Discovery, isolation and characterization of novel biomolecules with nematicidal activity.

Both, sesquiterpene lactones isolated from Ambrosia and trypsin inhibitors isolated from Cassia and from Mucuna are new potential tools to engineer transgenic plants with resistance to plant parasitic nematodes.Bioactive defense-related proteins include proteins such as protease inhibitors. Trypsin inhibitors have exhibited antiinsect, antifungal and nematicidal activities. The gene for trypsin inhibitor may protect plants from different pests and pathogens when expressed in transgenic plants. For this reason, we are involved in the isolation of novel trypsin inhibitors. Earlier studies indicated that seeds of Cassia fruticosa and Mucuna holdii, two tropical species, possess trypsin inhibitory activity. Trypsin inhibitors were isolated from both species by affinity chromatography. Isoforms of trypsin inhibitors were further purified by anion exchange chromatography.Three trypsin inhibitors were purified from Cassia. Five trypsin inhibitors were detected in Mucuna seed extract and these inhibitors exhibited high thermostability. Based on SDS-PAGE, the molecular weight of the purified trypsin inhibitors from Cassia was 17,700 Da and inhibitors from Mucuna were found to possess a molecular weight around 9,000 Da. Their molecular weight was also determined by MALDI-TOF laser mass spectrometry. MALDI-TOF mass analysis indicated that the molecular weights of Cassia and Mucuna inhibitors were 21,500 and 8,000 Da, respectively. Bioassays of trypsin inhibitors using a Caenorhabditis elegans feeding bioassay indicated that the trypsin inhibitors from both Cassia and Mucuna were capable of causing nematode mortality under the conditions of the bioassay.The purpose of this research was to discover novel biological substances with nematicidal activity. Plants contain a variety of bioactive natural products. Many of them may play a role in plant defense. Plants also contain bioactive proteins involved in defense. Two groups of plants were tested for nematicidal activity. The nematicidal activity found in the first group of plants was attributed to the presence of secondary metabolites, while the activity found in the second group was apparently due to the presence of active proteins. Aqueous extracts of Solidago missouriensis, Ambrosia psilostachya and Lespedeza stuevei, native plants of the United States, were tested for activity against M. incognita juveniles. Solidago and Ambrosia reduced gall formation in tomato infected with M. incognita in greenhouse experiments. In laboratory experiments, extracts of Solidago exerted a nernatistatic effect. Lespedeza inhibited nematode motility temporarily. Of the three, only A. psilostachya crude extract exhibited nematicidal activity, causing 100% mortality at a concentration of 20 mg/ml. Bioactivity-directed TLC fractionation of Ambrosia extract resulted in the isolation of three nematicidal secondary metabolites. The structures of two of these compounds, parthenin and coronopilin, were determined by NMR and mass spectrometry. Parthenin and coronopilin, are known sesquiterpenes and these compounds were reported previously to possess antitumor, antibacterial, antifungal, molluscicidal and insect antifeedant activities. However, this is the first report of their nematicidal activity. We proposed a model for the mode-of-action of parthenin in which the parthenin reacts with essential sulfhydryl groups of proteins. To test this hypothesis, parthenin was mixed with cysteine and then tested for nematicidal activity. Indeed, the nematicidal effect of parthenin was reduced 95% by mixing the compound with equimolar concentrations of cysteine. This suggests that the lethal effect of parthenin on nematodes is due to reaction with sulfhydryl groups present in essential proteins

Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nomicotine and N\u27-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N\u27-nitrosonomicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nomicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Sequence and Spatiotemporal Expression Analysis of CLE-Motif Containing Genes from the Reniform Nematode (Rotylenchulus reniformis Linford & Oliveira)

