Progress 10/01/06 to 09/30/12
Outputs
OUTPUTS: Throughout this project experiments were performed to identify and characterize toxins produced by various plant pathogens, and to understand the interactions between the wheat nuclei and its mitochondria. Using new isolates, different media for their growth, and various chromatographic methods, several new toxins were identified in Pyrenophora tritici-repentis and Stagonospora nodorum and initial purifications were performed. In addition a method was developed to detect mutations in P. tritici-repentis that result in resistance to the strobulurin type fungicides. The mitochondrial genome of Triticum trugidum, Triticum tauschii, Triticum longissimum, and the alloplasmic line [(lo)scs(ti)scs(d)], which contains the T. longissmum mitochondria in a durum background, were sequenced, assembled and compared. In addition the mitochondrial proteome of two alloplasmic lines, [(lo)scs(ti),scs(d)] and [(lo)scs(d) __], were compared and differences identified. Most recently experiments were performed to investigate the effect of nuclear cytoplasmic interactions on the resistance/susceptibility of wheat to foliar diseases. Fertile alloplasmic lines were innoculated with two isolates of P. tritici-repentis, BR15 which contains all previously identified toxins and the most virulent isolate in our collection Pti2, and disease rated on a 1-5 scale five days after infection. The same alloplasmic lines were also inoculated with a mixture of three rust races (Puccinia recondita f. sp. tritici) and disease ratings taken at 10-12 days after infection. From the research performed in this project at total of 11 publications were produced and 17 posters or presentations were given at 8 different national and international meetings. PARTICIPANTS: The S. nodorum research was performed in collaboration with Dr. T. L. Friesen and J. D. Faris of the USDA Northern Crops Research Center on the NDSU campus. Research on the P tritici-repentis tox A mutation was performed by Ms. Christine Tandeski (technician) and Dr. Kevin Gyolai (graduate student). In collaboration with Dr. T Adhikari (Dept of Plant Pathology NDSU) and Jaimin Patel (graduate student), additional toxins in P. tritici-repentis are being identified and characterized. Drs. S. Meinhardt and K. Kianian are PIs on the nuclear mitochondrial interaction project leading the design of the experiments. Dr. Anantharama Rishi is a post doctoral researcher has performed the mitochondrial isolation, electron microscopy and mass spectrometry studies. Andrzej Noyszewski (graduate student) preformed the mitochondrial genome sequencing and assembly. Ryan burciaga (graduate student) performed the experiments testing the alloplasmic lines for resistance to the foliar pathogens. TARGET AUDIENCES: The target audience for this research has been other researchers studying the interactions between P. tritici-repentis and S. nodorum. The project to investigate nuclear cytoplasmic interactions is targeted towards researchers working to understand how the mitochondrial genome interacts and communicates with the nucleus. In addition this project will help our understanding of the role of mitochondria in disease resistance/susceptibility. With further work, this information will be of aid to breeders generating more resistant wheat lines and may be of importance in understanding the role of mitochondria in human diseases. Eventually this research will provide information that will help farmers to be more productive and healthy. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Additional toxins were identified in S. nodorum most notable Snn2 which is a proteinaceous toxin. Two non-proteinaceous toxins were identified in P. tritici-repentis, the first causing chlorosis and the second necrosis. The necrosis toxin was partially purified through selective partitioning between different solvents. Characterization of the necrosis toxin indicates that it is a non-host-selective toxin. Still there appears to be some resistance since the upper leaves of some wheat lines show little response to the toxin while other lines show complete necrosis. The mitochondrial genome of the alloplasmic line [(lo)scs(ti)scs(d)] showed significant rearrangements compared to its mitochondrial donor T. longissimum and to T. turgidum. Several new open reading frames were identified that were composed of pieces of other known mitochondrial genes. The rearrangement of the mitochondrial genome appeared to occur in a block fashion where certain groups of genes retained their order. A total of 7 large gene blocks and four pairs of genes were found be conserved in T. turigum, T. longissimum, and the alloplasmic line. A few genes found in the T .turgidum mitochondrial genome were absent from either T. longissimum or the alloplasmic line. Preliminary screening of 48 fertile alloplasmic lines for increased resistance or sensitivity to the P. tritici-repentis isolates BR15 and Pti2 when compared to the nuclear donor. Fourteen lines showed a statistically significant differential response to BR15 and seven show a differential response to Pti2. None of the lines showed complete resistance. While most lines showed increased resistance, two lines showed increased senitivity to BR15 and two to Pti2. Six alloplasmic lines exhibited increased resistance to both fungal isolates. Twenty one alloplasmic lines were then tested for resistance to a combination of three leaf rust races common to North Dakota. Nine of these lines gave a resistant phenotype while their parent was susceptible. Only one line showed decreased sensitivity to both P. tritic-repentis isolates and to the rust isolates. Screening to identify additional alloplasmic lines with disease resistance will be continued. The line showing increase resistance to the pathogens tested will be investigated to determine the mechanism of this resistance.
Publications
- Jaimin S. Patel, Neil C. Gudmestad, Steven Meinhardt, and Tika B. Adhikari (2012) Pyraclostrobin Sensitivity of Baseline and Fungicide Exposed Isolates of Pyrenophora tritici-repentis. Crop Protection 34:37-41
- Dong, X., Meinhardt, S.W., and Schwarz, PB (2012) Isolation and characterization of two endoxylanases from Fusarium graminearum. J. Agri. Food Chem. 60:2538−2545.
- Delgado, J. A., Meinhardt, S., Markell, S.G., Goswami, R.S., (2012) Gene Cloning Using Degenerate Primers and Genome Walking. In: Plant Fungal Pathogens, Methods in Molecular Biology (Bolton, M. D.; Thomma, B. P. H. J., Eds.) Vol. 835, pp. 611-622.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Investigation of nuclear-mitochondrial interaction in the alloplasmic line containing the Triticum logissimum cytoplasm in a Triticum durum nuclear background has continued. Assembly of the mitochondrial genomes of Triticum logissimum, Triticum turgidum, Triticum taushii, the (lo)scsti scsd alloplasmic line (durum(lo)) have been completed and many of the differences identified have been investigated. Differential gel electrophoresis have been used to identify mitochondrial proteins with statistically significant changes in concentration of the weak durum wheat [(lo) scsd __ ] compared to the strong [(lo)scstiscsd] durum wheat. Work has continues on the identification of factors important for the interaction of wheat and Pyrenophora tritici-repentis. A method to measure the appearance of several different mutations in the mitochondrial cytochrome b gene that result in resistance to Qoi fungicides in P. tritici repentis was developed. In addition, a method was developed for the partial purification of a newly identified toxin from P. tritici repentis. PARTICIPANTS: Work on the mitochondrial genome is being performed in collaboration with Dr. S. Kianian. A postdoctoral researcher, Dr. A. Rishi, has performed the proteomics investigation of the mitochondria. Studies of the P. tritici-repentis toxins and development of the resitance assay was performed in collaboration with Drs. T. Adhikari and J. Patel. TARGET AUDIENCES: This project will support the efforts of researchers to understand the physiology of wheat and its interaction of plant pathogens, which may lead to the development of healthier and disease resistant line. Ultimately this research should help wheat farmers throughout the united states. PROJECT MODIFICATIONS: The research involving the understanding of nuclear mitochondrial interactions has been added.
