maize and MSV.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: The Tomato yellow leaf curl virus, Sardinian isolate (TYLCSV) C4 protein is a putative transcriptional gene silencing (TGS) suppressor.

Authors: E. A. RODRIGUEZ-NEGRETE, R. LOZANO-DURAN, A. G. CASTILLO, AND E. R. BEJARANO

Address: Departamento de Genética. Facultad de Biología. Campus de Teatinos. IHSM, UMA-CSIC. 29010 Málaga, Spain.

Abstract: Transcriptional gene silencing (TGS) constitute an important plant defense mechanism against invader molecules as transposons and DNA viruses. TGS guides epigenetic modifications such as DNA and histones methylation with the purpose of impair viral transcription and replication processes. DNA viruses, including Geminiviridae family are able to evade TGS and produce successful infections. Recently, it has been described that C2/L2 Geminivirus-derived gene product act as TGS suppressor by interacting and inactivating the adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor (Buchmann et al., 2009). However, no other geminiviral protein with TGS suppressor activity has been identified so far. Our group is interested in the identification and characterization of gene silencing suppressors derived from Tomato yellow leaf curl disease (TYLCD)- related viruses. In this study, we described the pathogenic factor C4 encoded by Tomato yellow leaf curl Sardinia virus (TYLCSV, family Geminiviridae) as a possible TGS suppressor. After sequencing of bisulfite-treated DNA, highly methylated viral-derived genomes were detected in TYLCSV C4 mutant infected Nicotiana benthamiana plants; whereas hypomethylated progeny was present in wild-type virus infected plants. Interestingly, wild-type virus genomes present mainly non CpG methylation (CpNpG and CpHH context) and residual CpG methylation, whereas genomes derived from C4 mutant virus infected plants showed high methylation levels in both cytosine contexts. These results suggest an active role of C4 protein in TGS suppression. The biological implications of these observations will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Interaction between Geminivirus Replication Protein and the SUMO Conjugating Enzyme Is Required for Viral Infection

Authors: MIGUEL A. SÁNCHEZ-DURÁN1, MARY B. DALLAS2, JOSÉ T. ASCENCIO-IBAÑEZ2, JAVIER RUIZ-ALBERT1, LINDA HANLEY-BOWDOIN2 AND EDUARDO R. BEJARANO1

Address: 1 Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM- UMA-CSIC), Dept. Biología Celular, Genética y Fisiología, Universidad de Málaga, Campus Teatinos, 29071 Málaga, Spain. 2 Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA

Abstract: Geminiviruses are small DNA viruses that replicate in nuclei of infected plant cells using plant DNA polymerases. These viruses encode a protein designated as AL1, Rep, or AC1 that is essential for viral replication. AL1 is an oligomeric protein that binds to double-stranded DNA, catalyses cleavage and ligation of single-stranded DNA, and induces the accumulation of host replication machinery. It also interacts with several host proteins, including the cell cycle regulator retinoblastoma (RBR), the host DNA replication protein PCNA (Proliferating cellular nuclear antigen), and the sumoylation enzyme that conjugates SUMO to target proteins (SUMO conjugating enzyme SCE1). The SCE1-binding motif was mapped by deletion to a region encompassing AL1 amino acids 85-114. Alanine-mutagenesis of lysine residues in the binding region either reduced or eliminated the interaction with SCE1, but no defects were observed in other AL1 functions such as oligomerization, DNA binding, or interaction with RBR. AL1 K68A and K96A mutations reduced or abolished virus infectivity in plants, respectively. The mutations also reduced viral DNA accumulation in transient replication assays in tobacco cells, suggesting that AL1-SCE1 interaction is required for viral DNA replication. Ectopic AL1 expression did not result in broad changes in the sumoylation pattern of plant cells, but specific changes were detected indicating that AL1 modifies the sumoylation state of selected host proteins. These results established the importance of AL1-SCE1 interactions during geminivirus infection in plants and suggested that AL1 interferes with sumoylation of selected host factors to create an environment suitable for viral infection.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: The infective cycle of Cabbage leaf curl virus (CaLCuV) is affected by CRUMPLED LEAF (CRL) gene in Arabidopsis thaliana.

Authors: D. L. TREJO-SAAVEDRA, J. P. VIELLE-CALZADA AND R. F. RIVERA- BUSTAMANTE.

Address: Departamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6 Libramiento Norte, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México.

Abstract: Geminiviruses are single-stranded DNA viruses that cause serious crop losses worldwide. Successful infection by these pathogens depends extensively on virus-host intermolecular interactions that allow them to express their gene products, to replicate their genomes and to move to adjacent cells and throughout the plant. To identify host genes that show an altered regulation in response to Cabbage leaf curl virus (CaLCuV) infection, a screening of transposant Arabidopsis thaliana lines was carried out. Several genes were identified to be virus responsive and one, Crumpled leaf (CRL) gene, was selected for further characterization. CRL was previously reported by Asano et al., (2004) to affect the morphogenesis of all plant organs and the division of plastids. We report here that CRL expression, during CaLCuV infection shows a short but strong induction at an early stage (3-5 days post inoculation, dpi). To study the role of CRL in CaLCuV infection, CRL over- expressing and silenced transgenic plants were generated. We compared the replication, movement and infectivity of CaLCuV in transgenic and wild type plants. Our results showed that CRL over-expressing plants showed an increased susceptibility to CaLCuV infection (as compared to wt plants) whereas CRL- silenced plants, on the contrary, presented a reduced susceptibility to viral infection. The possible role of CRL in the CaLCuV infection cycle will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus Host Interaction

Title: Arabidopsis synaptotagmins: their roles in virus cell-to-cell movement and plant development

Authors: Asako Uchiyama, Harumi Shimada-Beltran, Sondra G. Lazarowitz

Address: Cornell University, Dept. of Plant Pathology and Plant-Microbe Biology, Ithaca, NY 14853. USA

Abstract: Synaptotagmins are calcium sensors that regulate synaptic vesicle exocytosis and endocytosis. Thought to be exclusive to animals, they have recently been characterized in Arabidopsis, in which they comprise a five gene family (SYTs A, B, C, D and E). We have shown that Arabidopsis SYTA regulates endocytosis and movement protein-mediated trafficking of plant virus genomes through plasmodesmata. Our studies suggest that distinct virus movement proteins transport their cargos to plasmodesmata for cell-to-cell spread via an endosome recycling pathway. SYTA has also been reported to have a role in responses to osmotic stress and calcium-dependent freezing tolerance. To further define the roles of Arabidopsis SYTs in biotic and abiotic stress we are examining virus infectivity on syt mutant plants, and the spatial and temporal regulation of SYT gene expression using appropriate promoter::GUS reporter genes. We find that SYTA and SYTE appear to be ubiquitously expressed in Arabidopsis. In contrast, SYTB is highly expressed in stems, siliques, flowers and anthers, while SYTC is specifically expressed in stomatal guard cells. Fitting with these findings, the onset of virus infection is delayed in sytb null mutants, but not in sytc null mutants. The detailed patterns of expression for SYTA, SYTB, SYTC and SYTE will be presented, and their roles in the responses of Arabidopsis to biotic and abiotic stresses will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Gene expression profile of Nicotiana benthamiana infected with South African cassava mosaic virus using a cross-species cDNA microarray approach

Authors: F. VAN SCHALK and M.E.C. REY

Address: School of Molecular and Cell Biology, University of the Witwatersrand, Private Bag 3, South Africa, 2050

Abstract: Cassava Mosaic Disease (CMD) is one of the main biotic and economically important constraints of cassava cultivation in southern Africa. South African cassava mosaic virus (SACMV) in particular is endemic to South Africa. Compatible virus infection induces and suppresses host gene expression at the global level. These gene-expression changes are the molecular basis of symptom development and general stress and defence-like responses of the host. Currently, only limited amounts of expression data are available for cassava. A cDNA microarray study was conducted in order to elucidate host gene responses to SACMV in a susceptible host, Nicotiana benthamiana, at four different time points (7, 14, 21 and 35 days post inoculation (dpi). A high degree (>70%) of nucleotide conservation in cross-species hybridization (CSH) between several crop species, including Arabidopsis and N. benthamiana, has been demonstrated, and facilitated a CSH cDNA microarray study in the absence of a cassava EST chip. This was achieved by using a custom made Arabidopsis microarray “gene chip” that was printed with 5000 pathogen-inducible ESTs in duplicate. Evaluation of symptoms during the infectivity study revealed that symptoms appeared at 14dpi, and plants were fully symptomatic at 35 dpi (1.05 X 107 molecules SACMV DNA A/ng TNA using quantitative real-time PCR), with the appearance of characteristic disease symptoms such as leaf distortion, chlorosis and stunting. Three biological replicates were conducted for microarray hybridizations. Following hybridization and scanning, data was analysed using Limma (Bioconductor package), Carmaweb and TIGR Mev. By using stringent selection criteria ( ≥2 or ≤ -2 -fold) transcripts were identified to be up- and down-regulated. Ten genes were selected and validated independently using semi-quantitative Real-time PCR. The expression levels of many transcripts encoding proteins involved in the regulation of transcription and translation, metabolism, cell-wall biogenesis, chloroplast functions and photosynthesis were repressed at 14dpi and were associated with the increasing levels of SACMV titre in the host cells. Genes were grouped into Gene Ontologies (GO), and comparisons with gene expression changes induced by geminiviruses (TYLCV and CaLCuV), and other RNA viruses in solanaceous species, will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Virus-induced gene silencing (VIGS) in plants of commercial and scientific interest

Authors:Villanueva-Alonzo,H.1, Guerra-Peraza,O.2, Robertson,D.2, López- Ochoa,L.1, and Moreno-Valenzuela O1. Address: 1. Unidad de Bioquímica y Bilogía Molecular de Plantas, Centro de Investigación Científica de Yucatán. Calle 43 No. 130, Colonia Chuburná de Hidalgo, Mérida, Yucatán, México. 2. North Carolina State University, Raleigh, NC 27695.

Abstract: A silencing vector for plants of commercial and scientific interest was developed from the begomovirus Euphorbia mosaic virus-Yucatan Peninsula (EuMV-YP). The EuMV-YP coat protein was replaced by up to 500 bp to DNA of homologous or heterologous gene magnesium chelatase subunit I gene (chlI) isolated from Nicotiana benthamiana. The third to fourth leaf stage of Arabidopsis thaliana and N. benthamiana plants were bombarded with the modified viral vector. Chlorosis due to silencing was observed in both species. In Nicotiana plants, chlorosis was observed nine days after the bombardment, beginning in the vascular tissue, and after two weeks, chlorosis was observed in leaf tissue; silencing persisted throughout the life of the plant. In A. thaliana silencing could be observed at the 35 dpi, only in the vascular tissue. Experiments are carrying on in Capsicum annuum, Capsicum chinense, Jatropha curcas, and Solanum lycopersicon. Our vector will be used in the study of gene function of these plants, especially during plant pathogen interaction.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Functional analysis of the NIK-mediated antiviral signaling pathway in tomato plants.

Authors: A.A. SANTOS1, J.A. CONDORI-APFATA2, O.B. BRUSTOLINI2, F.M. ZERBINI, 3 E. P. B. FONTES2

Address: 1INCT em Interações Planta-Praga/Fapemig/CNPq/MCT 2Departamento de Bioquímica e Biologia Molecular/BIOAGRO 3Departamento de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, Brasil, 36570-000

The begomovirus NSP (Nuclear Shuttle Protein) facilitates the transport of viral DNA from the nucleus to the cytoplasm and cooperates with the movement protein MP to promote the translocation of viral DNA to the adjacent, uninfected cells through plasmodesmata. NSP interacts with members of the LRR-RLK (“leucine-rich repeat receptor like kinase”) family, designated NIKs (“NSP- Interacting Kinase”). Binding of NSP to the activation loop of NIK inhibits kinase activity and hence the viral protein suppresses receptor autophosphorylation and defense responses. Mutagenesis assays in the activation loop of NIK have demonstrated that the threonine 474 residue is phosphorylated in vitro and plays a crucial role in the kinase activity that is required for signaling. Replacement of Thr- 474 with aspartate acid produces the T474 mutant, which exhibits constitutive activation, enhanced substrate phosphorylation activity and less inhibitory effect by NSP binding. The goal of this investigation was to analyse the NIK kinase domain in defense responses against begomovirus in tomato. The NIK normal and mutant T474D cDNA was placed under the control of 35S promoter into a binary vector for plant transformation (35S-AtNIK and 35S-AtNIK-T474D). Primary transformants were selected by PCR and the expression of the transgene was confirmed by normal and quantitative RT-PCR in independently transformed lines. Infectivity assays were carried out in AtNIK- and AtNIK-T474D-overexpressing lines, with the virus ToYSV-[MG-Bi2]. Overexpression of super active AtNIK-T474D altered the infection rate by ToYSV, and interfered in symptom development. As compared to untransformed plants and NIK-overexpressing 35S-AtNIK1-6 transgenic lines, independent transgenic AtNIK-T474D lines displayed a lower infection rate and attenuated symptoms. These results confirmed in planta the essential role for phosphorylation of the Thr-474 residue for NIK function and underlined the possibility for the development of more efficient tolerance strategies against geminiviruses. We also performed global expression profiling on geminivirus- infected leaves and on T474D-overexpressing tomato leaves. The present geminivirus- and T474D-induced transcriptional studies demonstrated a clear predominance of shared responses over stimulus-specific positive changes on tomato leaves, suggesting that virus infection is the trigger of the NIK activation.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus-Host Interactions

Virus Family: Geminiviridae

Category: Virus-host interactions

Title: Downstream components of NIK-mediated antiviral signaling.

Authors: C. ZORZATTO, J.P.B. MACHADO, K.V.G. LOPES, A.A. SANTOS AND E.P.B. FONTES

Address: INCT em Interações Planta-Praga/CNPq/Fapemig/MCT Departamento de Bioquímica e Biologia Molecular, BIOAGRO, Universidade Federal de Viçosa, 36570.000 Viçosa, MG, Brazil.

Abstract: The NSP-interacting kinase (NIK) was first identified as a virulence target of the begomovirus nuclear shuttle protein (NSP) and associated with defense signaling against geminivirus. Subsequently, the ribosomal protein L10 (rpL10A), a QM-like protein, was identified as a specific partner and substrate of NIK1 that functions as an effector of the antiviral signaling through an as yet unknown mechanism. To identify potential targets for rpL10, we performed yeast two-hybrid screens using a cDNA library from Arabidopsis thaliana. Potential cDNAs encoding L10-interacting proteins were first selected on selective medium (absence of Leu, Trp and His and supplemented with 10 mM 3AT). We isolated one clone that displayed His prototrophy and contained a full-length cDNA from the At5g05800 gene which encodes an unknown protein. The interaction of the cDNA- encoded protein with rpL10 was further confirmed by monitoring β-galactosidase activity in yeast protein extracts. To determine the subcellular localization of the At5g05800, we utilized an Agrobacterium-mediated transient expression assay in epidermal cells of tobacco leaves. Confocal microscopy revealed that At5g05800 fused to GFP localizes in the cytoplasm and in the nucleus. We are currently confirming the in vivo and in vitro biochemical interaction between L10 and At5g05800 and assessing the biological significance of complex formation.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Disease Epidemiology

Title: Distribution of Begomovirus associated with tomatoes in the Sultanate of Oman

Authors: A.J. KHAN, A.O. AL-MATRUSHI AND A. AL-SHIHI

Address: Department of Crop Sciences, College of Agricultural & Marine Sciences, Sultan Qaboos University, P.O. Box-34, Al-Khod 123, Sultanate of Oman

Abstract: Tomato leaf curl virus (TYLCV) is an important begomovirus distributed in in Oman mainly on tomatoes and other solanaceous crops. Tomato (Solanum esculentum L.) is cultivated in Al-Batinah, Musandum, Al-Bureimi, Muscat, Dhakhiliya and Sharqiyah regions and governorate in the Sultanate of Oman with Al-Batinah being the largest tomato producer. The crop is grown both in shade house and open field with average annual production of 50,000 tones. The main biotic limiting factor to the tomato production in Oman is a whitefly-transmitted begomovirus that induces leaf curling, yellowing and plant stunting symptoms. To assess the distribution of begomovirus associated with tomatoes in different regions of Oman, all tomato grown areas were surveyed during November to April of 2009 & 2010 and symptomatic leaf samples were collected for laboratory test. Total nucleic acids isolated from these tomato samples were used as templates in PCR amplification of core coat protein gene of begomoviruses using degenerate primers. All symptomatic samples tested PCR positive with core coat protein specific primer, FD-CP-382/ RD-CP-1038. The disease incidence of begomoviruses varied from 5 – 100% depending on cultural practices of the framers. Crops covered for 10 – 15 weeks with Agryl from planting showed very low disease incidence. Highest disease incidence was recorded in Al-Batinah region followed by Musandam, Al-Bureimi, Muscat, Dhakhiliya and Sharqiyah regions.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Epidemiology/Transmission

Title: Distribution and molecular variability of begomovirus affecting the main crops in Cuba: Epidemiological elements.

Authors: Y. Martínez-Zubiaur; M. Quiñonez, E. Fiallo-Olive, Y. Marrero, M. de los A. Martínez, I. Palenzuela, I. Miranda.

Address: Departamento de Fitopatología, Centro Nacional de Sanidad Agropecuaria. Apdo 10, San José de las Lajas. Habana, Cuba.

Abstract: Since the late 80´s, begomoviruses have affected tomato production in Cuba. This paper is aimed at showing the results about the national surveys in order to know the distribution of these viruses in the major growing regions of tomato production in the country, identifying the most affected areas, the epidemiological understanding of the main causes affecting the high percentages of incidence and severity, and monitoring and demonstrating the adaptability of the TYLCV-IL(CU) in tomato agroecosystems. Further researches about their molecular evolution were carried out, demonstrating their potential ecological habitats in other crops such as pepper, beans and squash, which act as natural reservoirs of TYLCV-IL(CU). The DNA study from the plants collected in national surveys has shown the presence of new bipartite begomovirus species infecting tomato, pepper, tobacco and weeds. The sequencing of tobacco plant DNA with symptoms also detected the presence of Euphorbia Mosaic Virus. The results showed factors related to changes in agricultural, biological and molecular systems which manage the emergence of a viral disease, its development and increasing severity in the natural and productive ecosystem interface.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission

Multiplex PCR for the simultaneous detection of DNA (geminivirus) and RNA (potyvirus) viruses infecting cassava

MARUTHI MN1, ABARSHI MM1, Mohammed IU1, Kumar L2, and Legg JP2.