The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globodera, and Meloidogyne genera of sedentary endoparasites. Here, we describe the isolation, sequence analysis, and spatiotemporal expression of three R. reniformis genes encoding putative CLE motifs named Rr-cle-1, Rr-cle-2, and Rr-cle-3. The Rr-cle cDNAs showed .98% identity with each other and the predicted peptides were identical with the exception of a short stretch of residues at the carboxy(C)-terminus of the variable domain (VD). Each RrCLE peptide possessed an amino-terminal signal peptide for secretion and a single C-terminal CLE motif that was most similar to Heterodera CLE motifs. Aligning the Rr-cle cDNAs with their corresponding genomic sequences showed three exons with an intron separating the signal peptide from the VD and a second intron separating the VD from the CLE motif. An alignment of the RrCLE1 peptide with Heterodera glycines and Heterodera schachtii CLE proteins revealed a high level of homology within the VD region associated with regulating in planta trafficking of the processed CLE peptide. Quantitative RT-PCR (qRT-PCR) showed similar expression profiles for each Rr-cle transcript across the R. reniformis life-cycle with the greatest transcript abundance being in sedentary parasitic female nematodes. In situ hybridization showed specific Rr-cle expression within the dorsal esophageal gland cell of sedentary parasitic females

Alternation of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) inNicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Alteration of Tobacco Alkaloid Content through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

Compositions and methods for reducing the level of nornicotine and N′-nitrosonornicotine (NNN) in Nicotiana plants and plant parts thereof are provided. The compositions comprise isolated polynucleotides and polypeptides for cytochrome P450s that are involved in the metabolic conversion of nicotine to nornicotine in these plants. Expression cassettes, vectors, plants, and plant parts thereof comprising inhibitory sequences that target expression or function of the disclosed cytochrome P450 polypeptides are also provided. Methods for the use of these novel sequences to inhibit expression or function of cytochrome P450 polypeptides involved in this metabolic conversion are also provided. The methods find use in the production of tobacco products that have reduced levels of nornicotine and its carcinogenic metabolite, NNN, and thus reduced carcinogenic potential for individuals consuming these tobacco products or exposed to secondary smoke derived from these products

Sequence and Spatiotemporal Expression Analysis of CLE-Motif Containing Genes from the Reniform Nematode (Rotylenchulus reniformis Linford & Oliveira)

The reniform nematode, Rotylenchulus reniformis, is a sedentary semi-endoparasitic species with a host range that encompasses more than 77 plant families. Nematode effector proteins containing plant-ligand motifs similar to CLAVATA3/ESR (CLE) peptides have been identified in the Heterodera, Globodera, and Meloidogyne genera of sedentary endoparasites. Here, we describe the isolation, sequence analysis, and spatiotemporal expression of three R. reniformis genes encoding putative CLE motifs named Rr-cle-1, Rr-cle-2, and Rr-cle-3. The Rr-cle cDNAs showed .98% identity with each other and the predicted peptides were identical with the exception of a short stretch of residues at the carboxy(C)-terminus of the variable domain (VD). Each RrCLE peptide possessed an amino-terminal signal peptide for secretion and a single C-terminal CLE motif that was most similar to Heterodera CLE motifs. Aligning the Rr-cle cDNAs with their corresponding genomic sequences showed three exons with an intron separating the signal peptide from the VD and a second intron separating the VD from the CLE motif. An alignment of the RrCLE1 peptide with Heterodera glycines and Heterodera schachtii CLE proteins revealed a high level of homology within the VD region associated with regulating in planta trafficking of the processed CLE peptide. Quantitative RT-PCR (qRT-PCR) showed similar expression profiles for each Rr-cle transcript across the R. reniformis life-cycle with the greatest transcript abundance being in sedentary parasitic female nematodes. In situ hybridization showed specific Rr-cle expression within the dorsal esophageal gland cell of sedentary parasitic females.This article is published as Wubben, Martin J., Lily Gavilano, Thomas J. Baum, and Eric L. Davis. "Sequence and spatiotemporal expression analysis of CLE-motif containing genes from the reniform nematode (Rotylenchulus reniformis Linford & Oliveira)." Journal of nematology 47, no. 2 (2015): 159.</p