Impacts
Analysis of the mitochondrial genomes from Triticum turgidum, Triticum logissimum, durum (lo), and Triticum tauschii indicated multiple differences. The assembly of the T. turgidum mitochondrial genome was collinear with Triticum aesitivum. Within the genomes there are a large number of rearrangements. Seven blocks of four or more genes, represent 39 of the 61 genes identified, are moved together maintaining the relative gene order. In addition there are five groups of two genes that appear to move together. Four ORFs (273, 240, 349, and 359) were present in T. turgidum and T. longissimum. Only ORF 359 was absent from durum (lo). Multiple additional OFRs have been identified and are being analyzed. Analysis for small nucleotide polymorphisms is in progress. Differential two dimensional polyacrylamide gels electrophoresis of the proteins found in mitochondria from weak and strong plants showed 13 protein spots showed with statistically significant changes. All but one spot was increase in the weak plants. Three protein spots increased by 1.2 to 2.7 fold were identified as heat shock like proteins. Five protein spots contained proteins involved in the mitochondrial portion of photorespiration increased 1.3 to 1.6 fold. One protein spot was a previously identified male sterility restorer protein, mitochondrial aldehyde dehydrogenase. Two protein spots contained at least two proteins one of which had been previously identified as involved in photorespiration. One protein spot also contained chloroplast ATP synthase alpha subunit (increased 1.3 fold) and the other RUBISCO large subunit (decreased 1.4 fold). The changes in these two chloroplast proteins suggest that the difference was not due to different levels of chloroplasts contamination. Photorespiration provides a means for plants to remediate of phosphoglycolate formed when Ribulose bisphosphate carboxylase reacts with oxygen rather than carbon dioxide and involves enzymes in the chloroplast, mitochondria and peroxisomes. The net result is the loss of one carbon dioxide molecule and the use of 8.25 ATP equivalents. This process can reduce the ability of the plants to fix carbon dioxide reducing plant growth and creating an energy deficiency. Resistance to the strobilurin (Qoi) type fungicides has become a significant problem for multiple fungal plant pathogens worldwide. Resistance to the Qoi fungicides in P. tritici-repentis has been present in Europe for several years but has not appeared in the United States. A simple two-step method using PCR followed by restriction digestion was developed to screen for the point mutations in P. tritici-repentis and Pyrenophora terres, G143A and F129L, that result in a resistant isolate. This method can now be used to monitor for the apearnace of resistant isolates of P. tritici-repentis in the United States. A protocol for the partial purification of a new necrosis toxin from P. tritici-repentis has been developed. It consists of a series of partitioning steps followed by reverse phase chromatography. The host specificity of this toxin has been drawn into question and this is being characterized at this time.
Publications
- Patel, JS, Meinhardt, SW, Sierotzki, H, Stammler, G, Gudmestad, NC and Adhikari, TB. 2011. A Two-Step Molecular Detection Method for Pyrenophora tritici-repentis Isolates Insensitive to QoI Fungicides. Plant Dis. 95:1558-1564.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The project investigating nuclear mitochondrial interaction using the alloplasmic lines with Triticum logissimum cytoplasm in a Triticum durum nuclear background has continued. The mitochondrial genomes of Triticum logissimum, Triticum turgidum, Triticum taushii, the (lo)scs-ti scs-d alloplasmic line have been sequenced by 454 sequencing, are being assembled and the differences studied. Two dimensional gels were made from mitochondria purified by differential centrifugation and density gradient centrifugation. Differential spots were excised, trypsin digested and identified by LC tandem mass spectrometery. Mitochondria purification from wheat leaves has proved difficult due to the large number of chloroplasts present in the leaves. In an attempt to obtain purer mitochondria samples, antibodies to the Transfer of Outer Membrane 20 protein (TOM20 protein), which is part of the protein complex that translocates proteins through the outer mitochondrial membrane, have been generated. Cross reactivity with chloroplast proteins was checked by western blot. These antibodies were used to further purify the isolated mitochondria by affinity chromatography. Purity of the preparation was checked using western blotting and anti-RUBISCO antibodies to probe the samples. Investigation of the newly identified toxin from Pyrenophora tritici-repentis has continued. The toxin was extracted from the aqueous phase and then extracted a second time from the organic phase to the aqueous phase by changing the extraction conditions. Further purification using solid phase extraction has been tried. PARTICIPANTS: Drs. S. Meinhardt and K. Kianian are PIs on the nuclear mitochondrial interaction project leading the design of the experiments. Anantharama Rishi is a postdoctoral researcher has performed the mitochondrial isolation, electron microscopy and mass spectrometry studies. Studies of the toxins from P. tritici-repentis are in collaboration with Dr. T Adhikari. TARGET AUDIENCES: The groups that this information is pertenent to are the scientist interested in germplasm enhancement of wheat and scientists interested in understanding nulcear mitochondrial interactions PROJECT MODIFICATIONS: After teaching Drs. Friesen and Faris' laboratory groups about protein purification they have become relatively independent of my group for protein purification. I continue to consult on purification processes but I am not actively working on purification of toxins that they are identifying. The project with Dr. Kianian arose about the same time and uses both my expertise in proteomics and my knowledge of mitochondrial function. Therefore my efforts have moved more to this project. Both projects are involved in understanding wheat and its health. I have continued to work on P. tritici-repentis with Dr. Adhikari.
