In vivo subcellular localization of Mal de Río Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

The in vivo subcellular localization of Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and α-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.Instituto de BiotecnologíaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Development of nanobodies against Mal de Río Cuarto virus major viroplasm protein P9‑1 for diagnostic sandwich ELISA and immunodetection

Mal de Río Cuarto virus (MRCV) is a member of the genus Fijivirus of the family Reoviridae that causes a devastating disease in maize and is persistently and propagatively transmitted by planthopper vectors. Virus replication and assembly occur within viroplasms formed by viral and host proteins. This work describes the isolation and characterization of llama-derived Nanobodies (Nbs) recognizing the major viral viroplasm component, P9-1. Specific Nbs were selected against recombinant P9-1, with affinities in the nanomolar range as measured by surface plasmon resonance. Three selected Nbs were fused to alkaline phosphatase and eGFP to develop a sandwich ELISA test which showed a high diagnostic sensitivity (99.12%, 95% CI 95.21–99.98) and specificity (100%, 95% CI 96.31–100) and a detection limit of 0.236 ng/ml. Interestingly, these Nanobodies recognized different P9-1 conformations and were successfully employed to detect P9-1 in pull-down assays of infected maize extracts. Finally, we demonstrated that fusions of the Nbs to eGFP and RFP allowed the immunodetection of virus present in phloem cells of leaf thin sections. The Nbs developed in this work will aid the study of MRCV epidemiology, assist maize breeding programs, and be valuable tools to boost fundamental research on viroplasm structure and maturation.Instituto de BiotecnologíaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Monti, Demian Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Monti, Demian Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Aduriz Guerrero, Matí­as. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romão, Ema. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Dumon, Analia Delina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Dumon, Analia Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wigdorovitz, Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Wigdorovitz, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Muyldermans, Serge. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Muyldermans, Serge. Dalian University of Technology. School of Bioengineering. Liaoning Key Laboratory of Molecular Recognition and Imaging; ChinaFil: Vincke, Cécile. Vrije Universiteit Brussel. Lab of Cellular and Molecular Immunology; BélgicaFil: Vincke, Cécile. VIB Center for Inflammation Research. Myeloid Cell Immunology Lab; BélgicaFil: Parreño, Viviana. Instituto Nacional de Tecnología Agropecuaria (INTA). INCUINTA. Instituto de Virología e Innovaciones Tecnológicas; ArgentinaFil: Parreño, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Study on the plant-pathogen interaction between plants and Mal de Río Cuarto virus (MRCV)

