Progress 10/01/04 to 09/30/08
Outputs
OUTPUTS: The majority of this research was conducted as part of a PhD program in Cereal and Food Sciences. This provided the student with the opportunity to gain skills in mycology, enzymology, protein biochemistry, and data analysis. The final results of this work were presented at the 2007 National Fusarium Head Blight Forum (Kansas City, MO. December 2007). The audience at the NFHBF includes most North American researchers who are currently working on aspects of Fusaurium head blight of wheat and barley, and represents academics, government, and private industry. PARTICIPANTS: Principal Investigator: Paul B. Schwarz, Professor, Department of Plant Sciences, North Dakota State University. Developed original project concept and supervised research and administrative aspects of the project. Steven Meinhardt, Associate Professor, Department of Plant Pathology, North Dakota State University. Provided expertise and equipment associated with protein purification. Partner Organizations: North Dakota Barley Council, State Board of Agricultural Research (ND SBARE), provided additional financial support for the project. Collaboration and Contacts: Jan Delcour, Laboratory of Chemistry and Biochemistry at the Catholic University Leuven, Belgium. Conducted inhibition studies using TAXI and XIP. Training or Professional Development: Xinrong Dong, graduate research assistant. Conducted much of the research associated with the project as part of her PhD program. TARGET AUDIENCES: The xylanases produced by Fusarium graminearum have a potential role in pathogenesis. The audience for this work was researchers working on various aspects of Fusarium head blight of wheat and barley. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
This work demonstated a number of new facts regarding the xylanases of Fusarium graminearum. The production of xylanase by F. graminearum was found to be highest when using a solid state culture that consisted of wheat bran an hydrated with a synthetic medium. Wheat essentially represents the natural substrate of the pathogen. Two xylanase were isolated and purified from this culture. The purified enzymes were identified by LC-MS/MS as the products of genes FG06445 and FG03624 with 61% and 51% sequence coverage. These are subsequently referred to as high (HMW) and low molecular weight (LMW) xylanase, respectively. Our work has confirmed the results of previous proteomics studies that suggested these are the primary xyalanases produced by F. graminearum. The purification protocol developed here, employed ion-exchange, gel filtration, and hydrophobic interaction chromatography, and resulted in 125 and a 268-fold purification of the HMW and LMW xylanases, respectively. Basic biochemical characterization of the enzymes estimated molecular weight of the HMW xylanase at 41,000 and LMW xylanase at 17,000. Both enzymes showed high activity between pH 5.5 and 8, with maximal activity at pH 6. Both enzymes had similar temperature dependencies, with 60% of maximal activity between 35 and 60C, and maximal activities at 45- 50C. Kinetic studies using both wheat arabinoxylan and beechwood xylan, showed xylan to be the preferred substrate. The enzymes were tested against the endogenous proteinaceous endoxylanase inhibitors, TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibiting protein). Their work has confirmed that the LMW xylanase is largely inhibited 90% by TAXI but is not significantly inhibited by XIP. The HMW xylanase was tested only with XIP and was not inhibited by this protein. These results support the suggestion that TAXI may have a role in the plants defense against the Fusarium pathogen.
Publications
- No publications reported this period
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Progress 10/01/06 to 09/30/07
Outputs
Research activities focused on the isolation and characterization of xylanase from Fusarium graminearum, The in-vitro production of xylanase(s) by F. graminearum was found to be optimal in cultures using wheat bran, 1.66 mU/mg-protein, as opposed to 0.74 mU/mg-protein with agar plates containing peptone as the carbon source. Two xylanases were initially identified following ion-exchange chromatography (SP-Sepharose). The two xylanases were separated by the first ion-exchange step, and were then processed individually through subsequent steps. Subsequent purification of the two xylanases was 52- and 40- fold, respectively by a combination gel filtration (Superdex 75), HPLC ion-exchange (SP) and HPLC hydrophobic interaction chromatography (Phenyl Sepharose). The purity and the relative molecular weights of the xylanases were estimated by SDS-PAGE to be 20 and 40 KDa, respectively. Only a single band was observed for each enzyme. The sequences of the two xylanases were analyzed by trypsin digestion followed by LC-MS/MS. Results of this work were presented at the 2007 National Fusarium Head Blight Forum.
Impacts
Fusarium Head Blight of wheat and barley has caused annual losses in excess of $170 million to wheat and barley growers of North Dakota. Results from this work will be useful in addressing the interaction between the Fusarium pathogen and the host plant, an area that has previously received little attention. Establishing the importance of Fusarium cell wall degrading enzymes in the disease will assist breeders, geneticists and pathologists in identifying resistance sources, such as inhibitors of the cell wall degrading enzymes of Fusarium. Information from the LC-MS/MS work has strongly suggested that the two xylanases are products of two previously identified putative genes, FG03624 and FG06445. For the high molecular weight xylanase, FG06445, 84% of the sequence was observed while for the low molecular weight xylanase, FG03624, 65% of the sequence was identified. After removal of the predicted signal sequence, the predicted molecular masses were 22 and 38 KDa, which is in fact very close to what was observed by SDS-PAGE. The inhibitory effect of endogenous xylanase inhibitors (TAXI, XIP) from wheat on the two xylanases is currently under evaluation.
