Kirsten Lehtoma

Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin.

The complete cDNAs corresponding to two distinct gypsy moth (Lymantria dispar) larval gut aminopeptidases, APN1 and lambda APN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac delta-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The lambda APN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.

STRUCTURE OF THE GYPSY MOTH VITELLOGENIN GENE

Genomic clones containing the vitellogenin (Vg) gene from the gypsy moth were isolated from two genomic libraries and characterized. The nucleotide sequence of a 16,132 bp region of the gypsy moth genome was determined which included a 3,666 bp region upstream from the transcription initiation site and 499 bp region downstream from the transcribed region. Primer extension analysis was performed to identify the transcription initiation site. Gene sequence confirmed the sequence of VgmRNA recently reported [Hiremath and Lehtoma, J. Insect Biochem. Mol. Biol. (1997) 27:27-35] and indicated that the gypsy moth Vg gene contains seven exons interrupted by six introns. Sequence analysis of the promoter region revealed presence of several motifs associated with sex-specific and developmentally regulated genes in other systems. The nucleotide sequence comparison analyses showed that the gypsy moth Vg gene had considerably similarity with the Bombyx mori Vg gene but not with those from Anthonomous grandis and Aedes aegypti.

Native mycorrhizal fungi replace introduced fungal species on Virginia pine and American chestnut planted on reclaimed mine sites of Ohio

Plant-microbe community dynamics influence the natural succession of plant species where pioneer vegetation facilitates the establishment of a distantly related, later successional plant species. This has been observed in the case of restoration of the American chestnut (Castanea dentata) on abandoned mine land where Virginia pine (Pinus virginiana) facilitated the establishment of chestnut seedlings. This was apparently due to the natural mycorrhizal networks of pine, which aided the survival and growth of chestnut seedlings. In this study, we assessed the survival and propensity of introduced mycorrhizal fungi on Virginia pine to colonize pure American and backcrossed American chestnut. Seedlings were planted in Perry State Forest located in southeastern Ohio. This area was mined for coal in the 1950s and had very little reclamation done aside from experimental tree plantings. The selected site, with little topsoil or organic matter, was characterized by high concentrations of Al, high soil temperatures, and a pH of 3.6. Virginia pine seedlings were inoculated using ectomycorrhizal (ECM) cultures of Amanita rubescens, Laccaria laccata, and Pisolithus tinctorius via liquid media. After three months, roots were tested for the presence of mycorrhizae. They were then transplanted and grown for two years in the greenhouse. After verifying mycorrhizal colonization, 600 pines were out planted in May of 2005. Chestnut seedlings (100 one-year-old seedlings) inoculated with P. tinctorius by the Ohio state tree nursery had been planted by other researchers at the same time. After eight growing seasons, pines and chestnuts were measured and sampled for ECM colonization. Growth measurements showed that pines and hybrid chestnuts had significantly more aboveground biomass compared to pure American chestnut (P = 0.01). Eleven fungal species were detected using DNA sequencing. With the exception of Amanita, the inoculum that were out planted with both chestnut and Virginia pine were replaced after 8 field seasons by fungi native to the site. More fungal species were sampled from the Virginia pines than from chestnut roots, which contributed to the significant differences in ECM fungal community composition between the two species (P = 0.005).

Pyrolusite Process ® to Remove Acid Mine Drainage Contaminants from Kimble Creek in Ohio: A Pilot Study

The Kimble Creek abandoned coal mine site, located on Wayne National Forest in southeastern Ohio, is among several abandoned coal mine sites that have been responsible for the acid mine drainage (AMD) polluting ground and surface water. Materials released by AMD include iron, aluminum, manganese, other hazardous substances, and acidity that are harmful to aquatic life. We tested at the Kimble Creek site the Pyrolusite Process®, a microbiological water treatment system that employs metal-oxidizing bacteria adsorbed on limestone rocks. The pilot treatment plant constructed was inoculated with a mixture (five strains) of metal-oxidizing bacteria. Water was examined at regular intervals for pH, concentrations of metals (Fe, Al, Mn, Mg, Zn, Ca), dissolved oxygen, temperature, conductivity, hardiness, sulfate and chloride contents, acidity, alkalinity, and dissolved solids. Results showed that the Prylosite Process consistently increased the pH of effluent water to above-neutral levels.

Identification of a small heat-shock protein associated with a ras-mediated signaling pathway in ectomycorrhizal symbiosis

Initiation, development, and establishment of a functional ectomycorrhiza involve a series of biochemical events mediated by a number of genes from the fungus as well as the host plant. We have identified a heat shock protein gene from Laccaria bicolor (Lbhsp) that appears to play a role in these events. The size and characteristics of Lbhsp suggest that it belongs to the family of small heat-shock proteins described in the literature. Nucleotide sequencing of an almost full length cDNA indicated that the Lbhsp mRNA is about 611 nucleotides long and codes for a single protein of ~ 17 kDa. Isolation and characterization of the Lbhsp gene showed that it was made up of three exons separated by two small introns. Southern analysis suggested that the L. bicolor genome contains at least two copies of the Lbhsp gene. Temporal expression analyses revealed that the gene is expressed within 4 to 12 hours after interaction with red pine roots. The yeast two-hybrid studies showed that the Lbhsp was closely associated with the ras gene (Lbras) described earlier. The data suggest that Lbhsp may play a supporting role in ras-mediated mycorrhizal signaling pathways during various stages of ectomycorrhizal development.