In VitroTranscripts from Cloned cDNAs of the Lettuce Infectious Yellows Closterovirus Bipartite Genomic RNAs Are Competent for Replication inNicotiana benthamianaProtoplasts

AbstractFull-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 benthamianaprotoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derivedin vitrotranscripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG)in vitrotranscripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only cappedin vitrotranscripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived cappedin vitrotranscripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectiousin vitrotranscripts can be derived from the cloned cDNAs of a closterovirus genome