Replication of Heterologous Combinations of Helper and Defective RNA of Citrus Tristeza Virus

Abstract

Citrus tristeza virus (CTV) populations are among the more complex of plant RNA viruses with unusual mixtures of strains and defective RNAs (dRNAs). Citrus plants infected with different CTV isolates contain multiple dRNA molecules that differ in size and relative abundance within and between isolates. Additionally, we found mixtures of heterologous dRNAs in populations. To examine the replication of CTV dRNAs, the protoplast system had to be extended to support helper-assisted amplification of input dRNAs. The use of freshly extracted sap of CTV-infected tissue as inoculum increased the infection of Nicotiana benthamiana protoplasts sufficiently to result in accumulation of high levels of CTV RNAs as well as dRNAs within 2 or 3 days postinoculation. 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 deletion 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 genomic or the subgenomic RNAs of the helper virus. To investigate the maintenance of dRNAs in CTV populations, we examined heterologous interactions between dRNAs and helper viruses. Wild-type populations of heterologous strains T68 and T3, as well as the homologous T36, supported replication of synthetic T36 dRNAs. Replacement in the T36 dRNA of the 5′ region, which is most 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. The differential specificities of different CTV replicase complexes with dRNA replication signals is one possible factor that affects the maintenance of dRNA population structures.