Progress 03/01/10 to 02/28/14
Outputs
Target Audience: Target audience includes: The research conducted under this grant will have high impact on the fungal community at large and should attract the attention of scientists in multiple plant science-related disciplines; for example, cell biology, molecular plant-microbe interactions, protein biochemistry, evolutionary biology, plant breeding and plant pathology in general. Changes/Problems: Due to the decrease in recommended funds, we will not be able to conduct all components of the three objectives listed in the original submission. This reduction in the work proposed is due to the high cost of the microarray studies and the intensive amount of work needed for microarray analyses and biochemical analyses proposed. It was necessary to reduce the amount of funds requested for supplies and services. It was also necessary to reduce the amount of funds requested to support the people necessary to conduct these studies. Therefore, we have reduced the amount of work to be conducted for Objective 1. Objective 1: Multifaceted analyses of ToxA interactions. 1A. Comparative analyses of the effects of ToxA on chloroplast function: It will be necessary to reduce the number of time point for control and treatment to 3 time points rather than 4. 1B. Evaluation of early signaling associated with ToxA endocytosis. It will be necessary to eliminate this entire component of Objective 1. Changes to proposed methods under objective 2: Microscopic evaluation and qPCR quantification of fungal mass in infected leaf samples suggested that a low percentage of fungal tissue would be a limiting factor to the originally proposed microarray approach; therefore, RNA-seq was investigated as an alternative. To determine if RNA-seq could detect fungal transcripts in early time point samples, a transcript library of infected leaves was prepared, and 51-mer single-end sequences acquired on an Illumina HiSeq2000 sequencer. Of 216M reads generated, a total of 1.8% mapped to the Ptr reference genome. We utilized reference genome-guided transcript assembly and quantification and compared this with an assembly of a similar number of sequence reads obtained from a library of transcripts expressed under culture conditions. Similar numbers of transcripts associated with previously annotated reference loci were detected in both libraries, of which 6283 were shared; 1003 and 1101 transcripts were differentially detected in the in planta and culture library, respectively. Functional annotations of the differentially detected-in planta transcripts reflect the requirements of the pathogen for host penetration, cell wall degradation and the need to counteract the host response to infection. Those differentially detected in the culture library reflect the regulation of genes required for rapid growth, including those involved in protein and DNA biosynthesis. Therefore, we determined that the number of fungal specific sequences present in early samples was sufficient to define differences in transcripts that are produced in response to differing growth conditions (manuscript in preparation). Changes to proposed approaches under objective 3: In addition to solving the structure of ToxB for insights into possible function, studies were conducted to determine if ToxB acts extracellularly or in an intracellular manner, as does ToxA. Briefly, studies showed that ToxB remains in the apoplast several hours after treatment. Additionally, intracellular expression of ToxB does not lead to symptom development. These data strongly support the hypothesis that ToxB remains in the apoplast and acts as an extracellular apoplastic effector (manuscript complete awaiting review, Figueroa, etal. 201X). What opportunities for training and professional development has the project provided? Mr. Ashley Chu, Postbaccalaureate researcher, contributed to the work described under Objective 1. Dr. Afua Nyarko, Postdoctoral Associate, Department of Biochemistry and Biophysics, Oregon State University; NMR data collection and analysis under Objective 3. Dr. Melania Figueroa Betts, Postdoctoral Fellow, Department of Botany and Plant Pathology, Oregon State University; Dr. Figueroa Betts contributed to the production in Pichia pastoris and the purification of 13C/15N-labeled ToxB for structural studies under Objective 3. Dr. Genevieve Weber, Postdoctoral Associate, contributed to the work on ToxA effects on plant responses in the presence of NAC under Objectives 1 and on determining fungal biomass in inoculated plants at different times after inoculation under Objective 2. Dr. Iovanna Pandelova, Research Associate, Department of Botany and Plant Pathology, Oregon State University; contributed to Objectives 1, 2, and 3. Ms. Viola Manning, Senior Research assistant, Department of Botany and Plant Pathology, Oregon State University; contributed to Objectives 2 and 3. Collaborations: Drs. Elisar Barbar and Afua Nyarko, Department of Biochemistry and Biophysics, Oregon State University; NMR data collection and analysis. Dr. Kiran Kumar Singarapu, Center for NMR and Structural Chemistry, Indian Institute of Chemical Technology; NMR data analysis. How have the results been disseminated to communities of interest? Invited Speaker, 2010 American Phytopathological Society Annual Meeting, Special Session: Small Molecules in Phytopathology: From Determinants of Disease to Modulators of Defense, title of talk: Unraveling the site- and mode-of-action of protein host-selective toxins, Lynda M. Ciuffetti, Oregon State University. Invited Speaker, 2011 The 26th Fungal Genetics Conference, Asilomar, Concurrent Session: Host Selective Toxins, title of talk: Same Fungus, Two Different Host-Selective toxins: Perceptions and Outcomes, Lynda M. Ciuffetti, Oregon State University. Invited speaker, 2011 RR Nelson Memorial Lecture, Penn State. Invited speaker, 2014 Dept. of Botany and Plant Pathology Purdue University: Multicomponent Analyses of Virulence in the Wheat Pathogen Pyrenophora tritici-repentis L.M. Ciuffetti, Oregon State University. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Multifaceted analyses of Ptr ToxA interactions including comparative analyses of the effects of Ptr ToxA on chloroplast function under objective 1 completed. Mode-of-action of host-selective toxins (HSTs) produced by P. tritici-repentis (Ptr) is essential to completely understand how these pathogenicity factors condition plant disease susceptibility. Data indicate that ToxA treatment leads to photosystem (PS) dysfunction, which in turn, leads to light-dependent generation of ROS, a contributor to cell death. In the presence of light and therefore ROS, dramatic decreases in protein levels of PSI and PSII correlate with a decrease in their transcripts. To evaluate ToxA in the absence of light and therefore ROS a biochemical approach was employed and microarray analysis was performed. No dramatic changes in protein levels of PS complexes or transcripts were detected in the dark. ToxA regulated fewer total genes in the dark, including defense-related genes. Ethylene biosynthesis genes were shown to be up-regulated and play a significant role in ToxA-induced symptom development in light only. Results suggest that for some genes ToxA-regulated gene expression is also light dependent. Antioxidant NAC inhibits symptom development induced by ToxA in the light. To further dissect pathways and understand ROS role in ToxA-induced effects on plants gene expression in the presence of NAC genes representing groups of interest were evaluated by qPCR. (manuscript in preparation). We evaluated the potential of consistent host responses associated with susceptibility by comparative analysis of ToxA- and ToxB-induced transcriptional changes. We found clear similarities in induction of defense responses. However transcriptional and biochemical responses and symptom development occur more rapidly for ToxA than ToxB, which could be explained by differences in perception as well as by differential activation of specific processes; e.g., ethylene biosynthesis in ToxA treatment. Suggesting that perception of HSTs result in activation of defense responses further supporting the hypothesis that necrotrophic fungi exploit defense responses to induce cell death (published, Pandelova et al. 2012). Evaluation of host impact on gene expression in P. tritici-repentis under objective 2 was completed. The interaction between a plant and microbe involves a complex array of signals resulting in a diversity of adaptations and corresponding changes of gene expression in both partners. Most studies focused on the host by examining transcriptional changes induced by pathogens or their virulence factors. Fewer focused on how pathogens are influenced by the host, especially fungal pathogens. The goal here was to investigate the influence of the host, wheat, on gene expression of Ptr during saprophytic and parasitic growth. GFP-labeled fungal tissue was beneficial to monitor early stages of disease development and allowed visualization of a short biotrophic phase on the susceptible and resistant cultivars at 24 and 48 hpi. Preliminary microscopy studies allowed optimization of inoculation conditions; estimation of fungal biomass by qPCR determined the experiments with highest percent of fungal tissue. Preliminary Illumina run determined if samples could be multiplexed. RNA was isolated from 24 susceptible cultivars, resistant cultivar and dead leaves inoculated with a race 1 isolate and collected at 30, 72 and 102 hpi. Libraries were prepared and sequences acquired. Read mapping to a reference genome and comparative analyses were performed. Read mapping showed high correlation between percent of fungal tissue estimated using DNA samples by qPCR and percent of RNA reads mapped to the Ptr reference genome. Percent of fungal transcripts in all samples was sufficient for further analyses. Difference in severity of symptoms between cultivars correlated with percent of fungal specific reads. At all times susceptible and ToxA-sensitive cultivars had higher percent of Ptr-specific reads than susceptible, but not ToxA-sensitive cultivars or resistant cultivars. Expression of ToxA was time-and cultivar-dependent. To further characterize genes that play a role in pathogenesis, we developed a reverse genetic approach of gene deletion in Ptr. We tested three methods for gene deletion of the HST-gene ToxA. Methods included genetic transformation of race 1 protoplasts with: 1) a linear minimal element of ToxA fused to a hygromycin resistance cassette (hygR), 2) a large PCR-generated fragment of ToxA 5’ and 3’ sequence surrounding hygR, and 3) two PCR-generated fragments from (2) but overlapping in the hygR cassette, also known as a split-marker approach. The split-marker approach generated a large number of hygR transformants and was most efficient, with 60% of transformants having properly integrated the hygR cassette. Inoculation of ToxA-expressing isolates on ToxA-sensitive cultivars typically leads to the production of necrotic lesions. Testing the impact of gene replacement of ToxA by inoculation of ToxA-sensitive cultivars gave surprising and exciting results. In one ToxA-sensitive cultivar, the lack of ToxA production resulted in similar levels of symptom production with a slightly different phenotype, indicating that this cultivar contains a sensitivity locus to an additional HST. Another ToxA-sensitive cultivar showed a much greater reduction in the size of the necrotic lesions. The third cultivar tested had an increase in symptom development, with similar-sized necrotic lesions and the appearance of spreading chlorosis, indicating this cultivar contains two additional sensitivity loci to HSTs produced. Additionally, data indicated that ToxA symptom development was epistatic to development of these other symptoms in this cultivar. The epistatic effect of ToxA perception by this cultivar was recapitulated by heterologous expression of ToxA in another isolate and repression of symptoms induced by that isolate in the absence of ToxA. This is the first report of necrotrophic effector epistasis. An additional unexpected outcome from this study suggested ToxA has virulence function in the absence of its cognate recognition partner. (Manuscript complete, awaiting review, Manning, V.A. and Ciuffetti, L.M. 201X). Determination of the three dimensional structure of the Ptr ToxB under objective 3 was completed. Ptr ToxB (ToxB), a HST produced by P. tritici-repentis, is a low molecular weight protein that appears to have a complex interactive interface with its host plant. A homolog of ToxB, toxb, shares 86% identity with ToxB, yet it does not induce chlorosis. Activity assessment of ToxB mutant proteins indicated that the ability to elucidate the interaction interface of ToxB with its plant partners would benefit from solving the three-dimensional structure. Therefore, we proposed a comparative structural analysis of ToxB and toxb and also