RNA transcripts made from a single from the clones were infectious following transfection with the vulnerable cell lines. Infection was confirmed by CPE and immuno electron microscopy. Virions had been purified from infected cells and fed to bird cherry oat aphids, Rhopalosiphum padi. Aphids tested positive for infection by the RhPV clone by RT PCR, western blot analysis and immuno localization by light microscopy, two weeks just after acquisition in three replicate experiments. The cDNA clone from the RhPV genome was inserted in to the genome of Autographa californica multiple nucleopolyhedrovirus to create the recombinant baculovirus AcRhPV6. Expression in the RhPV genome in Sf21 cells resulted in formation of RhPV virus like particles whose capsids are structurally and immunologically indistinguishable from the native virions. The presence of genomic RhPV RNA in recombinant baculovirus infected cells and in VLPs was confirmed by RT PCR.
Assembly of RhPV VLPs in the nucleus of baculovirus contaminated cells suggests that in Sf21 cells both the five and IGR IRES of RhPV are energetic, the virus encoded protease is functional selleckchem SB 525334 for processing of RhPV polyproteins, and replication of RhPV is not necessary for encapsidation of RNA. For analysis of the infectivity zafirlukast of baculovirus expressed RhPV6, virions purified from baculovirus infected Sf21 cells had been fed to R. padi. Aphids were tested for infection through the baculovirus generated RhPV clone by RT PCR and western blot evaluation, 4 weeks just after acquisition. Baculovirus expression of RhPV in lepidopteran cell lines that do not help replication of RhPV gives a probable option method for in vitro production of clones of this virus. Essential interactions of Bacillus thuringiensis toxins with membrane receptors and their part in insect resistance A.
Bravo, I. Gomez, L. Pardo, C. Muoz, C. P?rez, L. Fernandez Departamento de Microbiolog?a Molecular, Instituto de Biotecnolog?a Universidad Nacional Aut?noma de M?xico, Cuernavaca Morelos, The insecticidal proteins developed by Bacillus thuringiensis, Cry toxins, are applied to manage insect pests. The main action of Cry harmful toxins would be to lyse midgut epithelial cells by forming lytic pores within the apical membrane. Cry harmful toxins are modular proteins comprised of 3 domains, domain I is concerned in pore formation and domains II and III in receptor interactions. Here we summarize recent findings about the Cry receptor interactions and their purpose in toxin action. Cry toxins interact sequentially with many receptors. In lepidopteran insects, Cry1A harmful toxins interact initially which has a cadherin receptor. This interaction includes three diverse epitopes and promotes a last proteolytic processing on the toxin that induces the formation of a 250 kDa pre pore structure, which has been advised to become the responsible for the ionic pore formation.