Impacts
The T. aestivum mitochondria genome was previously sequenced and is available in Genbank. Assembly of the mitochondrial genomes of Triticum logissimum, Triticum turgidum, Triticum taushii, the (lo)scs-ti allowpalsmic line was initially performed with T. aesitivum as the reference assembly but only T. turgidum was similar to T. aestivum. The other genomes were subjected to de novo assembly. Assembly has progress with nearly complete assembly of the T. tugidum and (lo)scs-ti mitochondrial genomes. Unfortunately T. logissimum sequence was significantly contaminated with bacterial DNA and the removal of these sequences prior to assembly is required. Still most of the known mitochondrial genes from T. logissimum could be assembled. All samples were also contaminated with chloroplast DNA but sequenced at only 5X coverage. By comparison to the T. aestivum chloroplast genome, it has been possible to remove these sequences as well as assemble a copy of the chloroplast genomes with 5X coverage for each of these species. Coverage of all assembled mitochondrial areas was at least 20X and up to 40X. A complete circle of the DNA could not be made for any species with the present data. The mitochondrial genome of T. turgidum was almost identical to that of T. aestivum with only a few single nucleotide polymorphisms. The alloplasmic line (lo)scs-ti scs-d showed significant rearrangements of the genes when compared to T. aestivum. Comparison of the changes in the amino acid sequences is now being made. Comparison of two dimensional electrophoresis gels of the weak and strong mitochondrial proteins indicated 12 proteins that were consistently different in three experiments. These were excised and sent for mass spectrometry analysis. The majority of these proteins were of chloroplast origin. This indicating that in our preparations the differences in chloroplast contamination was a major factor and would most likely mask any changes in mitochondrial proteins. Western blots of samples from purified chloroplasts indicated that the antibody made to TOM20 did not cross react with any protein in the chloroplast. Purification of mitochondria using these antibodies and anti-rabbit antibodies coupled to magnetic beads resulted in what appeared to be a significant purification of the mitochondria. Western blot analysis comparing the amplitude of the RUBISCO protein signal present to that of the TOM 20 in both the affinity purified mitochondria and the starting material showed only an approximately 50% difference. This suggests that either our antibodies do bind to a chloroplast surface membrane protein or that RUBISCO is sticking to the outside of the mitochondria. We are currently comparing through differential gel electrophoresis the affinity purified mitochondria to purified chloroplast in an attempt to identify chloroplast proteins. After this differential gel electrophoresis will be used to compare (lo)scs-ti __ mitochondrial proteins (weak plants) to those of (lo)scs-ti scs-d mitochondria (strong plants). Extraction studies, temperature stability, and protease sensitivity indicate that the newly identified P. tritici-repntis toxin is not proteinaceous in nature.