continued with studies on the site- and mode-of-action of ToxB. Our laboratory optimized heterologous protein production and isotopic-labeling protocols to provide sufficient quantities of isotopically-labeled ToxB and toxb to our collaborator, Dr. Elisar Barbar (Biochemistry and Biophysics, OSU), to solve the three-dimensional structures for both proteins via NMR studies. Solution structures indicate that ToxB and toxb form a predominantly β-sheet topology composed of six β-strands oriented in an antiparallel fashion to form a β-barrel-like structure. The solution structures of ToxB and toxb are virtually identical. However, toxb displays increased conformational heterogeneity, which together with increased propensity of toxb to disulfide bond reduction, and residue differences in a putative active site, suggest that absence of toxin activity is correlated with amino acid differences that increase the internal dynamics of the protein and abolish interactions with putative targets (revised manuscript submitted, Afua Nyarko etal. 201X).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
I. Pandelova, M. Figueroa, L. J. Wilhelm, V. A. Manning, A. N. Mankaney, T. C. Mockler, L. M. Ciuffetti (2012) Host-selective toxins of Pyrenophora tritici-repentis induce common responses associated with host susceptibility. Plos One, doi:10.1371/journal.pone.0040240.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Afua Nyarko et al., 2014. Solution NMR Structures of Active Ptr ToxB and its Inactive Homolog Identify Determinants of Toxin Activity. Submitted to: Journal of Biological Chemistry.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
M. Figueroa, V. A. Manning, I. Pandelova and L. M. Ciuffetti (2014) Characterization of the host-selective toxin Ptr ToxB as an apoplastic effector. Submitted to: MPMI
- Type:
Journal Articles
Status:
Other
Year Published:
2014
Citation:
Manning, V.A. and Ciuffetti, L.M. 2014. Necrotrophic effector epistasis in the Pyrenophora tritici-repentis-wheat interaction. For submission to: Phytopathology.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
The 26th Fungal Genetics Conference, Asilomar, CA, March 15-20, 2011; Poster Session: Biochemistry and Metabolism, Poster title: The elucidation of the three-dimensional structure of ToxB, a chlorosis-inducing host-selective toxin produced by Pyrenophora tritici-repentis. Melania Figueroa, Afua Nyarko, Lynda M. Ciuffetti1, Elisar Barbar. Department of Botany and Plant Pathology, Department of Biochemistry and Biophysics. Oregon State University.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Pandelova I, Manning VA, Ciuffetti LM, 2013. Who is to blame: defining the host responses that lead to ToxA-induced susceptibility. 27th Fungal Genetics Conference, Fungal Genetics Reports, S60:460.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Manning VA, Pandelova I, Ciuffetti LM, 2013. You turn me on: Pyrenophora tritici-repentis genes differentially regulated early during infection of wheat. 27th Fungal Genetics Conference, Fungal Genetics Reports, S60:561.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2010
Citation:
Unraveling the site- and mode-of-action of protein host-selective toxins. Lynda M. Ciuffetti, Viola A. Manning, Iovanna Pandelova, and Melania Figueroa Betts, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, American Phytopathological Society Annual Meeting, Charlotte, NC, August 7-11, 2010.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2011
Citation:
Same Fungus, Two Different Host-Selective toxins: Perceptions and Outcomes.
L.M. Ciuffetti, V.M. Manning, I. Pandelova, and M. Figueroa, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, The 26th Fungal Genetics Conference, Asilomar, CA, March 15-20, 2011.
|
Progress 03/01/12 to 02/28/13
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Due to the decrease in recommended funds, we will not be able to conduct all components of the three objectives listed in the original submission. This reduction in the work proposed is due to the high cost of the microarray studies and the intensive amount of work needed for microarray analyses and biochemical analyses proposed. It was necessary to reduce the amount of funds requested for supplies and services. It was also necessary to reduce the amount of funds requested to support the people necessary to conduct these studies. Therefore, we have reduced the amount of work to be conducted for Objective 1. Objective 1: Multifaceted analyses of ToxA interactions. 1A. Comparative analyses of the effects of ToxA on chloroplast function: It will be necessary to reduce the number of time point for control and treatment to 3 time points rather than 4. 1B. Evaluation of early signaling associated with ToxA endocytosis. It will be necessary to eliminate this entire component of Objective 1. What opportunities for training and professional development has the project provided? Mr. Ashley Chu, Postbaccalaureate researcher, contributed to the work described under Objective 1. Dr. Afua Nyarko, Postdoctoral Associate, Department of Biochemistry and Biophysics, Oregon State University; NMR data collection and analysis under Objective 3. Dr. Melania Figueroa Betts, Postdoctoral Fellow, United States Department of Agriculture, Agricultural Research Service, Forage Seed and Cereal Research Unit, Oregon State University, Corvallis, Oregon; Dr. Figueroa Betts contributed to the production in Pichia pastoris and the purification of 13C/15N-labeled ToxB for structural studies under Objective 3. Dr. Iovanna Pandelova, Research Associate, Department of Botany and Plant Pathology, Oregon State University; contributed to Objectives 1, 2, and 3. Ms. Viola Manning, Senior Research Assistant, Department of Botany and Plant Pathology, Oregon State University; contributed to Objectives 2 and 3. How have the results been disseminated to communities of interest? I. Pandelova, M. Figueroa, L. J. Wilhelm, V. A. Manning, A. N. Mankaney, T. C. Mockler, L. M. Ciuffetti (2012) Host-selective toxins of Pyrenophora tritici-repentis induce common responses associated with host susceptibility. Plos One, doi:10.1371/journal.pone.0040240. M. Figueroa, V. A. Manning, I. Pandelova and L. M. Ciuffetti (201X) Characterization of the host-selective toxin Ptr ToxB as an apoplastic effector. (submitted) What do you plan to do during the next reporting period to accomplish the goals? continue with objectives as outlined
Impacts
What was accomplished under these goals?
