Replication of heterologous combinations of helper and defective RNA of citrus tristeza virus

Citrus tristeza virus (CTV) populations are among the more complex of plant RNA viruses with unusual mixtures of strains and defective RNAs (dRNAs). C itrus plants infected with different CN isolates contain multiple dRNA mole cules that differ in size and relative abundance within and between isolate s. Additionally, we found mixtures of heterologous dRNAs in populations. To examine the replication of CTV dRNAs, the protoplast system had to be exte nded to support helper-assisted amplification of input dRNAs. The use of fr eshly extracted sap of ON-infected tissue as inoculum increased the infecti on of Nicotiana benthamiana protoplasts sufficiently to result in accumulat ion of high levels of CN RNAs as well as dRNAs within 2 or 3 days postinocu lation. A series of dRNA-like molecules, each with a single large internal deletion, were created from an infectious cDNA clone of the CTV T36 isolate and examined for amplification in N. benthamiana protoplasts using a CTV d eletion mutant as the helper virus. Of 12 synthetic dRNAs, only three with sizes of 3650, 3819, and 4460 nucleotides were efficiently replicated. CTV dRNA replication did not appreciably affect levels of accumulation of the g enomic or the subgenomic RNAs of the helper virus. To investigate the maint enance of dRNAs in CTV populations, we examined heterologous interactions b etween dRNAs and helper viruses. Wild-type populations of heterologous stra ins T68 and T3, as well as the homologous T36, supported replication of syn thetic T36 dRNAs. Replacement in the T36 dRNA of the 5' region, which is mo st variable among CTV strains, with the corresponding sequences from VT,T68 , T3, or T30 resulted in chimeric dRNAs that failed to be replicated by the T36 helpers but were replicated to detectable levels by the T68 helper. Th e differential specificities of different CTV replicase complexes with dRNA replication signals is one possible factor that affects the maintenance of dRNA population structures. (C) 2000 Academic Press.