Generación de plantas transgénicas de Naranjo Dulce que expresan la proteína de cubierta viral del virus de la psorosis de los cítricos (CPsV) para la inducción de resistencia mediada por proteína

MARIA CECILIA ZANEK, CARINA REYES, EDUARDO PEÑA, MARIA INÉS PLATA, NORMA COSTA, OSCAR GRAU Y MARIA LAURA GARCÍA

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

Psorosis a serious disease affecting citrus in Argentina and Uruguay. It seems to be spread by an unknown vector, causing important losses. The causal agent, Citrus psorosis virus (CPsV), type member of the genus Ophiovirus, is multipartite and its genome consists of at least three RNAs of negative polarity. RNA 1, which contains two ORFs, codes for the putative RNA polymerase (280K), and for a 24K polypeptide 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 (CP) of 48 KDa. To obtain disease-resistant or tolerant transgenic Sweet orange plants by expressing the coat protein viral gene. Internodal stem segments, cut out from young seedlings, were transformed by inoculation with Agrobacteriumtumefaciens (EHA105)carrying the CP gene of CPsV under the control of the 35S promoter of CaMV (Cauliflower Mosaic Virus). Shoot tips were selected by GUS expression, and their regeneration was performed in vitro by grafting into Troyer citrange(Citrus sinensis x Poncirustrifoliata) seedlings. The presence of the viral transgenes was tested by PCR using portions of leaf tissue from the regenerated plants. The transgenic plants were multiplied by grafting in Rough Lemon seedlings in order to be challenged with CPsV. Fifteen transgenic plants expressing 48K protein were obtained, and nine of them were evaluated by Southern blot and TAS-ELISA. These lines contain one to three integrated copies of the coat gene. The expression of the 48K protein were from 2 to 12 times higher than the non transgrenic citrus control when tested by TAS-ELISA. The level of expression of the coat protein in trangenic plants was lower than in infected citrus in greenhouse conditions. There was no correlation between the copy number of the integrated 48K gene and its protein expression. Transgenic plants expressing the 48K have been obtained and some of those lines have been also multiplied to evaluate the acquired resistance to CPsV. The challenge is now in progress.