Objective 1. We hypothesized that ToxA treatment leads to photosystem (PS) dysfunction, which leads to light-dependent generation of ROS and ROS accumulation and is a contributor of cell death. After 24 hpi in the dark, ToxA treatment does not largely effect the overall amount of photosystem complex proteins, however, there is an accumulation of PSI and II-intermediate complexes. Sensitive wheat leaves were infiltrated with ~1 mM of ToxA or water and incubated in light or dark. Leaves then were collected at 3, 9 and 14 hpi for either thylakoid fraction isolation (in the light or dark) or RNA extraction (only in the dark) and samples stored at -80oC for further processing. Blue native-gel (BN-gel) electrophoresis was performed on thylakoid membrane isolated from water or ToxA-infiltrated samples that had been incubated in light or dark. These anlyses indicated that all protein complexes are reduced in leaves exposed to light at 14 hpi with less noticeable changes at 9 hpi. Samples from ToxA-infiltrated leaves incubated in the dark had a slight decrease in supercomplexes compared to control water-treated leaves at both 9 and 14 hpi. Further analysis of the thylakoid membrane samples by 2D-gel electrophoresis indicate a decrease in intensities of the protein bands that correspond to supercomplexes is detected in ToxA-treated samples at 9 hpi and is more pronounced in leaves incubated in the light than in the dark. In the presence of light ToxA induces global transcriptional reprogramming, leads to up-regulation of defense response genes and down-regulation of genes involved in photosynthesis. Parallel experiments to detect transcriptional changes were performed with water and ToxA-infiltrated sensitive wheat leaves incubated in the dark. RNA was extracted, processed and hybridized on to microarrays. A statistically significant data set was obtained using four different statistical methods. Up- and down-regulated probesets showed fewer genes are differentially regulated in the dark upon ToxA treatment as compared to light (2479 ps vs 7065 ps). Similar to ToxA treatment in the light, some defense-related genes were up-regulated, supporting ToxA’s role as elicitor of defense responses, although the number of probesets was considerably smaller compared to light treatment. Additionally, ethylene biosynthesis genes, which were shown to be up-regulated and play significant role in symptom development in ToxA-treated leaves incubated in the light are not effected in ToxA-treated samples incubated in the dark. In contrast to the ToxA-induced effect in the presence of light on the regulation of genes for PSI and II and chlorophyll a/b-binding proteins, no statistically significant down-regulated genes were detected in leaves incubated in the dark. Results suggest that regulation of expression due to ToxA treatment for some genes is light dependent. Objective 2. The goal is to investigate the influence of the host, wheat, on gene expression of Ptr during saprophytic and parasitic growth. Furthermore, to determine whether there is a differential effect of different wheat cultivars on fungal gene expression when the plant is undergoing either a resistance or susceptible response. For parasitic growth, fungal inoculations with the reference isolate Pt-1C-BFP, which produces the HSTs ToxA and ToxC, have been conducted on three different host genotypes including Auburn (resistant), Glenlea (ToxA-sensitive) and 6B365 (ToxC-sensitive). For saprophytic growth, dead leaves of Auburn where inoculated. Leaves were collected at early, intermediate and late stages of infection. Microscopic evaluation and qPCR quantification of fungal mass in infected leaf samples suggested that a low percentage of fungal tissue in the total sample could be a limiting factor if the originally proposed microarray approach was used, despite optimization of inoculations to increase fungal biomass, especially at early time points. Therefore, RNA-seq was considered as another option, as it has proven to be more sensitive than microarrays in detecting low level transcripts. To investigate if RNA-seq could detect fungal transcripts in early time point samples and estimate what percent of fungal transcript is present in these samples, RNA was prepared from leaves collected 30 hpi, a transcript library prepared, and 51-mer single-end sequences acquired on an Illumina HiSeq 2000 sequencer. A total of 216,650,780 reads were generated that contained 3,878,905 reads that mapped to the Ptr reference genome, suggesting that approximately 1.8% of the sequenced reads present in the sample are fungal in origin. To determine if these reads assemble into transcripts that can be used in downstream differential expression analyses, we utilized components from the Tuxedo suite for reference genome-guided transcript assembly and quantitation. For comparison, the same pipeline was used to process a similar number of sequence reads obtained from a library produced from transcripts expressed under culture conditions. Similar numbers of transcripts associated with previously annotated reference loci were detected in the In planta and culture libraries (7286 and 7384, respectively) of which 6283 were shared; 1003 and 1101 transcripts were differentially detected in the In Planta and culture library, respectively. Functional annotations of the differentially detected-In Planta transcripts reflect the requirements of the pathogen for host penetration, cell wall degradation and the need to counteract the host response to infection and include a large number of differentially detected Carbohydrate Active Enzymes , especially glycosyl hydrolases, peptidases, and proteins with oxido-reductase activities, including peroxidases. The differentially detected transcripts in the culture library reflect the regulation of genes required for rapid growth, including those involved in protein and DNA biosynthesis. The number of fungal specific sequences present in 30 hpi samples is sufficient to define differences in transcripts that are produced in response to differing growth conditions. Both transcript libraries contain transcripts, some of which are shared and some of which are differentially detected, which do not overlap with the reference annotation. These are likely to be genes that were previously missed by ab initio gene prediction programs employed by the Broad Institute during the initial genome annotation process and will assist the genome reannotation process. Objective 3. We have continued our progress on the determination of the three dimensional structure of Ptr ToxB. NMR data of well-resolved, intense peaks were collected, data necessary for backbone and sidechain resonance assignments were collected. Data for assigning the NOEs were collected and proton chemical shift assignments finalized . NOEs formed between side aliphatic protons were completed, but these data were not optimal to produce a high definition structure. To optimize these data, we have been successful in collecting high quality NMR data and better spectra at 800 MHz suitable for structural determination. Efforts are underway to analyze these high quality, new data for active ToxB. We already know, through several analyses, that ToxB is a single domain protein composed of six beta strands. Approximately one third of all amino acid residues are buried in the protein core. Additionally, a Heteronuclear NOE (HetNOE) experiment indicates that ToxB is a rigid and tightly folded protein. The structure obtained for toxb (inactive protein) is the first structure for this protein family. The toxb structure consists of six beta strands (β1- β6) oriented in an antiparallel fashion to form a β-barrel-like structure with ~10 Å diameter, and a backbone heavy atom rmsd of 0.73 Å. There are two disulfide bonds involving, Cys2-Cys43; and Cys18-Cys65 important in stabilizing the overall structure.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2012
Citation:
Pandelova, M. Figueroa, L. J. Wilhelm, V. A. Manning, A. N. Mankaney, T. C. Mockler, L. M. Ciuffetti (2012) Host-selective toxins of Pyrenophora tritici-repentis induce common responses associated with host susceptibility. Plos One, doi:10.1371/journal.pone.0040240.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
M. Figueroa, V. A. Manning, I. Pandelova and L. M. Ciuffetti (201X) Characterization of the host-selective toxin Ptr ToxB as an apoplastic effector. (submitted)
|
Progress 03/01/11 to 02/29/12
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Due to the decrease in recommended funds, we will not be able to conduct all components of the three objectives listed in the original submission. This reduction in the work proposed is due to the high cost of the microarray studies and the intensive amount of work needed for microarray analyses and biochemical analyses proposed. It was necessary to reduce the amount of funds requested for supplies and services. It was also necessary to reduce the amount of funds requested to support the people necessary to conduct these studies. Therefore, we have reduced the amount of work to be conducted for Objective 1. Objective 1: Multifaceted analyses of ToxA interactions. 1A. Comparative analyses of the effects of ToxA on chloroplast function: It will be necessary to reduce the number of time point for control and treatment to 3 time points rather than 4. 1B. Evaluation of early signaling associated with ToxA endocytosis. It will be necessary to eliminate this entire component of Objective 1. What opportunities for training and professional development has the project provided? Mr. Ashley Chu, Postbaccalaureate researcher, contributed to the work described under Objective 1. Dr. Afua Nyarko, Postdoctoral Associate, Department of Biochemistry and Biophysics, Oregon State University; NMR data collection and analysis under Objective 3. Dr. Melania Figueroa Betts, Postdoctoral Fellow, Department of Botany and Plant Pathology, Oregon State University; Dr. Figueroa Betts contributed to the production in Pichia pastoris and the purification of 13C/15N-labeled ToxB and toxb for structural studies under Objective 3. Dr. Iovanna Pandelova, Research Associate, Department of Botany and Plant Pathology, Oregon State University; Objective; contributed mainly to studies under Objectives 1, and 2. How have the results been disseminated to communities of interest? Invited Speaker, 2011 The 26th Fungal Genetics Conference, Asilomar, Concurrent Session: Host Selective Toxins, title of talk: Same Fungus, Two Different Host-Selective toxins: Perceptions and Outcomes, Lynda M. Ciuffetti, Oregon State University. The 26th Fungal Genetics Conference, Asilomar, CA, March 15-20, 2011; Poster Session: Biochemistry and Metabolism, Poster title: The elucidation of the three-dimensional structure of ToxB, a chlorosis-inducing host-selective toxin produced by Pyrenophora tritici-repentis. Melania Figueroa, Afua Nyarko, Lynda M. Ciuffetti, Elisar Barbar. Department of Botany and Plant Pathology, Department of Biochemistry and Biophysics. Oregon State University. What do you plan to do during the next reporting period to accomplish the goals? continue with objectives as outlined
Impacts
What was accomplished under these goals?
