A Small RNA Resembling the Beet Western Yellows Luteovirus ST9-Associated RNA Is a Component of the California Carrot Motley Dwarf Complex

Abstract

Virions were purified from Anthriscus cerefolium or Coriandrum sativum plants infected with the viruses that cause California carrot motley dwarf. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virion preparations yielded a single prominent protein species of approximately 28,000 molecular weight; however, denaturing agarose gel electrophoresis showed that virions contained three prominent single-stranded RNAs of approximately 5.6, 4.2, and 2.8 kb. Northern hybridization analyses, using transcripts generated from cloned cDNAs that corresponded to each of the virion RNAs, showed that the 5.6- and 4.2-kb RNAs were the genomic RNAs of the carrot red leaf luteovirus (CRLV) and the carrot mottle umbravirus (CMoV), respectively. Virions also contained an approximately 1.3-kb RNA related to the CMoV genomic RNA. The 2.8-kb RNA did not hybridize with CRLV or CMoV cRNA probes. Analysis of naturally infected carrot (Daucus carota) plants showed that CRLV, CMoV, and the 2.8-kb RNA were always present in carrot motley dwarf—affected plants. Greenhouse aphid- and mechanical-transmission experiments showed that the 2.8-kb RNA was consistently present in plants also infected by both CRLV and CMoV, but never in plants infected by only CMoV. Near full-length cloned cDNAs corresponding to the 2.8-kb RNA were prepared, and the complete nucleotide sequence was determined to be 2,835 nucleotides. Two large open reading frames (ORFs), 1a and 1b, were present within the sequence and were separated by an amber (UAG) stop codon. A third ORF (ORF 2), capable of encoding a protein of 4,289 molecular weight, was located near the 3′ terminus. BLASTP results showed that the 2.8-kb RNA was most closely related to the beet western yellows luteovirus (BWYV) ST9-associated RNA. Based on its biological and molecular characteristics, we have named the 2.8-kb RNA the CRLV-associated RNA (CRLVaRNA).