Vanilla viruses and coat protein mediated resistance

Abstract

Vanilla is becoming an increasingly important crop in the South Pacific. Two different vanilla potyviruses infect the two commercial vanilla species grown in the region, Vanilla fragrans, which is grown mainly in Tonga, Cook Islands, Fiji and Vanuatu, is infected by vanilla necrosis potyvirus (VNV) (Pearson and pone 1987), while Vanilla tahitensis, found mainly in French Polynesia with small amounts in the Cook Islands, is infected by vanilla mosaic potyvirus (VaMV) (Wisler et al.1987). I have shown by serological and host range analysis that VaMV and VNV are distinct viruses. Antiserum to VaMV from V. tahitensis did not react with VNV-infected V. fragrans plants, and VaMV from V. tahitensis plants did not react with a polyclonal antiserum to VNV from V. fragrans. In transmission tests (sap and/or aphid transmission), VaMV did not infect 20 different plant species, which contrasts with VNV where mechanical inoculation produced local lesions in Chenopodium amaranticolor and C. quinoa and systemic infection in N. benthamiana, N. clevelandii and V. fragrans. These results show that there are at least two distinct potyviruses of vanilla in the Pacific. The coat protein (CP) gene of VNV has been cloned and sequenced. Comparisons of this gene with other potyviral CP sequences revealed 97% nucleotide sequence homology (98% amino acid homology) to a US isolate of watermelon mosaic virus II (WMV-Il), 93% nucleotide sequence homology (96% amino acid homology) to an Australian isolate of WMV-II and 81% nucleotide sequence homology (88% amino acid homology) to soybean mosaic virus-N (SMV-N). Serological analysis, by both Western blot and ELISA, confirmed the close relationship between VNV and WMV-II. I suggest that VNV be renamed WMV-II (Tonga). In order to obtain medium to long term control the possibility of coat protein mediated-resistance was examined. Four constructs of the full-length VNV CP gene were engineered for expression in transgenic plants of Nicotiana benthamiana as a resistance model. After mechanical inoculation with a high concentration of WMV-II, one plant line containing a full-length sense CP gene (construct-18) exhibited immunity to WMV-II infection and other three constructs (construct-10R, full-length CP gene, antisense; construct-9, full-length CP gene, with kozak's ATG, sense; construct-4R, full-length CP gene, with kozak's ATG, antisense) showed a short delay in infection or were sensitive. After analysis of both the resistant and sensitive plant lines by Southern, Northern and Western blotting, we confirmed that potyvirus CP gene can be very effective in protecting against VNV infection and that construct-18 may be useful for introduction into Vanilla fragrans plants in the future. A hypothesis of transgenic viral genes and the effect of the insert position was proposed to account for these observations.