Objective 1. Understanding the mode-of-action of the host-selective toxins (HSTs) produced by P. tritici-repentis (Ptr) is essential for a complete understanding of how these pathogenicity factors condition plant disease susceptibility. Numerous data support the developing hypothesis that ToxA binds to an extracellular receptor whereupon it is internalized through endocytosis and translocated to the chloroplast. In the chloroplast, ToxA interacts with a specific chloroplast protein (ToxABP1) leading to PS dysfunction. In turn, PS dysfunction leads to light-dependent generation of ROS and ROS accumulation is a contributor of cell death. Current data indicate that when ToxA-treated leaves are exposed to light, all proteins in both PSI and PSII are extremely reduced after 24 hpi. We also found that after 24 hpi in the dark, ToxA treatment leads to the accumulation of PSI and II-intermediate complexes. To address this objective: 1) a biochemical approach was employed to establish earlier time points when changes in protein content of PS complexes are detected; 2) microarray analysis was performed in order to determine the impact of ToxA on gene regulation in the absence of chloroplastic ROS. Sensitive wheat leaves were infiltrated with ~1 mM of ToxA or water and incubated in light or dark. Leaves then were collected at 3, 9 and 14 hpi for either thylakoid fraction isolation or RNA extraction and samples stored at -80oC for further processing. Considerable progress has been made to complete both experimental approaches proposed. In our initial progress report (2010) we reported that blue native-gel (BN-gel) electrophoresis was performed on thylakoid membrane isolated from water or ToxA-infiltrated samples that had been incubated in light or dark. Analysis of samples using BN-gel indicated that all protein complexes are reduced in leaves exposed to light at 14 hpi with less noticeable changes at 9 hpi. Samples from ToxA-infiltrated leaves incubated in the dark had a slight decrease in supercomplexes compared to control water-treated leaves at both 9 and 14 hpi. In this year of the project, further analysis of the thylakoid membrane samples by 2D-gel electrophoresis was conducted. Data indicate that a decrease in intensities of the protein bands that correspond to supercomplexes is detected in ToxA-treated samples at 9 hpi and is more pronounced in the leaves incubated in the light than in the dark. In our earlier microarray study we showed that in the presence of light ToxA induces global transcriptional reprogramming, leads to up-regulation of defense response genes and down-regulation of genes involved in photosynthesis. Parallel experiments to detect transcriptional changes were performed with water and ToxA-infiltrated sensitive wheat leaves incubated in the dark. RNA was extracted, processed and hybridized on to microarrays (GeneChip Wheat Genome Array). A statistically significant data set was obtained. Preliminary analysis of the newly obtained data set suggests that fewer genes are differentially regulated in the dark upon ToxA treatment as compared to light. In contrast to the ToxA-induced effect in the presence of light on the regulation of genes for photosystem I and II and other chloroplast-related proteins, no statistically significant down-regulated genes were detected in leaves incubated in the dark. Similar to experiments conducted in the light, some defense-related genes were up-regulated in the dark, supporting ToxA’s role as elicitor of defense responses. These results suggest that some regulation of the gene expression due to ToxA treatment is light dependent while others are not. Objective 2. Interaction of a plant and microbe involves a complex array of signals resulting in a diversity of adaptations and corresponding changes of gene expression in both partners. The vast majority of the studies of these interactions have focused on the host by examining transcriptional changes induced by pathogens or their virulence factors. Far fewer have focused on how plant pathogenic fungi are influenced by their host. The goal of this objective is to investigate the influence of the host, wheat, on gene expression of Ptr during saprophytic and parasitic growth. Furthermore, during parasitic growth, to determine whether there is a differential effect of wheat cultivars on fungal gene expression when the plant is undergoing either a resistance or susceptible response. Experimental work on this objective is in progress. For parasitic growth, fungal inoculations are conducted on different host genotypes. Leaves are collected at different periods of infection and analyzed. Objective 3. We have continued our progress on Objective 3 of the grant: Determination of the three-dimensional structure of Ptr ToxB. Ptr ToxB (ToxB), a chlorosis-inducing HST produced by P. tritici-repentis, is a low molecular weight protein (6.5 k-Da) that appears to have a complex interactive interface with its host plant. A homolog of Ptr ToxB, Ptr toxb (toxb), shares 86% identity with ToxB, yet it does not induce chlorosis. Activity assessment of ToxB mutant proteins, which had been produced by 1) site-directed mutagenesis based on differences between these two proteins and 2) chimera formation of different regions of these two proteins, indicated that the ability to elucidate the interaction interface of ToxB with its plant partners will require solving the three-dimensional structure. In addition, these studies suggested that determination of the three-dimensional structure for toxb will be instrumental for comparative analyses. Nuclear Magnetic Resonance (NMR) methods were determined to be appropriate for structure determination because the small protein size is well within the range of NMR capabilities and methods for recombinant expression have been developed that could easily be adapted for preparation of the 13C/15N labeled protein required for these methods. The elucidation of the three-dimensional structure of ToxB, as proposed in Objective 3, will help to design future experiments to characterize the interaction interface of ToxB, and unravel the site- and mode-of-action of this HST. In our initial progress summary (2010), we reported that we had optimized the protein production and isotopic-labeling protocol and produced sufficient quantities of 15N-labeled & 13C/15N -isotope labeled ToxB for NMR. NMR data of well-resolved, intense peaks was collected on both 15N- & 13C/15N doubly-labeled ToxB samples. All data necessary for backbone and sidechain resonance assignments was collected and all backbone and sidechain, and some side chain proton chemical shifts were assigned. All data for assigning the NOEs (distance constraints are obtained from estimates of nuclear Overhauser effect [NOE] intensities) were also collected. In this year of the project (2011), finalizing proton chemical shift assignments of side chains and assigning all the NOEs formed between side aliphatic protons was completed. Having all the assignments in hand, a preliminary structure has been determined. A combination of analyses indicated that ToxB is a single domain protein composed of six beta strands. Approximately one third of all amino acid residues are buried in the protein core. In addition, a Heteronuclear NOE (HetNOE) experiment, which identifies stretches of residues that are flexible or rigid, indicates that ToxB is a rigid and tightly folded protein. Progress has also been made in solving the structure of the inactive homolog of ToxB, toxb. Sufficient isotope-labeled protein was produced, and similar NMR methods used, to begin structure elucidation. Initial data suggests that toxb has only four beta strands, as opposed to the six beta strands of ToxB. Residues at the core of the protein are similar to those in ToxB.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2011
Citation:
Invited Speaker, 2011 The 26th Fungal Genetics Conference, Asilomar, Concurrent Session: Host Selective Toxins, title of talk: Same Fungus, Two Different Host-Selective toxins: Perceptions and Outcomes.