1Natural Resources Institute, University of Greenwich, Chatham Maritime, Kent ME4 4TB, United Kingdom. E-mail: [email protected] 2Internatioanl Institute for Tropical Agriculture (IITA), Ibadan, Nigeria

Cassava in Africa is affected by two important viral diseases; cassava mosaic disease (CMD) and cassava brown streak virus disease (CBSD). CMD is caused by eight species of ssDNA viruses of the family Geminiviridae, genus Begomovirus, of which variants of African cassava mosaic virus (ACMV) and East African cassava mosaic virus (EACMV) are most common in Africa. CBSD is caused by two species of the +ve sense ssRNA viruses; Cassava brown streak virus (CBSV) and Cassava brown streak Uganda virus (CBSUV) that belong to the family Potyviridae, genus Ipomovirus. Both these groups of viruses are transmitted by the whitefly, Bemisia tabaci, family Aleyrodidae. Until recently CMD is widespread and more damaging while CBSD was benign and confined to the coastal and lake shore areas in eastern and southern Africa at altitudes below 1000 metres above sea level. However, in the last 5-6 years CBSD has spread to mid to high altitudes as it invaded areas that were previously affected by CMD. This has created multiple infections of both the diseases, the diagnosis of which is a challenge because of the nature of the viruses involved (RNA and DNA). In order to address this, we have developed a suite of nucleic acid extraction protocols, virus-specific and generic protocols as well as uniplex and multiplex reverse transcription polymerase chain reaction (RT-PCR) assays for the simultaneous detection of both DNA and RNA viruses. The cetyl trimethyl ammonium bromide (CTAB) method was optimised for the simultaneous extraction of DNA and RNA molecules. Novel primers were designed (CBSVF3&R3, CBSVF5&R3) which were twice as efficient in detecting CBSV/CBSUV compared to the currently available CBSV10&11 primers in field-collected samples, while CBSVF2&R7 and CBSVF2&R8 differentiated CBSV and CBSUV. Using the multiplex RT-PCR we were able to detect simultaneously CBSV/CBSUV either with ACMV or EACMV, which increased the efficiency of virus detection while reducing the associated costs and minimised sample handling. As well as their utility for virus diagnosis, development of these protocols has provided new insights into the population dynamics of CBSV/CBSUV in which widespread mixed infections of these viruses were found in the field, implications of which will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission

Virus Family: Geminiviridae

Category: Epidemiology/transmission

Title: Studies on single and double infection of bipartite begomoviruses by whitefly transmission

Authors: M. A. MACEDO1,2, V. N. GUIMARÃES1,2, T. G. PEREIRA1,2, P. P. F. LEMOS1,2, A. K. INOUE-NAGATA1,2.

Andress: 1Universidade de Brasília, 70910-900 Brasília, Brazil; 2Embrapa Vegetables, Brasília/Anápolis, BR 060 Km 09, C.P. 218, 70359-970, Brasília, Brazil.

Abstract: Tomato severe rugose virus (ToSRV) and Tomato yellow vein streak virus (ToYVSV) are bipartite begomoviruses, transmitted by whiteflies (Bemisia tabaci biotype B), frequently found infecting tomato (Solanum lycopersicum) crops in Brazil. To more fully understand the interaction begomovirus/whitefly, as well as how occurrence of mixed infection may influence virus emergence and dominance in an infected plant, a study on begomovirus transmission was carried out focusing on determination of transmission efficiency of two begomoviruses, ToSRV and ToYVSV, on single and double infection. At first, the transmission methodology was optimized under our conditions. By using one insect per plant and clip cages we were able to obtain a transmission efficiency of ca. 70%, when using ToSRV and the susceptible “Viradoro” tomatoes. Then, transmission tests were done with single and double infections, using the ToSRV 1164 and ToYVSV- TGVV isolate. Whitefly colonies were maintained on virus-free cabbage or tobacco plants in a greenhouse. The virus isolates were inoculated on tomato plants ca. 30 days prior to transmission tests, when they showed clear interveinal chlorosis and leaf distortion symptoms. Infection was confirmed by PCR using species specific primers. A large population of whiteflies was allowed to mass feed on virus-infected source plants for 48h acquisition access periods (AAP). Following virus acquisition, the whiteflies were transferred to clip-cages (one insect per cage for single virus inoculation, and two individuals, each one fed on each virus source, for double inoculation) and allowed an inoculation access period (IAP) of 48h. Insects were then manually killed, and only those with one alive insect on singly inoculated plant and two insects for doubly inoculated one were used for further analysis. Plants were incubated for three weeks and analyzed for the presence of ToSRV and ToYVSV by PCR or rolling circle amplification (RCA) followed by RFLP using MspI. Total DNA from plants infected with ToSRV and ToYVSV subjected to RCA-RFLP generated different patterns, enabling detection of each species and occurrence of mixed infection. The percentage of plants infected with both viruses was low in all trials. This indicated that a type of cross protection may exist on begomovirus-tomato interaction. In the double virus inoculation, transmission efficiency of ToYVSV was higher than of ToSRV in two out of three tests. This was an unexpected result since apparently there is a natural predominance of ToSRV over ToYVSV in the field. However, trials are being repeated with larger numbers of replicates to confirm this result.

Financial support: UnB, CAPES, Embrapa, National Research Institute for Plant-Pest Interactions

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Epidemiology

Title: EPIDEMIOLOGY AND ECO-FRIENDLY MANAGEMENT OF YELLOW MOSAIC VIRUS OF GRAIN LEGUMES IN INDIA

Author: YPS Rathi (Emeritus Scientist)

Address: B-55, Pallavpuram - I, Meerut - Meerut - 250110, UP, India.

Mungbean yellow mosaic virus (MYMY) also called as a” yellow plague”of grain legumes is vectored by whitefly (Bemisia tabaci Genn) in a persistent circulative manner. Currently, it is a number one problem causing substantial yield losses in urdbean (Vigna mungo), mungbean (V. radiata), mothbean (V.aconotifolia) and soybean (Glycine max) in India. In case a susceptible genotype gets infected at an early stage of crop growth yield losses may even go up to 100%. The foliage of host crop exhibits bright yellowing and the disease field can be recognized from far distance .A better understanding of the epidemiology and application of plant and cow products may provide new approaches to develop eco-friendly management practices to manage the disease. Studies on epidemiology in the Tarai region of Uttarakhand state revealed that besides the population of whiteflies, weather parameters like temperature, rainfall and humidity play important role in the development of the disease epidemic.

Whitefly population increased with the rise in temperature. High relative humidity, heavy shower and strong winds in rainy season were found detrimental to whitefly adults. Further studies on host range revealed that ratoon crop of pigeonpea and/ or some other weeds might be serving as primary source of inoculum.

Experiments were conducted to contain this disease through agronomic practice (manipulation in planting dates and intercropping with barriers (non-host) crops, insecticides (foliar and soil application, seed treatment),spray of cow products (milk, buttermilk ,urine),neem oil and host resistance. Low disease incidence was recorded in early (June) and late (August) planting as well as low plant spacing (5cm). Seed dressing with carbofuran 3-G or phorate 10-G with two foliar sprays of 1.0% neem oil+1.0% butter milk +2.0% cow urine + 1.0% detergent significantly reduced the disease incident. Inter-cropping of non host crops with grain legumes were not found very effective barriers. Some genotypes viz. Pant U 19, 30, 35 and PU-1 GD (urdbean), PM 1,2,3 & 4 (mungbean) and PK 416, PK 564,PK 1029, SL 142(Soybean) resistant to MYMV have been identified.

None of the individual management approach was found highly effective against the vector / virus due to high vector population and wide range of host plants serving as initial foci of the whitefly vector and the virus through out the year. Moreover, a single whitefly adult is sufficient to initiate the infection. Hence the development of integrated management strategy having tolerant varieties in core is the only promising way to manage this “yellow plague” of grain legumes effectively.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Epidemiology / Transmission

Title: Infectivity of blackgram isolate of Mungbean yellow mosaic India virus on cowpea.

Authors: Rouhibakhsh1, A. and V. G. Malathi2

Address: 1. Department of Horticulture, Agricultural Faculty, Ilam University, Ilam, Iran. 2. Advance Center for Plant Virology, Division of Plant Pathology, IARI, New Delhi, 110012, India.

Abstract: Yellow mosaic disease of grain legumes is caused by four virus species under the genus Begomovirus. Mungbean yellow mosaic India virus (MYMIV) is more prevalent in northern India. The blackgram isolate (Bg) of MYMIV is known not to infect cowpea through whitefly inoculation. In the present study, agroinoculation of Bg isolate was performed on three cultivars of cowpea and the results were compared with that of cowpea (Cp) isolate of MYMIV. The Bg isolate was infectious on cowpea, and produced leaf curl symptoms only in cv Pusa Komal. The isolate was replication incompetent as only trace levels of viral replicative forms were detected in all the three cultivars tested. The Cp isolate produced severe leaf curl symptom in cv. Pusa Komal besides the golden mosaic symptoms. Agrobacterium mediated delivery of viral components clearly established that, blackgram isolate is capable of infecting cowpea and causes atypical leaf curl and not yellow mosaic symptoms.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Epidemiology/transmission

Title: Study of weeds as begomovirus source to tomato plants in Brazil.

Authors: S. S. BARRETO1, M. A. REIS2, P. P. F. LEMOS1 AND A. K. INOUE- NAGATA1,2

Address: 1Universidade de Brasília, 70910-900 Brasília, Brazil; 2Embrapa Vegetables, BR 060 Km 09, C.P. 218, 70359-970, Brasília, Brazil.

Abstract: Weeds can potentially act as virus sources for many diseases, favoring their spread in economically important crops. The begomoviruses, transmitted by whiteflies, cause worldwide epidemics on the tomato crop and there is enough evidence that they can also infect various weed species, including those that occur frequently in cultivated tomato fields. The study of potential alternative hosts of begomovirus is essential to contribute for the formulation of effective control measures. The objective of this study was to identify the begomoviruses present on weeds and evaluate their ability to infect tomato plants. Initially, some begomovirus infected weed samples (total DNA) from the begomovirus collection of Embrapa Vegetables were selected for inoculation to tomato plants by particle bombardment. Total DNA of samples from Crotalaria sp. (#768), Euphorbia heterophylla (#3539), Nicandra physaloides (#1646) and Sida sp. (#780) were subjected to rolling circle amplification (RCA) and inoculated by particle bombardment in tomato plants and in the original (or closely related species) weed seedlings: Crotalaria incana, E. heterophylla, N. physaloides and Sida santaremnensis. Inoculated plants were kept in the greenhouse and, after 20-30 days, they were tested for begomovirus infection by PCR with universal begomovirus primers. Infection was confirmed in all plant species demonstrating that these samples contained begomoviruses able to infect tomato plants. All infected plants showed typical begomovirus infection symptoms, such as mosaic, leaf distortion and stunting, with the exception of tomato plants inoculated with #768 preparation. Then, the DNA-A was cloned from the infected plants for begomovirus identification. From infected C. incana and N. physaloides plants, clones of Tomato severe rugose virus (ToSRV) were obtained; from E. heterophylla, clones of Euphorbia yellow mosaic virus (EuYMV); from S. santaremnensis, Sida micrantha mosaic virus (SiMMV); and ToSRV clones were isolated from all tomato plants, but in those inoculated with Crotalaria sp. (#768) ToSRV and EuYMV clones, while with E. heterophylla (#3539) ToSRV and SiMMV clones were found. Primers specific for each begomovirus species were produced and confirmed the presence of each species in the original DNA sample. This result suggested that all four weed species are potential begomovirus source for tomato plants, and that ToSRV was, in general, preferentially replicated in tomato cells. As the begomoviruses are transmitted by whiteflies in nature, we are now testing these plants as begomovirus source by vector transmission. Preliminary results indicate that N. physaloides is a better source to tomato begomovirus than Sida sp.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Epidemiology/Transmission Virus Family: Geminiviridae

Category: Disease Epidemiology, Transmission

Title: Molecular characteristics of Cotton leaf curl Multan virus and its associated satellite DNA infecting Hibiscus rosa-sinensis and okra in China.

Authors: Zifu He1, 2, Dong Di1, Mingjie Mao1, Xiao-man She1, Judith K Brown2

Address: 1 Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, Guangdong, 510640, China; 2 School of Plant Sciences, The University of Arizona, Tucson, AZ, 85721, USA.

Cotton leaf curl disease, caused by the genus Begomovirus (family, Geminiviridae), is a major constraint to sustainable cotton production in Asia (Pakistan and India) and Africa (Egypt and Sudan). In Asia the disease is caused by a complex of begomoviruses that includes Cotton leaf curl Alabad virus (CLCuAV), Cotton leaf curl Kokhran virus (CLCuKV), Cotton leaf curl Multan virus (CLCuMV), Cotton leaf curl Rajasthan virus (CLCuRV), Cotton leaf curl Bangalore virus (CLCuBV), Papaya leaf curl virus (PaLCuV), Tomato leaf curl Bangalore virus (ToLCBV) and Cotton leaf curl Burewala virus (CLCuBuV). In Egypt and Sudan, the disease is caused by several strains or variants of Cotton leaf curl Gezira virus (CLCuGV). Begomoviruses cause similar symptoms in cotton and symptoms are characterized by upward curling of leaves, vein swelling, and formation of enations on the undersides of leaves from or near the main and minor veins. In 2005, a begomovirus- associated leaf curl disease was observed for the first time in China rose (Hibiscus rosa- sinensis Linn.) in China. The viral genome for isolate G6, from Guangdong, was PCR amplified from DNA extracts of symptomatic China rose leaves, cloned, and sequenced. The complete nucleotide (nt) sequence of DNA-A was found to be 2737 nt in length, and to contain six predicted ORFs. Sequence alignment (Clustal W in DNASTAR (Lasergene) and BLASTn analysis revealed that the DNA-A shared more than 89% nt sequence identify with the published sequences for CLCuMV isolates, and the highest nt sequence identify, at 96.1%, with isolate 62 of CLCuMV [GenBank Accession AJ002447]. The DNA- A shared its next highest nt identity, at 87.1%-89.8%, with the isolates of CLCuRV, and less than 87% identity with all other begomoviruses. A DNA β type satellite was amplified from DNA extracts using the PCR primers β01 and β02 (Briddon et al., 2001). The DNA β was 1346 nt in size and encoded a predicted C1 ORF in the complementary strand. The DNA β shared its highest sequence identity with the CLCuMV and CLCuRV associated DNA β at 92.1% and 88.7%, respectively, and less than 80% identity with other begomovirus-associated satellites. In 2008, yellow vein and leaf curl symptoms were observed in okra (Abelmoschus esculentus (L.) Moench) plants in Guangdong. An apparently full-length begomoviral genome was cloned and sequenced from symptomatic okra plants (okra06 isolate), revealing the presence of a DNA-A component (2737 nt) and an associated DNA β type satellite (1346 nt). The okra06 DNA-A sequence and DNA β shared their highest sequence identities, at 99.0%, respectively, with CLCuMV–[G6] and its associated satellite. Collectively, these results indicate that the begomovirus-satellite complex infecting China rose and okra plants in Guangdong, China is most like the strain of CLCuMV having its extant origin in Pakistan.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression Virus Family: Geminiviridae

Category: Replication/ Gene expression

Title: Two strains of Euphorbia mosaic virus are incompatible in replication and contain short Rep sequences in the BV1 promoter region.

Authors: B. BAÑUELOS-HERNÁNDEZ, J. GREGORIO-JORGE, G.R. ARGÜELLO-ASTORGA

Address: Instituto Potosino de Investigación Científica y Tecnológica, A.C. Camino a la Presa San José 2055, Colonia Lomas 4ta. Sección, San Luis Potosí, S.L.P., México C.P. 78216. E-mail: [email protected]

Abstract: Euphorbia mosaic virus (EuMV) is a member of the SLCV clade, a lineage of New World begomoviruses that display distinctive features in their replication-associated protein (Rep) and virion-strand replication origin. Two different strains of EuMV have been isolated in Mexico, one in the Yucatan Peninsula (YP) and another in the Jalisco State. These strains display different putative replication specificity determinants, and we decided to investigate whether or not these begomoviruses are compatible in replication. Pseudorecombination experiments with EuMV-Jal and EuMV-YP genomic components demonstrated that they do not form infectious reassortants in Nicotiana benthamiana, hence indicating that this begomovirus species probably is not a replicating lineage in natural ecosystems. Sequence analysis of the DNA-B intergenic region (IR) of EuMV strains led to the unexpected discovery of short sequences ranging from 35- to 51-nt in length that are identical to a segment of the rep gene in the cognate viral DNA-A. Similar short rep sequences were identified in three distinct viruses from South America related to EuMV. These short rep sequences in the DNA-B IR are positioned downstream to a ~160-nt non-coding domain highly similar to the CP promoter of viruses belonging to the SLCV clade. This assemblage of DNA-A- related sequences within the DNA-B IR is reminiscent of polyomavirus microRNAs and could be involved in the posttranscriptional regulation of the cognate viral rep gene, an intriguing possibility that is currently being examined in our laboratory.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression Virus Family: Geminiviridae

Category: Replication/Gene expression

Title: The impact of geminiviral proteins on the nuclear architecture

Authors: Krenz B. 1, Neugart F. 2, Jeske H. 1 and Kleinow T. 1

Address: 1University of Stuttgart, Institute of Biology, Dpt. of Molecular Biology and Plant Virology, Stuttgart, Germany 2University of Stuttgart, Institute of Cell Biology, Stuttgart, Germany

Abstract: Geminiviruses constitute a large group of plant viruses whose genome is packed as single- stranded DNA circles in a small, twinned isometric particle. The New World geminivirus Abutilon mosaic virus (AbMV) possesses a bipartite genome consisting of DNA A and DNA B, which are replicated via double-stranded intermediates within plant cell nuclei. DNA A encodes proteins which are necessary for viral replication, transcription and encapsidation, and among those proteins the multifunctional replication-associated protein (Rep; syn. AC1or AL1) is the only one which is essential for replication. The two DNA B- encoded proteins, the nuclear shuttle protein (NSP; syn. BV1 or BR1) and the movement protein (MP; sy. BC1 or BL1), facilitate systemic spread of viral genomes within the host plant and have both an impact on viral pathogenicity. Earlier studies have shown that a geminiviral infection induces characteristic changes in the plant nuclear architecture, e.g. hyperthrophy of nucleus and nucleolus, segregation of nucleoli into discrete granular and fibrillar regions and redistribution of the plant chromatin to the nuclear margin. To investigate this nuclear remodeling in closer detail, we overexpressed AbMV Rep and/or the movement-associated proteins (MP and NSP) simultaneously with fluorescent protein- tagged marker proteins for the nuclear enevelope (NE) and/or nucleoli/cajal bodies. Solely overexpression of Rep in leaf tissues in AbMV DNA B-transgenic Nicotiana benthamiana led to intense necking, budding and nearly division of the nuclei as monitored by the NE marker. Even more intense alterations of the organization from the plant nuclei/NE were observed upon overexpression of Rep, MP and NSP. NE-coated vesicles invaginated into the nucleus as well as budded from the nuclear surface. Some vesicles, contained nucleoplasm and seem to move to the cell periphery. In summary, these data points out a putative alternative route for geminiviral intracellular movement.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression

Virus Family: Geminiviridae

Category: Replication/Gene expression

Visualization of Abutilon mosaic virus replication-associated protein self-interaction by bimolecular fluorescence complementation in planta

Authors: Krenz B. 1, Neugart F. 2, Kleinow T. 1 and Jeske H. 1

Address: 1University of Stuttgart, Institute of Biology, Dpt. of Molecular Biology and Plant Virology, Stuttgart, Germany 2University of Stuttgart, Institute of Cell Biology, Stuttgart, Germany

Abstract: Geminiviruses are small DNA viruses that replicate in the nuclei of infected plant cells. The viral multifunctional replication-associated protein (Rep) catalyzes the initiation of rolling-circle replication and is the only viral-encoded protein essential for virus replication. In vitro studies revealed that Rep contains an oligomerization domain leading to self-interaction, which is essential for the protein's functionality. This, furthermore, is also correlated with its localization within cells. To investigate the subcellular localization and the homooligomerization potential of Abutilon mosaic virus (AbMV) Rep in living plant cells in detail, we used the bimolecular fluorescence complementation (BiFC) approach and confirmed self-interaction and nuclear as well as subnuclear localization domains in the epidermal tissue of N. benthamiana plants. Furthermore, RCA/RFLP- and Southern Blot-analyses were performed to prove functionality of split-YFP tagged AbMV Rep in viral DNA replication.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression Virus Family: Geminiviridae

Category: Replication/ Gene expression

Title: The begomoviral conserved late element (CLE) is a functional target of plant transcription factors

Authors: M. CANTÚ-IRIS 1, A. JUÁREZ-REYES 1, R. RIVERA-BUSTAMANTE, R. 2, Y G. ARGÜELLO-ASTORGA1

Address: 1Instituto Potosino de Investigación Científica y Tecnológica, A.C. Camino a la Presa San José 2055, Colonia Lomas 4ta. Sección, San Luis Potosí, S.L.P., México C.P. 78216. 2Departamento de Ingeniería Genética, Cinvestav Irapuato, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México. E-mail: [email protected]

Abstract: The genes cp, mp and nsp of bipartite begomoviruses are expressed at late stages of the infective cycle, and their expression is regulated by the transactivation protein, TrAP. The extant evidence indicates that there are TrAp- responsive elements in the promoter region of begomovirus late genes, but none of them has been unequivocally identified at present. The conserved late element (CLE) is an octameric motif (GTGGTCCC) present in the intergenic region of many begomoviruses, for this reason we intended to elucidate its function in the regulation of late genes. In order to explore this question we generated tobacco transgenic plants harboring constructs in which synthetic DNAs containing either multimers of CLE or combinations of CLEs with other viral regulatory elements, like the G-box motif, were cloned upstream of a minimal 35S promoter fused to the reporter gene uidA. Hystochemical assays to detect β-glucuronidase activity in the transgenic plants revealed that the CLEs are recognized by plant transcription factors, because multimers of these elements activate the reporter gene expression in mesophyll but not in vascular tissues in absence of any viral proteins. The number of CLE elements and the associated cis-regulatory elements clearly influenced the activity of the synthetic promoters. The experimental infection of the transgenic plants with begomoviruses displaying different tissue tropism will make feasible to determine whether or not the CLEs are actually involved in the transactivation by TrAP.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression Virus Family: Geminiviridae

Category: Replication/Gene Expression

Title: Analysis of trans-replication of a betasatellite by a begomovirus

Authors: Khadim Hussain, Mazhar Hussain, Shahid Mansoor and Rob W. Briddon

Address: National Institute for Biotechnology and Genetic Engineering (NIBGE), Jhang Road, P.O. Box 577. Faisalabad Pakistan

Abstract: Begomoviruses (genus Begomovirus, family Geminiviridae) are whitefly transmitted viruses with circular, single-stranded (ss)DNA genomes that are encapsidated in twinned icosahedral particles. These viruses cause serious diseases of cultivated crops across the tropics and, increasingly, more temperate regions. In contrast to the view held earlier by geminivirologists, that the majority of begomoviruses have genomes consisting of two ssDNA components (known as DNA A and DNA B), it has become evident that in the Old World the vast majority of begomoviruses have genomes consisting of a single ssDNA component and associate with a newly identified class of ssDNA satellites known as betasatellites. In the 10 years since their first identification, significant advances have been made in determining the functions and interactions of betasatellites. Nevertheless, significant questions remain to be answered. The study described here was designed to investigate the transreplication of betasatellites by begomoviruses. The begomovirus Cotton leaf curl Multan virus (CLCuMV) and its cognate betasatellite Cotton leaf curl Multan betasatellite (CLCuMB) were used as the model system for these studies. Deletion mutagenesis was used to identify betasatellite sequences important for transreplication of CLCuMB by CLCuMV. This study showed that two regions of sequence are likely important, although no sequence deletion appeared to entirely abolish transreplication. The first contains a predicted hairpin structure with similarity to a hairpin structure in all geminiviruses that is nicked by Rep to initiate DNA replication. The second encompasses a hypervariable sequence that contains motifs with similarity (but not identity) to the iterons of CLCuMV. Based on this finding, and the results published by others, a model of the origin of replication (ori) of betasatellites is proposed and this model is compared to the ori of begomoviruses.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Replication/ Gene expression Virus Family: Geminiviridae

Category: Replication/Gene expression

Title: Characterization of strong promoters from American begomoviruses

Authors: A. LONDOÑO, L.R. RIEGO-RUIZ, G.R. ARGUELLO-ASTORGA

Address: División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica. Camino a la Presa San José 2055, Col. Lomas 4 sección, P.O. Box 78216, San Luis Potosí, SLP, México.

Abstract: Several works point out the usefulness of geminiviral genomes as source of material for biotechnological applications. From these elements promoters got especial interest because recent reports of highly active promoters in Old-World begomoviruses and other ssDNA viruses infecting plants. In this work we use the beta-glucuronidase reporter system to quantitatively evaluate the strength of the Rep promoter of representatives of the two American begomoviruses lineages (typical and Squash leaf curl virus´s clade). After a preliminary screening to test the experimental system we have identified a promoter from a virus of the SLCV clade that is three times more efficient than CaCMV P35S. A second member of this clade does not exhibit such activity, suggesting that the capacity of the promoter to drive the transcription of the reporter gene is not related with lineage-conserved elements. In fact, when the promoter was reduced to its minimal composition (only a TATA-box and the iterated sequences) both promoters behaved equal and had an activity similar to its equivalent in typical begomoviruses. The elements that might be responsible of the activity of the promoter have been identified by sequences analysis and are pending to be characterized experimentally.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution

Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Genetic diversity by extensive recombination in the complex of monopartite begomoviruses infecting sweet potato in Brazil

Authors: L.C. ALBUQUERQUE1,2,4, B. PINHEIRO1,2, S.G. RIBEIRO3, R.O. RESENDE2, E. MORIONES4, A.K. INOUE-NAGATA1,2, AND J. NAVAS- CASTILLO4

Address: 1Embrapa Vegetables, C.P. 218, Brasília, DF, 70.359-970, Brazil. 2Universidade de Brasília, Brasília, DF, 70910-970, Brazil. 3Embrapa Cenargen, C.P. 02372, Brasília, DF, CEP 70970-900, Brazil. 4Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), 29750 Algarrobo- Costa, Málaga, Spain.

Abstract: Sweet potato (Ipomoea batatas) is one of the most important root crops in the world and has been cultivated through vegetative propagation for food, animal feed and industrial raw material. Sweet potato plants are usually infected by a number of pathogens, especially viruses. Recently, a number of Ipomoea- infecting monopartite begomoviruses (genus Begomovirus) have been described that could pose a threat to sweet potato production. Here, we present data on the molecular diversity of a number of begomovirus isolates collected in Brazil from the Sweet Potato Embrapa Germoplasm Bank (SPEGB) and from commercial sweet potato fields. Total DNA was extracted from leaf samples and subjected to rolling cycle amplification (RCA) using φ29 DNA polymerase. The RCA products were digested with a set of restriction enzymes to identify unique sites for cloning of full- length genomes. Fifty-eight restricted fragments corresponding to putative full- length genome components were cloned and sequenced. Only the 36 sequences with <99% nucleotide identity among each other were considered for further analysis. We found the presence of a novel species and four new strains of the previously described species Sweet potato leaf curl virus (SPLCV), Sweet potato golden vein-associated virus (SPGVaV), Sweet potato leaf curl Spain virus (SPLCESV) and Sweet potato leaf curl Lanzarote virus (SPLCLaV), and named here as SPLCV-Pernambuco (SPLCV-PE), SPGVaV-Rondônia (SPGVaV-RO), SPLCESV-Bahia (SPLCESV-BA) and SPLCLaV-São Paulo (SPLCLaV-SP). Our results indicated that sweet potato plants cultivated in Brazil are infected by monopartite begomoviruses with a high degree of genetic variability, both in the SPEGB and field samples. Phylogenetic and recombination analyses revealed extensive evidence of intra- and inter-species genetic exchange.

Financial support: CAPES, CNPq, Embrapa, FAP-DF th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus family: Geminiviridae

Category: Virus Diversity and Evolution

Title: The first monopartite begomoviruses discovered in Western Hemisphere endemic eudicots: evidence for swepovirus diversification in ‘wild’ and ‘cultivated’ hosts Authors: J. K. Brown1, H. Delatte2, J. Bird,3 and Ali M. Idris1,4 Address: 1School of Plant Sciences, The University of Arizona, 85721, Tucson, AZ, USA; 2CIRAD, UMR PVBMT, Pôle de Protection des Plantes, 7 chemin de l’IRAT, 97410 Saint Pierre, La Réunion, France; 3College of Agricultural Sciences, University of Puerto Rico, Rio Piedras, PR 00928; 4Plant Stress Genomics and Technology Research Center, King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia

Abstract: The first extant Western Hemisphere monopartite begomovirus genomes were discovered infecting two uncultivated eudicots, Merremia quinquefolia and M. aegyptii (L.) Urb endemic to the Caribbean Basin. One virus, herein, Merremia leaf curl virus (MeLCV), is a previously unreported recombinant species, whereas, the other virus is a new, distinct strain of Sweet potato leaf curl virus (SPLCV). Phylogenetic analyses of the cloned genomes for multiple sweet potato infecting isolates and swepovirus-like relatives grouped as two major clades within the genus, Begomovirus, whose members comprise viruses and strains isolated from the cosmopolitan sweet potato plant (Ipomoea batatas) (L.) and blue morning glory (Ipomoea indicata (Burman) (Merrill), a perennial vine native to the U.S. and American Tropics but has established elsewhere in mild climate zones worldwide. Recombination analysis revealed that MeLCV is a double recombinant. In contrast SPLCV-PR is not a recombinant genome but diverges from SPLCV at 92%, making it a new begomoviral strain. All attempts to detect (beta) satellite DNAs or (alpha) DNA-1 or DNA-2 nano-like DNA molecules in purified nucleic acid preparations by Southern hybridization or PCR using various primer pairs failed to reveal these types of molecules in symptomatic source plants. However, defective interfering DNAs (one quarter unit length) were frequently detected in Merremia species infected by MeLCV and SPLCV-PR. Phylogenetic (maximum likelihood and Bayesian), recombination analysis, and molecular clock studies were undertaken to investigate the evolutionary genesis of SPLCV-PR and MeLCV isolates extant in eudicots endemic to the Caribbean region, in relation to their closest begomovirus relatives. Results thus far provide evidence of two divergent swepovirus lineages and molecular clock analysis suggests that the two monopartite viruses from wild eudicots in Puerto Rico have diversified since establishing there. Sweet potato is known to be native to the Caribbean region and South America and is thought to have been carried from there to the eastern hemisphere (Africa, Asia) by European explorers. Sweet potato has been radiocarbon-dated in the Cook Islands to 1000 AD, and current thinking is that sweet potato (kamura) was brought to central Polynesia circa 700 AD, possibly by Polynesians who had traveled to South America, and then was distributed across Polynesia to Hawaii and New Zealand from there. The implication is that sweet potato either was infected with swepo-like viruses prior to its worldwide distribution and that these viruses are of New World origin, or else that sweet potato became infected in Eastern Hemisphere locations following its introduction there, with the subsequent introduction into the Americas via infected sweet potato tubers at a later date. If either scenario, in Puerto Rico, the whitefly vector would have been crucial for virus transmission from sweet potato to Merremia spp. and seemingly for subsequent spread and maintenance of the viruses in the landscape and/or cultivated sweet potato. Likewise, for swepoviruses to spread to blue morning glory in Spain (from unknown source plants), a whitefly vector would have been required. Follow-on analysis is underway to apply more sophisticated sequence analyses to more succinctly elucidate the evolutionary relationships among swepoviruses from wild eudicot species in the Caribbean region where sweet potato and blue morning glory also has their centers of diversification.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Genetic structure of tomato-infecting begomovirus populations in two growing regions of Southeastern Brazil

Authors: G.P. CASTILLO-URQUIZA1, P. ALFENAS-ZERBINI1, J.E.A. BESERRA- JUNIOR1, E.S.G. MIZUBUTI1, A. VARSANI2, D.P. MARTIN3 AND F.M. ZERBINI1 Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, 36570-000, Viçosa, MG, Brazil; 2School of Biological Sciences, University of Canterbury, Private bag, Christchurch, 8140, New Zealand; 3Computational Biology Group, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, South Africa

Abstract: The incidence of begomoviruses has sharply increased in Brazil since the mid 1990’s, after the introduction of the B biotype of the whitefly Bemisia tabaci. It is believed that the insect vector transferred indigenous viruses infecting wild and weed hosts to tomato. After a rapid evolutionary process, novel species adapted to the new host became prevalent in the field. The objective of this work was to determine the genetic structure of begomovirus populations infecting tomatoes and associated weeds in two major tomato growing regions of Southeastern Brazil. The genetic structure of populations refers to the degree of genetic variability and its distribution within and between subpopulations, reflecting the evolutionary history and the potential of the population to evolve. A total of 119 tomato samples were collected in Paty do Alferes, Rio de Janeiro state, in May 2005. In Coimbra, Minas Gerais state, 17 tomato samples and 43 weed samples were collected in July 2007. Total DNA was extracted from each sample and full-length begomovirus genomes were amplified using the phage phi29 DNA polymerase, cloned into plasmid vectors and completely sequenced. Sixty-nine DNA-A clones were completely sequenced and the sequences were used for the determination of the genetic structure of viral populations. A total of eight viral species were detected, including six which were detected for the first time. Two species were prevalent in tomatoes: Tomato yellow vein streak virus (ToYVSV), detected only at Paty do Alferes, and Tomato common mosaic virus (ToCmMV), detected at both locations. Analysis of the genetic structure of ToYVSV and ToCmMV subpopulations indicated a higher degree of genetic variability for ToCmMV. Furthermore, comparison between ToCmMV subpopulations from Coimbra and Paty do Alferes indicates greater variability in Coimbra. Considering that the presence of begomoviruses in tomatoes has been verified in Coimbra since 2001, but was verified for the first time in Paty do Alferes only in 2005, these results are consistent with a recent introduction of viral populations in Paty do Alferes, followed by a rapid population expansion. In Coimbra, the virus and its host may have been co-evolving for a longer time.

Financial support: National Research Institute for Plant-Pest Interactions and Pronex- Fapemig

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Virus diversity and evolution

Title: Epidemiology and molecular characterization of Tomato curly stunt virus and its insect vector Bemisia tabaci in South Africa

Authors: L.L. ESTERHUIZEN1, S.W. VAN HEERDEN2, M.E.C. REY3 and H. VAN HEERDEN4

Address: 1 Department of Biochemistry, University of Johannesburg, Johannesburg, South Africa. 2Sakata Vegenetics RSA (Pty) Ltd., Lanseria, South Africa. 3 Department of Cell and Molecular Biochemistry, University of Witwatersrand, Johannesburg, South Africa. 4 Department of Veterinary Tropical Diseases, University of Pretoria, Pretoria, South Africa.

Abstract:

Tomato curly stunt virus (ToCSV) is a tomato yellow leaf curl disease present in southern Africa. Since its introduction in South Africa (1998), ToCSV has become one of the most destructive tomato viral diseases in the country. The distribution and genetic diversity of ToCSV and its whitefly vector Bemisia tabaci were monitored from 2006 to 2009. ToCSV specific primers developed in this study was used to screen samples for ToCSV infection and representative viral genomes (2.7 kbp) were sequenced. The survey results indicated that ToCSV distribution has gradually radiated north- and south-wards and the virus is now well established and widespread in 5 of the 9 provinces in South Africa. Sequence analysis of 45 viral isolates revealed potential recombination events and limited genetic variation among isolates (95%). The highest mutational frequencies were in the IR, V1 and C1 open reading frame. Phylogenetic analysis detected two genetic clusters at 4-5% nucleotide divergence, with geographical but not temporal variation. Construction of agroinfectious viral clones of the two genetic clusters indicated that ToCSV has a monopartite genome and was also used to investigate an apparent link between genotype and symptom severity. Mitochondrial cytochrome oxidase I sequences used to genotype B. tabaci populations from different localities in South Africa identified three genotypes: the B biotype, the Q biotype and a distinct non-B haplotype with a restricted distribution. The B biotype was the most extensively distributed biotype and largely responsible for the increase in ToCSV incidence. Although the Q biotype is a recent introduction and of limited geographical range in South Africa, it is already associated with the ToCSV epidemic.Furthermore, two new begomovirus species closely related to ToCSV (81% and 84% nucleotide homology) and associated with yellowing and leaf curling symptom phenotype was identified, based on the 89% begomovirus species demarcation criteria.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Bipartite and monopartite begomoviruses infecting Ipomoea spp. and Merremia spp. in Venezuela

Authors: E. FIALLO-OLIVÉ1,2, B. MÁRQUEZ-MARTÍN1, F. GERAUD-POUEY3, D. T. CHIRINOS3, J. NAVAS-CASTILLO1 AND E. MORIONES1

Address: 1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM- UMA-CSIC), 29750 Algarrobo-Costa, Málaga, Spain. 2Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba. 3Universidad del Zulia, Maracaibo, Zulia , Venezuela.

Abstract: Wild plants play an important role in maintaining a high diversity of pathogens, including viruses, potentially hazardous to crops. We have conducted extensive surveys in several regions of Venezuela to characterize the genetic diversity of begomoviruses present in symptomatic wild plants. Rolling circle amplification (RCA) using φ29 DNA polymerase was used to amplify full-length begomovirus genomes from DNA extracts. RCA products were analyzed by restriction enzyme analysis and fragments corresponding to putative full-length genome components (DNA-A or DNA-B) were cloned and sequenced. Here we present the results obtained with samples of wild species of the genera Ipomoea and Merremia (family Convolvulaceae). Sequence analysis allowed to identify three novel bipartite begomovirus species and a novel strain of Merremia mosaic virus, all of them with the typical genome structure of the New World begomoviruses. One of the samples infected with a bipartite begomovirus was also infected with a “swepovirus”, i.e., an Ipomoea-infecting monopartite begomovirus. A unique DNA satellite molecule, far related to those recently found associated with Ipomoea-infecting begomoviruses in Spain, was also found in the aforementioned sample. These results support a high genetic diversity of the begomovirus populations infecting wild Ipomoea spp. and Merremia spp. plants in Venezuela and highlight the possible relevance of these wild plants as reservoirs of begomoviruses that could pose a threat to cultivated crops, including sweet potato (I. batatas).

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Begomovirus diversity and recombination in the Caribbean, a hot ‘melting pot’ in the New World

Authors: E. FIALLO-OLIVÉ1,2, E. MORIONES2, J. NAVAS-CASTILLO2 AND Y. MARTÍNEZ-ZUBIAUR1

Address: 1Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba. 2Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), Estación Experimental “La Mayora”, 29750 Algarrobo-Costa, Málaga, Spain.

Abstract: Solanaceous crops in the Caribbean have shown in recent years an increase in symptoms suggestive of viral diseases. Many of these diseases are transmitted by the whitefly Bemisia tabaci and most of them are probably caused by begomoviruses (genus Begomovirus, family Geminiviridae). Thus, several novel begomoviruses have been found recently in Cuba causing diseases in relevant crops such as tomato, tobacco and pepper [1, 2]. Moreover, many weeds are also prone to be infected by begomoviruses in the Caribbean, standing out the triad Euphorbia spp., Sida spp. and Rhynchosia minima, which have been shown to constitute a continuous source of novel begomovirus species [e.g., 3]. It is obvious the importance that these reservoirs can have in the emergence of new begomoviruses with unpredictable pathological consequences for horticultural crops. In order to understand the molecular diversity of the begomoviruses found in the Caribbean and the factors that could have been involved to generate that diversity, we have conducted a thorough phylogenetic and recombination analysis of the recently discovered begomovirus species in this region, comparing them with isolates representative of all the New World begomovirus species available in the databases. As it has been shown in other areas, recombination is confirmed as a major source of variation conducting to the appearance of novel begomovirus species. Also, it is worth noting that a number of recombinant genomes exhibit as putative parental begomoviruses closely related to species reported from the Caribbean islands, Florida (USA), and Yucatan peninsula (Mexico), making this region a true “melting pot” contributing to the generation of begomovirus diversity.

[1] Fiallo-Olivé E, Martínez-Zubiaur Y, Rivera-Bustamante RF (2009) Plant Pathol 58: 785. [2] Fiallo-Olivé E, Rivera-Bustamante RF, Martínez-Zubiaur Y (2009) Plant Pathol 58: 785. [3] Fiallo-Olivé E, Martínez-Zubiaur, Y Moriones E, Navas-Castillo J (2010) Arch Virol (in press).

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Virus diversity and Evolution

Title: Diversity and evolutionary dynamics of begomovirus infecting a wild crop relative, Capsicum annuum var. glabriusculum

Authors: P. GONZÁLEZ-JARA1, M. RODELO-URREGO1, A. FRAILE1, D. PIÑERO2 AND F.

GARCÍA-ARENAL1

Address: 1Centro de Biotecnología y Genómica de Plantas, UPM-INIA, Universidad Politécnica de Madrid. Campus de Montegancedo, 29223 Pozuelo de Alarcón, Spain. 2Instituto de Ecología, Universidad Nacional Autónoma de México, AP 70-275, México, D.F, Mexico.

Abstract: Pathogens may condition the population dynamics and genetics of their hosts. In opposition to wild animal hosts, evidence on the role of virus infections in the population dynamics and genetics of wild plants is limited, and the potential impact of viruses has rarely been considered in the conservation of wild plant biodiversity. We have approached these topics focussing on a potentially endangered plant species, the chiltepin (Capsicum annuum var. glabriusculum), which is the wild ancestor of domesticated pepper. Chiltepin is distributed naturally in dry tropical forests of Mexico, where some populations may be threatened due to human over-exploitation and habitat degradation. Here we have analysed the impact of begomovirus infection on the chiltepin population dynamics and its relationship with host ecological factors and population genetics. To this end, we sampled for three years chiltepin populations throughout the distribution range of the species in Mexico, in its wild habitat and in habitats with different degrees of anthropogenic influence. Plants were analyzed for the presence of viruses known to have a broad host range or previously reported infecting pepper in México, as begomoviruses. Demographic analyses indicated that virus infection had a negative effect on chiltepin fecundity and survival, therefore impacting on its population dynamics. Begomoviruses showed the highest incidence, and symptomatic plants were mainly infected by Pepper golden mosaic (PepGMV) or Pepper huasteco yellow vein (PHYVV) viruses. Genetic analyses revealed that while host population was highly structured by geography this was not so for either PepGMV or PHYVV, which population structure suggests a recent epidemic expansion on the host population. Analysis of the association between begomovirus incidence and habitat degradation showed that incidence was higher and more variable among years in the anthropic habitats than in the wild ones. This higher incidence was associated with decreased biological diversity and with increased host density, according to theory, in anthropic habitats, but not with the host genetic variability. Therefore viral infections, mainly begomoviruses, may have a role in the population dynamics of a wild plant and may be considered an additional risk factor for the survival of endangered species.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution

Virus Family: Geminiviridae Category: Genetic diversity/Evolution

Title: Molecular characterization of Sweet potato leaf curl virus (SPLCV) Korea isolates

Authors: Jungan Park1, Hyejung Lee1, Yuchul Jung1, Jaelim Yu1, Hong-Soo Choi2, and Sukchan Lee1 (E-mail: [email protected])

Address: 1Department of Genetic Engineering, Sungkyunkwan University, Korea, 2Agricultural Microbiology Division, National Academy of Agricultural Science, Korea

Abstract: Sweet potato plants showing typical disease symptoms such as leaf curling, vein yellowing and stuntings were collected from eight areas (Haenam, Yeojoo, Chungju & Nonsan) of Korea in 2002-2004. Full length DNA-A component of SPLCV were amplified by polymerase chain reaction (PCR) and charaterized. DNA-A component of SPLCV Korea group 1 consists of 2828 nucleotides and SPLCV Korea group 2 has 2829 nucleotides. SPLCV Korea has a genome organization similar to that of monopartite begomoviruses. The DNA-A had two ORFs (AV1 & AV2) in the virion sense and four ORFs (Ac1, AC2, AC3 & AC4) in the complementary sense, seperated by an intergenic region (IR) containg a conserved stem-loop motif. Sequence comparisons showed that the SPLCV Korea isolates were closely related to those of SPLCV Brazil isolates (FJ969834, FJ969835 & FJ969836), SPLCV Japan (AB433788) and SPLCV USA (AF104036) with nuclotide sequence similarity ranging from 96-98%. The relationships between SPLCV Korea isolates and other begomoviruses were investigated by using phylogeny of derived DNA-A, AC1 and AV1 nucleotide sequences. In all phylogenetic trees, SPLCV Korea isolates clustered with SPLCV Brazil isolates (FJ969834, FJ969835 & FJ969836). Recombination detection program (RDP3) analysis providse the recombination events. Taken together, these data indicated that two or three recombination events were identified between SPLCV Korea isolates and other begomoviruses.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Agroinoculation of cloned Honeysuckle yellow vein virus (HYVV) isolated from Lonicera japonica and its recombination study

Authors: Hyejung Lee, Gunsup Lee, Jungan Park, Seungchan Cho, Jisook Chae, Eunbyul Ji, Kangsan Roh, Taejune Jun, Sukchan Lee (E-mail: [email protected])

Address: Department of Genetic Engineering, Sungkyunkwan University, Korea,

Abstract: A New Honeysuckle yellow vein geminivirus (HYVV) was isolated and characterized from symptomatic leaves of Lonicera japonica with yellow net mosaic on leaves in Korea. Complete genome of HYVV was amplified with two sets of primers for HYVV, which were specifically designed for detection and cloning any begomoviruses infected on tomatoes. HYVV has a 2763 bp in size as a monopartite genome DNA-A. Grafting challenging of a virus-free L. japonica (scion) on HYVV-infected L. japonica showed that virus-free plants produced typical HYVV disease symptoms on newly developed leaves. pMON521 plasmids containing 1.3 copies and 2.0 copies respectively of the cloned HYVV were infectious and produced disease symptoms on Nicotiana benthamiana via agroinoculation, the causal agent of L. japonica which induced the yellow net mosaic is a new HYVV Korea isolate. Electron microscopy of HYVV-infected L. japonica leaves revealed the presence of crystalline inclusion showing geminivirus specific granular structures in some of the nuclei in phloem cells. Phylogenetic analysis of HYVV with other 30 begomoviruses shows that HYVV were closed to HYV (M) V-UK and HYVV Japan. Recombination detection program 3 (RDP3) found evidence for recombination TbLCV Japan (AB055008) as a major parent and unknown as minor parent. Based on recombination analysis, we suggest that the HYVV Korea isolates could be from the recombination with HYVV UK isolate and TbLCV Japan isolate (AB055008). In conclusion, HYVV recombination analysis indicated that this is the first reports of HYVV Korea isolates with known TbLCV and the first evidence of recombination between HYVV Korea isolate and TbLCV Japan isolate (AB055008).

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity / Evolution

Title: Investigations towards understanding the evolution and global movement of Abutilon mosaic virus

Authors: SIMONA KRABERGER1, DAISY STAINTON1, KARYNA ROSARIO2, HOLGER JESKE3, DARREN P MARTIN4, DAVID COLLINGS1 AND ARVIND VARSANI1, 5

Address: 1School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; 2College of Marine Science, University of South Florida, St Petersburg, Florida; 3Department of Molecular Biology and Plant Virology, Institute of Biology, University of Stuttgart, Stuttgart, Germany; 4Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 5Electron Microscope Unit, University of Cape Town, Cape Town, South Africa

Abstract: Abutilon mosaic virus (AbMV) is a bipartite begomovirus which is transmitted by whitefly (Bemisia tabaci), and causes striking leaf mosaic patterns in Abutilon sp. It is this attractive variegated foliage phenotype, first reported in the 19th century when imported from the West Indies to London, which has led to the extensive, worldwide propagation of AbMV-infected plants for the commercial/home market. Here we investigate the worldwide diversity of AbMV, and analyse the genomes for evidence of recombination as a result of the extensive propagation and generation of hybrids. Infected samples of Abutilon megapotimicum, A. striatum and A. pictum were collected from various locations in Australasia and North America. Components A and B of the viral genomes were cloned, sequenced and compared against AbMV isolates found in the GenBank database. Pairwise nucleotide comparison of Australasian AbMV DNA-A isolates showed a ~95% - 99% nucleotide identity amongst themselves, and ~94% - 99% identity to AbMV DNA-A isolates from elsewhere in the world while Australasian AbMV DNA-B isolates share ~93% - 99% nucleotide identity, and ~90% - 99% identity to AbMV DNA-B isolates from elsewhere in the world. Fixed effects likelihood (FEL) analysis using HyPhy software identified positive selection in four open reading frames (ORF) of DNA-A, the AV1 coat protein at codon 192, the AC2 transcriptional activator protein at codon 82, the AC3 replication enhancer at codon 21 and AC4 at codon 20. Analysis showed a single site under positive selection in DNA-B, codon 203 at the C-terminal end of the movement protein ORF BC1. Recombination analysis revealed recombination in DNA-A, specifically in the C-terminal region of ORF AV1-CP. This recombination event was detected between Sida golden mosaic virus (SiGMV) and three Australasian AbMV isolates collected from Abutilon megapotimicum, but not in any other AbMV isolates. Analysis of larger AbMV sample sets, including archived plant samples, will give us high resolution data for dating the viruses accurately and thus inference into AbMV evolution and global movement of AbMV infected plants. th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Discovering circomics! Deep insights into plant viral circular genomes

Authors: Björn Krenz1, Tobias Paprotka2, Judith Horn1, Benjamin Schäfer1, Patricia Wyant1, Stephan Strohmeier1 and Holger Jeske1

Address: 1University of Stuttgart, Institute of Biology, Dpt. of Molecular Biology and Plant Virology, Stuttgart, Germany 2Viral Mutation Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA.

Abstract: Geminiviridae and Nanoviridae are plant pathogen families which, due to their small circular genomes, can be investigated by circomics in an ideal manner. They comprise diverse viruses of monocotyledonous and dicotyledonous hosts and cause devastating crop losses, mainly in tropical and subtropical countries, but also in temperate zones. During the past forty years many new diseases have emerged due to the high mutation and recombination frequency of the viruses. The interplay of replication, recombination and repair (RRR connection) is the basis of these epidemics. Circomics applied on gemini- and nanoviral DNA populations in order to explore the quasispecies space will help to understand the evolutionary and epidemic potential of these pathogens. Circomics profits from recent progress in rolling circle amplification (RCA) and deep sequencing techniques. Previous work has demonstrated the capability of RCA for characterizing new and known viruses convincingly. We have characterized new begomoviruses infecting weeds in Bolivia. Furthermore, we have found the first evidence for an evolutionary connection between geminiviruses and nanoviruses in the New World by identifying nanovirus-like α- satellites in conjunction with begomoviruses. Pyrosequencing batches of 50 viral isolates with over 40,000 reads of 300 nts average length was able to identify geminiviruses within two weeks, a so far unprecedented efficiency for geminiviruses. In this context, a bioinformatic tool named WiMSeEx has been developed to explore sequence data on the basis of geminiviral genome peculiarities.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae Category: Virus diversity and Evolution Title : Phylogenetic analysis of TbLCV and its evidence for recombination with TYLCV

Authors: Hyejung Lee1, Jungan Park1, Eui-Joon Kil¹, Mangyong Jung1, Jin-gweon Yang¹ , Hong-Soo Choi2, and Sukchan Lee1* Address: 1Department of Genetic Engineering, Sungkyunkwan University, Korea 2Agricultural Microbiology Division, National Academy of Agricultural Science, Korea

Abstract Tobacco leaf curl virus (TbLCV) is a member of Begomovirus, Geminiviridae, that characterized by closed circular single stranded DNA molecules of 2.7~2.8 kb in length. In 2007-2008, TbLCV Korea isolates were isolated & identified in Iksan, Sunchang, Gimje, and Jeju in Korea. Phylogenetic analysis of Rep, CP, IR, and full sequences was analyzed using the bayesian analysis and neighbor joining. Phylogenetic analysis indicated that all Korea TbLCV isolates were closed to HYVV and TbLCV Japan isolates but being away from China isolates. Recombination detection program 3 (RDP3) found the evidences for recombination between 4 TbLCV Korea isolates and the recombination event 1 showed a recombination between an unknown as a major parent and TbLCV Korea Sunchang (HM164550) as a minor parent. Event 2 was analyzed between TbLCV Korea Iksan (HM164541) as the major parent and PaLCV Guangdong (FJ869907) as the minor parent. Third recombination event was observed between TbLCV Yunnan (AF240674) as major parent and TYLCV Korea Jeju (HM130914) as minor parent. Fourth recombination event was identified between EpYVV Japan (NC_003556) as major parent and unknown as minor parent. Based on recombination analysis, we suggest that the TbLCV Korea isolates were involved in recombination with TYLCV.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Phylogeny and recombination analysis of Tomato yellow leaf curl virus (TYLCV) and Bemisia tabaci in Korea

Authors: Hyejung Lee¹, Jungan Park¹, Eui-Joon Kil¹, Jin-gweon Yang¹, Kyeong-yeoll Lee², Hong-Soo Choi³, Sukchan Lee¹ (E-mail: [email protected])

Address: ¹Department of Genetic Engineering, Sungkyunkwan University, Korea, 2School of Applied Biosciences, Kyungpook National University, Korea, ³Agricultural Microbiology Division, National Academy of Agricultural Science, Korea

Abstract: Tomato yellow leaf curl virus (TYLCV) is a member of the genus Begomovirus, members of which are characterized by closed circular single-stranded DNA genomes of 2.7–2.8 kb in length, and include viruses transmitted by the Bemisia tabaci whitefly. No reports of TYLCV in Korea are available prior to 2008, after which TYLCV spread rapidly to most regions of the southern Korean peninsula (Gyeongsang-Do, Jeolla-Do and Jeju- Do). Fifty full sequences of TYLCV were analyzed in this study, and the AC1, AV1, IR, and full sequences were analyzed via the Bayesian analysis and neighbor joining in MEGA 4.0. Phylogenetic analysis demonstrated that the Korea TYLCVs were divided into two subgroups. TYLCV Korea 1 group (Masan) originated from TYLCV Japan (Miyazaki) and the TYLCV Korea 2 group (Jeju/Jeonju) from TYLCV Japan (Tosa/Haruno). A B. tabaci phylogenetic tree was constructed with 16S rRNA and mitochondria cytochrome oxidase I sequences. The sequence data of 16S rRNA revealed that Korea B. tabaci was closely aligned to B. tabaci isolated in Iran and Nigeria. Recombination detection program 3 found evidence for recombination between unknown and TYLCV Iran (AJ132711 and FJ355946) as a major parent; and between the TYLCV Israel isolate (X76319) and unknown as minor parents, in three different independent recombination events. Based on recombination analysis, we suggest that the TYLCV Korea isolates were involved in recombination with the Israel and Iran isolates. Therefore, we suggest that two TYLCV Japan isolates were introduced to Korea via different routes, and then transmitted by native B. tabaci.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Diversity of tomato begomoviruses in the three major tomato growing states of Brazil

