Genetic engineering of wood color in plants

The invention relates to genetically engineering the wood color of woody plants by incorporation of the lignin pathway gene O-methyltransferase into the genome of the plants.https://digitalcommons.mtu.edu/patents/1068/thumbnail.jp

Genetic transformation and regeneration of plants

An Agrobacterium-mediated transformation and regeneration method for plants including a transformation method to produce transgenic plants with an altered lignin composition.https://digitalcommons.mtu.edu/patents/1067/thumbnail.jp

Using Deep RNA Sequencing for the Structural Annotation of the Laccaria Bicolor Mycorrhizal Transcriptome

BACKGROUND: Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. METHODOLOGY: We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. CONCLUSIONS: 69% of expressed mycorrhizal JGI "best" gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided that there is a sequenced genome and a set of gene models

Cloning of Protocatechuate 3,4-Dioxygenase Genes from Bradyrhizobium japonicum USDA110

A heterologous gene probe encoding the α and β subunits of the Pseudomonas cepacia protocatechuate 3,4-dioxygenase (PCD) was used to detect its homolog in the genome of Bradyrhizobium japonicum USDA110. Three cosmid clones carrying a 2.2-kb BamHI insert showed high levels of PCD activity. SacI digestion of one of the genomic clones, pBjG17, produced a 2.5-kb insert DNA that complemented a PCD mutant of P. cepacia

Differential expression of a malate synthase gene during the preinfection stage of symbiosis in the ectomycorrhizal fungus Laccaria bicolor

The ectomycorrhiza is a symbiotic organ formed between a filamentous fungus and a plant root, mainly tree roots. Root colonization involves significant shifts in gene expression resulting in metabolic and structural changes in the fungus, including growth toward the plant root, penetration and establishment of the symbiotic organ. The preinfection stage of the association is crucial as changes that occur throughout mycorrhiza formation are set in motion. Using an in vitro system for identifying preinfection stage symbiosis-regulated genes from the Laccaria bicolor-Pinus resinosa interaction we have identified a malate synthase from L. bicolor (Lb-MS). The glyoxylate pathway, of which malate synthase is an enzyme, acts as a tricarboxylic acid pathway bypass generating four-carbon compounds for biosynthesis. While it is anticipated that malate synthase would be a part of the genetic and metabolic machinery of any fungus, Lb-MS is of interest because it is symbiosis regulated. Lb-MS is regulated through interaction between the fungus and the host, by glucose and by the presence of other carbon sources in the medium. Its proposed role in the symbiosis is in the utilization of two carbon compounds formed from catabolic processes in early interaction facilitating hyphal net growth

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes. © 1994 Springer-Verlag

Cloning and identification of symbiosis-regulated genes from the ectomycorrhizal Laccaria bicolor

An in vitro conifer ectomycorrhizal model system was developed to study changes in gene expression during the establishment of symbiotic relationship. Using the model system and the mRNA differential display technique, we have identified cDNA clones from Laccaria bicolor that appear to be involved in establishing and maintaining the symbiotic relationship between L. bicolor and red pine. These differentially expressed cDNA clones were obtained from mRNA extracted from the fungus 6-24 h after initiation of interaction. BLAST analysis showed that sequences of these clones did not match with any of the previously characterized genes. The cDNAs had regions with varying degrees of similarity to genes involved in signal transduction and transcriptional activation

Effects of mycorrhizal-associated streptomycetes on growth of Laccaria bicolor; Cenococcum geophilum, and Armillaria species and on gene expression in Laccaria bicolor

Coplate assays were conducted to determine effects of two mycorrhizal-associated Streptomyces species, strains A and H, on growth of the ectomycorrhizal fungi Laccaria bicolor and Cenococcum geophilum, and on the pathogenic fungi Armillaria ostoyae and A. gallica. Strain A stimulated growth of the ectomycorrhizal fungi, but inhibited growth of the Armillaria species. Strain H inhibited growth of all fungi. The effects of the streptomycetes on L. bicolor gene expression were also examined. For this, the fungus was grown in Roux bottles containing culture filtrates at 50% concentration from strains A or H. At 24, 48, and 72 h, the fungus was harvested, mRNA was isolated, and in vitro translation protein products were analyzed by gel electrophoresis. Noticeable differences were seen in protein profiles among the treatments and the control. New protein bands were observed when the fungus was exposed to filtrate from strain A compared to the untreated control. In contrast, filtrate from strain H tended to suppress fungal gene expression as evidenced by the disappearance of protein bands. The use of streptomycetes for enhancement of mycorrhizal formation and improvement of plant growth is discussed