Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes.

Grapefruit (Citrus paradisi Macf. cv Duncan) plants were transformed with several sequences from citrus tristeza closterovirus (CTV) that varied in terms of position in the CTV genome and virus strain origin in an attempt to obtain resistant plants. The sequences included the capsid protein gene from three different strains, a nontranslatable version of the capsid protein gene, the replicase (RdRp), the minor capsid protein (p27), a highly transcribed gene of unknown function (p20) and the more conserved 3' end of the genomic RNA. Transgenic plants were generated from all of the constructs, except from the p20 and p27 genes. Southern and Western blot analyses demonstrated that stably transformed grapefruit plants were obtained and that at least some transgenes were expressed. In a first effort at virus challenge, 25 transgenic lines were graft inoculated with a severe strain of CTV. Although some transgenic plants averaged lower titers of virus than controls, there was great variability in titer in both controls and transgenic plants, and all were apparently susceptible to the virus.

Progress on strain differentiation of Citrus tristeza virus and its application to the epidemiology of citrus tristeza disease.

Citrus tristeza virus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region.

Characterization of the 3' proximal gene of the citrus tristeza closterovirus genome.

The 3' proximal open reading frame (ORF 11) in the citrus tristeza virus (CTV) genome potentially encodes a protein of 209 amino acids with an estimated molecular weight of 23 kDa (p23). The p23 ORF from the severe Florida strain T36 of CTV was expressed in Escherichia coli, and the expressed protein was used to raise polyclonal antibodies in a rabbit. Using these antisera on a Western blot, a protein of expected size (23 kDa) was detected in tissue extracts from CTV-infected citrus but not from uninfected citrus. Most of the p23 protein was found in the soluble, cytoplasmic fraction. Comparison of the sequence of p23 genes from several biologically and geographically diverse CTV isolates indicated a high degree of conservation for this gene and for the RNA binding motif in particular. A cluster dendrogram of the deduced amino acid sequences correlated with the biological properties of the isolates, forming distinct groups of mild, quick decline on stem pitting-inducing isolates. Therefore it is possible that, in addition to the capsid protein gene, the p23 gene also may serve as an indicator for disease severity.

The diverged copy of the citrus tristeza virus coat protein is expressed in vivo.

Nucleotide sequence analysis of a portion of the citrus tristeza closterovirus (CTV) genome revealed an open reading frame immediately upstream of the coat protein gene that can encode a protein with a calculated M(r) of 27,360 (p27). The deduced amino acid sequence indicated that this putative nonstructural gene product is highly homologous to the coat protein. To investigate whether p27 was expressed in CTV-infected plants, a fusion protein of p27 produced in Escherichia coli was used to raise polyclonal antibodies. Western blot analysis using the p27 antibodies indicated that p27 is expressed in CTV-infected citrus, but not in uninfected plants. Tissue fractionation studies revealed that p27 accumulates in cell wall enriched fractions.

Nucleotide sequence and organization of eight 3' open reading frames of the citrus tristeza closterovirus genome.

The citrus tristeza closterovirus (CTV) RNA genome was cloned as cDNA generated from both CTV-specific double-stranded RNA and genomic RNA, and the sequence of the 3' 7292 nucleotides was determined. The sequenced portion contained eight open reading frames potentially encoding, in the 5' to 3' direction, proteins with the apparent molecular weights of 65, 61, 27, 25 (capsid protein, CP), 18, 13, 20, and 23 kDa, and a potential noncoding region of 277 nucleotides. The 65-kDa protein is a viral homolog of cellular hsp70 heat shock proteins (hsp), the 61-kDa protein is distantly related to the hsp90 proteins, and the 27-kDa protein is a diverged copy of the CP. Database searches did not identify any protein sequences of significant similarity to the remaining four ORFs downstream of the CP. A specific four-gene module consisting of the hsp70 protein, the hsp90-related protein, the diverged copy of the CP, and the CP itself was found to be common in organization between CTV and beet yellows closterovirus. All four proteins in this module were highly conserved, indicating that these viruses probably have evolved from a common ancestor.