Authors: A.T.M. LIMA1, F.Y.B. NAITO2, F.N. SILVA1, C.S. ROCHA1, D.R. BARROS1, G.P.C. URQUIZA1, A.W.O. MALTA1, M.F. BASSO1, M.T. GODINHO1, E.S.G. MIZUBUTI1, F.M. ZERBINI1 AND A.K. INOUE-NAGATA2,3

Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; 2Dep. de Fitopatologia/Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil; 3Embrapa Hortaliças, BR 060 Km 09, C.P. 218, Brasília, DF, 70359-970, Brazil Abstract: Nowadays, begomoviruses are the most economically important tomato viruses in Brazil. These viruses became widespread on this crop since the introduction of the B biotype of Bemisia tabaci in the mid-1990's. Production of tomato fruits destined for processing is concentrated in three states: Goiás (GO), São Paulo (SP) and Minas Gerais (MG). Fresh market tomatoes are planted throughout the country. However, the three above mentioned states plus the state of Rio de Janeiro acount for the majority of the production. Although begomoviruses are present throughout the growing areas, incidence is variable according to the region and growing season. We are interested in determining the diversity and distribution of tomato begomoviruses in the major tomato growing states of GO, SP and MG. Six municipalities were selected for sample collection and begomovirus genotyping: Ribeirão Preto, SP (low incidence of begomovirus); Luziânia, GO (low incidence) and Morrinhos, GO (high incidence); Florestal, Carandaí and Jaíba, all in MG and with high incidence. Samples of symptomatic tomato plants were collected, total DNA was extracted and used as a template for RCA-based amplification of viral genomes. For the samples from SP and GO, the RCA-RFLP profile was analyzed. Samples from Luziânia were the most uniform with 3 RFLP profiles within 42 samples, followed by Morrinhos with 4/48 and Ribeirão Preto with 14/120. From Luziânia samples, 90.4% had one specific profile (A profile), also found in 91.6% of Morrinhos samples. This A profile may represent the most prevalent virus in Goiás state. Conversely, in São Paulo state, 24.1% of the samples displayed the A profile, 45% displayed a distinct B profile, and the remaining 30.8% displayed 12 different profiles. Direct sequencing of RCA products, using a primer directed to the 5’ end of the coat protein and the intergenic region, was carried out on 15 samples and compared with the RCA-RFLP result. Indication of mixed infection was found in two samples, whereas the other 13 sequences were classified as Tomato severe rugose virus related viruses. Samples from Minas Gerais state were processed in the same fashion, and 40 full-length viral genomes were cloned and sequenced. The following viruses were detected: Tomato chlorotic mottle virus (ToCMoV; 18 isolates from Florestal and 2 from Carandaí), Tomato severe rugose virus (ToSRV; 6 isolates from Florestal and 11 from Carandaí), Tomato mottle leaf curl virus (ToMLCV; 2 isolates from Jaíba), and Tomato yellow spot virus (ToYSV; 1 isolate from Jaíba). Analysis of the genetic structure of the ToCMoV population indicated a lower degree of genetic diversity compared to populations of two other tomato-infecting begomoviruses (Tomato yellow vein streak virus, ToYVSV, and Tomato common mosaic virus, ToCmMV) also present in Minas Gerais state. Financial support: National institute for Plant-Pest Interactions and Pronex-Fapemig.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Begomoviruses infecting weeds in Bolivia

Authors: A.T.M. LIMA1, F.N. SILVA1, C.S. ROCHA1, T.F.S. ANTUNES1, D.R. BARROS1, G.P. ROSALES2, N. ORTUÑO2, A. GANDARILLAS2, R.O. RESENDE3 AND F. M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; 2Fundación PROINPA, Av. Meneces s/n. Km. 4, Zona El Paso, Cochabamba, Bolivia; 3Dep. de Biologia Celular/Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil.

Abstract: The genus Begomovirus (family Geminiviridae) includes dicot-infecting, whitefly-transmitted viruses with the genome comprised of one or two molecules of circular, single-stranded DNA. Begomoviruses cause serious damage to crops in several Latin American countries, and are also associated with the wide range of wild and weed hosts. However, information on the occurrence of begomoviruses is lacking in some countries, such as Bolivia. Therefore, the aim of this study was to assess the presence of begomoviruses infecting weeds in Bolivia. Total DNA was extracted from 35 weed samples collected in March of 2009 in tomato, pepper and bean fields in the area known as the "Mesothermal Valleys" (towns of Comarapa, Saipina, San Isidro and Pulquina in the Province of Manuel M. Caballero, Department of Santa Cruz). The RCA-amplified DNA was cleaved with BamH I and Hind III, cloned and completely sequenced. Sequences were compared to those of previously characterized begomovirus species, and the ICTV-established 89% DNA-A identity threshold was used to determine taxonomic placements. Out of nine clones sequenced so far, four obtained from Sida spp. and one from Physalis sp. displayed 95% identity with the recently described begomovirus Sida mosaic Brazil virus (SiMBV). Bean golden mosaic virus (BGMV) was found in four unidentified (most likely leguminous) weeds. These initial results demonstrate the existence of begomoviruses infecting wild hosts in Bolivia, and suggest that there is a low species diversity among weeds. We are currently analyzing the presence of begomoviruses in tomato, pepper and bean samples collected at the same fields.

Financial support: PROSUL-CNPq, National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae Category: Genetic diversity/Evolution

Title: Genetic analysis and phylogeographic history of MSV reveals the spatial and temporal origins of contemporary MSV-A recombinant lineages and identifies southern Africa as the ultimate origin of Maize streak disease

Authors: A. L. Monjane, G. W. Harkins, D. P. Martin, P. Lemey, D. N. Shepherd, P. Lefeuvre, E. P. Rybicki , B. E. Owor , R.S. Ali, B. Flett, M. Ramusi, S. Oluwafemi, and A. Varsani

Address: Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory, Cape Town, 7925, South Africa

The unrelenting threat of maize streak disease (MSD) to sustainable maize production in Sub-Saharan Africa, has motivated research aimed at elucidating the ecological and genetic factors driving the continent-wide spread of MSV-A – the maize streak virus strain that cause the disease. Determining the geographical origin of MSV-A and retracing its historical spread could yield important insights into how and why this important pathogen emerged. Similarly, determining when and where the major extant MSV-A lineages arose could identify geographical hotspots for epidemic strain genesis and guide the formulation of better strategies aimed at curtailing the continental spread of MSD. Here, we use model- based phylogeograpgic analyses (implemented within the Bayesian inference framework of the molecular evolution analysis program ,BEAST), in tandem with recombination analysis of publicly available MSV sequences (including 204 new full length MSV genomes) to reconstruct a plausible history of MSV-A movements over the past 150 years . Our results suggest that MSV arose somewhere in southern Africa around 1863 (95 % highest probability density (HPD) intervals 1809-1935), a few decades prior to the first credible reports of MSD in South Africa. The virus then dispersed trans-continentally at mean rates of 33.6 km/yr (95 % HPD intervals 1.4-97.6 km/yr). While characterising the contribution of recombination to MSV-A diversification we found no evidence of inter-species recombination with other African streak viruses, and only a few instances of inter inter- MSV strain recombination. We use distinctive patterns of nucleotide variation caused by 20 unique intra- MSV-A recombination events to tentatively classify the MSV-A genotypes into 18 different haplotypes. Despite many of these haplotypes displaying distinct, geographical distributions it is apparent that they have mostly emerged within the past four decades from either East Africa or southern Mozambique. Collectively, our results suggest that persistent sampling and analysis of MSV-A genomes within these diversification hotspots could be used to effectively monitor the emergence and track the spread of future epidemiologically important MSV-A lineages.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Diversity of Dicot-infecting Mastreviruses

Huma Mumtaz1, Safaa G. Kumari2, Nazia Nahid1, Darren P. Martin3, Shahid Mansoor1 and Rob W.Briddon1

1Agriculture Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan 2Virology Laboratory, International Center for Agricultural Research in the Dry Areas (ICARDA), Aleppo, Syria 3Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa

Until recently the diversity of dicot-infecting mastreviruses (DIMs; family Geminiviridae, genus Mastrevirus) was believed to be low, with only two species, Tobacco yellow dwarf virus from Australia and Bean yellow dwarf virus (BeYDV) from South Africa, being reported in the literature. The recent identification of a distinct species of DIM (Chickpea chlorotic dwarf Pakistan virus [CpCDPKV]) in Pakistan, and the release of the sequence of a further species occurring in Sudan (Chickpea chlorotic dwarf Sudan virus [CpCDSDV]), indicated that across southern Asia, the Middle East and North Africa, the DIM diversity is likely to be higher than initially anticipated. Additionally it was shown that BeYDV, which is closely related to CpCDPKV and CpCDSDV, occurs in Pakistan and likely has its origins in this region. This study presents the sequences of two further DIMs. The first originated from chickpea in Syria and is equally related to the other DIMs in the regions (between 81 and 84% nucleotide sequence identity) and thus likely represents a newly identified species for which we propose the name Chickpea chlorotic dwarf Syria virus. The second virus was isolated from chickpea growing at Bahawalnagar (Pakistan), near the border with India. The sequence of the latter virus is more closely related to CpCDPKV (~89% identity) than the other species and thus likely represents a distinct strain of this species. Phylogenetic analysis of the viruses isolated from chickpea suggests that there is a gradient of relatedness across South Asia, the Middle East and North Africa which may indicate that speciation occurred during the geographic spread of their common ancestor. BeYDV is most closely related to CpCDSDV and the Syrian virus, suggesting that its origin may lie in the western part of the Middle East or North Africa and was fairly recently introduced into southern Africa. The significance of these findings will be discussed.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Genetic diversity of DNA satellites associated with Ipomoea-infecting monopartite begomoviruses

Authors: H.P. TRENADO1, E. FIALLO-OLIVÉ1,2, G. LOZANO1, D. CHIRINOS3, F. GERAUD-POUEY3, E. MORIONES1, R.W. BRIDDON4, AND J. NAVAS- CASTILLO1

Address: 1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM- UMA-CSIC), 29750 Algarrobo-Costa, Málaga, Spain. 2Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba. 3Universidad del Zulia, Maracaibo, Zulia, Venezuela. 4Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.

Abstract: Single-stranded DNA satellites, including the betasatellites, are frequently associated with begomovirus (family Geminiviridae) infections. The first ssDNA satellite identified was that associated with tomato leaf curl virus (ToLCV) originating from Australia. The ToLCV satellite (ToLCV-sat) sequence includes a hairpin structure containing the nonanucleotide loop sequence (that is conserved between most geminiviruses and betasatellites), a second hairpin, and a region with sequence rich in adenine (A-rich). The ToLCV-sat is believed to be a betasatellite deletion mutant. In screening sweet potato (Ipomoea batatas) and I. indica samples collected in Spain for the presence of begomoviruses, we identified circular molecules with a structure similar to the ToLCV-sat. The size of these molecules ranged from 662 to 750 nt. They appear to encode no open reading frames and contain a geminivirus-like stem-loop structure with the nonanucleotide sequence in the loop, a second stem-loop, and an A-rich region. However, the sequence of these molecules show less than 50% identity to ToLCV- sat with the exception of an approximate 100 bp fragment which is conserved in both sequences (greater than 90% sequence identity). Moreover, in a survey searching for begomoviruses in wild plants carried out in Venezuela, a Merremia dissecta plant was found infected with a monopartite begomovirus closely related to the Ipomoea-infecting begomoviruses so far reported. This plant also contained DNA satellite-like molecules of 733 nt. Sequencing of five clones (99.9-100% nt identity between them) showed that these molecules have a structure similar to that of ToLCV-sat and the related DNA satellites from Spain. Phylogenetic analysis showed a relationship between ToLCV-sat, the DNA satellites of sweet potato and I. indica from Spain and the DNA satellite of M. dissecta from Venezuela. Confirmation of trans-replication of these novel molecules by the Ipomoea/Merremia-infecting begomoviruses and of putative interference with their helper viruses to modify symptoms, awaits availability of infectious clones.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution

Virus Family: Geminiviridae

Category: Genetic Diversity/ Variability

Title: Begomovirus species diversity in South American weeds

Authors: T.G. PEREIRA1,2, F.Y.B. NAITO1,2, R.S. FONTENELE1,3, R. BLAWID1,3, F.R. FERNANDES4, A.T.M. LIMA4, F.N. SILVA4, C.S. ROCHA4, G.P. ROSALES5, C.L. OLIVEIRA1,2, R.O. RESENDE1, F.M. ZERBINI4, S.G. RIBEIRO3, F. J. L. ARAGÃO3, A.K. INOUE-NAGATA1, 2

Address: 1Universidade de Brasília,CEP 70910-900 Brasília, Brazil. 2Embrapa 3 Vegetables, BR 060 Km 09, C.P. 218, CEP 70359-970, Brasília, Brazil. Embrapa Recursos Genéticos e Biotecnologia, Pq Estação Biológica, Brasília, Brazil. 4Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570- 000, Brazil. 5Fundación PROINPA, Av. Meneces s/n. Km. 4, Zona El Paso, Cochabamba, Bolivia

Abstract: Begomoviruses (family Geminiviridae) threaten economically important plants worldwide and also infect weeds and wild plants, which may act as virus reservoirs for crop species due to their wide distribution, especially in the tropics. The objective of this study was to assess the diversity of begomoviruses that infect a number of relevant weed species in South America. Samples from four different weed genera (Abutilon sp., Blainvillea sp., Euphorbia heterophylla and Sida spp.) were collected in Brazil and in neighbouring Bolivia from 2004 to 2009. Total DNA was extracted from leaf samples and subjected to rolling circle amplification (RCA) using Phi-29 DNA polymerase. RCA products were cleaved with restriction enzymes, cloned and sequenced. Twenty-five full-length DNA-A clones were obtained. Comparison with sequences from public databases grouped these sequences within six viral species. All 13 E. heterophylla samples were infected by Euphorbia yellow mosaic virus (EuYMV), whereas one Blainvillea sp. sample was infected by Blainvillea yellow spot virus (BlYSV) and one Abutilon sp. sample was infected by Abutilon mosaic virus (AbMV). Samples of Sida spp. were infected by Sida micrantha mosaic virus (SiMMV, one sample), EuYMV (two samples), Sida mosaic Brazil virus (SiMBV, two samples) and two additional novel viruses (three samples). The sequences of these two novel viruses shared 86 to 87% nucleotide identity with each other (depending on the isolate), a maximum of 82-83% nucleotide identity with Tomato leaf distortion virus (EU710749), and exhibited a closer phylogenetic relationship with okra, tomato and sida-infecting begomoviruses isolated from Brazil. Thus, begomovirus species diversity seems to be low in E. heterophylla, whereas it is much higher in Sida spp. Interestingly, with the exception of SiMMV, none of the viruses detected in these weeds have been found so far infecting crop species in Brazil. Financial support: Embrapa, CNPq, FAP DF, National Research Institute for Plant-Pest Interactions. th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Begomovirus occurring on tomatoes in Brazil and their diversity in Goiás state Authors: B. PINHEIRO1,2, L. C. ALBUQUERQUE1,2, D. P. MARTIN3, A. VARSANI3, AND NOUE-NAGATA AK1,2

1 2 Address: Universidade de Brasília, 70910-900, Brasília, Brazil. Embrapa Vegetables, BR 060 Km 09, C.P. 218, 70359-970, Brasília, Brazil. 3University of Cape Town, Rondebosch, Cape Town, 7701, South Africa

One of the main factors affecting tomato production worldwide is the disease caused begomoviruses. In Brazil although many different begomovirus species have found naturally infecting tomato plants, the precise economic relevance of each species is still not well understood. We analyzed the diversity of begomoviruses infecting tomatoes in the Brazilian state, Goiás - one of the most important tomato growing regions in South America. Initially, the 14 most divergent begomovirus isolates evident within the Embrapa begomovirus collection (sampled from 2002 to 2004 throughout the country) based on sequence analysis of PCR amplified fragments were selected for full DNA-A genome component sequencing. These 14 sequences were classifiable into four distinct species: Tomato severe rugose virus (ToSRV), Tomato mottle leaf curl virus (ToMLCV), and two possible new species (collected in the North-East region of Brazil). For analysis of the relative population prevalence of the different strains in tomatoes, and to assess intra- and inter-specific genetic variability in Goiás, samples were collected within a 500Km area encompassing the municipalities of Goiás State and the Federal District. From 44 samples collected between 2002 to 2004 we obtained fifty DNA-A clones which were fully sequenced. While most of the cloned viruses (41 clones of 36 samples) were ToSRV isolates, 8 clones (from 7 samples) were identified as Tomato yellow vein streak virus, and one as ToCMoV. The low genetic diversity of the dominant ToSRV population suggests that it may have either been founded relatively recently or that there exists a very rapid rate of population turnover and virus spread throughout the Goiás region. The data we have generated will in the future enable precise estimation of rates and directions of ToSRV dissemination: information that will have a strong influence on determining future control strategies.

Financial support: CNPq, Embrapa, National Research Institute for Plant-Pest Interactions, Unilever

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Genetic structure of a population of the begomovirus Bean golden mosaic virus (BGMV) that infects lima bean (Phaseolus lunatus L.) in the state of Alagoas, Brazil

Authors: R. RAMOS-SOBRINHO1,2, S.J.C. SILVA1,3, T.A.L. SILVA4, S.G. RIBEIRO4, G.S.A. LIMA2, I.P. ASSUNÇÃO2 AND F.M. ZERBINI 1

Address: 1Dep. de Fitopatologia/BIOAGRO, UFV, Viçosa, MG, 36570-000, Brazil; 2Dep. de Fitopatologia, CECA, UFAL, Maceió, AL, 57072-970, Brazil; 3Dep. de Fitopatologia, UFRPE, Recife, PE, 52171-900, Brazil; 4Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, 70770-917, Brazil.

