Progress 02/15/12 to 02/14/16
Outputs
Target Audience:Target audiences for this foundational research project are the scientific community studying plant diseases, teachers educating the next generation of scientists, and the general public with a need to better understand the impact of plant diseases on global food production. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This research was mainly performed by Postdoctoral Research Associates Mihwa Yi and Ely Oliveira Garcia, and by graduate Student Pierre Migeon. Pierre has now decided to continue his Genetics PhD program by specializing in bioinformatics. He will be an author on at least one publication from this project, and his exposure to laboratory research in fungal biology and molecular plant-fungal interactions will provide him with valuable grounding in biology and strengthen his abilities in bioinformatics. Research technician Melinda Dalby has assisted in various aspects of this project. Graduate Student Stuart Sprague worked together with research technician Kim Park and Dr. Sunghun Park (KSU) on producing rice labeled with the plasmodesmata marker protein. Graduate Student Xu Wang worked on this project together with our collaborator Dr. Jung-Youn Lee at the University of Delaware. I have initiated a new collaboration with Drs. Jie Zhou and Zonghua Wang of Fujian Agriculture and Forestry University, Fuzhou, China. As part of this collaboration, Dr. Huakun Zheng, a visiting scientist from Fujian Agriculture and Forestry University has been working on related research in my lab at KSU. How have the results been disseminated to communities of interest?Project results have been disseminated by presentations to academic, government, and industry researchers at professional meetings and at universities in 2016 including at the Rice and Wheat Blast Symposium, 36th Rice Technical Working Group Meeting, March 1-4, Galveston, Texas; and the Comparative genomic approaches to understanding the evolution of Magnaporthales-Symposium and Workshop, Rutgers University, January 7-8, New Brunswick, New Jersey. In 2015, results were presented at 2nd National Wheat Blast Workshop, December 6, St. Louis, Missouri; the 36th New Phytologist Symposium on Cell biology at the Plant-Microbe Interface, November 29 to December 1, Munich, Germany; the 4th International Conference on Biotic Plant Interactions, August 1-3, Nanjing, Chiina; the NRI-AFRI Microbial Programs Awardee Meeting, July 23-24, Washington, D.C.; the Plant Biosecurity Course entitled "Plant Biosecurity in Theory and Practice", May 18, Manhattan, Kansas; the Department of Plant and Microbial Biology, University of California, Berkeley, April 29, Berkeley, California; Department of Plant Pathology, April 6, the University of Nebraska, Lincoln, NE (Invited by Plant Pathology Graduate Student Association); the 28th Fungal Genetics Conference, March 17-22, Asilomar, Pacific Grove, California; and the Gordon Conference on Chemical & Biological Terrorism Defense: Research Synergies for Chemical and Biological Defense, March 8-13, Ventura, California. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Major activities completed: Objective 1: Perform detailed microscopic characterization of the hyphal cell-to-cell penetration junctions including localization of plasmodesmal markers and putative fungal movement proteins: We performed Correlative Light and Electron Microscopy (CLEM) with collaborators Jung-Youn Lee and Jeffery Caplan (University of Delaware) to determine where blast invasive hyphae (IH) crossed the rice cell wall relative to rice pit fields (regions with clustered plasmodesmata). With detailed analysis of 42 crossing sites, we found that IH had obviously crossed in pit fields in 17 percent of the crossing points, and surrounding plasmodesmata and pit field cell walls appeared undamaged. Forty-three percent of IH crossing points showed pit fields plasmodesmata nearby, and 40 percent of crossing points lacked visible plasmodesmata. In CLEM images as well as previous electron micrographs, pit fields were between 700 and 1300 micrometers in diameter, and IH that had crossed the wall were around 0.50 micrometers in diameter. These results suggest that IH do seek out regions with pit fields to cross the wall. These data suggest IH don't always cross in pit fields, although it is not possible to prove that PD were totally absent at the crossing points lacking visible plasmodesmata. We localized the Bas53:RFP protein (labeled with monomeric red fluorescent protein) at the cell wall crossing points in both lateral (epidermal to epidermal cells) and vertical (epidermal to mesophyll cells) cell invasions. In these studies, Bas53:RFP fluorescence identified double rings where IH had crossed to new cells, located on each cytoplasmic side of the wall at the crossing points. Through live cell confocal imaging of cell wall crossing, Bas53:RFP was first seen in a linear pattern across the wall before the IH crossed and then the fluorescence wrapped the base of the IH after crossing. Bas53:FP appeared again at the crossing points where IH move into the third ring of rice cells. Bas2 and the new putative lipase Bas169 also wrapped the base of the IH after crossing. The putative apoplastic effector Bas79:FP (various fluorescent proteins) shows a more diffuse localization pattern spreading out in the cell walls in areas of the rice cell wall in contact with IH, before crossing. Bas79:FP was then detected in 2nd BICs after cell entry. In addition to a dramatic funnel shaped-pattern in the appressorium, Bas150:FP showed the punctate pattern in the rice cell wall after IH crossed. It