Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence. Approach (from AD-416): Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques. The research group at Northern Illinois University is investigating genetic mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including aflatoxin-producing Aspergillus (A.) flavus. Our major interest is the study of the global fungal regulatory gene VeA (or velvet) and connected regulatory networks. This regulator is unique to fungi and it is conserved in other fungal species, particularly in the group called Ascomycetes. VeA is known to play a role in regulation of fungal development, secondary metabolism, and virulence and therefore, it has a high potential to be used to control plant diseases caused by Aspergillus and other fungi. We have investigated the effects of oxidative stress (type of stress involving reactive oxygen) in A. flavus and the role that VeA plays in the response to this stress. Our study revealed a reduction in survivability in the veA mutant as the concentration of the reactive oxygen species increased in the medium, compared to the control strain. This result indicates that the presence of veA is necessary for a normal adaptive response under exposure to oxidative stress. Furthermore, ribonucleic acid (RNA) analysis of veA mutant and control strain under oxidative stress indicated alterations in expression profiles of several genes involved in the HogA oxidative response signaling pathway (a type of network of proteins that is activated in response to external stresses on a cell) in the absence of veA. A manuscript reporting this work is also being prepared for publication. Also during this reporting period the study of A. flavus NsdD and NsdC regulatory genes and their role in morphogenesis (biological process involving development) and aflatoxin production has been published in Eukaryotic Cell. This work addresses research objectives 2 and 3 of Project Plan 6435-41420-005-00D. Studies have continued on the connection between veA and starch degradation by A. flavus. It is known that starch degrading enzymes are necessary for successful infection of plant tissues in corn. We observed that amylase activity (a type of enzyme activity needed for starch degradation) is significantly reduced in the veA mutant compared to the control strain in starch medium and also in corn medium. We have also detected alterations of protease activity, another enzyme possibly involved in successful fungal invasion, in the veA mutant compared to an A. flavus control. Culture supernatants of the veA mutant and control strain growing on corn or starch medium were analyzed for differences in proteins secreted into the growth medium. An initial set of data has been obtained and proteins putatively associated with hydrolytic activity in A. flavus have been identified. This work addresses research Objective 2 of Project Plan 6435-42000-020-00D. Additionally, the goal of performing in silico analysis (performed via computer simulation) of A. flavus secondary metabolism gene clusters #27 and #39 has also been accomplished. This analysis included structural genomics and comparative genomics using genomic (entirety of an organism�s hereditary information) databases from other fungal species. A manuscript on this analysis is now being prepared for publication. Furthermore, we have identified gene clusters similar to A. flavus cluster #39 that are partially conserved in other Aspergillus species. In collaboration with SRRC scientists we have also identified the compound produced by gene cluster #27. The compound is a pigment that is found in mature sclerotia (over wintering bodies or survival structures of fungi) of A. flavus and it appears to contribute to survival of sclerotia exposed to intense sunlight or drought conditions. This work has been recently submitted for publication in Fungal Genetics and Biology. Another two A. flavus secondary metabolite gene clusters are currently under study, clusters #23 and #14. This work addresses research outlined in Objective 1 of Project Plan 6435-41420-005- 00D.
Impacts
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence. Approach (from AD-416): Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques. The research group at Northern Illinois University is investigating genetic regulatory mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including aflatoxin- producing Aspergillus (A.) flavus. Our major interest is the study the global fungal regulatory gene VeA (or velvet) and connected regulatory networks. This regulator is unique to fungi and it is conserved in other fungal species, particularly in the group called Ascomycetes. VeA has high potential to be used to control plant diseases caused by Aspergillus and other fungi. We further characterized the initial findings related to the connection between veA and starch degradation by A. flavus. It is known that starch degrading enzymes are necessary for successful infection of plant tissues in corn. Through high performance liquid chromatography analysis we observed that amylase activity (type of enzyme activity) is significantly reduced in the veA mutant compared to the control strain in starch medium and also in corn medium. Furthermore, preliminary experiments indicated that A. flavus mutants lacking LaeA, a gene that produces a protein described in A. nidulans to interact with VeA protein in nuclei, also presented a decrease in amylase activity compared to the control strain. In addition, we have also detected a reduction of protease activity, another enzyme possibly involved in successful fungal invasion, in the veA mutant compared to A. flavus wild- type levels. Culture supernatants of the veA deletion mutant and control strain growing on corn medium were analyzed for differences in proteins secreted into the growth medium. An initial set of data has been obtained and proteins putatively associated with hydrolytic activity in A. flavus have been identified. We have also investigated the effects of oxidative stress (type of stress involving reactive oxygen) in A. flavus and the role that VeA plays in the response to this stress. Our study revealed a reduction in survivability in the veA mutant as the concentration of the oxygen species increased in the medium, compared to the wild type strain. This result indicates that the presence of veA is necessary for a normal adaptative response under exposure to oxidative stress. Furthermore, ribonucleic acid analysis of veA mutant and wild type strain under oxidative stress indicated alterations in expression profiles of several genes involved in the HogA oxidative response signaling pathway (a type of network of proteins involved in specific effects in a cell) in the absence of veA. Interestingly, preliminary results indicated protein-protein interaction between VeA and some proteins participating in this signaling pathway. The goal of performing in silico analysis (perfomed via computer simulation) of A. flavus secondary metabolism gene clusters #27 and #39 has also been accomplished. This analysis included structural genomics and comparative genomics using genomic (entirety of an organism hereditary information) databases from other fungal species. Furthermore, collaborative efforts yielded the identification of the compound associated with cluster #39, partially conserved in other Aspergillus species. Also importantly the dark pigmented compound found in mature sclerotia (over wintering bodies or survival structures of fungi) produced by gene products of cluster #27 appears to contribute to A. flavus survival under dry conditions. Also during this reporting period the study of A. flavus NsdD and NsdC genes and their role on morphogenesis (biological process involving development) and aflatoxin production has been finalized and was resubmitted after a positive review to Eukaryotic Cell.
