Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Fellow participants (scientists and industry stakeholders) in the NC-213 multistate project Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two undergarduates have worked on portions of this project, and have been trained in general microbiology and molecular biology techniques. How have the results been disseminated to communities of interest? A poster was presented at the Fungal Genetics meeting in Pacific Grove, CA, in March, 2013; additionally, I attended the annual meeting of the NC-213 multistate project in Kansas City, MO, in February, 2013, and discussed our research and results with other participants. What do you plan to do during the next reporting period to accomplish the goals? Conclude the gene expression study of fungus and wheat; write up and publish results. Continue supervising undergraduates in the study of latent wheat infection, shifting funding to other sources (i.e. Nebraska Wheat Board) as this funding for me on this project is now at an end.
Impacts
What was accomplished under these goals?
The above goals are those of the overall project, not all of which are relevant to each individual investigator. Under goal 2 (and, to a certain extent, goal 3), my lab is concluding greenhouse studies of the interaction between susceptible and resistant wheat and the wheat scab fungus,Fusarium graminearum, in the first several days of plant infection, before visible symptoms in the plant. We are doing this by analyzing the gene expression of both plant and fungus. For the fungus, gene expression analysis is complete and shows, hour by hour, where the fungius is in the wheat head relative to the inoculation point and whether the fungus is producing the virulence factor and mycotoxin deoxynivalenol. Plant gene expression analysis is ongoing, and is expected to provide a better understanding of specific genes involved in the wheat-fungus interaction than has been provided by other methods (such as QTL mapping). This knowledge, in turn, could inform wheat breeding practices to better produce scab-resistant plants without loss of valuable agronomic traits. Additionally, my lab has begun evaluation of several hundred wheat accessions used by Nebraska breeders for latentFusariuminfection in the seed. Understanding of latent, asymptomatic carriage ofFusariumby wheat can better explain the ecology of the wheat-fungus interaction, while carriage of asymptomaticFusariummay protect wheat from infection with virulent strains, as has been observed in other crop-pathogen systems (such as corn-nontoxigenicAspergillus flavus, or perennial ryegrass and nontoxigenic lolitreme endophytes).
Publications
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Progress 10/01/10 to 09/30/13
Outputs
Target Audience: Fellow participants (scientists and industry stakeholders) in the NC-213 multistate project Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two undergraduates have worked on portions of this project, and have been trained in general microbiology and molecular biology techniques. How have the results been disseminated to communities of interest? A poster was presented at the Fungal Genetics meeting in Pacific Grove, CA, in March, 2013; additionally, I attended the annual meeting of the NC-213 multistate project in Kansas City, MO, in February, 2013, and discussed our research and results with other participants. What do you plan to do during the next reporting period to accomplish the goals? Conclude the gene expression study of fungus and wheat; write up and publish results. Continue supervising undergraduates in the study of latent wheat infection, shifting funding to other sources (i.e. Nebraska Wheat Board) as this funding for me on this project is now at an end.
Impacts
What was accomplished under these goals?
The above goals are those of the overall project, not all of which are relevant to each individual investigator. Under goal 2 (and, to a certain extent, goal 3), my lab is concluding greenhouse studies of the interaction between susceptible and resistant wheat and the wheat scab fungus, Fusarium graminearum, in the first several days of plant infection, before visible symptoms in the plant. We are doing this by analyzing the gene expression of both plant and fungus. For the fungus, gene expression analysis is complete and shows, hour by hour, where the fungus is in the wheat head relative to the inoculation point and whether the fungus is producing the virulence factor and mycotoxin deoxynivalenol. Plant gene expression analysis is ongoing, and is expected to provide a better understanding of specific genes involved in the wheat-fungus interaction than has been provided by other methods (such as QTL mapping). This knowledge, in turn, could inform wheat breeding practices to better produce scab-resistant plants without loss of valuable agronomic traits. Additionally, my lab has begun evaluation of several hundred wheat accessions used by Nebraska breeders for latent Fusarium infection in the seed. Understanding of latent, asymptomatic carriage of Fusarium by wheat can better explain the ecology of the wheat-fungus interaction, while carriage of asymptomatic Fusarium may protect wheat from infection with virulent strains, as has been observed in other crop-pathogen systems (such as corn-nontoxigenic Aspergillus flavus, or perennial ryegrass and nontoxigenic lolitreme endophytes).
Publications
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Winter wheat of Fusarium head blight (FHB)-susceptible cultivar Wesley and resistant cultivar Wesley-FHB was grown in the greenhouse at the University of Nebraska-Lincoln and inoculated with the pathogenic fungus Fusarium graminearum. Beginning 120 hours post-inoculation, and hourly until 168 hours post-inoculation, kernels at defined distances from the inoculation point were harvested. RNA was extracted from these kernels for quantitative reverse transcript PCR (qRT-PCR) to confirm 1) presence of fungus (detection of fungal housekeeping gene transcripts) and 2) whether or not fungus was producing the mycotoxin and virulence factor deoxynivalenol (DON). Based on these findings, RNA was prepared for Affymetrix Wheat GeneChips, which would provide a comprehensive profile of wheat gene expression during fungal infection. Timepoints for data collection were empirically determined on the basis of the previous year's results. The project results, from its inception through March 2012, were presented at the annual NC-213 meeting, held in Minneapolis, MN, March 6-7, 2012. Whate maturation was not synchronous and RNA quality was found insufficient for Affymetrix GeneChip analysis. Consequently, new plantings have been made using spring wheat, susceptible cultivar Wheaton and resistant cultivar Alsen, with which I have extensive past experience. PARTICIPANTS: PI Heather Hallen-Adams inoculated wheat, harvested all wheat samples, participated in the RNA extraction and qRT-PCR and was responsible for data analysis. Graduate student Maria de Jesus Quintero (has since left graduate studies) performed qRT-PCR, and was taught to do so as part of her graduate training. Undergraduate Richard Spinner (has since graduated) transplanted wheat and performed RNA extraction. This was part of his training in molecular biology. TARGET AUDIENCES: Target audiences include wheat growers and breeders, and plant pathologists. PROJECT MODIFICATIONS: After two years, winter wheat is not displaying the same degree of synchronous development I have come to expect with spring wheat, and RNA extractions, while suitable for qRT-PCR, have not been of sufficient quality for Affymetrix GeneChip analysis. For the final year of the project, I am therefore shifting to spring wheat, which can be planted year-round, has a shorter growth cycle (not requiring vernalization), and which in my past experience has exhibited highly synchronous development and from which I have not had difficulty extracting high quality RNA. Additionally, I am performing all RNA extractions myself to minimize variation caused by differing personnel.
Impacts
Fungal housekeeping genes, and therefore fungal presence, was detected from the kernels nearest the inoculation point in the earliest time points monitored (as early as 93 hours post-inoculation, although hourly sampling did not begin until 120 hours post-inoculation), in both FHB-susceptible and -resistant winter wheat. Mycotoxin gene expression was also detected at the earliest time points, with a slight lag for the Tri8 gene (near the end of the DON biosynthetic pathway) compared with the Tri5 gene (the first step in DON biosynthesis). Differences in the timing of DON biosynthetic gene expression must be confirmed by further study and in different wheat cultivars; if verified, they may suggest targets for control.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: This project contributes to the understanding of the genetic mechanisms of Fusarium head blight (FHB) in wheat, with an especial effort to characterize the "cross-talk" between plant and fungus during the early stages of infection in order to identify wheat genes/proteins directly inductive of or responsive to the mycotoxin and virulence factor deoxynivalenol (DON). 250 FHB-susceptible plants and 250 FHB-resistant plants derived from winter wheat var. "Wesley" were inoculated with Fusarium graminearum. Visible disease progress was noted over a 25 day period. Kernels immediately adjacent to, and 4 spikelets below, the inoculation point were harvested daily from 4-8 and 17-25 days post-inoculation, and every three hours from 5-7 days post-inoculation. RNA was extracted from the kernels and real-time PCR was used to determine 1) fungal presence or absence and 2) expression of DON genes in the presence of fungus. This data will be used to determine the timing of the initiation of DON gene expression and thus the optimal time frame for hourly sampling of kernels; and select samples within five hours of DON gene expression initiation will be analyzed using the Affymetrix Wheat GeneChip microarray. PARTICIPANTS: This project was performed by the PI, Heather Hallen-Adams, and by three undergraduate research assistants at the University of Nebraska-Lincoln, Mr. Alex Hlavaty, Mrs. Geraldine Spinner and Mr. Richard Spinner. These students, juniors at the start of the project, were trained in safe handling of plant pathogenic fungi; wheat inoculation; safe and hygienic laboratory procedures; RNA isolation and purification; cDNA synthesis, and quantitative reverse-transcript PCR. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Results from real-time PCR identify a period of 160-180 hours post-inoculation as the time when DON mycotoxin gene expression begins. This information will be used to collect hourly samples and confirm (to within the hour) the start of DON gene expression. These samples will be used for wheat gene expression profiling, and next years' impacts/outcomes will include the identification of candidate wheat genes responsible for eliciting DON production, and for responding to DON.
Publications
- Hallen-Adams HE, Wenner N, Kuldau GA, Trail F (2011) Deoxynivalenol biosynthesis-related gene expression during wheat kernel colonization by Fusarium graminearum. Phytopathology 101:1091-1096.
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