Cellular and molecular interactions of banana bunchy top virus (BBTV) with the aphid vector pentalonia nigronervosa

Abstract

Banana bunchy top virus (BBTV), a plant DNA virus in the family Nanoviridae, is transmitted by the banana aphid, Pentalonia nigronervosa, in a circulative nonpropagative manner. Information available on the interactions between BBTV and its vector is very limited. The goal of this work is to foster a basic understanding of BBTV-aphid interaction that will allow the generation of new options for the management of the disease. I have developed an immunofluorescence assay that allowed us to specifically localize BBTV into the anterior midgut (stomach) and into specific cells forming the principal salivary glands within the aphid vector. In order to examine the translocation and retention of BBTV into the aphid vector, I used timecourse experiments and transmission assays coupled with real-time PCR and immunofluorescence assays on dissected aphid tissues. These results indicate that BBTV is internalized into the anterior midgut in which it accumulates and is retained at concentrations higher than either in the haemolymph or in the principal salivary glands. I document that the anterior midgut and principal salivary glands can have direct contact within the aphid body, suggesting a possible haemolymph-independent translocation path. Through additional experiments, I have analyzed the interaction between GroEL proteins produced by the endosymbiotic bacterium Buchnera aphidicola, harbored within the aphid, and BBTV viral particles. Interaction assays were performed using immunocapture PCR, dot-blot, and far-western blot assays. However, I failed to obtain evidence of BBTV-GroEL interaction. Furthermore, I have used transmission assays, real-time PCR and immunofluorescence to analyze the localization, accumulation, and transmission efficiency of BBTV across 4 different lineages of Pentalonia, including Pentalonia caladii, a species colonizing heliconias, ginger and taro. Immunofluorescence assays showed similar BBTV translocation pattern among the 4 Pentalonia lineages. The results obtained showed for the first time that P. caladii is a competent vector of BBTV. Finally, I used fluorescent markers to study the process of cellular internalization in guts and salivary glands of P. nigronervosa. The data obtained suggests that BBTV does not use receptor-mediated endocytosis as described for aphid-trasnmitted luteoviruses. Therefore, I discuss the possible alternative mechanisms of virus internalization.