Impacts
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence. Approach (from AD-416) Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques. This collaboration addresses work described in objectives 1, 2 and 3 of the project plan to identify the molecular mechanisms that regulate the fungus-plant interaction and the production of aflatoxin. One goal of this reporting period was to determine why starch degradation is reduced in the Aspergillus (A.) flavus veA mutant (fungus where veA gene is defective) as production of starch degrading enzymes is required for successful infection of plant tissues in corn. The goals of this project were substantially met as assays for various enzymes involved in starch metabolism showed that an alpha-amylase or possibly a glucoamylase (enzymes that breakdown starch) was not as active in the A. flavus veA mutant strain and this indicates that VeA may control expression of genes involved in amylase degradation. Further enzyme assays are being conducted to better elucidate the enzyme(s) whose production is dependent on VeA. A second goal was to determine the effects of osmotic and oxidative stress in A. flavus mycotoxin production and the role that VeA plays in governing these effects. The research goals of this project have been partially met as growth experiments showed that the A. flavus veA mutant exhibits differential growth and developmental characteristics compared to the wild-type A. flavus under conditions of osmotic or oxidative stress. Gene expression studies are underway to identify which key genes involved in osmotic and oxidative stress are differentially regulated in the veA mutant compared to wild-type. A third goal of this reporting period was to generate A. flavus strains that can be used to study the interaction of key regulators of A. flavus development and secondary metabolism such as the genes LaeA and VeA with other transcription factors (genes) such as AflR, NsdD and NsdC. This goal has been substantially met as all needed plasmid vectors (vehicles for transportating genes into cells) were constructed and transformed into A. flavus. Research is currently underway to determine which of these regulatory factors physically interact with one another. A fourth goal of this reporting period was to study the role of nitric oxide in A. nidulans development and toxin production. This goal was substantially met as we found that nitric oxide induces resistant structure formation as well as changes in the biosynthesis of mycotoxins. A manuscript has been submitted for publication regarding this work. Research progress was monitored through teleconferencing, frequent email communications and reports.
Impacts
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Identify key genes involved in regulation of oxidative stress, nitric oxide production, and nitrogen metabolism in A. nidulans and A. flavus whose expression are dependent on the presence of regulatory genes such as veA. Determine role of these genes in regulation of biological activities such as fungal toxin production, morphogenesis, and virulence. Approach (from AD-416) Data acquired from A. flavus whole genome microarray/two-hybrid studies will be used to identify key genetic components of signaling pathways that control aflatoxin production and fungal morphogenesis. Using both A. flavus and the model fungus, A. nidulans, gene inactivation studies will determine the role of novel as well as previously characterized genes involved in fungal response to oxidative stress on toxin production and morphogenesis. The role of VeA and other regulatory proteins on production of hydrolytic enzymes involved in fungal pathogenesis of crop plants will be determined using molecular and biochemical techniques. The research group at Northern Illinois University (NIU) is investigating genetic regulatory mechanisms that control the detrimental impact of fungal species that are of agricultural importance, including Aspergillus flavus. One of our major interests is the study a novel regulatory system, called velvet (VeA), unique to fungi like Aspergillus flavus, with high potential to control plant diseases. We have demonstrated that velvet is vital for the production of aflatoxin, cyclopiazonic acid and aflatrem mycotoxins by Aspergillus flavus. We also showed that VeA is indispensable for the production of sclerotia, resistant structures formed by Aspergillus flavus. Furthermore, recent data indicated that VeA is necessary for maximum pathogenicity by the fungus. Virulence of Aspergillus flavus on peanut and corn seeds was reduced in the absence of the veA gene product. Our studies indicate that production of key enzymes necessary for infection of plant tissue is decreased if this veA gene is deleted compared to the control strains, particularly amylase (starch breakdown) activity. In addition, parallel functional genomic studies (studies to determine the functionality of genes) comparing Aspergillus flavus wild type (infact gene) with a veA deletion fungal strain were performed to elucidate veA-affected regulatory pathways that might be involved in controlling toxin production, sporogenesis (spore or seed formation), or formation of sclerotia (fungal survival structures). The results from this study indicated that genes involved in nitrogen metabolism are regulated by veA, including niaD, encoding a nitrate reductase (involved in nitrogen metabolism). In this experiment, niaD expression was significantly lower in the veA mutant compared to the wild- type. Microscopic examination of sclerotia showed that the niaD mutant produced fewer and smaller sclerotia than the wild-type strain. Analysis of conidial (spore) formation and aflatoxin production showed that the niaD mutant (where this gene is rendered non-functioanl) produced about 10-fold more conidia than the wild-type, but less aflatoxin. These results indicate that nitrogen metabolism plays a role in both secondary metabolism and development in Aspergillus flavus and that expression of some of the genes involved are veA-dependent. We are also charactering a new secondary metabolism gene cluster containing a gene whose expression, according to our microarray analysis, is veA-dependent. Other selected genes from these functional genomic studies will be characterized in the near future. In addition, we are evaluating the effect of osmotic and oxidative stress in Aspergillus flavus mycotoxin production. Research progress was monitored through teleconferencing, frequent email communications, and reports.
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