Molecular Characterization of Citrus tatter leaf virus Historically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts

Abstract

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3′ end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wild-type virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22°C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5′ positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction.