Papaya resistant to papaya ringspot virus

Papaya lines 63-1 was developed using recombinant DNA techniques to resist infection by papaya ringspot virus (PRSV), a major limiting factor in papaya production.

This papaya line was developed by inserting virus-derived sequences that encode the PRSV coat protein (CP). The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology.

PRSV belongs to the potyvirus group and is an aphid-transmissible RNA virus that commonly infects papaya, causing serious disease and economic loss.

Cultivar: Sunset

pGA482GG/cpPRV-4

The Agrobacterium tumefaciens binary plasmid pGA482GG/cpPRSV-4 used for the transformation contained three plant-expressible genes, the PRSV CP, neo, and uidA genes. The plasmid also had two genes encoding resistance to tetracycline and gentamycin antibiotics, respectively, but their associated DNA regulatory sequences enabled expression only in bacteria. The plasmid included the right- and left-border regions derived from the A. tumefaciens T-DNA.

Expression of the PRSV CP gene was controlled by including promoter and transcription termination and polyadenylation signal sequences derived from the 35S transcript of cauliflower mosaic virus (CaMV).

In addition, the CP gene sequences were fused to the 5' untranslated sequence and the first 39 nucleotides from the cucumber mosaic virus (CMV) CP to enhance translation of the transgene mRNA. The inclusion of these additional sequences was necessary because PRSV naturally encodes its CP as part of a polyprotein and, therefore, the CP coding region normally lacks a translation initiation ATG codon.

Expression of the neo gene was under control of the promoter and terminator sequences from the nopaline synthase (nos) gene of A. tumefaciens. The second marker gene, uidA, was modified for plant expression by the addition of 35S promoter region from CaMV and the nos 3'-termination region.

Southern blot analyses indicated that line 63-1 contained intact, functional genes encoding the PRSV CP and NPTII, and did not contain the GUS encoding gene. Genomic hybridization with probes to the gentamycin resistance gene and the Ori T/Tet region indicated that either all or part of the genes for gentamycin and tetracycline resistance had integrated into the papaya genome. However, these genes were not functional since their bacterial promoters cannot drive expression of these genes in plants.