Publications
- Faris JD, Zhang Z, Lu H, Lu S, Reddy L, Cloutier S, Fellers JP, Meinhardt SW, Rasmussen JB, Xu SS, Oliver RP, Simons KJ, and Friesen TL. (2010) A unique wheat disease resistance-like gene governs effector-triggered susceptibility to necrotrophic pathogens. Proc. Natl. Acad. Sci. 107(30) p13544-13549
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: A project to understand the coordinated regulation of the mitochondrial and nuclear genomes in wheat was initiated. An alloplasmic durum line containing the T. logissimum cytoplasm exhibits normal growth when the scs(ti) gene, the species cytoplasmic specific gene from T. timopheevii, is present but stunted growth when a homologous durum gene scs(d) is present. Using these two lines, and the parents used to generate them, we have investigate the proteome and development of the mitochondria. Electron microscopy studies to determine the number and size of the mitochondria were performed on plants grown for 6 weeks and on plant obtained after only 2 weeks of growth. Preliminary shotgun proteomics studies were performed on mitochondria obtained from the strong plants in collaboration with the University of Minnesota Center for Mass Spectrometry and Proteomics. The isolated mitochondria were sonnicated for 5 min and then treated with trypsin. The membranes were pelleted by centrifugation and the supernatant recovered. The peptides present in the supernatant were analyzed by liquid chromatography tandem mass spectrometry. The spectra were analyzed using the viridiplantae and wheat unigene databases using various assignment packages. Two-dimensional gel electrophoresis has been started on the mitochondria preparation. Further work to develop a more complete protein database will continue. Several media were investigated to provide for the routine in vitro production of a newly identified necrosis toxin found in a Pyrenophora tritici-repentis isolate recovered in Arkansas. PARTICIPANTS: Drs. S. Meinhardt and K. Kianian are PIs on the nuclear mitochondrial interaction project leading the design of the experiments. Anantharama Rishi is a post doctoral researcher has performed the mitochondrial isolation, electron microscopy and mass spectrometry studies. The University of Minnesota Mass Spectroscopy and Proteomics Center has performed all LC-tandem MS studies and assisted in protein identification. TARGET AUDIENCES: The projects described here are directed towards the improvement of wheat for producers. The information gained will also improve our understanding of the impediments to cross species gene transfer for wheat breading. PROJECT MODIFICATIONS: The interaction between nuclear and mitochondria genomes has been added since it provides a basic understanding of speciation which may lead to easier introgression of foreign disease resistance genes to improve wheat disease resistance. The weak plants observed are significantly more sensitive to disease indicating that incompatibility of the nuclear and mitochondrial genomes affects disease resistance.
Impacts
The electron microscopy studies showed no difference between the number and volume of the mitochondria found in the stunted and normal progeny after 6 weeks of growth. Light microscopy studies also indicated no difference in the cell size or development of the cells between the normal and stunted 6 week old plants. After 2 weeks of growth there is a two fold increase in the number of mitochondria found the stunted plants versus the normal plants. Preliminary shotgun proteomic studies identified only 110 different proteins within the sample identifying only 15% of the spectra obtained. Most spectra were of sufficient intensity to suggest that the difficulty in identification was not due to the sample or data acquisition. These results indicate that a more complete protein database will be necessary to use a shotgun approach to study the mitochondrial proteome of the wheat. The two dimensional gel electrophoresis studies indicate multiple differences in the proteome between the stunted and normal plants. These proteins will be excised and attempts will be made to identify them by LC-MS/MS. The in vitro production of the toxin will now allow further characterization of the toxin as well as genetic studies to determine the location of the sensitivity/resistance gene.
Publications
- Adhikari, Tika B., Bai, Jianfi, Meinhardt, Steven W., Gurung, Suraj, Myrfield, Mary, Patel, Jaimin, Ali, Shukate, Gudmestad, Neil C., Rasmussen, Jack B. (2009) Tsn1-Mediated Host Responses to ToxA from Pyrenophora tritici-repentis. Molec. Plant-Microb. Interact. 22(9):1056-1068.