Abstract: The lima bean (Phaseolus lunatus) is one the main cultivated legumes in the Brazilian Northeast, consisting in a major source of protein to the population. However, despite its economic importance, it is neglected by breeding programs and most of the available germplasm is highly susceptible to viruses, particularly begomoviruses (family Geminiviridae). In the field, begomovirus-infected plants display an intense yellow or golden mosaic, and yield is drastically reduced. The objective of this work was to characterize begomovirus populations infecting lima beans in the state of Alagoas, Brazil. Lima bean plants with typical symptoms of begomovirus infection were collected in a number of areas around the state during 2005. A small number of samples were also collected in the neighboring state of Pernambuco. Total DNA was extracted and the presence of a begomovirus was confirmed by PCR. The viral genomic components were amplified using the DNA polymerase from phage phi29, and then cleaved with either BamH I or Hind III. Clones were submitted to cleavage with Hae III and, based on their restriction pattern, 23 DNA-A clones were selected for sequencing (20 from samples collected in Alagoas, and three from samples collected in Pernambuco). Phylogenetic analysis was carried out with MEGA 4.0, and the genetic diversity of the population was evaluated using the DnaSP program, version 5. Analysis of the 23 sequences revealed that they all belonged to a single begomovirus species, Bean golden mosaic virus, with 90 to 94% nucleotide sequence identity with a BGMV isolate from soybean (FJ665283). All sequences clustered with other Brazilian BGMV isolates from common bean and soybean in the phylogenetic tree, although the three isolates from Pernambuco are closer to the soybean isolate than the 20 isolates from Alagoas. Thus, the lima bean isolates segregated based on their geographical origin. Analysis of the population indicated a high degree of genetic variability, significantly higher than that observed for two begomovirus populations from tomato obtained in Southeastern Brazil. Financial support: National Research Institute for Plant-Pest Interactions, Pronex- Fapemig and FAPDF.

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Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Recombination and pseudorecombination driving the evolution of Tomato severe rugose virus and Tomato rugose mosaic virus: Two distinct DNA-As sharing the same DNA-B.

Authors: F.N. SILVA1, A.T.M. LIMA1, C.S. ROCHA1, M. ALVES-JÚNIOR, M.1 HALLWASS2, A.K. INOUE-NAGATA3 AND F. M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil; 2Dep. de Biologia Celular/Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, 70910-900, Brazil; 3Embrapa Hortaliças, BR 060 Km 09, C.P. 218, Brasília, DF, 70359-970, Brazil

Abstract: The genus Begomovirus (family Geminiviridae) includes dicot-infecting, whitefly-transmitted viruses with the genome comprised of one or two molecules of circular, single-stranded DNA. In Brazil, tomato-infecting begomoviruses have emerged as serious pathogens in recent years due to the introduction of a new biotype of the whitefly vector. Tomato rugose mosaic virus (ToRMV) and Tomato severe rugose virus (ToSRV) are often found in tomato fields. The complete DNA- B sequences of ToSRV (GenBank RefSeq NC_009612) and ToRMV (NC_002556) show an identity of 98.2%. Additionally, the high nucleotide identity (97.5%) between their common regions suggests the possibility that the two viruses share the same DNA-B. The objectives of this study were to assess the formation of viable pseudorecombinants between ToRMV and ToSRV in tomato plants, and to study how recombination and pseudorecombination modulated the evolution of these two begomoviruses. Infectious clones corresponding to the DNA-A and DNA- B of the isolates ToSRV-[BR:PG1:Pep:03] and ToRMV-[BR:Ub1:96] were used for biolistic inoculation in all possible combinations between DNA-A and DNA-B (including single and mixed infections), and were detected by RCA following RFLP analysis at 28 days after inoculation. Pseudorecombination was verified in all cases. Recombination analysis showed that part of the common region and most of the Rep (AC1) ORF were transferred from ToSRV to ToRMV. Symptom severity was equivalent in single or mixed infections, indicating that synergism does not occur between these two viruses. However, ToRMV negatively affected the accumulation of ToSRV when the two viruses were co-inoculated, indicating that the recombinant is better adapted than the parental. These results are consistent with a scenario in which ToRMV captured the DNA-B from ToSRV after having received its Rep coding region and CR in a recombination event, thus highlighting the major role that both recombination and pseudorecombination have played in the emergence and evolution of novel tomato-infecting begomoviruses in Brazil. Financial support: National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

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Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic Diversity/Evolution

Title: Low diversity of Begomovirus species infecting Cleome affinis in Northeastern Brazil

Authors: S.J.C. SILVA1,2, G.P. CASTILLO-URQUIZA1, G. PIO-RIBEIRO2, I.P. ASSUNÇÃO3, G.S.A. LIMA3, F.M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, UFV, Viçosa, MG, 36570-000, Brazil; 2Dep. de Fitopatologia, UFRPE, Recife, PE, 52171-900, Brazil; 3Dep. de Fitopatologia, CECA, UFAL, Maceió, AL, 57072-970, Brazil.

Abstract: The genus Begomovirus (family Geminiviridae) includes viruses with a circular, single- stranded DNA genome encapsidated in twinned icosahedral particles. They are vectored by the whitefly Bemisia tabaci and cause serious diseases in several economically important crops. Begomoviruses are also associated with a wide range of weed plants, which in some cases act as reservoirs for cultivated plants. Cleome affinis is a weed that belongs to the family Capparaceae, and which is frequently associated with lima bean (Phaseolus lunatus) crops. In the years of 2007 to 2010, a total of 24 samples of Cleome affinis were collected in the states of Alagoas, Bahia, Paraíba, Pernambuco and Sergipe, all in Northeastern Brazil. Total DNA was extracted from each sample and viral genomes were RCA-amplified from all 24 samples. The amplified DNA was cleaved with Cla I, Hind III and Pst I, cloned and sequenced. The ICTV-established 89% DNA-A identity threshold was used to determined taxonomic placements. All nine DNA-A clones sequenced so far correspond to isolates of Cleome leaf crumple virus, displaying 95% identity with a recently described ClLCrV isolate from Mato Grosso do Sul, Brazil (FN435999). A careful examination of all samples did not indicate the presence of alphasatellites in association with ClLCrV (as recently reported for the isolate from Mato Grosso do Sul by Paprotka et al., Virology 404:148, 2010). In a phylogenetic tree, all ClLCrV isolates formed a monophyletic cluster with tomato- and weed-infecting begomoviruses from Brazil. These and previous results suggest a low begomovirus species diversity in this weed.

Financial support: National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

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Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: High diversity of Begomovirus species infecting Macroptilium spp. in Northeastern Brazil

Authors: S.J.C. SILVA1,2, G.P. CASTILLO-URQUIZA1, G. PIO-RIBEIRO2, I.P. ASSUNÇÃO3, G.S.A. LIMA3, F.M. ZERBINI1

Address: 1Dep. de Fitopatologia/BIOAGRO, UFV, Viçosa, MG, 36570-000, Brazil; 2Dep. de Fitopatologia, UFRPE, Recife, PE, 52171-900, Brazil; 3Dep. de Fitopatologia, CECA, UFAL, Maceió, AL, 57072-970, Brazil.

Abstract: Begomoviruses (family Geminiviridae) have a circular, single- stranded DNA genome encapsidated in twinned icosahedral particles. They are vectored by the whitefly Bemisia tabaci and cause serious diseases in several economically important crop plants. Begomoviruses are also associated with a wide range of weed and wild hosts. Virus diversity on weed and wild hosts is a major concern, since they may act as reservoir hosts. Macroptilium spp. is a leguminous weed which is frequently associated with Phaseolus lunatus (lima bean) crops. In the years of 2005 to 2010 a total of 41 samples of Macroptilium spp. were collected in the states of Alagoas, Paraíba, Pernambuco and Sergipe, all in Northeastern Brazil. Total DNA was extracted from each sample and viral genomes were RCA- amplified from all 41 samples. The amplified DNA was cleaved with BamH I, Hind III, Kpn I and Spe I, cloned and sequenced. The ICTV-established 89% DNA-A identity threshold was used to determined taxonomic placements. Out of seven DNA-A clones analyzed so far, three clones corresponded to isolates of Bean golden mosaic virus (BGMV), displaying 89-90% identity with a BGMV isolate from common bean. These identity values indicate that these three isolates comprise a distinct BGMV strain, named BGMV-Ml. One clone corresponded to an isolate of Euphorbia mosaic virus (EuMV), showing 97% identity with a EuMV isolate from Euphorbia heterophylla. Two clones corresponded to a novel species, for which the name Macroptilium yellow spot virus (MaYSV) is proposed, and one clone corresponded to another novel species for which the name Macroptilium yellow net virus (MaYNV) is proposed. In a phylogenetic tree, both novel species clustered with Brazilian begomoviruses, indicating their indigenous origin. The detection of four distinct viruses (including two novel ones) out of seven clones indicates a high diversity of begomoviruses infecting Macroptilium spp. in Northeastern Brazil.

Financial support: National Research Institute for Plant-Pest Interactions and Pronex-Fapemig

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Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family:Geminiviridae

Category: Genetic Diversity/Evolution

Title: The perspective for the use of genetically modified common beans in Brazil based on genetic diversity of Bean golden mosaic virus

Authors: T. A. L. SILVA, R. S. FONTENELE, J. C. FARIA, F. J. L. ARAGÃO, AND S. G. RIBEIRO

Address: Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica – Brasília, DF, Brazil

Abstract: Bean golden mosaic is the most important viral disease affecting common beans (Phaseolus vulgaris) in Brazil. It is caused by Bean golden mosaic virus (BGMV) and is prevalent in all bean producing areas. Aiming to control the disease, a transgenic bean elite line was developed with an intron-hairpin construction to silence the AC1 viral gene. This line shows high resistance to BGMV with the capacity to contain the replication of the virus both in the green house and in the field. The silencing through small interfering RNAs (siRNA) is sequence specific and a high identity to the transgene is required to assure resistance since gene silencing can be inefficient when the identity is lower than 90%. To guarantee the broad use of transgenic cultivars in the different regions of the country, we have analyzed the sequence of begomoviruses isolated from bean samples from several states. A total of 85 bean samples were collected. Total DNA from dried leaves was extracted with Extract-N-Amp™ Plant PCR Kit followed by RCA reaction using Phi-29 polymerase. PCR reaction with primers BGMV-HPXHO and PAR1c484 amplified a ~1.3Kb fragment encompassing part o AC1 gene, the entire common region and part of the coat protein gene. Sequences obtained from 25 isolates were analyzed and compared with corresponding sequences from BGMV-[BR:GO 87-1] (M88686). The identity was between 85,2% and 97,4% indicating that most likely only BGMV is infecting common beans in the field. Although some isolates have sequence identities lower than 90% over the entire PCR fragment, the portion corresponding to that used in the RNAi construction is highly identical (between 90,5 and 98,8%). These results will warrant the large scale use of transgenic cultivars resistant to BGMV based on RNAi over the Brazilian territory.

Financial support: Embrapa/Monsanto, FAPDF

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Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Nanoviridae

Category: Genetic Diversity / Evolution

Title: Genetic Diversity of Banana bunchy top virus (BBTV) in the South Pacific Tongan Archipelago.

Authors: Daisy Stainton1, Simona Kraberger1, Karyna Rosario2, Milen Marinov1, Elizabeth Wiltshire1, Matthew Walters1, Samieluela Lolohea3, Mana’ia Halafihi4, Ika Katoa4, Waikato Aholelei 4 Luseane Taufa4, David Collings1, Darren P Martin5and Arvind Varsani1, 6.

Address: 1School of Biological Sciences, University of Canterbury, Christchurch, New Zealand; 2College of Marine Science, University of South Florida, St Petersburg, Florida; 3Tonga College, Tonga’tapu, Kingdom of Tonga; 4Ministry of Agriculture and Food, Forests and Fisheries, Kingdom of Tonga; 5Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 6Electron Microscope Unit, University of Cape Town, Cape Town, South Africa

Banana bunchy top virus (BBTV) is a six component, single-stranded DNA virus of the Nanoviridae family that can cause major losses in banana (Musa sp.) due to severe stunting of the plants. Although bananas are an important crop in many South Pacific Islands and BBTV symptoms have been identified in the region, there has been limited molecular analysis of BBTV in these islands. Here we report the first genomic analysis of all six BBTV components from the Kingdom of Tonga. In 2010, we sampled infected banana plants from the three main Tongan islands, (Tongatapu, Ha’apai and Vava’u, which are separated from each other by approximately 150-200 km). We isolated, cloned and sequenced all six BBTV components from twelve Tongan samples. Phylogenetic analysis indicates that all the Tongan BBTV sequences cluster with the South Pacific group as expected. Pairwise distance analysis revealed that the components encoding the capsid (DNA-S), the master replication (DNA-R), nuclear shuttle (DNA-N), and cell cycle link (DNA-C) proteins share approximately 98 - 99% similarity. The other two components, encoding the movement (DNA-M) and unknown (DNA-U3) proteins, share 91 - 99% similarity. When comparing Tongan BBTV components to all the sequences available in GenBank the range of nucleotide identities varied depending on the component ( DNA-R 88.6 - 99.9%, DNA-U3 73.7 - 99.6%, DNA- S 87 - 99.9%, DNA-M 81.6 - 99.8%, DNA-C 85.5 - 99.6% and DNA-N 83.9 - 99.9%). We found evidence of inter-component recombination and Fixed effects likelihood (FEL) estimates indicated that all the open reading frames are under negative selection. However, there are a few codons in the N terminal domain of the nuclear shuttle protein and the movement protein that are under positive selection. This study allows a better understanding of the evolutionary history and movement of BBTV in the Pacific region. This is part of an ongoing study in the Pacific region looking at BBTV.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Genetic Diversity/Evolution Virus Family: Geminiviridae

Category: Genetic diversity/Evolution

Title: Genetic analysis and phylogeographic history of MSV reveals the spatial and temporal origins of contemporary MSV-A recombinant lineages and identifies southern Africa as the ultimate origin of Maize streak disease

Authors: A. L. Monjane, G. W. Harkins, D. P. Martin, P. Lemey, D. N. Shepherd, P. Lefeuvre, E. P. Rybicki , B. E. Owor , R.S. Ali, B. Flett, M. Ramusi, S. Oluwafemi, and A. Varsani

Address: Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Observatory, Cape Town, 7925, South Africa

The unrelenting threat of maize streak disease (MSD) to sustainable maize production in Sub-Saharan Africa, has motivated research aimed at elucidating the ecological and genetic factors driving the continent-wide spread of MSV-A – the maize streak virus strain that cause the disease. Determining the geographical origin of MSV-A and retracing its historical spread could yield important insights into how and why this important pathogen emerged. Similarly, determining when and where the major extant MSV-A lineages arose could identify geographical hotspots for epidemic strain genesis and guide the formulation of better strategies aimed at curtailing the continental spread of MSD. Here, we use model- based phylogeograpgic analyses (implemented within the Bayesian inference framework of the molecular evolution analysis program ,BEAST), in tandem with recombination analysis of publicly available MSV sequences (including 204 new full length MSV genomes) to reconstruct a plausible history of MSV-A movements over the past 150 years . Our results suggest that MSV arose somewhere in southern Africa around 1863 (95 % highest probability density (HPD) intervals 1809-1935), a few decades prior to the first credible reports of MSD in South Africa. The virus then dispersed trans-continentally at mean rates of 33.6 km/yr (95 % HPD intervals 1.4-97.6 km/yr). While characterising the contribution of recombination to MSV-A diversification we found no evidence of inter-species recombination with other African streak viruses, and only a few instances of inter inter- MSV strain recombination. We use distinctive patterns of nucleotide variation caused by 20 unique intra- MSV-A recombination events to tentatively classify the MSV-A genotypes into 18 different haplotypes. Despite many of these haplotypes displaying distinct, geographical distributions it is apparent that they have mostly emerged within the past four decades from either East Africa or southern Mozambique. Collectively, our results suggest that persistent sampling and analysis of MSV-A genomes within these diversification hotspots could be used to effectively monitor the emergence and track the spread of future epidemiologically important MSV-A lineages.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Gene Expression Virus Family: Geminiviridae

Category: Gene Expression

Title: The V2 protein of Monopartite begomovirus determines virulence, hypersensitive response and suppression of posttranscriptional gene silencing.

Authors: PRADEEP SHARMA, AND MASATO IKEGAMI*

Address: Division of Crop Improvement, Directorate of Wheat Research, Karnal 132 001, India *Graduate School of Agricultural Sciences, Tohoku University, Sendai 981-8555, Japan

Abstract: Tomato leaf curl Java virus-A (ToLCJV-A[ID]) from Southeast Asia is a new member of the emerging group of monopartite begomoviruses that require a betasatellite component for symptom induction. In this study, the role of V2 in viral pathogenesis and posttranscriptional gene silencing (PTGS) was studied. The subcellular localization of V2 was also determined. Our results showed that V2 of ToLCJV-A[ID] elicits a reaction resembling the hypersensitive response (HR) associated with the induction of necrosis and a systemic burst of H2O2 production when expressed from a potato virus X vector in Nicotiana species and tomato. Transient expression of ToLCJV-A[ID] V2 after agroinfiltration of N. benthamiana and tomato also triggered HR-like cell death, demonstrating that ToLCJV-A[ID] V2 is a target of host defense responses. We further showed that deletion of 58 amino acids (aa) from the N-terminus did not affect the HR, suggesting that this region has no role in the HR, while deletion of 60 aa from the C-terminus of V2 abolished both the HR response and V2 silencing suppressor activity, suggesting that these sequences are required for the HR-like response and suppression of PTGS. Visualization of the subcellular localization of ToLCJV-A[ID] V2 showed that this protein is localized to the cell periphery and perinuclear region, where it is co- localized with the endoplasmic reticulum. This finding demonstrated that ToLCJV- A[ID] V2 is a pathogenicity determinant that elicits an HR-like response, which has been identified for the first time in an infectious ssDNA virus.

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Poster Session Geminiviridae Virus induced gene silencing Virus Family: Geminiviridae

Category: Virus induced gene silencing

Title: Silencing of PPR gene in garlic (Allium sativum L) “blanco perla” resistant to white rot using V-VIGS derived of PHYVV.

Authors: L. GUEVARA-OLVERA, G. ACOSTA-GARCÍA, F. DELGADILLO- SANCHEZ, R. F. RIVERA-BUSTAMANTE AND L. CHAIREZ-RODRÍGUEZ.

Address: Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y A. García Cubas s/n, C.P. 38020, Celaya, Guanajuato, México. Departamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6 Libramiento Norte, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México. INIFAP, Campo experimental Bajío, km 6 Carr. Celaya-San Miguel de Allende, Apdo. Postal 112, Celaya, Guanajuato CP 38000

Abstract: White rot caused by S. cepivorum Berk, is the predominant disease of garlic (A. sativum L) crops worldwide. Several strategies for white rot control are now being used, based on successful, stable, widespread garlic resistance. In concordance, A. sativum cv “blanco perla” has shown resistance to white rot. Several gene fragments (ESTs) expressed during resistance of garlic “blanco perla” to white rot have been identified and isolated using suppression subtractive hybridization (SSH). PPR clone is expressed specifically during garlic resistance, and this PPR gene in concordance with database could be involved in RNA stabilization. The aim of this research is demonstrate the role of PPR gene during garlic resistance by using RNAi, based on use of double-stranded RNA to target specific mRNAs for degradation. One of the major tools to generate transient loss- of-function is the virus induced gene silencing (VIGS). Two fragments (292 and 200 bp) of PPR gene were inserted into the pepper huasteco yellow veins virus (PHYVV)-derived vector. Reduction of mRNA level and a susceptible phenotype in garlic “blanco perla” after PHYVV::PPR inoculation could provide new information to design strategies to control of white rot disease.