El virus del Mal de Río Cuarto (MRCV, Fijivirus, Reoviridae) causa la principal enfermedad delmaíz en nuestro país provocando grandes pérdidas económicas. El virus es transmitido porchicharritas de la familia Delphacidae e infecta además diversas gramíneas como trigo, cebada,avena y sorgo, dando lugar a síntomas severos. El genoma del MRCV está formado por diezsegmentos de ARN doble cadena (ARNdc) que codifican para trece proteínas. Durante el ciclode infección, el MRCV replica en cuerpos de inclusión citoplasmáticos virales denominadosviroplasmas, y nuestro grupo demostró que están compuestos principalmente por la proteínano estructural P9-1 (Maroniche, 2011). Para otros reovirus se ha demostrado que losviroplasmas contienen además ARN viral y proteínas virales minoritarias junto concomponentes celulares del hospedante. Con el fin de avanzar en la comprensión de las bases moleculares de la enfermedad en primerlugar se analizaron las interacciones de las proteínas del MRCV entre sí utilizando la técnica dedoble híbrido de levaduras (Y2H). Se identificaron cuatro interacciones positivas: P6 con P6 ycon P9-1, P9-1 con P9-1, y P9-2 con P9-2. Se definieron además las regiones de P6 y de P9-1involucradas en las interacciones utilizando mutantes de deleción y se encontró que la regióncentral de P6 con un posible dominio “coiled-coil” es necesaria para la interacción consigomisma y con P9-1, mientras que los últimos 24 residuos de P9-1 intervienen en la formación dedímeros de P9-1. Estos resultados, junto con resultados previos del grupo (Maroniche, 2011),permitieron postular a P6 como componente minoritario del viroplasma e indicaron que laregión C-terminal de P9-1 sería necesaria para la formación del viroplasmas. Adicionalmente se evaluó la interacción de las proteínas virales P6, P7-2, P9-1, P9-2 y P10 conproteínas celulares que son candidatas a cumplir roles relevantes en la interacción MRCVhospedante. Ente otros resultados, se encontró que P7-2 interactúa con la proteína SKP1 (SPhase Kinase Associated Protein 1) componente del complejo E3 ligasa del sistema ubiquitinaproteasoma (UPS). Estos resultados son de gran relevancia ya que sugieren que P7-2 tiene lacapacidad de interferir con el UPS. Para continuar con la caracterización de las proteínas virales P9-1 y P6 e identificar posiblescomponentes celulares asociados con los viroplasmas, se realizaron relevamientos de una biblioteca de ADN copia (ADNc) de hoja de trigo por Y2H. Se encontró que P9-1 es capaz deinteractuar con una proteína con posible función de aldosa 1-epimerasa y otra con posiblefunción de aciltransferasa. Por su parte, P6 mostró interacción con una posible tiorredoxina. Dado que las proteínas identificadas intervienen en el metabolismo de la planta, se postulaque su interacción con proteínas virales podría estar asociada con la generación de síntomas. Finalmente se determinó que tanto P6 como P9-1 contienen potenciales motivos PEST dedegradación vía proteasoma. Se construyeron mutantes sin estos motivos y se observó unincremento en los niveles de acumulación de P6 y P9-1 mutadas en plantas, sugiriendo que los PEST serían funcionales. Si bien aún resta comprender los mecanismos precisos que subyacen a las interaccionesproteína-proteína encontradas en este trabajo de Tesis y sus consecuencias biológicas, estosresultados significan un importante avance en el conocimiento de la función y rol de lasproteínas codificadas por el MRCV, en especial de P6 y P9-1.Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) causes the most important maize diseasein Argentina leading to great economic losses. The virus is transmitted by planthoppers fromthe Delphacidae family and also infects other grasses like wheat, rye, oat and sorghum, causingsevere symptoms. The MRCV genome is composed of ten double stranded RNA (dsRNA)segments that code for thirteen proteins. During its infection cycle, MRCV replicates incytoplasmic inclusion bodies called viroplasms, and our group has found that are mainlycomposed by the viral non-structural protein P9-1 (Maroniche, 2011). In other reovirus, theviroplasms also contain viral RNA and minor viral proteins as well as cellular components of thehost. To gain insight into the molecular basis of the disease, MRCV protein-protein interactions wereanalyzed using the yeast two-hybrid (Y2H) technique. Four positive interactions were found: P6with itself and with P9-1, P9-1 with P9-1, and P9-2 with P9-2. The regions of P6 and P9-1involved in such interactions were also identified by using deletion mutants. Through theseanalyses, it was established that the central region of P6 containing a possible coiled-coildomain is necessary for P6/P6 and P6/P9-1 interactions, whereas the last 24 residues of P9-1are involved in P9-1 dimmer formation. These results, together with previous results of ourgroup (Maroniche, 2011), allowed us to postulate P6 as a minor viroplasm component andthey also suggest that P9-1 C-terminal region is necessary for viroplasm assembly. Additionally, the interactions of viral proteins P6, P7-2, P9-1, P9-2 and P10 with cellularproteins that are proposed to have important roles in MRCV-host interaction were analyzed. Among other results, it was found that P7-2 interacts with SKP1 (S-Phase Kinase Associated Protein 1), component of E3 ligase complex of the ubiquitin-proteasome system (UPS). Theseresults are relevant because they suggest that P7-2 is able to interfere with the UPS. To further characterize P6 and P9-1 and to identify possible cellular components of theviroplasms, Y2H screenings of a wheat copy DNA (cDNA) library where carried out. Theseassays showed that P9-1 interacts with a putative aldose 1-epimerase and with a putativeacyltransferase. In addition, P6 interacts with a protein with a predicted thiorredoxin activity. Given that the identified proteins are involved in the plant metabolism, their interaction withviral proteins might be associated with symptom development. Finally, it was identified that P6 and P9-1 contain potential PEST motives for degradationthrough the UPS. P6 and P9-1 mutants without these motives showed increased proteinaccumulation in plants, suggesting that their PEST motives are functional. Although the mechanisms underlying the protein-protein interactions found in this Thesis, aswell as their biological consequences remain unknown, this study broadens the currentknowledge of the function and role of MRCV encoded proteins, in particular those of P6 and P9-1.Fil: Llauger, Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Interaction of Mal de Río Cuarto virus (Fijivirus genus) proteins and identification of putative factors determining viroplasm formation and decay