L.M. Ciuffetti, V.M. Manning, I. Pandelova, and M. Figueroa, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, The 26th Fungal Genetics Conference, Asilomar, CA, March 15-20, 2011.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2011
Citation:
Posters:
The 26th Fungal Genetics Conference, Asilomar, CA, March 15-20, 2011; Poster Session: Biochemistry and Metabolism, Poster title: The elucidation of the three-dimensional structure of ToxB, a chlorosis-inducing host-selective toxin produced by Pyrenophora tritici-repentis. Melania Figueroa, Afua Nyarko, Lynda M. Ciuffetti, Elisar Barbar. Department of Botany and Plant Pathology, Department of Biochemistry and Biophysics. Oregon State University.
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Progress 03/01/10 to 02/28/11
Outputs
Target Audience:
Nothing Reported
Changes/Problems: Due to the decrease in recommended funds, we will not be able to conduct all components of the three objectives listed in the original submission. This reduction in the work proposed is due to the high cost of the microarray studies and the intensive amount of work needed for microarray analyses and biochemical analyses proposed. It was necessary to reduce the amount of funds requested for supplies and services. It was also necessary to reduce the amount of funds requested to support the people necessary to conduct these studies. Therefore, we have reduced the amount of work to be conducted for Objective 1. Objective 1: Multifaceted analyses of ToxA interactions. 1A. Comparative analyses of the effects of ToxA on chloroplast function: It will be necessary to reduce the number of time point for control and treatment to 3 time points rather than 4. 1B. Evaluation of early signaling associated with ToxA endocytosis. It will be necessary to eliminate this entire component of Objective 1. What opportunities for training and professional development has the project provided? Mr. Ashley Chu, Postbaccalaureate researcher, contributed to the work described under Objective 1. Dr. Afua Nyarko, Postdoctoral Associate, Department of Biochemistry and Biophysics, Oregon State University; NMR data collection and analysis under Objective 3. Dr. Melania Figueroa Betts, Postdoctoral Fellow, Department of Botany and Plant Pathology, Oregon State University; Dr. Figueroa Betts contributed to the production in Pichia pastoris and the purification of 13C/15N-labeled ToxB for structural studies under Objective 3. How have the results been disseminated to communities of interest? Presentations: Invited Speaker, 2010 American Phytopathological Society Annual Meeting, Special Session: Small Molecules in Phytopathology: From Determinants of Disease to Modulators of Defense, title of talk: Unraveling the site- and mode-of-action of protein host-selective toxins, Lynda M. Ciuffetti, Oregon State University. Meeting Abstracts: Unraveling the site- and mode-of-action of protein host-selective toxins. Lynda M. Ciuffetti, Viola A. Manning, Iovanna Pandelova, and Melania Figueroa Betts, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, American Phytopathological Society Annual Meeting, Charlotte, NC, August 7-11, 2010. What do you plan to do during the next reporting period to accomplish the goals? continue with objectives as outlined
Impacts
What was accomplished under these goals?