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Virus induced gene silencing Virus family: Geminiviridae

Category: Virus induced gene silencing

Title: Development of an efficient system for assessing gene function in the cotton plant using virus-induced gene silencing (VIGS)

Authors: Cecilia Hernandez-Zepeda, ZiFu He, Judith K. Brown

Address: School of Plant Sciences, The University of Arizona, 85721, Tucson, AZ. USA Abstract: A vector for virus induced gene silencing (VIGS) based on the Cotton leaf crumple virus (CLCrV) DNA-A, was previously cloned and shown to be infectious and to silence host genes when biolistically delivered to cotton seedlings (Idris and Brown, 2004). The CLCrV VIGS vector is approximately 1.9 Kbp in size, and comprises the CLCrV DNA-A component that lacks most of the coat protein (CP) ORF, and the DNA-B component, which has not been modified (wild type). In this study, we used the CLCrV VIGS vector to transiently silence several candidate cotton genes involved in fatty acid synthesis and adaptation to cold stress, fiber initiation, fiber elongation, and cellulose synthesis in primary and secondary cell walls in upland cotton (Gossypium hirsutum): 1) fatty acid desaturase (FAD2-4); 2) a steroid 5-reductase (GhDET2); 3) actin (GhACT1); 4) two chitinase like genes (GhCTL1 and GhCTL2). ClustalV alignment of the cotton mRNAs was carried out using available sequences of each target gene. Aligned sequences were used to design specific primers for each gene. Total RNA was isolated from cotton (leaves, roots, cotton boll) and used as template to amplify (by RT-PCR) fragments of the different target genes. These fragments were cloned into the VIGS CLCrV vector. VIGS CLCrV carrying the FAD-2-4, DET2, ACT1, CTL1 and CTL2 gene fragments were constructed and sequenced to confirm the integrity of the cloned gene fragment in relation to published sequences available in GenBank used to guide primer design. Cotton seedlings (G. hirsutum cv Deltapine 5415) were bombarded with 1.1-μm diameter tungsten microprojectiles (BioRad) coated with a mixture of 1 μg each of the following CLCrV DNA-A plasmid and 1 μg of the wild type CLCrV DNA-B component: (1) VIGS CLCrV – FAD2-4, (2) VIGS CLCrV –DET2, (3) CLCrV wild type virus, and (4) VIGS ‘empty’ (no insert control) plasmid. Inoculated plants were maitained in the growth room at 28C with a 16/8h photoperiod and ~ 80% RH. Mock-inoculated (water without of DNA template), virus-free cotton plants were maintained as controls. Results showed that cotton plants inoculated with the VIGS vector carrying the FAD2-4, DET2, ACT1, CTL1 and CTL2, each co-inoculated with CLCrV DNA B dimer developed a range of mild symptoms four weeks post inoculation (PI). In comparison, wild type CLCrV-inoculated plants developed typical, severe CLCrV disease symptoms about 12 days PI. Analysis of gene expression and assessment of gene silencing is underway.

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Poster Session Geminiviridae Management / Control Virus Family: Geminiviridae

Category: Management / Control

Title: RNAi-resistance to bipartite Begomoviruses in genetically modified Tomatoes

Authors: L. M. ALMEIDA, T. M. CIPRIANO, K. BONFIM, E. O. P. L. NOGUEIRA, S. G. RIBEIRO, F. J.F.L. ARAGÃO

Address: Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, 70770-900, Brasília, DF, Brasil

Abstract: Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetable crops in Brazil. Sixty thousand hectares are cultivated annually for production of fresh fruit and for processing industry. A large variety of begomoviruses affect the crop in the producing areas. The control is generally carried out with the use of pesticides, causing problems for the environment. The use of resistant plants is the best alternative to manage this problem. Transgenic tomatoes were engineered to express an intron-hairpin quimeric gene with a Rep- derived fragment from Tomato rugose mosaic virus (ToRMV). This construction aimed to silence the expression of the Rep gene and generate resistant plants. Transgenic tomatoes R1 plantlets from lines L1 and L4 were inoculated by particle bombardment with infectious clones of ToRMV, Tomato severe rugose virus (ToSRV), Tomato yellow vein streak virus (ToYVSV) and Tomato chlorotic mottle virus (ToCMoV). Both tested lines were resistant to ToRMV and to the close related ToSRV but not to ToCMoV or ToYVSV, which have less than 69% identity to the transgene, confirming the specificity of RNAi mediated resistance. Polymerase chain reaction analysis of R5 progeny of family 4 (Line 1) revealed the presence of the transgene in 17 out of 23 plants (segregation rate 3:1). Northern blot analysis detected the presence of small interfering RNAs (siRNA) in the transgenic plants. The R5 plants were tested for virus resistance by biolistics inoculation with ToRMV. Although not all the plants from R5 progeny of line 1 were resistant, the rate of resistance was significantly higher (92%) than in the R1 progeny (70%).

Financial support: Embrapa, National Research Institute for Plant-Pest Interactions

th 6 International Geminivirus Symposium

Poster Session Geminiviridae Management / Control Virus Family: Geminiviridae

Category: Management/Control

Title: Broad-spectrum resistance to Brazilian bipartite Begomovirus species in tomato lines carrying the Ty-1 or tcm-1 loci

Authors: R. BLAWID1,2, R.F. FONTENELE1, C. LACORTE1, R.C.P. CARVALHO2, R.O. RESENDE2, L.S. BOITEUX3, M.E.N. FONSECA3, A. C. M. BRASILEIRO1, AND S.G. RIBEIRO1

Address: 1Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica - Brasília-DF, Brazil; 2Departamento de Biologia Celular, UNB, Brasília- DF, Brazil; 3Embrapa Hortaliças, Rodovia Brasília/Anápolis BR 060 Km 09, Gama- DF, Brazil

Abstract: In this study we report on the selection of tomato lines resistant to a broad range of tomato-infecting begomoviruses from Brazil. Two introgression lines named LAM 157 and LAM 144, which carry the resistance loci tcm-1 and Ty-1, respectively, were recently obtained. Stable resistance was observed in both lines by inoculating infectious clones of Tomato chlorotic mottle virus (ToCMoV), Tomato severe rugose virus (ToSRV), Tomato yellow vein streak virus (TYVSV) and Tomato rugose mosaic virus (ToRMV) via biolistics. Plants were evaluated for virus symptoms and for the presence of viral DNA by Southern blot as well as PCR analysis. In addition, due to its higher sensitivity over conventional PCR, quantitative real-time PCR (qPCR) was employed to quantify viral DNA-A of ToCMoV in the two introgression lines and the susceptible ‘Santa Clara’. For this purpose, four different primer pairs from the ToCMoV DNA-A were designed. The amplified fragments ranged from 59 bp to 196 bp. The best primer pair was used to prepare a standard curve using a plasmid containing the full-length of ToCMoV genome. Only few plants from the lines LAM 157 and LAM 144 showed begomovirus symptoms and they were very mild in comparison to the chlorotic mottling observed in ‘Santa Clara’. Analysis of total DNA by Southern blotting showed much less viral DNA in resistant plants than found in ‘Santa Clara’. Moreover, results obtained from qPCR analysis showed that the susceptible cultivar accumulated 12- to 43-fold more copies of viral DNA-A compared to the resistant lines. Susceptible plants presented a mean copy number of 2.05×105 of viral DNA-A in 2.24 ng of total DNA extracts, whereas resistant plants showed values varying from 6 copies to 2.1×104. Our results confirm the potential use of the introgression lines carrying the loci tcm-1 and Ty-1 as resistance sources against the main disease complex of begomovirus infecting tomato plants in Brazil.

Financial support: Embrapa/Monsanto, National Research Institute for Plant-Pest th 6 International Geminivirus Symposium

Poster Session Geminiviridae Management / Control Interactions; CNPq, FAP-DF Virus Family: Geminiviridae

Category: Management and Control

Title: The biosafety of siRNA transgenic common beans resistant to Bean golden mosaic virus.

Authors: J. C. FARIA1, F. J. L. ARAGÃO2

Address: 1Embrapa Arroz e Feijão, Caixa Postal 179, Santo Antônio de Goiás, GO CEP 75375-000 - Brasil; 2Embrapa Recursos Genéticos e Biotecnologia, Caixa Postal ----, Brasília, DF CEP - Brasil.

Abstract: Bean golden mosaic virus (BGMV) is an important Begomovirus distributed in most of the bean growing areas in Brazil. Disease losses can be up to 100% in a singly early affected field. We recently showed that transgenic beans expressing a small interfering RNA derived from the rep gene successfully prevents plants from infection by BGMV both under high inoculation pressure in the greenhouse or in the field. According to the Brazilian law regulating on the biosafety of Genetically Modified Organisms the biosafety of GMOs shall be based on the Molecular Characterization (including protein biosafety), Agronomic and Environmental evaluation, and Substantial Equivalence. The transgene is present as two copies in a single locus, and was inherited as a single gene in a Mendelian way. Up to 30% of heterozygous plants may be infected by BGMV thus showing gene dosage effect. However, homozygous plants are completely resistant. Except for resistance to BGMV the transgenic line Olathe 5.1 was essentially like its counterpart Olathe Pinto in all required analysis. The complete set of data will be submitted to The National Technical Biosafety Commission (CTNBio) by November 2010 for analysis and a possible authorization for commercial release.

Financial support: Embrapa/Monsanto

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Poster Session Geminiviridae Management / Control Virus Family: Geminiviridae

Category: Management/Control

Title: Segregation pattern of genetic resistance to Tomato yellow leaf curl virus - Israel (TYLCV-IL) in F2:3 families derived from the tomato hybrid ‘TyKing’

Authors: R.C. Pereira-Carvalho1,2,3, L.S. Boiteux1, M.E.N. Fonseca1, J.A. Díaz- Pendón3, E. Moriones3, R. Fernández-Muñoz3, AND R.O. Resende2

Address: 1National Center for Vegetable Crops Research (CNPH), Embrapa Vegetable Crops, Brasilia-DF, Brazil; 2Departments of Plant Pathology and Cell Biology, UnB, Brasilia-DF, Brazil; 3Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC), 29750 Algarrobo-Costa, Málaga, Spain. E-mail: [email protected]

Abstract: The tomato (Solanum lycopersicum L.) line ‘TX-468-RG’ (derived from the F1 hybrid ‘Tyking’) was identified as a source of resistance to distinct bipartite Begomovirus species in Brazil and to a number of monopartite Begomovirus species of the ‘Tomato yellow leaf curl disease’ (TYLCD) complex (1). The phenotypic expression of this resistance, even under a high virus pressure, is manifested by reduced viral accumulation associated with either absence of symptoms or very mild symptoms restricted to apical leaves. Inheritance of resistance to the bipartite Tomato chlorotic mottle virus using an F2 population indicated a recessive gene/locus model (tentatively named as tcm-1) (2). An additional study to verify the inheritance of ‘TX-468-RG’ resistance to Tomato yellow leaf curl virus - Israel (TYLCV-IL) was conducted in Spain using an F2 population. Segregation ratio indicated also a recessive resistance, but with an apparent interaction with other minor genes with epistatic effects (1). Further experiments (using F2:3 families) were established aiming to clarify if the resistance of ‘TX-468-RG’ to both monopartite and bipartite viruses is controlled by identical genetic factors. In a first study, progenies of 171 F2:3 families derived from the cross between ‘Ohio 8245’ (susceptible) x ‘TX-468-RG’ (resistant) were evaluated via agroinoculation using one infective TYLCV-IL clone. The frequency distribution observed (41 families of resistant homozygous plants, 87 families of susceptible heterozygous plants, and 43 families of susceptible homozygous plants) and chi-square test confirmed the 1:2:1 segregation model expected. The analysis of 16 plants within each family indicated a good fit to the 1(R):3(S) segregation ratio in all susceptible heterozygous families assessed. These results reinforce the hypothesis of a recessive monogenic inheritance, minimizing the effects of other epistatic genes. Additional studies will be conducted evaluating the same sample of F2:3 families against Begomovirus species with bipartite genome. Literature cited

García-Cano et al. 2008. Phytopathology 98:618-627 Giordano et al. 2005. Euphytica 143:27-33.

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Poster Session Geminiviridae Management / Control Virus Family: Other

Category: Management

Title: SUSTAINABILITY OF INDIAN AGRICULTURE SANS PESTICIDES

Author: YPS Rathi (Emeritus Scientist)

Address: B-55, Pallavpuram - I, Meerut - Meerut - 250110, UP, India.

In this era of Liberalization, Privatization & Globalization there is heavy international competition within India as well as abroad on all fronts and agriculture in particular. Dr. Norman Borlaug, Noble Laureate described the phenomenon of the growing population of IIIrd world countries as “Frightening situation”. Hence, the priority is clear cut before us as our population surpassed one billion. We have not only to sustain the food production but to continue to achieve higher and higher production to ensure that every mouth gets sufficient food.

Introduction of high yielding semi dwarf varieties of wheat and rice with basket of agrochemicals and ensured irrigation resulted in Green Revolution. During this era of green revolution many contact and systemic pesticides were developed and widely used. Indiscriminate use of agro chemicals caused health hazards, environmental pollution and damage to eco-system. Quite often we used to read in newspaper and magazines sensational headings such as “ Flow of banned pesticides to third world countries”, “Pesticides induce cancer and sterility”, “ Pesticides: a guest that overstayed”, “Pesticides, a death harvest”, etc. After reading such sensational news, we are forced to think why the pesticides should not be banned. However, at the same time we are not ready to forget the history of mankind filled with miseries of diseases / insect pest epidemics.

In recent years, the organic movement and awareness of environmental hazards, some people of the developed countries started talking of “Organic farming”. But the question arises can the farmers of 3rd world countries afford and sustain the organic farming? Can this “Kitchen garden strategy” of organic produce fill up the belly of our bulging population? The only way out is to minimize the gap between the genetical / potential yield through integrated crop management. In this approach Integrated Pest Management (IPM) is an integral part wherein eco-system disturbance is minimized. It will not be an exaggeration that the pesticides are indispensable for tropical and subtropical countries because of the climate of these countries is highly congenial to the pests. The adoption of monoculture is highly prone to outbreak of pathogens / insects. Moreover, the cash crops always need pesticide umbrella. However, these arguments do not justify at all that we should use the pesticides to maximum. In fact the pesticides should be used in targeted judicious manner and hence IPM strategies should be developed against more and more diseases and insect pests. Besides, the farmers should be educated about the pesticides awareness and safe & effective application technology.

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Poster Session Geminiviridae Mixed Infection

Virus Family: Geminiviridae

Category: Virus identification

Title: Association of two begomoviruses with severe golden mosaic disease of cowpea

Authors: NAIMUDDIN AND MOHAMMAD AKRAM

Address: Division of Crop Protection, Indian Institute of Pulses Research, Kanpur-208 024, India

Abstract: Golden mosaic disease of cowpea in India is known to be caused by Mungbean yellow mosaic India virus (MYMIV). A severe yellow mosaic disease of cowpea in and around Kanpur was prevalent in Kharif (rainy season) 2009. Primer pairs were designed to get a fragment of DNA A containing coat protein gene of four begomoviruses (viz. Mungbean yellow mosaic India virus, Mungbean yellow mosaic virus, Horsegram yellow mosaic virus, Dolichos yellow mosaic virus) known to infect various leguminous crops in India. In PCR tests, all the ten samples assayed gave positive results with MYMIV specific primer pairs and three of them yielded amplicon of expected size with primer pair specific to Dolichos yellow mosaic virus (DoMYV) also, indicating the presence of mixed infection of MYMIV and DoYMV in these cowpea samples. No amplification was obtained with primer pairs specific to Mungbean yellow mosaic virus and Horsegram yellow mosaic. The sequences of amplified coat protein genes of MYMIV and DoYMV contained a single open reading frame with 774 nucleotides and 257 amino acids and were designated as MYMIV-[CpKn] and DoMYV-[CpKn]. Nucleotide and deduced amino acids sequences of coat protein gene of MYMIV-[CpKn] had respectively, 95-99 % and 96-100% similarity with other isolates of MYMIV. Nucleotide and deduced amino acids sequences of coat protein gene of DoYMV-[CpKn] had respectively, 95-96 % and 97-98% similarity with other isolates of DoYMV. This appears to be the first report of mixed infection of MYMIV and DoYMV in naturally infected cowpea.

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Poster Session Geminiviridae Etiology aVirus Family: Geminiviridae

Category: Etiology

Title: Is tomato leaf deformation disease caused by the DNA-A component of a novel begomovirus from Peru?

Authors: B. MÁRQUEZ-MARTÍN1, L. ARAGÓN-CABALLERO2, E. FIALLO- OLIVÉ1,3, J. NAVAS-CASTILLO1, AND E. MORIONES1

Address: 1Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM- UMA-CSIC), Estación Experimental “La Mayora”, 29750 Algarrobo-Costa, Málaga, Spain; 2Facultad de Agronomía, Universidad Nacional Agraria La Molina, La Molina, Lima, Perú; 3Centro Nacional de Sanidad Agropecuaria (CENSA), La Habana, Cuba.

Abstract: Begomoviruses (genus Begomovirus, family Geminiviridae) cause serious damage to tomato (Solanum lycopersicum) crops in the New World. Since early 2000's a severe disease associated with Bemisia tabaci (Hemiptera: Aleyrodidae) infestations is damaging tomato crops throughout Peru. Affected plants exhibit symptoms of curling and deformation of leaves and up to 100% yield losses are observed when infections occur in early growth stages. A survey of diseased plants was conducted at major tomato growing regions of Peru. Rolling circle amplification using φ29 DNA polymerase and restriction fragment length polymorphism analysis suggested that a begomovirus was present in symptomatic plants. The full-length sequence of a begomovirus DNA-A-like component was determined. It comprises 2591 nucleotides and has the typical genomic structure of a New World begomovirus DNA-A. Less than 89% nucleotide sequence identity to known begomoviruses was found, indicating that it corresponded to an isolate of a novel begomovirus species for which the name tomato leaf deformation virus (ToLDeV) is proposed. Different stretches of the genomic component have the highest percentages of sequence identity with different begomoviruses, suggesting a recombinant origin. Closest relatives are isolates of soybean blistering mosaic virus, tomato yellow spot virus, and tomato chino La Paz virus. This is the first confirmation of a begomovirus infection in tomatoes in Peru. Efforts to identify and clone a putative DNA-B component from infected tomato samples have been fruitless. Infectivity analysis will determine if ToLDeV DNA-A is able to induce symptoms and accumulate systemically in tomato plants in the absence of a cognate DNA-B.