Mal de Río Cuarto virus (MRCV) is a member of the Fijivirus genus, within the Reoviridae family, that replicates and assembles in cytoplasmic inclusion bodies called viroplasms. In this study, we investigated interactions between ten MRCV proteins by yeast two-hybrid (Y2H) assays and identified interactions of non-structural proteins P6/P6, P9-2/P9-2 and P6/P9-1. P9-1 and P6 are the major and minor components of the viroplasms respectively, whereas P9-2 is an N-glycosylated membrane protein of unknown function. Interactions involving P6 and P9-1 were confirmed by bimolecular fluorescence complementation (BiFC) in rice protoplasts. We demonstrated that a region including a predicted coiled-coil domain within the C-terminal moiety of P6 was necessary for P6/P6 and P6/P9-1 interactions. In turn, a short C-terminal arm was necessary for the previously reported P9-1 self-interaction. Transient expression of these proteins by agroinfiltration of Nicotiana benthamiana leaves showed very low accumulation levels and further in silico analyses allowed us to identify conserved PEST degradation sequences [rich in proline (P), glutamic acid (E), serine (S), and threonine (T)] within P6 and P9-1. The removal of these PEST sequences resulted in a significant increase of the accumulation of both proteins.Fil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: de Haro, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Alfonso, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentin

The autographa californica multiple nucleopolyhedrovirus Ac12: A non-essential F box-like protein that interacts with cellular SKP1 component of the E3 ubiquitin ligase complex

The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac12 gene, which is conserved in ten other baculovirus, codes a predicted 217 amino acid protein of unknown function. In this study, we investigated the role of ac12 during baculovirus infection, by generating an ac12 knockout virus. The transfection of the recombinant genome in insect cells resulted in unaltered viral dispersion and occlusion body production when compared to the control bacmid. This finding demonstrates that ac12 is a non-essential gene. Transmission and scanning electron microscopy (SEM) analyses showed that ac12 knockout virus produced occlusion bodies morphologically similar to those obtained with the control and capable to occlude virions. However, a slight but significant size difference was detected by SEM observation of purified occlusion bodies. This difference suggests that ac12 may be involved in regulatory pathways of polyhedrin production or occlusion body assembly without affecting either viral occlusion or oral infectivity in Rachiplusia nu larvae. This was evidenced by bioassays that showed no significant differences in the conditions tested. A qPCR analysis of viral gene expression during infection evidenced regulatory effects of ac12 over some representative genes of different stages of the viral cycle. In this study, we also showed that ac12 is transcribed at early times after infection and remains detectable up to 72 hours post-infection. The mRNA is translated during the infection and results in a protein that encodes an F-box domain that interacts in vivo and in vitro with S phase kinase associated protein 1 (SKP1) adaptor protein, which is potentially involved in protein ubiquitination pathways.Instituto de BiotecnologíaFil: Costa Navarro, Guadalupe Soledad. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Amalfi, Sabrina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Lopez, Maria Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Arneodo Larochette, Joel Demian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Development of a novel set of Gateway-compatible vectors for live imaging in insect cells

Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high-throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway-compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C-terminal or the N-terminal ends. Additionally, a collection of organelle-specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self-interaction of the MRCV viroplasm matrix protein P9-1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high-throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins.Instituto de BiotecnologíaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Alfonso, Victoria. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Functional and biochemical properties of Mal de Río Cuarto virus (Fijivirus, Reoviridae) P9-1 viroplasm protein show further similarities to animal reovirus counterparts

Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.Instituto de BiotecnologíaFil: Maroniche, Guillermo Andrés. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Mongelli, Vanesa Claudia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Peralta, Andrea Veronica. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Distefano, Ana Julia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Distefano, Ana Julia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Llauger, Gabriela. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Taboga, Oscar Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Hopp, Horacio Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Complete genome sequence of maize yellow striate virus, a new cytorhabdovirus infecting maize and wheat crops in Argentina