Understanding the mode-of-action of the host-selective toxins (HSTs) produced by P. tritici-repentis is essential for a complete understanding of how these pathogenicity factors condition plant disease susceptibility. Ptr ToxA (ToxA), the effects it induces and the manner in which it induces them, is the subject of Objective 1 and entails an integrated analysis of the early effects of ToxA and its effects upon delivery to the chloroplast. Numerous data support the developing hypothesis that ToxA binds to an extracellular receptor kinase whereupon it is internalized through endocytosis and translocated to the chloroplast. In the chloroplast, ToxA interacts with a specific chloroplast protein (ToxABP1) leading to photosystem (PS) dysfunction. In turn, PS dysfunction leads to light-dependent generation of ROS and ROS accumulation is the ultimate cause of cell death. The purpose of this objective is to determine early signaling events induced by ToxA including if and when ToxA is detected by the host as an effector and further, to establish how ToxA interaction with ToxABP1 leads to PS dysfunction and ultimate cell death. Current data indicates that when ToxA-treated leaves were exposed to light, all proteins in both PSI and PSII are extremely reduced after 24 hpi. We also found that after 24 hpi in the dark, ToxA treatment leads to the accumulation of PSI and II-intermediate complexes. To obtain large amounts of ToxA to perform the experiments proposed in Objective 1, we utilized the Pichia pastoris expression system. The coding sequence of ToxA, under the control of a methanol-inducible promoter, was transformed into P. pastoris. Exposure of these cultures to methanol resulted in the secretion of ToxA into the culture filtrate. We had previously found that passage of the filtrate through a strong anion exchange column was sufficient to remove secreted P. pastoris proteins from the culture filtrate. Silver staining of ToxA-containing filtrates after strong anion exchange chromatography revealed that the only protein detectable after this purification step corresponds to ToxA. The activity of ToxA produced and purified from P. pastoris culture filtrates was tested and found to be similar to that of native ToxA. To establish earlier time points when changes in protein content of PS complexes are detected, sensitive wheat leaves were infiltrated with ~1 mM of ToxA or water and incubated in light or dark. Leaves then were collected at 9 and 14 hpi and the thylakoid fraction was prepared and stored at -80oC for further Blue native-gel (BN-gel) electrophoresis followed by SDS-PAGE analysis (2D-gel analysis). Preliminary analysis of samples using BN-gel indicates that all proteins are reduced in leaves exposed to light at 14 hpi with less noticeable changes at 9 hpi. Samples from ToxA-infiltrated leaves incubated in the dark have slight decreases in supercomplexes compared to control water-treated leaves at both 9 and 14 hpi. Further analysis of these samples by 2D-gel electrophoresis is underway to confirm these findings and obtain detailed information. Thus far, most of our efforts have been concentrated on Objective 3 of the grant. The elucidation of the three-dimensional structure of Ptr ToxB (ToxB), as proposed in Objective 3, will help to design future experiments to characterize the interaction interface of ToxB, and unravel the site- and mode-of-action of this HST. The structure of ToxB is currently being solved using Nuclear Magnetic Resonance (NMR). A protocol to heterologously express ToxB in Pichia pastoris was optimized to yield approximately 10 mg of protein per liter of P. pastoris culture filtrate. The experimental optimization of the conditions to acquire NMR spectra was established such that we could proceed with studies requiring 13C/15N -isotope labeled ToxB. A pilot experiment using 15N-labeled ToxB confirmed that NMR was a suitable technique to acquire the tertiary structure of ToxB. Efficient double 13C/15N-labeling of ToxB in P. pastoris was achieved by modification of the media described in the Invitrogen manual. Briefly, P. pastoris was grown in buffered minimal glycerol medium and buffered minimal methanol medium. The methanol-induction phase lasted four days, and aliquots of 10% 13C-methanol were added every 24 h to maintain a 0.5% final concentration of 13C-methanol in the P. pastoris culture. The 13C/15N-isotope labeled ToxB was purified to homogeneity via application of the P. pastoris culture filtrate to an anion exchange column. We have collected NMR data on two ToxB samples: one that is 15N uniformly labeled and another that is 13C/15N doubly labeled. Both samples were at high protein concentrations and gave well-resolved intense peaks. For the 15N sample, we have collected a 3D 15N edited NOESY for assignments of NOEs, a 3D HNHA experiment for determination of three-bond J coupling constants between amide protons and Ha to estimate dihedral angle ranges, and a 3D 15N edited TOCSY for assignments of aliphatic proton chemical shifts. For the 13C/15N sample, we have collected all standard triple resonance NMR experiments necessary for backbone and side chain resonance assignments. These include HNCACB, which correlates the alpha and beta carbon resonances of residue (i), with the backbone amide 15N-1H of residue (i), and CBCAcoNH interresidue experiment, which correlates the alpha and beta carbon of residue (i-1) with the backbone amide 15N-1H of residue (i). An HCCCONH TOCSY, which correlates all carbon side chains of residue (i-1) with the backbone amide 15N-1H of residue (i) was also collected to assign 13C chemical shifts of the side chains. So far we have obtained all backbone chemical shifts assignments, (NH, CA, CB), side chain carbon chemical shifts, and some side chain proton chemical shifts (from 3D edited 15N TOCSY experiment). We are now working on finalizing proton chemical shift assignments of side chains and assigning all the NOEs formed between side aliphatic protons. Having all the assignments in hand, we will then proceed with structural determination.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2010
Citation:
2010 American Phytopathological Society Annual Meeting, Special Session: Small Molecules in Phytopathology: From Determinants of Disease to Modulators of Defense, title of talk: Unraveling the site- and mode-of-action of protein host-selective toxins, Lynda M. Ciuffetti, Oregon State University.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2010
Citation:
Meeting Abstracts:
Unraveling the site- and mode-of-action of protein host-selective toxins. Lynda M. Ciuffetti, Viola A. Manning, Iovanna Pandelova, and Melania Figueroa Betts, Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA, American Phytopathological Society Annual Meeting, Charlotte, NC, August 7-11, 2010.
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