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Poster Session Geminiviridae Etiology Virus Family: Geminiviridae

Category: Etiology

Title: The first infectious clone of a “swepovirus”

Authors: H.P. TRENADO, A.F. ORÍLIO, B. MÁRQUEZ-MARTÍN, E. MORIONES, AND J. NAVAS-CASTILLO

Address: Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora”, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM- UMA-CSIC), Estación Experimental “La Mayora”, 29750 Algarrobo-Costa, Málaga, Spain.

Abstract: Sweet potato (Ipomoea batatas) and related species as I. indica are frequently infected by monopartite begomoviruses, as it has been recently reported in Spain [Lozano et al. (2009) J Gen Virol 90: 2550–2562]. Ipomoea-infecting begomoviruses (“swepoviruses”) group in a cluster distinct from the Old World and New World begomoviruses, seeming to represent one of the earliest points of divergence among this genus. Obtention of begomovirus infectious clones is a relatively simple process that has been simplified in different ways after availability of rolling circle amplification (RCA) techniques. Nevertheless, genomes of Ipomoea-infecting begomoviruses have been recalcitrant to render infectious clones to date. Thus, Koch’s postulates have not been fullfilled for any of the begomoviruses in this group. Here we report the obtention of an infectious clone of an isolate of sweet potato leaf curl Lanzarote virus (SPLCLaV). DNA extracted from a sweet potato plant from Málaga (Spain) infected with SPLCLaV was submitted to RCA with φ29 DNA polymerase to amplify the complete virus genome. After partial digestion, dimers were cloned into a binary vector, transformed in Agrobacterium tumefaciens, and agroinoculated in sweet potato “Beauregard” and “Promesa”, I. nil “Scarlett O’Hara”, I. setosa and Nicotiana benthamiana plants. Infection was confirmed in inoculated plants of the four tested species by molecular hybridization analysis using a probe to the CP gene and by PCR amplification with specific primers. Infected I. nil, I. setosa and N. benthamiana plants exhibited symptoms consisting of leaf curl, yellowing and growth reduction. In addition, I. nil plants showed yellow vein symptoms at late infection stages. No symptoms were observed in sweet potato plants either infected by agroinoculation or by grafting with infected I. setosa scions. Moreover, the progeny of the infectious SPLCLaV clone was transmissible by Bemisia tabaci biotype Q from agroinoculated I. setosa plants to healthy I. setosa and I. nil plants, suggesting that a fully biologically active molecule was cloned. Furthermore, the genome of the progeny begomovirus present in an inoculated I. setosa plant was sequenced after RCA amplification and cloning, confirming identity with the originally inoculated begomovirus. Thus, for the first time Koch’s postulates were fullfilled for the disease caused by a “swepovirus” in Ipomoea spp. th 6 International Geminivirus Symposium

Poster Session Geminiviridae Survey

Virus Family: Geminiviridae

Category: Virus survey

Title: Survey on begomoviruses on cucurbit species in Brazil

Authors: M.F. LIMA AND A.K. INOUE-NAGATA

Address: Embrapa Vegetables, BR 060 Km 09, Caixa Postal 218 CEP 70359- 970, Brasília, Distrito Federal, Brazil

Abstract: Over the last two decades, begomoviruses have become one of the major problems in vegetable crops such as tomato and pepper in Brazil. Cucurbit- infecting begomoviruses resulting in severe losses have also been reported in several countries. Considering their importance and the damage these viruses have been causing, this work aimed to investigate the occurrence of begomoviruses in cucurbits in Brazil. A total of 797 symptomatic and/or asymptomatic cucumber, pumpkin, squash, melon, watermelon, chayote, loofah, gherkin and Fevillea cordifolia samples collected in fields over nineteen counties of nine Brazilian states and the Federal District were tested for begomoviruses using a pair of degenerate primers PAL1v1978/PAR1c496 (Pl.Dis.77:340-347, 1993) by PCR. In addition, serological tests (296 samples) were performed for cucurbit- infecting viruses commonly found in Brazil (Papaya ringspot virus – type watermelon - PRSV-W; Watermelon mosaic virus – WMV; Zucchini yellow mosaic virus – ZYMV; Cucumber mosaic virus - CMV) using polyclonal antibodies. A few samples were also tested for Melon yellowing-associated virus (MYaV) and Zucchini lethal chlorosis virus (ZLCV). All PCR tests resulted negative for begomoviruses. Potyviruses occurred in 157 (PRSV-W), 119 (WMV) and 110 (ZYMV) samples; CMV was found in 99 samples, and MYaV and ZLCV were considered as minor viruses infecting cucurbits. These results indicate an apparent absence of begomoviruses infecting cucurbits in Brazil, considering the wide collection effort done. However, further investigation using other molecular tools in addition to PCR tests is being undergoing. High-frequency detection (48.4%) of potyviruses indicates their prevalence on these crops in the country.

Financial support: Embrapa and CNPq.

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Poster Session Geminiviridae Other

Category: Other

Title: Use of Virus-Induced Gene Silencing (VIGS) to accelerate genetic gain.

Authors: ALVAREZ-VENEGAS, R., ZHANG, Y., KRALING, K., and TULSIERAM, L.

Address: Dr. Raúl Alvarez-Venegas, Departamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6 Libramiento Norte, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México.

Abstract: Shorter breeding cycles and the opportunity for enhanced genetic gains, together with the study of the molecular basis of vernalization, are essential areas of research in plant biology. Several approaches have been employed to achieve gene silencing in plants, but none so far reported in canola (Brassica napus), and particularly to induce flowering without vernalization in true winter lines by using sense DNA sequences in virus-induced gene silencing (VIGS) vectors. The present research provides the methods to transiently down-regulate, by VIGS technology, vernalization genes in winter annuals, specifically the family of Flowering Locus C (FLC) genes in winter canola (BnFLC1 to BnFLC5). Down- regulation of the BnFLC genes allows winter annuals to flower without vernalization and consequently provides the means for enhanced genetic gains. The proposed silencing system can be used to down-regulate gene families, to determine gene function, and to induce flowering without vernalization in winter Brassica lines as well as in many important winter crops.

Acknowledgements: This project was funded by Pioneer Hi-Bred International, Inc. Raúl Alvarez-Venegas was supported by a Pioneer’s Discovery Research Grant. Raúl Alvarez- Venegas also thanks CONACYT CB2006/55028.

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Poster Session Geminiviridae Other

Virus Family: Geminiviridae

Category: Other (Education)

Title: Analysis of geminivirus literature in the last decade. Favorite journals, most prolific authors, geographic distribution.

Authors: R. FAJARDO, M.A. GARCIA-NERIA, D.L. TREJO-SAAVEDRA, R.F. RIVERA-BUSTAMANTE

Address: Departamento de Ingeniería Genética, Cinvestav Irapuato, Km. 9.6 Libramiento Norte, P.O. Box 629, C.P. 36500, Irapuato, Guanajuato, México.

Abstract: The number of geminivirus sequences deposited in GenBank has increased almost exponentially in the last few years. Most new genomes are coming from tropical areas from Central and South America, Asia and Africa. As an exercise of data mining, we wanted to explore if the number of new sequences correlated with an increase in the number of papers published in international journals. Using the Thompson ISI-WEB page, we downloaded all references of those papers that included the word “geminivirus” in either the title or abstract. The references were then filtered to eliminate reviews and those papers that only marginally mentioned a geminivirus or a geminivirus disease (i.e. not “honest gemini papers”). The remaining references were then organized by several categories such as authors, geographic origin, journal, year published, scientific discipline. This analysis is certainly biased by many factors such as, using only a single database and the presence of multi-institutional collaborations, therefore should be considered only as indicative. Nevertheless, several interesting observations were organized and will be presented. For example, no obvious correlation was observed between the increase on geminivirus genomic sequences (and their origin) with the number of papers confirming that a new genomic sequence by itself does not warranty its publication in an international journal. However, when organized by continental origin an important increase is observed in the Orient. In general, USA, as a country, leads almost every year in the number of published papers, nevertheless, the most prolific authors are not from USA showing that USA publications are distributed among a larger number of laboratories whereas other countries rely on a small number of highly productive researchers. This exercise was carried out as a effort to bring together computer science undergraduate students and biotechnology graduate students to promote interactions towards the much needed bioinformatic interfase. Supported by Concyteg.

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Poster Session Geminiviridae Other

The spread of Tomato yellow leaf curl virus (TYLCV) from the Middle East to the world

Pierre Lefeuvre, Gordon Harkins, Darren P. Martin, Philippe Lemey, Alistair J. A. Gray, Sandra Meredith, Francisco Lakay, Dionne N. Shepherd, Hossain Massumi, Jahangir Heydarnejad, and Arvind Varsani

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV; Genus Begomovirus, Family Geminiviridae) represents a serious looming threat to tomato production in all temperate parts of the world. Whereas determining where and when TYLCV movements have occurred could help curtail its spread and prevent future movements of related viruses, determining the consequences of past TYLCV movements could reveal the ecological and economic risks associated with similar viral invasions. Towards this end we applied Bayesian coalescent based phylogeography and recombination analyses to available TYLCV sequences (including those of 16 new Iranian full TYLCV genomes) and reconstructed a plausible history of TYLCV’s diversification and movements throughout the world. In agreement with historical accounts our results suggest that the first TYLCVs probably arose somewhere in the Middle East between the 1930s and 1950s and that the global spread of TYLCV only began in the 1980s after the evolution of the TYLCV-Mld and –IL strains. Despite TYLCVs having existed outside the Middle East since this time, we found no convincing evidence anywhere other than the Middle East of epidemiologically relevant TYLCV variants arising through recombination. Although the region around Iran is both the centre of present day TYLCV diversity and the site of the most intensive ongoing TYLCV evolution, our evidence suggests the region is epidemiologically isolated and that novel TYLCV variants found there are therefore probably not direct global threats. We instead identify the region around Israel and the western Mediterranean as the main launch-pads of global TYLCV movements.

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Poster Session Geminiviridae Other

Virus Family: Geminiviridae

Category:

Title: USE of rolling circle amplification to enable rapid completion of Koch’s postulates for circular DNA viruses.

Authors: M. Lapidot1, J.E. Polston2, D. Guenoune-Gelbart1, T. Sufrin-Ringwald1, H. Capobianco2, V. Gaba3

Address: Departments of 1Vegetable Research and 3Plant Pathology, Volcani Center, ARO, P.O.Box 6, Bet Dagan 50250, Israel 2Department of Plant Pathology, University of Florida, Gainesville, FL 32611, U.S.A.

Abstract: A new system for inoculation of plants with begomoviral DNA without cloning or the use of insect vectors is described. Total DNA extracted from begomovirus-infected plants was amplified by rolling circle amplification (RCA) using the bacteriophage phi29 DNA polymerase, and inoculated to plants by particle bombardment. Infection rates of up to 100% were obtained using this technique. This technique successfully inoculated all the begomoviruses evaluated: six bipartite (Abutilon mosaic virus, Bean golden yellow mosaic virus, Cabbage leaf curl virus, Squash leaf curl virus, Tomato mottle virus, Watermelon chlorotic stunt virus) as well as one monopartite (Tomato yellow leaf curl virus). The success of the technique was not dependent upon plant species. Four species from three plant families [Phaseolus vulgaris (bean), Solanum lycopersicon (tomato), Cucurbita pepo (squash), and Citrullus lanatus (watermelon)], could all be inoculated by this technique. The success of the method was not dependent upon either the type or the age of the source of virus. Infectious DNA was successfully obtained from fresh, frozen or dried plant material, from squashes of plant leaves on FTA cards, as well as from the insect vector. Plant material collected and dried as long as 25 years ago yielded infectious DNA by this method. In summary, this method can be used to obtain infectious DNA of single-stranded circular DNA viruses that can be activated for purposes of completing Koch’s postulates, for preservation of pure virus cultures, and for many other applications where infectious DNA is required.

Virus Family: Geminiviridae

Category: Virus Diversity and Evolution

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Poster Session Geminiviridae Other

Title: Characterization of a New Virus Discovered by Vector-Enabled Metagenomics on the Whitefly Vector

Authors: J. E. POLSTON1, T. F.F. NG2, V. CAMPOVERDE1, H. CAPOBIANCO1,T. XIA1, M. BREITBART2

Address: 1 Dept of Plant Pathology, University of Florida, Gainesville, FL 32611, and 2 College of Marine Science, University of South Florida, 140 7th Avenue South, Saint Petersburg, Florida, USA

Abstract: Begomoviruses are a highly emergent genus of whitefly-transmitted plant viruses, increasing in number from a few in the 1980’s to more than 200 in 2010. In addition, many of these viruses have had their geographic range extended through agricultural trade. In order to more rapidly identify the diversity of begomoviruses in a region, a new approach was recently explored. Vector-enabled metagenomics (VEM), is the sampling of insect vectors (i.e., whiteflies), followed by purification of viral particles and metagenomic sequencing of the viral nucleic acid. Adult whiteflies which are highly mobile and feed on a wide range of hosts, can collect a number of begomoviruses over time and space, VEM takes advantage of these characteristics and leverages the capability of metagenomics for the discovery of novel viruses. The objective of this study was to establish the validity of this approach, by identifying the host plant virus which was the source of a partial novel viral sequence found using VEM. Whiteflies were collected from cucurbit plants at Citra, FL. Approximately 80 clones were obtained and sequenced using VEM. The virome was dominated by sequences of Cucurbit leaf crumple virus (CuLCrV), however two sequences of the DNA-B of Sida golden mosaic virus (SiGMV) and a third 243 nt sequence which only had 84% nucleotide identity to the DNA-A of Tobacco leaf rugose virus were also found. While CuLCrV and SiGMV have been reported from this location previously, these results suggested that a new virus was also present. PCR primers, based on the 243 nt sequence, were designed to amplify, clone, and sequence the entire DNA-A using total DNA extracted from the whiteflies. The complete sequence of the DNA-A was obtained (2,628 nt) and this whole DNA component was actually more closely related to a Mexican isolate of Desmodium leaf distortion virus (GenBank DQ875870) (81% nucleotide homology) rather than Tobacco leaf rugose virus. To confirm the existence of this virus, a collection of more than 200 samples of wild plants in the same area but two years after the whiteflies were collected. Plants were screened using broad-spectrum primers and for those plants which were positive by PCR, full length DNA-A and DNA-B clones were obtained by RCA, followed by cloning and sequencing. Sequences that were highly similar to the DNA-A found in the whitefly were found in Chenopodium ambrosiodes L. which displayed symptoms of leaf curling and distortion. Multiple DNA-A clones and DNA-B clones were obtained. The DNA-A clones were 99-100% identical with each other and 96% identical to the DNA-A sequence obtained from the whitefly. Infectivity studies were conducted and infectious clones were identified. This demonstrates that discovery of viruses through VEM is not constrained by the collection host or by time and that VEM is capable of identifying viruses from neighboring crops or weeds.

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Poster Session Geminiviridae Other

Virus Family: Geminiviridae

Category: Other (Diagnostic / mixed infections)

Title: Evidence of Begomovirus mixed infections affecting tomato (Solanum lycopersicum) plants in Colombia

Authors: J. C. VACA-VACA, J. F. BETANCUR-PEREZ, AND K. LOPEZ-LOPEZ.

Address: Facultad de Ciencias Agropecuarias, Universidad Nacional de Colombia. Palmira, Valle del Cauca, Colombia.

Abstract:

The tomato (Solanum lycopersicum) crop in Latin America has been greatly affected by viral diseases mainly caused by Begomovirus. Mixed infections with two or more Geminivirus infected the same plant are common in tropical and subtropical areas. This kind of interactions between two or more virus could result in a development a high plant complex disease. In order to verify the presence of mixed infections of Begomovirus affecting tomato crops in Colombia, we made a large national survey in the mayor tomato growing areas of the country. Tomato plants showing Begomovirus-like symptoms such as chlorosis, yellow or golden mosaic, mottling, leaf curling and distortion were collected in fields located in Cundinamarca, Santander and Valle del Cauca state. Total DNA was extracted from leaf samples. Viruses were detected by polymerase chain reaction (PCR) using universal primer from de genus Begomovirus (PAL1v1978/PAR1c496). These primers direct the amplification of a fragment of 1.2 kb DNA-A comprising the 5’-region of rep gene, entire common region (CR) and the 5’-region of cp gene. The 1.2kb PCR fragment was digested with restriction enzymes in order to detect polymorphisms (PCR-RFLP analysis). It was digested with PstI, SalI, HincII and RsaI enzymes and it was obtained three RFLP pattern. Analyzing this result we conclude that in tomato samples from Santander and Cundinamarca States there were affected by at least two Begomovirus while field samples of tomato Valle del Cauca state were affected just for one virus. Interestingly the restriction pattern of Begomovirus from Valle del Cauca coincides with one of those observed in mixed infections present in plant samples from Cundinamarca and Santander states. To our known this is the first report of mixed Begomovirus infections affecting tomato crops in Colombia.

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Poster Session Parvoviridae Epidemiology/transmission

Virus Family: Parvoviridae

Category: Epidemiology/transmission

Title: Human parvovirus 4 (PARV4): lessons from epidemiologic studies performed in south-eastern France.

Authors: M. TOUINSSI, P. DE MICCO, AND P. BIAGINI

Address: UMR CNRS 6578 Equipe « Emergence et co-évolution virale », Etablissement Français du Sang Alpes-Méditerranée et Université de la Méditerranée, 27 Bd. Jean Moulin, 13005 Marseille, France.

Abstract: Human parvovirus 4 (PARV4) is a recently discovered virus belonging to the family Parvoviridae. This virus had limited sequence homology with parvovirus B19 despite a conserved genomic organization showing two large non-overlapping ORFs. Studies dealing with PARV4 remain relatively limited up to now. First epidemiological studies have revealed that the virus was present in the blood from febrile patients, intravenous drug users and individuals positive for HCV or HIV (6% to 30%). Interestingly, PARV4 had been also identified in individuals without apparent pathology and in blood products negative for parvovirus B19 DNA as well (1% to 24%). The natural history of PARV4, its clinical role, and, very probably, its real genetic diversity remain largely unknown. We have investigated the distribution of the virus in diverse human cohorts from south-eastern France in several recent studies (Biagini et al., Emerg Infect Dis 2008; Touinssi et al., Emerg Infect Dis 2010; Touinssi et al., submitted). Epidemiological data are exposed.

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