Creation of an Infectious Clone of Cotton leafroll dwarf virus (CLRDV), a fluorescent clone of CLRDV, and the transmission determinants of CLRDV by Aphis gossypii

Abstract

Cotton leafroll dwarf virus is an emerging pathogen in the United States. This disease has caused significant damage to the cotton (Gossypium hirsutum L.) production in other countries. In order to avoid these significant losses in the United States, a strong molecular understanding of the virus should be developed in order to inform disease and vector management. Two of the most important aspects to understand about viral diseases is their replication and their transmission. The first aim of this thesis is to further our understanding of CLRDV replication and systemic spread in plants by developing an infectious clone of the virus. Two infectious clones were developed, a wild-type virus which infects both cotton and Nicotiana benthamiana systemically, and a virus clone which produces Green Fluorescent Protein on a protein produced by the subgenomic RNA. This CLRDV-GFP clone was imaged by fluorescent microscopy and was detected infecting cotton plants systemically 28 days post infiltration. The second aim is to identify the transmission determinants of CLRDV. Agrobacterium tumefaciens expression vectors containing the CLRDV coat proteins P3 and P3-5 were produced with GFP, Red Fluorescent Protein, Myc and Flag tags. The major coat protein tagged with GFP (GFP-P3) was infiltrated into cotton leaves and aphids were allowed to feed on the leaf, GFP was then successfully detected in the leaf and aphid by Western blot. The results show that cotton is a suitable host for both agrobacterium expression of proteins and as a method of feeding aphids these expressed proteins. Future work is required to determine which viral protein is responsible for CLRDV transmission by the cotton-melon aphid (Aphis gossypii Glover).