Búsqueda de la función de las proteínas virales 24K y 54K del virus de la psorosis de los cítricos (CPsV) mediante transformación genética de cítricos

CARINA REYES, MARIA CECILIA ZANEK, EDUARDO PEÑA, MARINA BIEDMA, VICTOR ROMANOWSKY, MARIA INÉS PLATA, NORMA COSTA, OSCAR GRAU Y MARIA LAURA GARCIA

Congreso; V Encuentro Latinoamericano y del Caribe de Biotecnología agrícola, Boca Chica, República Dominicana, REDBIO; 2004

Citrus plantations in Argentina are seriously damaged by Psorosis, a naturally spread disease, caused by Citrus psorosis virus (CPsV) which is the type member of the genus Ophiovirus. This virus is tripartite and of negative polarity. RNA 1 contains two ORFs, coding for both the RNA polymerase (280K) and a 24 KDa polypeptide (24K) of unknown function. RNA 2 contains one ORF coding for a polypeptide of 54 KDa of unknown function and RNA 3 codes for the coat protein (48K). To study the function of 24K and 54K proteins, their subcellular localization in citrus and herbaceous hosts, and how they are related to virus pathogenesis in its natural host (symptoms, cytopathic effects, etc). Citrus transgenic plants which expres 54K or 24K proteins of CPsV were obtained by internodal Pineapple sweet orange stem transformation with Agrobacterium tumefaciens. The transgenic shoot tips, which had been selected by GUS expression (54K) or by fluorescence GFP (24K) were grafted on ethyolated seedlings of Troyer citrange grown in vitro to regenerate. Specificantisera against 24K and 54K proteins were also developed. To obtain the proteins, the 24K ORF was cloned, fused to a His-tag and expressed in the heterologousBac-to-Bac system, and purified using an affinity column. The 54K gene was cloned, fused to a His-tag and expressed in E. coli (pET system). Rabbits and mice were immunized with these purified proteins. The antisera were tested by dot blots, using alkaline phosphatase as detection system. Fifteen transgenic plants were obtained, ten of them were analyzed by PCR showing positive results, and by Southern blot it was determined that they contain one to three integrated transgen copies. The specific antisera are able to detect up to 1 ng of protein by dot blot. The transgenic lines and antisera will be used as tools to study viral protein functions that could provide insights of the viral infection and spreading mechanisms which, in term, could be used to produce virus resistant citrus plant.