Functional Characterization of Monoterpenoid Indole Alkaloid (MIA) Biosynthetic Genes in Catharanthus roseus

The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) are known to be among the most important source of natural drugs used in various cancer chemotherapies. MIAs are derived by combining the iridoid secologanin with tryptamine to form the central precursor strictosidine that is then converted to most known MIAs, such as catharanthine and vindoline that dimerize to form anticancer vinblastine and vincristine. While their assembly is still poorly understood, the complex multistep pathways involved occur in several specialized cell types within leaves that are regulated by developmental and environmental cues. The organization of MIA pathways is also coupled to secretory mechanisms that allow the accumulation of catharanthine in the waxy leaf surface, separated from vindoline found within leaf cells. While the spatial separation of catharanthine and vindoline provides an explanation for the low levels of dimeric MIAs found in the plants, the secretion of catharanthine to the leaf surface is shown to be part of plant defense mechanisms against fungal infection and insect herbivores. The transcriptomic databases of Catharanthus roseus and various MIA producing plants are facilitating bioinformatic approaches to identify novel MIA biosynthetic genes. Virus-induced gene silencing (VIGS) is being used to screen these candidate genes for their involvement in iridoid biosynthesis pathway, especially in the identification of 7-deoxyloganic acid 7-hydroxylase (CrDL7H) shown by the accumulation of its substrate, 7-deoxyloganic acid and decreased level of secologanin along with catharanthine and vindoline. VIGS can also confirm the biochemical function of genes being identified, such as in the glucosylation of 7-deoxyloganetic acid by CrUGT8 shown by decreased level of secologanin and MIAs within silenced plants. Silencing of other iridoid biosynthetic genes, loganic acid O-methyltransferase (LAMT) and secologanin synthase (SLS) also confirm the metabolic route for iridoid biosynthesis in planta through 7-deoxyloganic acid, loganic acid, and loganin intermediates. This route is validated by high substrate specificity of CrUGT8 for 7-deoxyloganetic acid and CrDL7H for 7-deoxyloganic acid. Further localization studies of CrUGT8 and CrDL7H also show that these genes are preferentially expressed within Catharanthus leaves rather than in epidermal cells where the last two steps of secologanin biosynthesis occur.

An investigation into fungal metabolism of halogenated and related steroids

Fungal metabolism of halogenated and related steroids was investigated. The fungi Aspergillus niger ATCC 9142, Curvularia lunata NRRL 2380 and Rhizopus stolonifer ATCC6227b were studied in this regard. 2l-Fluoro-, 2l-chloro, 2l-bromo- and 2l-methyl-pregn-4-ene-3,20diones were prepared and incubated with ~ niger (a C-2l-hydroxylator) in order to observe the effect of the C-2l substituent on the metabolism of these substrates. In all four cases, the C-2l substituent prevented any significant metabolism of these substrates. llB-Fluoropregn-4-ene-3,20-dione was prepared and incubated with C. lunata (an llB-hydroxylator) and ~ stolonifer (an lla-hydroxylator). With ~ lunata, the ll-fluoro- substituent prevent hydroxylation at the 11 position, but diverted it to a site remote from the fluorine atom. In contrast, with ~ stolonifer the llB-fluoro- substituent, although slowing the apparent rate of hydroxylation, did not prevent its occurrence at the 11a- position. llB-Hydroxypregn-4-ene-3,20-dione was also incubated with R. stolonifer. The llB-hydroxy-;group did not appear to have any significant effect on hydroxylation at the lla- position. The incubation of a substrate, unsaturated at a favoured site of hydroxylation with Rhizopus arrhizus ATCC 11145 provided a complex mixture of products; among them were both the a and S epoxides. The formation of these products is rationalized as arising because of the lack of regio- and stereospecificity of the hydroxylase enzyme(s) involved.

