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Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum.

ABSTRACT: A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch's postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.

SUBMITTER: Srivastava A

PROVIDER: S-EPMC5498505 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum.

Srivastava Ashish A Agrawal Lalit L Raj Rashmi R Jaidi Meraj M Raj Shri K SK Gupta Swati S Dixit Ritu R Singh Poonam C PC Tripathi Tusha T Sidhu Om P OP Singh Brahma N BN Shukla Sudhir S Chauhan Puneet S PS Kumar Susheel S

Frontiers in plant science 20170706

A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed natur ...[more]

PMID: 28729873

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Project description:INTRODUCTION:Papaver somniferum is the commercial source of morphine and codeine. The isolation of effective genes involved in the morphine biosynthesis of P. somniferum is very important in the production of specific metabolites achieved using metabolic engi-neering techniques. In this pathway, the key enzyme COR is involved in the conversion of codeinone to codeine and morphinone to morphine. METHODS:the gene encoding of this enzyme was isolated using primers designed on the base of gene sequence available on (NCBI) for P. somniferum. This gene correct size around (960 bp) was first subcloned into pTZ57RIT vector then cloned into expression vectors (pBI121) between BamHI and SacI sites to allow the expression of cor gene driven by the cauliflower mosaic virus 35S pro-moter. The result was confirmed through different molecular methods e.g. PCR and en-zyme digestion by BamHI and SacI. The recombinant plasmid was transformed into the E. coli strain DH5? using a freeze-thaw method. Having selected positive colones on selection medium, plasmid was extracted by miniprep method and recombinant plasmids were selected based on PCR and digestion. The construct was then mobilized in Agrobacterium tumefaciens C58/pGV3850 (KmR RifR). After gene transformation to P. somniferum plants, the agroinfiltration method was also used for transient expression of COR enzyme. RESULTS:evaluation results showed that morphine and codeine were detectable in the leaves of transgenic plants containing cor transgene and there was significant difference in the final production. After completing this experiment for three times, results showed that in 11 sets from 15 sets of leaves experiment tested, main alkaloids (codeine, morphine, papaverin, noscapine and thebaine) were detectable. CONCLUSION:Whereas no signal was detected in non-infiltrated control leaves or in leaves infiltrated with non-recombinant bacteria for morphine and codeine, others such as thebaine and papaverine were detectable.

Dataset Information

Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum.

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<i>Ageratum enation virus</i> Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in <i>Papaver somniferum</i>.

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