Silencing of quinolinic acid phosphoribosyl transferase (QPT) gene for enhanced production of scopolamine in hairy root culture of Duboisia leichhardtii.

Scopolamine is a pharmaceutically important tropane alkaloid which is used therapeutically in the form of an anesthetic and antispasmodic drug. The present study demonstrates enhanced scopolamine production from transgenic hairy root clones of Duboisia leichhardtii wherein the expression of quinolinate phosphoribosyl transferase (QPT) gene was silenced using the QPT-RNAi construct under the control of CaMV 35 S promoter. The RNAi hairy roots clones viz. P4, P7, P8, and P12 showed the enhanced synthesis of scopolamine with significant inhibition of nicotine biosynthesis. Optimization of culture duration in combination with methyl jasmonate elicitor in different concentrations (50 µM-200 µM) was carried out. Maximum synthesis of scopolamine had obtained from HR clones P7 (8.84 ± 0.117 mg/gm) on the 30th day of cultivation. Conspicuously, elicitation with wound-associated hormone methyl jasmonate enhanced the yield of scopolamine 2.2 fold (19.344 ± 0.275 mg/gm) compared to the culture lacking the elicitor. The transgenic hairy roots cultures established with RNAi mediated silencing of quinolinate phosphoribosyl transferase gene provides an alternative approach to increase the yield of scopolamine in fulfilling the demand of this secondary metabolite.

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

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.

Molecular Characterization of a Begomovirus, α-Satellite, and ß-Satellite Associated with Leaf Curl Disease of Parthenium hysterophorus in India.

Parthenium hysterophorus plants exhibiting severe leaf curl and stunting symptoms were observed near agriculture fields in Lucknow, India. The association of a begomovirus, ß-satellite, and α-satellite with these symptoms of a Parthenium disease was investigated by sequence analyses of virus and satellite DNA amplified by rolling circle amplification and polymerase chain reaction. The highest sequence identities and closest phylogenetic relationships for the begomovirus, ß-satellite, and α-satellite detected in P. hysterophorus plants were to Tomato leaf curl virus (ToLCV), papaya leaf curl ß-satellite (PaLCuB), and Ageratum yellow vein India α-satellite (AYVIA), respectively. These findings identified the virus and satellites infecting the Parthenium sp. as ToLCV, PaLCuB, and AYVIA, respectively. P. hysterophorus and tomato seedlings infected with cloned ToLCV, PaLCuB, and AYVIA by agroinoculation developed leaf curl symptoms, whereas plants infected with ToLCV alone or with ToLCV and AYVIA developed mild yellowing. The results show that this complex infects and causes disease in P. hysterophorus and tomato. P. hysterophorus is an invasive weed commonly found around agricultural fields and along roadsides in India. These results indicate that P. hysterophorus plants infected with ToLCV and associated satellite DNA act as an alternate host (reservoir), and that could lead to increased incidence of tomato leaf curl disease.

Molecular characterization of a new begomovirus associated with leaf yellow mosaic disease of Jatropha curcas in India.

During a survey in June 2011, severe leaf yellow mosaic disease was observed on about 45 % plants of Jatropha curcas growing in the Katerniaghat wildlife sanctuary in India. An association of a begomovirus with disease was detected in 15 out of 20 samples by PCR using begomovirus genus-specific primers and total DNA isolated from symptomatic leaf samples. For identification of the begomovirus, the complete genome was amplified using a Phi-29 DNA-polymerase-based rolling-circle amplification kit and total DNA from five representative samples and then digested with BamHI. The linearized RCA products were cloned and sequenced. Their GenBank accession numbers are JN698954 (SKRK1) and JN135236 (SKRK2). The sequences of the two begomovirus isolates were 97 % identical to each other and no more than 86 % to those of jatropha mosaic India virus (JMIV, HM230683) and other begomoviruses reported worldwide. In phylogenetic analysis, SKRK1 and SKRK2 clustered together and showed distant relationships to jatropha mosaic India virus, Jatropha curcas mosaic virus, Indian cassava mosaic virus, Sri Lankan cassava mosaic virus and other begomoviruses. Based on 86 % sequence identities and distant phylogenetic relationships to JMIV and other begomoviruses and the begomovirus species demarcation criteria of the ICTV (<89 % sequence identity of complete DNA-A genome), the begomovirus isolates associated with leaf yellow mosaic disease of J. curcas were identified as members of a new begomovirus species and provisionally designated as jatropha leaf yellow mosaic Katerniaghat virus (JLYMKV). Agroinfectious clones of the DNA molecule of the begomovirus isolate were also generated, and the fulfillment of Koch's postulates was demonstrated in J. curcas plants.

Characterization of a novel begomovirus associated with yellow mosaic disease of three ornamental species of Jatropha grown in India.

Severe yellow mosaic disease was observed in three ornamental species of Jatropha: J. integerrima, J. podagrica and J. multifida grown in gardens at Lucknow, India, during a survey in 2013. The causal pathogen was successfully transmitted from diseased to healthy plants of these species by whitefly (Bemisia tabaci). The infection of begomovirus was initially detected in naturally infected plant samples by PCR using begomovirus universal primers. The begomovirus was characterized having a monopartite genome based on sequence analyses of the cloned ∼2.9kb DNA-A genome amplified by rolling circle amplification using Phi-29 DNA polymerase. The genome contained 2844 nucleotides that could be translated into seven potential open reading frames. The nucleotide sequences of DNA-A genome of the begomovirus isolates: JI (KC513823), JP (KF652078) and JM (KF652077) shared 94-95% identities together and 93-95% identities with an uncharacterized begomovirus isolated from J. curcas (the only sequences available in GenBank database as GU451249 and EU798996 under the name jatropha leaf curl virus). These shared highest identity of 61% and highly distant phylogenetic relationships with other begomoviruses reported worldwide. Based on 61% sequence identities (much less than 89%, the species demarcation criteria for a new begomovirus) the isolates under study were identified as members of a new Begomovirus species for which the name was proposed as "Jatropha mosaic Lucknow virus (JMLV)". The recombination analysis also suggested that JMLV was not a recombinant species, hence considered as unidentified Begomovirus species. Koch's postulates were also established by agroinfiltration assay of agroinfectious clone of JMLV. Characterization of JMLV associated with yellow mosaic disease of J. integerrima, J. podagrica and J. multifida is being reported for the first time.

Molecular identification of a new begomovirus associated with leaf crumple disease of Jatropha curcas L. in India.

Association of a new begomovirus with leaf crumple disease of Jatropha curcas was identified based on sequence analysis of cloned ~2.7-kb viral DNA genomes from three representative samples amplified by RCA using phi-29 DNA polymerase. Sequence analysis of these isolates SKJ1 (KM066975), SKJ2 (KM189818) and SKJ3 (KM189819)--showed the presence of a monopartite begomovirus-like genome of 2,735 to 2,738 nucleotides containing seven ORFs: V3, V2 and V1 in virion sense and C3, C2, C1 and C4 in the complementary sense. Pairwise alignment showed 99 % nucleotide sequence similarity to each other and less than 81 % identity to other begomoviruses reported worldwide. Based on begomovirus species demarcation criteria for a new species (sequence identity <89 %), the begomovirus isolates were identified as the members of a new Begomovirus species and provisionally designated as Jatropha leaf crumple India virus (JLCrIV).