Publications
- Meinhardt, S., Dong, X., and Schwarz, P. Isolation of two xylanases from Fusarium graminearum. 2007. Proceeding of the 2007 National Fusarium Head Blight Forum; Dec. 2-4, Kansas City, MO. East Lansing: Michigan State University. pp 27.
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Progress 10/01/05 to 09/30/06
Outputs
The objectives of this project are to: 1.) isolate and characterize xylanase(s) from Fusarium graminearum, 2.) investigate factors controlling the production of xylanase(s) by F. graminearum during the infection of barley, and to 3.) investigate the effect of endogenous xylanase inhibitors on the activity of F. graminearum xylanase(s). During the previous period, optimal culture conditions for the production of xylanase(s) by F. graminearum were established. Maximum xylanase production was observed on sterile wheat bran with trace-nutrients. Work in the current period has focused on purification of xylanase(s) from this media. Crude extracts are prepared from the solid state culture by extraction with sodium acetate buffer. Xylanase is assayed using a dyed cross-linked arabinoxylan, and protein determined with Commasie blue. Xylanase activity in the crude extracts is very stable, but activity significantly declines with purification, suggesting the co-purification of a
protease, or loss of an essential cofactor. Inclusion of a standard protease inhibitor cocktail had little effect on activity, but inclusion of Mg++ salts appears to stabilize activity. Preliminary purification efforts evaluated the use of ion-exchange (SP-Fast Flow) and gel filtration (Superdex 75) chromatography. While only modest gains in purification-fold have been achieved through these techniques, there appears to be two xylanase activities. The major activity is associated with a protein of approximately 20-30 kDa, and a minor activity with a smaller protein. We are currently evaluating the use of affinity chromatography using media that was prepared by cross-linking wood xylan with epichlorohydrin. Xylanase binds to this media and can be released with a salt gradient.
Impacts
Fusarium Head Blight of wheat and barley has caused annual losses in excess of $170 million to wheat and barley growers of North Dakota. This project addresses an area of the interaction between the Fusarium pathogen and the host plant that has previously received little attention. Establishing the importance of Fusarium cell wall degrading enzymes in the disease may assist breeders, geneticists and pathologists in identifying resistance sources, such as inhibitors of the cell wall degrading enzymes of Fusarium. Slow progress in the development of resistant barley and wheat cultivars is due to a limited number of resistance genes, difficulty in incorporating exotic resistance genes into commercially acceptable cultivars due to linkage with important quality and agronomic traits, the complex genetics of FHB resistance, and to a poor understanding of FHB disease mechanisms.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
The objectives of this project are to: 1.) isolate and characterize xylanase(s) from Fusarium graminearum, 2.) investigate factors controlling the production of xylanase(s) by F. graminearum during the infection of barley, and to 3.) investigate the effect of endogenous xylanase inhibitors on the activity of F. graminearum xylanase(s). Culture conditions for the production of xylanase(s) by F. graminearum were evaluated. Media evaluated included potato dextrose agar (PDA), potato agar (PA), synthetic agar (agar, peptone, micro-nutrients) and modified synthetic agar (synthetic agar plus trace-nutrients). Xylan or sterilized wheat bran were added as inducers. Culture was conducted at 20 degrees C, pH 5.0, for 7 days. Initial growth was more rapid with bran than with xylan. The greatest level of xylanase activity was observed with the modified synthetic medium including bran. The lowest level of production was with PDA. Liquid and solid-state cultures were compared using
the synthetic or modified synthetic media. Solid-state culture yielded a higher productivity per unit volume, but liquid cultures provided higher xylanase yields (units/g substrate).
Impacts
Fusarium Head Blight of wheat and barley has caused annual losses in excess of $170 million to wheat and barley growers of North Dakota. This project addresses an area of the interaction between the Fusarium pathogen and the host plant that has previously received little attention. Establishing the importance of Fusarium cell wall degrading enzymes in the disease may assist breeders, geneticists and pathologists in identifying resistance sources, such as inhibitors of the cell wall degrading enzymes of Fusarium. Slow progress in the development of resistant barley and wheat cultivars is due to a limited number of resistance genes, difficulty in incorporating exotic resistance genes into commercially acceptable cultivars due to linkage with important quality and agronomic traits, the complex genetics of FHB resistance, and to a poor understanding of FHB disease mechanisms.
Publications
- No publications reported this period
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