A rhabdovirus infecting maize and wheat cropsin Argentina was molecularly characterized. Through nextgeneration sequencing (NGS) of symptomatic leaf samples,the complete genome was obtained of two isolates of maizeyellow striate virus (MYSV), a putative new rhabdovirus,differing by only 0.4% at the nucleotide level. The MYSVgenome consists of 12,654 nucleotides for maize and wheatvirus isolates, and shares 71% nucleotide sequence identitywith the complete genome of barley yellow striate mosaicvirus (BYSMV, NC028244). Ten open reading frames(ORFs) were predicted in the MYSV genome from theantigenomic strand and were compared with their BYSMVcounterparts. The highest amino acid sequence identity ofthe MYSV and BYSMV proteins was 80% between the Lproteins, and the lowest was 37% between the proteins 4.Phylogenetic analysis suggested that the MYSV isolatesare new members of the genus Cytorhabdovirus, familyRhabdoviridae. Yellow striate, affecting maize and wheatcrops in Argentina, is an emergent disease that presents apotential economic risk for these widely distributed crops.Fil: Maurino, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dumón, Analía Delina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Llauger, Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Alemandri, Vanina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: de Haro, Luis Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Mattio, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: del Vas, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Laguna, Irma Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; ArgentinaFil: Giménez, María de la Paz. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Patología Vegetal; Argentin

Complete genome sequence of maize yellow striate virus, a new cytorhabdovirus infecting maize and wheat crops in Argentina

A rhabdovirus infecting maize and wheat crops in Argentina was molecularly characterized. Through next-generation sequencing (NGS) of symptomatic leaf samples, the complete genome was obtained of two isolates of maize yellow striate virus (MYSV), a putative new rhabdovirus, differing by only 0.4% at the nucleotide level. The MYSV genome consists of 12,654 nucleotides for maize and wheat virus isolates, and shares 71% nucleotide sequence identity with the complete genome of barley yellow striate mosaic virus (BYSMV, NC028244). Ten open reading frames (ORFs) were predicted in the MYSV genome from the antigenomic strand and were compared with their BYSMV counterparts. The highest amino acid sequence identity of the MYSV and BYSMV proteins was 80% between the L proteins, and the lowest was 37% between the proteins 4. Phylogenetic analysis suggested that the MYSV isolates are new members of the genus Cytorhabdovirus, family Rhabdoviridae. Yellow striate, affecting maize and wheat crops in Argentina, is an emergent disease that presents a potential economic risk for these widely distributed crops.Instituto de BiotecnologíaFil: Maurino, María Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dumon, Analia Delina Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Llauger, Gabriela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Alemandri, Vanina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: De Haro, Luis Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mattio, Maria Fernanda. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; ArgentinaFil: Del Vas, Mariana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Laguna, Irma Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gimenez Pecci, María de la Paz. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentin

Mal de Río Cuarto virus infection causes hormone imbalance and sugar accumulation in wheat leaves

Abstract Background Mal de Río Cuarto virus (MRCV) infects several monocotyledonous species including maize and wheat. Infected plants show shortened internodes, partial sterility, increased tillering and reduced root length. To better understand the molecular basis of the plant-virus interactions leading to these symptoms, we combined RNA sequencing with metabolite and hormone measurements. Results More than 3000 differentially accumulated transcripts (DATs) were detected in MRCV-infected wheat plants at 21 days post inoculation compared to mock-inoculated plants. Infected plants exhibited decreased levels of TaSWEET13 transcripts, which are involved in sucrose phloem loading. Soluble sugars, starch, trehalose 6-phosphate (Tre6P), and organic and amino acids were all higher in MRCV-infected plants. In addition, several transcripts related to plant hormone metabolism, transport and signalling were increased upon MRCV infection. Transcripts coding for GA20ox, D14, MAX2 and SMAX1-like proteins involved in gibberellin biosynthesis and strigolactone signalling, were reduced. Transcripts involved in jasmonic acid, ethylene and brassinosteroid biosynthesis, perception and signalling and in auxin transport were also altered. Hormone measurements showed that jasmonic acid, brassinosteroids, abscisic acid and indole-3-acetic acid were significantly higher in infected leaves. Conclusions Our results indicate that MRCV causes a profound hormonal imbalance that, together with alterations in sugar partitioning, could account for the symptoms observed in MRCV-infected plants