Gene cloning and genome structure of geminivirus causing yellow leaf curl disease of angled luffa

Geminivirus was shown to be associated with leaf curl, yellows, or yellow leaf curl symptoms of angled luffa and some cucurbitaceous plants including sponge gourd, wax gourd, pumpkin, cucumber, calabash gourd and ivory gourd. Dot blot hybridization tests using nucleic acid probe derived from the component A of Thailand-tomato yellow leaf curl geminivirus exhibited positive hybridization reaction with the total DNA extract of infected plant samples. Southern blot analysis revealed unique 2.7 kilobase DNA bands from all sample tested. The virus from angled luffa was mechanical transmitted to luffa and tobacco (Nicotiana benthamiana). Virus purification from either inoculated luffa or tobacco plant yielded 14 ng of virus from 50 g of infected tissues with A260/A280 ratio of 1.57. The coat protein contained single polypeptide with the molecular weight of about 33 kilodaltons as determined by SDS polyacrylamide gel electrophoresis. This protein reacted with geminivirus antiserum in Western blot analysis. Polymerase chain reaction (PCR) was employed to amplify geminivirus DNA frangments from infected plant extract and DNA products were cloned. Nucleotide sequencing and genome analysis indicated that the total of 2,746 nucleotides of viral DNA was similar to the organized structure of the A component of subgroup III geminivirus. The viral genome contained 274 nucleotides of intergenic region (IR) with the hairpin structure. There were six open reading frames (ORFs) encoding for the important proteins V1, V2, C1, C2, C3 and C4, with molecular weights of 29,640, 13,316, 40,865, 16,099, 16,043 and 17,960 daltons, respectively. Nucleotide and amino acid sequences were used to determine the genetic relationship between angled luffa and other selected subgroup III geminiviruses. Phylogenetic analysis using IR sequence, nucleotide and amino acid sequences of the V1 and C1 ORFs indicated that the angled luffa geminivirus is closely related to tomato leaf curl virus from India (TLCV-In) while it was farther related from TYLCV-Th, mungbean yellow mosaic virus (MYMV-Th) and pepper leaf curl virus (PLCV-Th). Results obtained from this study indicated that the virus associated with angled luffa yellow leaf curl disease belongs to subgroup III geminivirus. The virus is named angled luffa leaf curl virus (AILCV), and its genomic sequence was deposited in the GenBank database with accession no. AF061339.