RESUMO
Pokeweed antiviral protein (PAP) is a ribosome inactivating protein (RIP) that depurinates the sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. PAP depurinates viral RNA, and in doing so, lowers the infectivity of many plant viruses. The mechanism by which PAP accesses uncapped viral RNA is not known, impeding scientists from developing effective antiviral agents for the prevention of the diseases caused by uncapped RNA viruses. Kinetic rates of PAP interacting with tobacco etch virus (TEV) RNA, in the presence and absence of eIFiso4F, were examined, addressing how the eIF affects selective PAP targeting and depurination of the uncapped viral RNA. PAP-eIFs copurification assay and fluorescence resonance energy transfer demonstrate that PAP forms a ternary complex with the eIFiso4G and eIFiso4E, directing the depurination of uncapped viral RNA. eIFiso4F selectively targets PAP to depurinate TEV RNA by increasing PAP's specificity constant for uncapped viral RNA 12-fold, when compared to the depurination of an oligonucleotide RNA that mimics the SRL of large rRNA, and cellular capped luciferase mRNA. This explains how PAP is able to lower infectivity of pokeweed viruses, while preserving its own ribosomes and cellular RNA from depurination: PAP utilizes cellular eIFiso4F in a novel strategy to target uncapped viral RNA. It may be possible to modulate and utilize these PAP-eIFs interactions for their public health benefit; by repurposing them to selectively target PAP to depurinate uncapped viral RNA, many plant and animal diseases caused by these viruses could be alleviated.
Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Potyvirus/metabolismo , RNA Viral/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Traqueófitas/virologia , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/genética , Proteínas de Plantas/genética , Potyvirus/genética , Purinas/química , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , Proteínas Inativadoras de Ribossomos Tipo 1/antagonistas & inibidores , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Curcin, a type I ribosomal inhibiting protein-RIP, encoded by curcin precursor gene, is a phytotoxin present in Jatropha (Jatropha curcas L.). Here, we report designing of RNAi construct for the curcin precursor gene and further its genetic transformation of Jatropha to reduce its transcript expression. Curcin precursor gene was first cloned from Jatropha strain DARL-2 and part of the gene sequence was cloned in sense and antisense orientation separated by an intron sequence in plant expression binary vector pRI101 AN. The construction of the RNAi vector was confirmed by double digestion and nucleotide sequencing. The vector was then mobilized into Agrobacterium tumefaciens strain GV 3101 and used for tissue culture independent in planta transformation protocol optimized for Jatropha. Germinating seeds were injured with a needle before infection with Agrobacterium and then transferred to sterilized sand medium. The seedlings were grown for 90 days and genomic DNA was isolated from leaves for transgenic confirmation based on real time PCR with NPT II specific dual labeled probe. Result of the transgenic confirmation analysis revealed presence of the gene silencing construct in ten out of 30 tested seedlings. Further, quantitative transcript expression analysis of the curcin precursor gene revealed reduction in the transcript abundance by more than 98% to undetectable level. The transgenic plants are being grown in containment for further studies on reduction in curcin protein content in Jatropha seeds.
Assuntos
Regulação da Expressão Gênica de Plantas , Inativação Gênica , Jatropha/genética , RNA Mensageiro/antagonistas & inibidores , Proteínas Inativadoras de Ribossomos Tipo 1/antagonistas & inibidores , Sementes/genética , Toxinas Biológicas/antagonistas & inibidores , Agrobacterium tumefaciens/genética , Biocombustíveis , Técnicas de Transferência de Genes , Vetores Genéticos , Jatropha/crescimento & desenvolvimento , Jatropha/toxicidade , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/biossíntese , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Sementes/crescimento & desenvolvimento , Toxinas Biológicas/biossíntese , Toxinas Biológicas/genéticaRESUMO
PD-S2, type 1 ribosome-inactivating protein from Phytolacca dioica L. seeds, is an N-ß-glycosidase likely involved in plant defence. In this work, we purified and characterized an in vivo proteolytic form of PD-S2, named cutPD-S2. Spectroscopic characterization of cutPD-S2 showed that the proteolytic cleavage between Asn195 and Arg196 does not alter the protein fold, but significantly affects its thermal stability. Most importantly, the proteolytic cleavage induces a 370-fold decrease of PD-S2 capacity of inhibiting in vitro protein biosynthesis. Our data catch the turning point from a typical role of PD-S2 as a defence protein to that of supplier of essential amino acids during seedling development.
