Project description:We have determined the complete nucleotide sequence of RNA1 of the tobravirus pea early browning virus [PEBV] from an overlapping series of cDNA clones. The 7073 nucleotide sequence contains four open reading frames [ORFs]. The 5' proximal ORF encodes a 141K polypeptide, and readthrough of the opal [UGA] termination codon of this ORF would lead to the synthesis of a second, 201K polypeptide. Both of these polypeptides have extensive amino acid homology with the putative replicase proteins of tobacco rattle virus [TRV] and tobacco mosaic virus [TMV]. The third ORF encodes a 30K polypeptide which has homology with the TRV 29K and TMV 30K putative cell-to-cell spread proteins. The fourth, 3' proximal ORF encodes a 12K polypeptide which has extensive homology with the TRV 16K protein whose function is unknown. Examination of the amino acid sequences of the 12K and 16K gene products reveals in each the presence of two multiple-cysteine/histidine motifs, a finding which suggests that these proteins might have zinc and/or nucleic acid-binding properties.
Project description:Pea early browning virus (PEBV) is transmitted by soil-inhabiting trichodorid nematodes and via seeds. The transcriptome sequencing method, followed by de novo assembly, revealed the PEBV Libyan isolate LyV66-91 genome. Its RNA1 resembled that of UK isolate SP5 with 93.91% nucleotide identity, and its RNA2 had 63.32% nucleotide identity to that of Dutch isolate E116.
Project description:The 3' proximal portion of the gene encoding the 201-kDa putative replicase protein from the Tobravirus pea early browning virus (PEBV) can potentially be expressed separately as a 54-kDa protein. Nicotiana benthamiana plants transformed with the open reading frame (ORF) encoding the 54-kDa protein, designated 54K ORF, were resistant to infection by purified PEBV at inoculum doses of up to 1 mg/ml, the highest concentration tested. However, resistance was abolished by the introduction into the 54K ORF of mutations that would cause premature termination of translation. This suggests that the resistance mechanism requires the involvement of an intact 54-kDa protein. The 54K ORF-transformed plants were also resistant to infection by broad bean yellow band virus and an uncharacterized isolate of British PEBV (PGRO R) but were not resistant to infection by two other tobraviruses, pepper ringspot virus and the I6 isolate of tobacco rattle virus. Additionally, two variants of PEBV which overcame 54K ORF-mediated resistance have been isolated, the analysis of which might provide important information about both the resistance mechanism itself and the process of normal virus replication.
Project description:SnRK1 (sucrose-non-fermenting-1-related) protein kinases are involved in the regulation of plant metabolism controlling both gene expression and phosphorylation. The aim of the study was to investigate the role of SnRK1 in pea seed development. To study the effect of SnRK1 deficiency, transgenic pea plants were generated carrying a gene for VfSnRK1 in antisense orientation under control of seed specific vicilin promoter. Selected transgenic lines were characterized with decreased levels of PsSnRK1 mRNA and reduced up to 71% phosphorylation activity. Antisense inhibition of SnRK1 resulted in reduced seeds fresh weight, defect of pollen development. To dissect the SnRK1-antisense phenotype at the molecular level, a search for genes with differential expression patterns in transgenic plant versus wild type seeds has been performed using cDNA macroarray analysis. Radioactive labeled cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type (11, 13, 15, 17, 19, and 21 DAP) and transgenic SnRK1-antisense plant (13, 15, 17 and 19 DAP), which correspond to the transition phase of seed development, and hybridized to cDNA macroarrays.
Project description:Grass pea seeds and seedlings protein extracts were chromatographically fractionated. To identify the β-ODAP synthase enzyme, active fractions, as determined by a colorimetric assay that detects the presence of a derivative of free L-α,β-diaminopropionic acid (L-DAPA), were subjected to tryptic digestion and LC-MS/MS and searched against a database containing translated sequences from a long-read PacBio mRNA sequencing of grass pea seeds and seedlings.
Project description:Nitrogen application to legume seeds regulates seed metabolism and composition. In order to improve nitrogen flux into the embryo, the Vicia faba amino acid permease VfAAP1 (Miranda et al. Amino acid permeases in developing seeds of Vicia faba L.: expression precedes storage protein synthesis and is regulated by amino acid supply. Plant J 2001 28: 61-72) was expressed in pea under control of the seed-specific LeB4 promotor (Bäumlein et al. Cis-analysis of a seed protein gene promoter: the conservative RY repeat CATGCATG within legumin box is essential for tissue-specific expression of a legumin gene. Plant J 1992 2: 233-239). Several transgenic lines have been generated. Mature seeds of the homozygous marker gene-free line AAP14/10 showed an increase in amino acid supply, seed nitrogen and protein content due to higher globulin fraction. The effect of VfAAP1 was tested under field conditions in two growing periods, 2005 and 2006, and the data could be confirmed. Over-expression of VfAAP1 interferes with storage protein metabolism and alters fluxes of nitrogen during seed growth. The influence of VfAAP1 on altered gene expression in developing cotyledons was analysed using a 6k-Oligo-microarray. Four developmental stages (18, 22, 26 and 30 DAP) from seeds, grown 2006, of the transgenic line AAP14/10 were hybridized against wildtype control.
Project description:Changes in Composition and Diversity of Epiphytic Microbes on Field Pea Seeds, Partial Crop Peas, and Whole Crop Peas during Maturation and the Effect of Ensiling With or Without Adding a Lactic Acid Bacteria Inoculant
Project description:ABA regulates in plants a wide range of developmental events, mediates responses to environmental stress and is necessary to proceed through seed maturation and to acquire desiccation tolerance and dormancy. Immuno-modulation is a suitable means to study ABA functions during seed maturation. Anti-ABA single chain antibody was expressed in pea seed driving LeB4-promoter (Saalbach et al., High-level expression of a single chain Fv fragment (scFv) antibody in transgenic pea seeds J. Plant Physiol. 2001 158: 529-533), which produced only a weak phenotype with slightly decreased seed weight, globulin/albumin and total nitrogen content (aABA line 16 cultivar Erbi). In another approach with a stronger, improved USP-promoter used to express the anti-ABA antibody in pea seeds a different phenotype emerged (aABA line 7, cultivar Eifel). In this line individual seed weight increased by 20 to 30% together with higher globulin and albumin content. To dissect the aABA phenotype at the molecular level, a search for genes with differential expression patterns in transgenic plant versus wild type seeds has been performed using 6k-oligo microarray analysis. cDNA probes were prepared from RNA isolated from embryo of developing seeds of wild type (12, 18, and 22 DAP) and transgenic aABA plants (12, 18, and 22 DAP), which correspond to the transition phase of seed development, and 6k-oligo microarray.