IN-VITRO TRANSCRIPTS FROM CLONED CDNAS OF THE LETTUCE INFECTIOUS YELLOWS CLOSTEROVIRUS BIPARTITE GENOMIC RNAS ARE COMPETENT FOR REPLICATIONIN NICOTIANA-BENTHAMIANA PROTOPLASTS

Full-length cloned cDNAs of lettuce infectious yellows closterovirus ( LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthami ana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-de rived in vitro transcripts. Analysis of protoplasts inoculated with LI YV Virion RNAs or capped (m(7)GpppG) in vitro transcripts from LIYV RN A 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subge nomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgSRNA, indicating that LIYV RNA 1 can replicate i n the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 wa s not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-d erived capped in vitro transcripts. These data demonstrate that LIYV g enomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned c DNAs of a closterovirus genome. (C) 1996 Academic Press, Inc.