Generación de anticuerpos policlonales para la detección de la variante genotípica GIII de PVY, en cultivos de tomate de Árbol y papa de Colombia / Generation of policlonal antibodies for detection of the PVY genotypic variant GIII in tamarillo and potato crops from Colombia

El potyvirus PVY es uno de los agentes causales más frecuentemente asociados a problemas virales en cultivos de papa y tomate de árbol en Colombia. Dada la importancia económica de las enfermedades causadas por PVY y a la necesidad de generar material de siembra certificado por su sanidad viral, es fundamental la generación de herramientas de diagnóstico que permitan la detección temprana de este virus. En este trabajo se reporta la obtención de anticuerpos policlonales específicos, útiles para la detección del genotipo III de PVY (GIII), una de las tres variantes que recientemente han sido reportadas en cultivos de papa y tomate de árbol de la región Andina de Colombia. Como antígeno, se utilizó un péptido sintético diseñado a partir de la región variable del extremo N-terminal del gen de la cápside viral. La sensibilidad de los anticuerpos fue evaluada mediante pruebas de ELISA y dot-blot utilizando péptidos sintéticos. Se realizó una prueba piloto para validar el uso de los anticuerpos a partir de plantas sintomáticas y asintomáticas obtenidas de una región donde confluyen cultivos de ambas solanéceas, encontrándose que los anticuerpos generados ofrecen mayores niveles de detección que los anticuerpos comerciales comúnmente utilizados para detectar los serotipos PVY-O,C y PVY-N de este virus.

PVY is one of the potyvirus more frequently associated with viral infections in tomato and tamarillo crops in Colombia. Due to the economic impact of PVY and the need to certify seeds as virus-free it is important to develop diagnostic tools that allow its premature detection. In this work, the obtention of antibodies detecting the genotype III of PVY is reported. This genotype is one of the three PVY variants infecting tamarillo and potato in the Andean region of Colombia. The N-terminal variable region of the coat protein was chosen as antigen for antibody production. The sensibility of these antibodies was tested by ELISA and dot-blot using synthetic peptides. A pilot test was performed on symptomatic and non-symptomatic plants from a mixed orchard of tamarillo and potato. The generated antibodies showed higher detection levels than the commercial antibodies commonly used to detect the PVY-O,C and PVY-N serotypes.

Potato Virus Y mRNA Expression Knockdown Mediated by siRNAs in Cultured Mammalian Cell Line / 中国病毒学·英文版

RNA interference(RNAi)is a powerful tool for functional gene analysis which has been successfully used to downregulate the expression levels of target genes.The goal of this research was to provide a highly robust and concise methodology for in-vitro screening of efficient siRNAs from a bulk to be used as a tool to protect potato plants against PVY invasion.In our study,a 480bp fragment of the eapsid protein gene of potato virus Y(CP-PVY)was used as a target to downregulate PVY mRNA expression in-vitro,as the CP gene interferes with viral uncoating,translation and replication.A total of six siRNAs were designed and screened through transient transfection assay and knockdown in expression of CP-PVY mRNA was calculated in CHO-k cells.CP-PVY mRNA knockdown efficiency was analyzed by RT-PCR and real-time PCR of CHO-k cells co-transfected with a CP gene construct and siRNAs.Six biological replicates were performed in this study.In our findings,one CP gene specific siRNA out of a total of six was found to be the most effective for knockdown of CP-PVY mRNA in transfected CHO-k cells by up to 80%-90%.

Seleção de clones de batata com resistência múltipla à pinta preta e aos vírus X e Y / Selection of potato clones for multiple resistances to early blight, PVY and PVX

O objetivo do presente trabalho foi selecionar clones de batata com elevado desempenho agronômico e resistência à pinta preta e aos vírus X e Y. Para tanto, foram realizados 57 cruzamentos entre clones portadores dos alelos Ry adg e Rx1, e a cultivar 'Chiquita', resistente à pinta preta (Alternaria solani). Na safra das águas de 2004, 57 famílias clonais foram avaliadas em campo e 331 clones selecionados considerando a aparência de tubérculos. Desse total de clones, avaliados em mais dois experimentos no verão de 2005, 180 foram selecionados por meio do marcador SCAR RYSC3 como portadores do alelo Ry adg,. Também foram realizadas uma avaliação de desempenho agronômico na safra de inverno de 2006 e uma avaliação de resistência à pinta preta nas safras de verão de 2007 e 2008. Paralelamente, foi utilizado um marcador CAPS visando à seleção de clones portadores do gene Rx1. Dessa forma, combinando os resultados dos marcadores moleculares com os de campo, agrupados via índices de seleção, foi possível selecionar 20 clones de alto desempenho agronômico, resistentes à pinta preta e portadores do alelo Ry adg. Devido a problemas apresentados pelo marcador CAPS, apenas sete destes foram analisados e um identificado como portador do alelo Rx1.

The purpose of this study was to select potato clones with high agronomic performance and resistant to early blight, Potato Virus Y (PVY) and Potato Virus X (PVX). Crossings were done among progenitors carrying the Ry adg and Rx1 alleles for resistance to PVY and PVX and the cultivar 'Chiquita', which presents high levels of resistance to early blight (Alternaria solani). In the rainy season of 2004, 57 clonal families were evaluated in the field and 331 clones were selected based on tuber appearance. These clones were field evaluated in two trials in the rainy season of 2005 and 180 clones were selected for Ry adg allele with the SCAR marker designed RYSC3. Another agronomic evaluation was done in the winter season of 2006 and early blight was evaluated in the rainy seasons of 2007 and 2008. Simultaneously a CAPS marker was used to select for the presence of Rx1 allele. Combining the results from these experiments we were able to select 20 clones presenting high agronomic performance, resistance to early blight and carrying the Ry adg allele. The use of CAPS marker has practical difficulties due to production of poor amplification products to be digested with the DdeI enzyme and should be changed for another marker which shows more efficiency.