Gypsy moths (Lymantria dispar) in the Niagara Region : population density variation, introduction of an entomopathogenic fungus (Entomophaga maimaiga) and occurrence of nuclear polyhedrosis virus

The gypsy moth, Lymantria dispar, a major defoliator of broad leaf trees, was accidentally introduced into North America in 1869. Much interest has been generated regarding the potential of using natural pathogens for biological control of this insect. One of these pathogens, a highly specific fungus, Entomophaga maimaiga, was accredited with causing major epizootics in populations of gypsy moth across the north-eastern United States in 1989 and 1990 and is thought to be spreading northwards into Canada. This study examined gypsy moth population densities in the Niagara Region. The fungus, .E.. maimaiga, was artificially introduced into one site and the resulting mortality in host populations was noted over two years. The relationship between fungal mortality, host population density and occurrence of another pathogen, the nuclear polyhedrosis virus (NPV), was assessed. Gypsy moth population density was assessed by counting egg masses in 0.01 hectare (ha) study plots in six areas, namely Louth, Queenston, Niagara-on-the-Lake, Shorthills Provincial Park, Chippawa Creek and Willoughby Marsh. High variability in density was seen among sites. Willoughby Marsh and Chippawa Creek, the sites with the greatest variability, were selected for more intensive study. The pathogenicity of E. maimaiga was established in laboratory trials. Fungal-infected gypsy moth larvae were then released into experimental plots of varying host density in Willoughby Marsh in 1992. These larvae served as the inoculum to infect field larvae. Other larvae were injected with culture medium only and released into control plots also of varying host density. Later, field larvae were collected and assessed for the presence of .E.. maimaiga and NPV. A greater proportion of larvae were infected from experimental plots than from control plots indicating that the experimental augmentation had been successful. There was no relationship between host density and the proportion of infected larvae in either experimental or control plots. In 1992, 86% of larvae were positive for NPV. Presence and intensity of NPV infection was independent of fungal presence, plot type or interaction of these two factors. Sampling was carried out in the summer of 1993, the year after the introduction, to evaluate the persistence of the pathogen in the environment. Almost 50% of all larvae were infected with the fungus. There was no difference between control and experimental plots. Data collected from Willoughby Marsh indicated that there was no correlation between the proportion of larvae infected with the fungus and host population density in either experimental or control plots. About 10% of larvae collected from a nearby site, Chippawa Creek, were also positive for .E.. maimaiga suggesting that low levels of .E.. maimaiga probably occurred naturally in the area. In 1993, 9.6% of larvae were positive for NPV. Again, presence or absence of NPV infection was independent of fungal presence plot type or interaction of these two factors. In conclusion, gypsy moth population densities were highly variable between and within sites in the Niagara Region. The introduction of the pathogenic fungus, .E.. maimaiga, into Willoughby Marsh in 1992 was successful and the fungus was again evident in 1993. There was no evidence for existence of a relationship between fungal mortality and gypsy moth density or occurrence of NPV. The results from this study are discussed with respect to the use of .E.. maimaiga in gypsy moth management programs.

Permissivity of human HeLa cells to bovine adenovirus type 2 (BaV2 infection

Infection of hUlnan cells by bovine adenovirlls type 2 (BAV2) is abortive. To obtain a better understanding of this pllenomel1011, and in particular to identify Wllich steps in the viral replicative cycles are altered dllring this virlls-host cells interaction, we have llndertaken a detailed study of BAV2 infections of the nonpennissive hUlnan IIeLa cells. Using autoradiography and 3H-thymidine-labeled vvhole virus particles for infection of HeLa cells, vve determined that viral attachluent appears normal. Furthermore, Southern analysis revealed that internalization and transport to the nuclells occurs in BAV2 infected HeLa cells. To investigate viral DNi\ synthesis, infectivity assays involving hydroxyllrea, a viral DN-A synthesis inhibitor, were carried out. The results revealed that Bft:LV2 DNA synthesis does not occur in HeLa cells. Fllrtller investigations into viral early gene expression by northern blotting analyses indicated that HeLa cells fail to support expression of EIA. This suggested that abortive infection by BAV2 could be attributed to faiiure of EIA to express. To test the possibility that the failure to express ElA was due to the inability of the host cell to recognize the E lA prOlTIoter, ,ve carried out transient expression transfection experiments using plaslnids \vith the bacterial lacZ linder the control of either BAV2 or i\d5 EIA promoter. X-gal histochelIlical assays sho\ved expression of lacZ from the Ad5 ElA prOlnoter but no expression of lacZ [rOln the BAV2 EIA prOlTIoter. This further suggests that the abortive infection b:y BAV2 could be attributed to failure of EIA to express dlle to a nonfllnctional prOlTIoter in hlunan cells. Thus we speClllated that abortive infection of HeLa cells by adenoviruses may be averted by providing EtA functions in trans. To demonstrate this, we coinfected HeLa cells with Ad5 and BAV2, reasoning that Ad5 could cOlnpensate for EIA deficiency in BAV2. OUf results showed that BAV2 DNA synthesis was indeed Sllpported in HeLa cells coinfected with Ad5dlE3 as revealed by Southern analysis. In contrast, coinfection of HeLa cells \vith BAV2 and Ad5dlElE3 mutallt did not support BLt\V2 DNA synthesis. Interestingly, BAV2 failed to replicate in 293 cells which are constitlltively expressing the El genes. This could ilnply that El is necessary but not sufficient to avert the failllre ofBAV2 to undergo productive infection ofhulnan cells.