now appears that Bas83:FP, a symplastic effector that is not detected in appressoria or before cell wall crossing, strongly accumulates in BICs and in cytoplasm surrounding BICs in both first- and second- invaded cells. Bas83:FP fluorescence around the base of the IH showed recovery after photobleaching, indicating continued secretion while the IH grew in the cell. These results (the work of Mihwa Yi, Pierre Migeon, Xu Wang, Dr. Jung-Youn Lee and others) are described in a manuscript in preparation. Meanwhile, while performing live cell imaging with our new Zeiss LSM780 confocal microscope, Ely Oliveira-Garcia has improved our leaf sheath assay and imaging capabilities and obtained higher-resolution images that show symplastic effectors that appear packaged into vesicles in and around BICs. Symplastic effectors Bas1, Pwl1 and Pwl2 appear sorted into different micro-vesicles in BICs at the tips of primary hyphae. Bas83:FP is closely associated with these vesicles, and may be involved in BIC function instead of hyphal movement. Our current working hypothesis is that symplastic effectors are internalized into living rice cells via endocytosis. Objective 2: Analyze isolated cell wall fragments to determine if the fungus modifies PD channel size or pit field wall structure during crossing: We decided not to pursue this objective due to difficulties in isolating rice cell wall fragments and our success in visualizing cell wall crossing points by CLEM. The same questions are answered by the CLEM analysis. Objective 3: Determine if the fungus manipulates callose deposition at PD and how this impacts hyphal cell-to-cell movement. The callose staining dye, aniline blue, did not work to identify plasmodesmata-localized callose in our system in healthy, non-infected rice tissue as was expected. Specifically, the dye showed uniform staining of the rice cell wall rather than punctate staining of pit fields in the wall. We have shown that stained punctae do appear in the wall when the fungus appears ready to cross, and crossing points are stained with aniline blue. These results suggest that callose appears in the wall before and during crossing by the fungus. We are continuing our functional analysis of Bas169, the putative ortholog of the Fusarium graminearum lipase effector FGL1 that impacts callose deposition and innate immunity in wheat. The M. oryzae lipase-like effector is up-regulated during plant penetration and biotrophic development. Bas169:FP is focally secreted from the appressorial penetration pore into the appressorial O-ring and penetration pores before host invasion. Additionally, the lipase-like effector accumulates in plant cell wall crossing points during cell-to-cell colonization by the fungus. This accumulation of Bas169:FP at crossing points was observed during invasion of the 4th consecutive rice cells following initial successful colonization of the 1st cell by the fungus. These results suggest that infection structure-specific expression of this lipase-like effector supports appressorial functionality and fungal cell-to-cell movement. (Work of Ely Oliveira Garcia) Objective 4: Compare the PD size exclusion limit for movement of fluorescent reporters and fluorescent effectors in invaded and noninvaded rice cells. We have solid evidence that the fungus impacts the size exclusion limit (SEL) of rice plasmodesmata after infection. Specifically, we used particle bombardment transient expression of mCherry (28.8 kDa) and double-mCherry (57.6 kDa) in rice sheath epidermal cells and observed cell-to-cell movement of the fluorescent proteins. In uninoculated cells, mCherry moves into neighboring rice cells, but double-mCherry generally does not move out of bombarded cell. However, in infected tissue double-mCherry moves rapidly into neighboring rice cells. Therefore, it appears that fungal infection enlarges the SEL of plasmodesmata. (Work of Ely Oliveira Garcia, manuscript being planned.) Objective 5: Perform detailed functional analyses of putative fungal movement proteins. We will work to improve our ability for functional analyses of these effectors. Discoveries/Impacts:Co-localization and time course imaging showed distinct expression profiles and different micro-subcellular localization patterns for 6 putative effector proteins (Bas2, Bas3, Bas53, Bas79, Bas150 and Bas169) that localize to cell wall crossing points. This implicates finely tuned regulation of their expression and localization at both temporal and spatial levels. Our results also suggest a potential role of lipases for manipulation host cell channels, plasmodesmata, by the fungus. It appears that the fungus does manipulate the plasmodesmata SEL during biotrophic invasion, which would facilitate rapid cell-to-cell movement of cytoplasmic effectors. One of the original effectors in this study, Bas83, appears to have a role in the rice cytoplasm surrounding BICs, rather than in hyphal movement into new cells. In the course of this study, we have achieved higher resolution imaging of the BIC interfacial region. Images showing that various symplastic effectors appear packaged in vesicles in BICs and in the surrounding cytoplasm have lead us to pursue a working hypothesis that symplastic effectors are internalized into living rice cells via endocytosis.
Publications
- Type:
Journal Articles
Status:
Submitted
Year Published:
2016
Citation:
Zheng, H., S. Chen, X. Chen, S. Liu, X. Dang, C. Yang, M.C. Giraldo, J. Zhou, Z. Wang and B. Valent, The small GTPase MoSEC4 is essential for the vegetative development and pathogenicity by regulating the extracellular protein secretion in Magnaporthe oryzae. In preparation.
