Characterization of a tomato karyopherin that interacts with the Tomato Yellow Leaf Curl Virus (TYLCV) capsid protein

Abstract

cDNA from poly(A)+ RNA which has been extracted from A karyopherin a (LeKAPa1) cDNA was isolated from tomato Lycopersicon esculentum cv. MoneyMaker leaves and used as plants. The deduced LeKAPa1 protein sequence of 527 amino template for reverse transcription. The cDNA preparation was acids showed similarity to other plant karyopherin a proteins. then subcloned using Eco RI adapters into the Eco RI site of When LeKAPa1 was expressed in a yeast two-hybrid system the lgt10 phage vector (Stratagene). The recombinant l phage together with the gene coding for the capsid protein (CP) of the DNA was packaged in vitro using the Stratagene lambda packtomato yellow curl leaf virus (TYLCV), it interacted directly with aging extract Gigapack II according to the manufacturer’s CP. Thus, LeKAPa1 may be involved in the nuclear import of instructions, and used to infect the Escherichia coli C600 hfl’ TYLCV CP and, potentially, the TYLCV genomes during viral strain. The resulting cDNA library (2.0◊106 plaques) was screinfection of the host tomato cells. ened with the AtKAPa probe as described (Ausubel et al., 1994). Briefly, a 32P-labelled 2 kb fragment of AtKAPa cDNA containing the complete open reading frame was hybridized for 16 h