The 21-dehydroxylation of corticosteroids: evidence for an enol intermediate and the hydroxylation of steroids by fungal cyto-chrome P450: evidence for a stepwise mechanism

Two enzyme mechanisms were examined: the 21-dehydroxylation of corticosteroids by the anaerobe Eubacterium l en tum, and the hydroxylation of steroids by fungal cytochrome P450. Deuterium labelling techniques were used to study the enzymic dehydroxylation. Corticosteroids doubly labelled (2H) at the C-21 position were incubated with a culture of Eubacterium lentum. It was found that t he enzymic dehydroxylation proceeded with the loss of one 2H f rom C-21 per molecule of substrate. The kinetic isotope ef fect f or the reaction was found to be k~kD = 2. 28. These results suggest that enzyme/substr ate binding in this case may proceed via t he enol form of the substrate. Also , it appears that this binding is, at least in part, the rate determining step of t he reaction. The hydroxylation of steroids by fungal cytochrome P450 was examined by means of a product study. Steroids with a double bond at the A8 (9), ~( lO ), or ~ (ll) position were synthesized. These steroids were then incubated with fungal strains known to use a cytochrome P450 monooxygenase to hydroxylate at positions allylic to these doubl e bonds. The products formed in these incubations indicated that the double bonds had migrated during allylic hydroxylat ion. This suggests that a carbon centred radical or ion may be an intermediate i n the cytochrome P450 cat alytic cycle.

Biochemical and histological investigations of viral localisation in the hypersensitive reaction of Phaselous vulgaris L. var Pinto to tobacco mosaic virus infection

STOBBS, Lorne,W ABSTRACT Biochemical and Histological Investigations of viral localisation in the hypersensitive reaction of Phaseolus vulgaris L. var Pinto to tobacco mosaic virus infection. The infection of Phaseolus vulgaris L. var Pinto with tobacco mosaic virus (TMV) results in the production of distinct necrotic lesions confining the virus to restricted areas of the leaf surface. Biochemical and histological changes in the leaf tissue as a result of infection have been described. Trace accumulations of fluorescent metabolites, detected prior to lesion expression represent metabolites produced, by the cell in response to virus infection. These substances, are considered to undergo oxidation and in diffusing into adjacent cells, react with cellular constituents causing the death of these cells. Such cellular necrosis in advance of infection effectively limits virus spread. Chromatographic studies on extracts from TMV infected Pinto bean leaf tissue suggests that a number of extra-fluorescent metabolites produced on lesion'expression represent end products of phenolic oxidation r,eactionsoccurring earlier in these cells. Inhibition of phenolic oxidation by ascorbate infiltration or elevated temperature treatment resulted in the absence of extra-fluorescent metabolites and the continued movement of virus in the absence of necrosis. Further studies with i ascorbate infiltration indicated that irreversible necrotic events were determined as early as 12 tci 18 hrs after viral inoculation. Histochemical tests indicated that callose formation was initiated at this time, and occurred in response to necrotisation. Inhibition of necrosis by either ascorbate infiltration or elevated temp8rature treatment resulted in the absence of callose deposition. Scanning electron'micrographs of infected tissue revealed severe epidermal and palisade cell damage. Histochemical tests indicated extensive callose formation in cells bordering the lesion, and suggested the role of callose iTh the blockage of intercellular connections limiting virus movement. The significance of these cellular changes is discussed. ii