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The information obtained in this project has been presented as one publication and two posters (American Phytophathological Society and Plant Animal Genome conferences) over the last year. The information has also been provided to the breeders in the college to help with their selection processes. The research of one graduate student has been reported in his thesis and is now being written for publication. Methods for characterization of toxins are being taught to several graduate students in the department to be used as part of their research. PARTICIPANTS: The S. nodorum research was performed in collaboration with Dr. T. L. Friesen and J. D. Faris of the USDA Northern Crops Research Center on the NDSU campus. Research on the P tritici-repentis tox A mutation was performed by Ms. Christine Tandeski (technician) and Dr. Kevin Gyolai (graduate student). In collaboration with Dr. T Adhikari (Dept of Plant Pathology NDSU) and Jaimin Patel (graduate student), additional toxins in P. tritici-repentis are being identified and characterized. Work on the interaction of mitochondrial and nuclear genomes is in collaboration with Dr. Shahryar Kianian (Department of Plant Sciences). TARGET AUDIENCES: The target audience for this research will be wheat breeders and molecular plant pathologist study wheat diseases. This information will also be provided to extension plant pathologists to assist them in answering questions from farmers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research performed in the last reporting period has focused on the investigation of two wheat diseases Stagonospora nodorum and Pyrenophora tricitic-repentis. In collaboration with Drs. T. L. Friesen and J. D. Faris, the location of the sensitivity locus for the S. nodorum Toxin 2 was identified by molecular mapping (see publication). Several other toxins associated with the symptoms caused by S. nodorum infection have also been identified and are being characterized. At least one additional toxin has been identified in Pyrenophora tritici-repentis (tan spot) and is being investigated. Initial work indicated that the toxin was only present in intracellular wash fluids but continued variation in culture conditions and fungal isolate have allow the identification of the activity in culture filtrate solutions. Mutational analysis of Ptr. ToxA has continued and has been used to better define the region and nature of the interaction of this toxin with the host. In addition preliminary work on the genes expressed in wheat when infected with P. tritici-repentis has been completed. A new project investigating the interaction of the mitochondrial genome and the nuclear genome in wheat has been initiated with Dr. Shahryar Kianian. This project is in the initial stages but has the potential to provide basis for understanding the importance of this interaction in plant vigor and development.
Publications
- Reddy, Leela, Friesen, Timothy L., Meinhardt Steven W., Chao, Shiaoman, and Faris, Justin D. 2008. Genetic analysis and molecular mapping of a wheat gene (Snn2) conditioning sensitivity to a new semi-purified toxin (SnTox2) produced by Stagonospora nodorum. The Plant Genome 1:55-66.
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Progress 10/01/06 to 09/30/07
Outputs
Characterization of the toxins from Stagonospora nodorum and Pyrenophora tritici-repentis has continued. Several additional toxins from S. nodorum have been identified by Dr. Freisen. Purification of these toxins by chromatography is underway. An apparent chlorosis toxin was identified in P. tritici repentis based on the ability of partially purified culture filtrates to cause chlorosis on the wheat line ND495. We are currently working on the purification of this toxin. In collaboration with Dr. Paul Schwarz, Department of Cereal Sciences, NDSU, we have purified two xylanase enzymes secreted by Fusarium graminearum. Characterization of these enzymes is in progress.
Impacts
Through the use of fungal isolate characterization and column chromatography several proteinaceous toxins have been identified in Stagonospora nodorum. These are currently being investigated. Through the yeast two hybrid technique, the necrosis toxin from Pyrenophora tritici-repentis, Ptr ToxA, has been shown to interact with one of the electron transport proteins found in chloroplasts, plastocyanin. We are currently working on cloning and expressing plastocyanin to measure the strength of this interaction. Additional chlorosis toxins have been identified in P. tritici-repentis by the ability of crude culture filtrates to cause chlorosis on the wheat line ND495 which is resistant to the known chlorosis toxins. We have focused on one of these toxins and have purified it through two chromatographic steps. The purification has been hampered by the unexpected carry over of Ptr ToxA in the samples containing the chlorosis toxin. Two xylanases of Fusarium graminearum have been purified. The genes of these xylanases were identified by taking the purified proteins and separating them by SDS gel electrophoresis. The protein bands were excised from the gel digested with the protease trypsin, and the protein fragments sequenced by liquid chromatography tandem mass spectroscopy. The two xylanases are the products of the genes FG06445 and FG03624, which are secreted during growth of the fungus on bran and in the plant. The enzymatic properties of the two enzymes are being investigated.
Publications
- Tai,Yin Shai., Bragg, Jennifer, Meinhardt. Steven W. (2007) Functional characterization of ToxA and molecular identification of its Intracellular targeting protein in wheat. Amer. J. Plant Physiol. 2:76-89.
- Friesen T, Meinhardt S, Faris J. (2007) The Stagonospora nodorum-wheat pathosystem involves multiple proteinaceous host-selective toxins and corresponding host sensitivity genes that interact in an inverse gene-for-gene manner. The Plant Journal 51:681-692.
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