Cucumber mosaic virus and its 2b RNA silencing suppressor modify plant-aphid interactions in tobacco

The cucumber mosaic virus (CMV) 2b protein not only inhibits anti-viral RNA silencing but also quenches transcriptional responses of plant genes to jasmonic acid, a key signalling molecule in defence against insects. This suggested that it might affect interactions between infected plants and aphids, insects that transmit CMV. We found that infection of tobacco with a 2b gene deletion mutant (CMVD2b) induced strong resistance to aphids (Myzus persicae) while CMV infection fostered aphid survival. Using electrical penetration graph methodology we found that higher proportions of aphids showed sustained phloem ingestion on CMV-infected plants than on CMVD2b-infected or mock-inoculated plants although this did not increase the rate of growth of individual aphids. This indicates that while CMV infection or certain viral gene products might elicit aphid resistance, the 2b protein normally counteracts this during a wild-type CMV infection. Our findings suggest that the 2b protein could indirectly affect aphid-mediated virus transmission

Immunological analysis of chloroplast-derived HIV-1 antigens

The aim of this project was to assess the expression, purification and immunogenicity in mice of two important HIV antigens, the core protein p24 and the regulatory protein Nef, produced in transplastomic Nicotiana tabacum plants. A comparison of p24-expressing tobacco Petite Havana transplastomic plants, containing gene constructs with different 5’ untranslated regions (UTRs), demonstrated the importance of regulatory and stabilizing elements. Plants containing a p24 construct with a bacteriophage T7 gene 10 (T7g10) 5’ UTR and generating a protein with 5 extra amino acids at the N-terminus, accumulated almost twice as much protein as plants containing other constructs. Analysis of p24 accumulation in leaves of the high biomass tobacco variety Maryland Mammoth revealed a dramatic difference between a transplastomic line containing a codon-optimized construct encoding five extra N-terminal amino acids, which accumulated p24 in all leaves of the plant, and a transplastomic line containing a native p24 construct which accumulated p24 only in young leaves. To assess the subcutaneous and nasal immunogenicity of plant-derived p24, the protein was purified to ~95% homogeneity from Maryland Mammoth transplastomic plants. After immunization of experimental mice, ELISAs for p24-specific IgG and T-cell proliferation assays showed that chloroplast p24 could elicit a strong humoral response following subcutaneous administration and a cellular response following intranasal administration. Oral administration of partially purified tobacco p24-Nef as a booster, after subcutaneous priming with either p24 or Nef, elicited strong humoral and cellular responses, with both IgG1 and IgG2 subtypes found in sera. In addition, tobacco BiP was found to be as good an adjuvant as Vibrio cholerae toxin B and Mycobacterium tuberculosis Hsp70 for enhancing the immune response to p24.EThOS - Electronic Theses Online ServiceGBUnited Kingdo