Fungal biotransformation of morphine alkaloids

The purpose of the study was to determine the ability of certain fungi to biotransform morphine alkaloids into medicinally relevant intermediates. Fungal strains screened for their ability to affect biotransformation of morphine alkaloids include Cunninghamella echinulata, Helicostylum pirijorme, Pycnoporus sanguinea, Pycnoporus cinnabarina, Curvularia lunata and Sporotrichum sulfurescens. The research demonstrated that Cunninghamella echinulata N-demethylated thebaine, hydrocodone, codeine, oripavine and oxycodone into corresponding nor-compounds in varying yields. The study further focused on the characterization of the enzyme responsible for the biotransformation of thebaine into northebaine by Cunninghamella echinulata. The study clearly showed that incubation of the fungal culture with thebaine over a period of 48 hours was required to activate the biotransformation process. The biotransformation studies with [14C] labeled thebaine showed that Ndemethylation by Cunningham ella echinulata does not involve O-demethylation followed by methyl group transfer as suggested in previous studies.

Vitamin D status and latent tuberculosis infection : a preliminary study in a group of healthy Mexican agricultural workers

Vitamin D metabolites are important in the regulation of bone and calcium homeostasis, but also have a more ubiquitous role in the regulation of cell differentiation and immune function. Severely low circulating 25-dihydroxyvitamin D [25(OH)D] concentrations have been associated with the onset of active tuberculosis (TB) in immigrant populations, although the association with latent TB infection (LTBI) has not received much attention. A previous study identified the prevalence of LTBI among a sample of Mexican migrant workers enrolled in Canada's Seasonal Agricultural Workers Program (SA WP) in the Niagara Region of Ontario. The aim of the present study was to determine the vitamin D status of the same sample, and identify if a relationship existed with LTBI. Studies of vitamin D deficiency and active TB are most commonly carried out among immigrant populations to non-endemic regions, in which reactivation of LTBI has occurred. Currently, there is limited knowledge of the association between vitamin D deficiency and LTBI. Entry into Canada ensured that these individuals did not have active TB, and L TBI status was established previously by an interferon-gamma release assay (IGRA) (QuantiFERON-TB Gold In-Tube®, Cellestis Ltd., Australia). Awareness of vitamin D status may enable individuals at risk of deficiency to improve their nutritional health, and those with LTBI to be aware of this risk factor for disease. Prevalence of vitamin D insufficiency among the Mexican migrant workers was determined from serum samples collected in the summer of 2007 as part of the cross sectional LTBI study. Samples were measured for concentrations of the main circulating vitamin D metabolite, 25(OH)D, with a widely used 1251 250HD RIA (DiaSorin Inc.®, Stillwater, MN), and were categorized as deficient «37.5 nmoI/L), insufficient (>37.5 nmollL, < 80 nmol/L) or sufficient (2::80 nmoI/L). Fisher's exact tests and t tests were used to determine if vitamin D status (sufficiency or insufficiency) or 25(OH)D concentrations significantly differed by sex or age categories. Predictors of vitamin D insufficiency and 25(OH)D concentrations were taken from questionnaires carried out during the previous study, and analyzed in the present study using multiple regression prediction models. Fisher's exact test and t test was used to determine if vitamin D status or 25(OH)D concentration differed by LTBI status. Strength of the relationship between interferongamma (IFN-y) concentration (released by peripheral T cells in response to TB antigens) and 25(OH)D concentration was analyzed using a Spearman correlation. Out of 87 participants included in the study (78% male; mean age 38 years), 14 were identified as LTBI positive but none had any signs or symptoms of TB reactivation. Only 30% of the participants were vitamin D sufficient, whereas 68% were insufficient and 2% were deficient. Significant independent predictors of lower 25(OH)D concentrations were sex, number of years enrolled in the SA WP and length of stay in Canada. No significant differences were found between 25(OH)D concentrations and LTBI status. There was a significant moderate correlation between IFN-y and 25(OH)D concentrations ofLTBI-positive individuals. The majority of participants presented with Vitamin D insufficiency but none were severely deficient, indicating that 25(OH)D concentrations do not decrease dramatically in populations who temporarily reside in Canada but go back to their countries of origin during the Canadian winter. This study did not find a statistical relationship between low levels of vitamin D and LTBI which suggests that in the presence of overall good health, lower than ideal levels of 2S(OH)D, may still be exerting a protective immunological effect against LTBI reactivation. The challenge remains to determine a critical 2S(OH)D concentration at which reactivation is more likely to occur.

STRATEGIES FOR PREVENTION AND TREATMENT OF HEPATITIS C VIRUS INFECTION

Hepatitis C virus (HCV) is the causative agent of Hepatitis C, a serious global health problem which results in liver cirrhosis and hepatocellular carcinoma. Currently there is no effective treatment or vaccine against the virus. Therefore, development of a therapeutic vaccine is of paramount importance. In this project, three alternative approaches were used to control HCV including a DNA vaccine, a recombinant viral vaccine and RNA interference. The first approach was to test the effect of different promoters on the efficacy of a DNA vaccine against HCV. Plasmids encoding HCV-NS3 and E1 antigens were designed under three different promoters, adenoviral E1A, MLP, and CMV ie. The promoter effect on the antigen expression in 293 cells, as well as on the antibody level in immunized BALB/c mice, was evaluated. The results showed that the antigens were successfully expressed from all vectors. The CMV ie promoter induced the highest antigen expression and the highest antibody level. Second, the efficiency of a recombinant adenovirus vaccine encoding HCV-NS3 was compared to that of a HCV-NS3 plasmid vaccine. The results showed that the recombinant adenovirus vaccine induced higher antibody levels as compared to the plasmid vaccine. The relationship between the immune response and miRNA was also evaluated. The levels of mir-181, mir-155, mir-21 and mir-296 were quantified in the sera of immunized animals. mir-181 and mir-21 were found to be upregulated in animals injected with adenoviral vectors. Third, two recombinant adenoviruses encoding siRNAs targeting both the helicase and protease parts of the NS3 region were tested for their ability to inhibit NS3 expression. The results showed that the siRNA against protease was more effective in silencing the HCV-NS3 gene in a HCV replicon cell line. This result confirmed the efficiency of adenovirus for siRNA delivery. These results confirmed that CMV ie is optimum promoter for immune response induction. Adenovirus was shown to be an effective delivery vector for antigens or siRNAs. In addition, miRNAs were proved to be involved in the regulation of immune response.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

Variability in the Insect and Plant Adhesins, Mad1 and Mad2, within the Fungal Genus Metarhizium Suggest Plant Adaptation as an Evolutionary Force

Several species of the insect pathogenic fungus Metarhizium are associated with certain plant types and genome analyses suggested a bifunctional lifestyle; as an insect pathogen and as a plant symbiont. Here we wanted to explore whether there was more variation in genes devoted to plant association (Mad2) or to insect association (Mad1) overall in the genus Metarhizium. Greater divergence within the genus Metarhizium in one of these genes may provide evidence for whether host insect or plant is a driving force in adaptation and evolution in the genus Metarhizium. We compared differences in variation in the insect adhesin gene, Mad1, which enables attachment to insect cuticle, and the plant adhesin gene, Mad2, which enables attachment to plants. Overall variation for the Mad1 promoter region (7.1%), Mad1 open reading frame (6.7%), and Mad2 open reading frame (7.4%) were similar, while it was higher in the Mad2 promoter region (9.9%). Analysis of the transcriptional elements within the Mad2 promoter region revealed variable STRE, PDS, degenerative TATA box, and TATA box-like regions, while this level of variation was not found for Mad1. Sequences were also phylogenetically compared to EF-1a, which is used for species identification, in 14 isolates representing 7 different species in the genus Metarhizium. Phylogenetic analysis demonstrated that the Mad2 phylogeny is more congruent with 59 EF-1a than Mad1. This would suggest that Mad2 has diverged among Metarhizium lineages, contributing to clade- and species-specific variation, while it appears that Mad1 has been largely conserved. While other abiotic and biotic factors cannot be excluded in contributing to divergence, these results suggest that plant relationships, rather than insect host, have been a major driving factor in the divergence of the genus Metarhizium.

UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.

Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.