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Al Souhail, Q., Y. Hiromasa, M. Rahnamaeian, M.C. Giraldo, D. Takahashi, B. Valent, B., A. Vilcinskas and M.R. Kanost. 2016. Characterization and regulation of expression of an antifungal peptide from hemolymph of an insect, Manduca sexta. Article in Developmental and Comparative Immunology, 61: 258-268.
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Progress 02/15/14 to 02/14/15
Outputs
Target Audience:Target audiences for this foundational research project are the scientific community studying plant diseases, teachers educating the next generation of scientists, and the general public with a need to better understand the impact of plant diseases on global food production. Changes/Problems:No major changes to report. What opportunities for training and professional development has the project provided?Personnel working on this project: During this period, the research was performed by Graduate Student Pierre Migeon and Postdoctoral Research Associate Ely Oliveira Garcia. Research technician Melinda Dalby continues to assist in aspects of this project. Graduate Student Stuart Sprague is working with Dr. Sunghun Park (KSU) to on rice transformation rice. Research technician Kim Park is assisting with the rice transformations. We continue our collaborations with Dr. Jung-Youn Lee and Graduate Student Xu Wang at the University of Delaware. How have the results been disseminated to communities of interest?Project results have been disseminated to academic, government, and industry researchers at professional meetings and at universities in 2015 including the 28th Fungal Genetics Conference, March 17-22, Asilomar, Pacific Grove, California; and the Gordon Conference on Chemical & Biological Terrorism Defense: Research Synergies for Chemical and Biological Defense, March 8-13, Ventura, California. In 2014, results were presented at the Joint International Symposium on Mechanisms of Cellular Compartmentalization, Collaborative Research Center SFB 593 and Graduate School GRK 1216, Philipps Universität Marburg, September 24 - 26, Marburg, Germany; Department of Plant Pathology, Kansas State University, September 18, Manhattan, Kansas; Department of Plant Pathology, Pennsylvania State University, July 28-29, University Park, Pennsylvania; the Cellular & Molecular Fungal Biology Gordon Research Conference on Fungal Biology from the Integrated Perspective of Molecular Mechanisms, Systems and Evolution, June 15-20, Holderness, New Hampshire; Wheat Blast Workshop: knowing the pathogen to control the disease, Embrapa Wheat, June 3-4, Passo Fundo, Brazil; and at a Special Symposium: "Blast control - a moving target," 35th Rice Technical Working Group Meeting, February 18, New Orleans, Louisiana. What do you plan to do during the next reporting period to accomplish the goals?We will continue the experiments in progress, with no changes in strategy at this point.
Impacts
What was accomplished under these goals?
Major activities completed: Objective 1a: Detailed microscopic analyses of invasive hyphal cell-to-cell movement. Seed from transformed rice plants expressing the plasmodesmata marker gene encoding PDLP5 fused to yellow fluorescent protein have been obtained and analysis is underway. (Work of Stuart Sprague, Melinda Dalby, Kim Park and Dr. Sunghun Park). Objective 1b: Localization dynamics of effectors during early infection stages. During this period, our department acquired a Zeiss LSM780 confocal microscope similar to the one we were using at the University of Delaware, which has expedited analyses of BAS proteins (putative effectors) dynamics in first- and second-invaded rice cells. With previous support from USDA-NIFA, we showed that the fungus undergoes a characteristic morphological switch immediately after entry into a host cell, and the IH cells that undergo this switch are associated with a specialized interfacial structure, the biotrophic interfacial complex (BIC). We also showed that cytoplasmic effectors accumulate in BICs via a Golgi-independent (BFA-insensitive) secretion system involving the exocyst complex, while apoplastic effectors are secreted via the classical Golgi-dependent secretion system. Therefore, we also tested BFA-sensitivity of secretion of our new BAS proteins. The 6 Bas proteins showed distinctive localization patterns throughout infection related development in addition to the signals at the cell wall crossing points (Manuscript in preparation). Bas53 showed faint fluorescence in appressoria and in BICs in first-invaded cells (1st BICs) and later in BICs in second-invaded cells (2nd BICs). During cell wall crossing, Bas53:FP was first seen in a linear pattern across the wall before the IH crossed and then the fluorescence wrapped the base of the IH after crossing. Bas53:FP appeared again at the crossing points where IH move into the third ring of rice cells. Bas2 showed a localization pattern similar to Bas53. Bas79:FP was not detected during appressorium formation, penetration, and growth in the first-invaded cells. Fluorescence was first detected spreading out in the cell walls in areas of the rice cell wall in contact with IH, before crossing. Fluorescence was then detected in 2nd BICs after cell entry. Despite lack of Bas79 fluorescence while IH were growing in 1st-invaded cells, treatment with BFA caused retention of fluorescence inside the IH, indicating that Bas79 was being secreted at low levels in these cells. Sensitivity to BFA treatment showed that Bas79 is secreted by classical golgi-dependent secretion similar to apoplastic effectors. Bas83:FP was not detected in appressoria, but it showed BFA-insensitive secretion into BICs. In the 2nd-invaded cells, Bas83 again localized to BICs, and it showed strong, irregular fluorescence around IH after crossing. Bas83:FP fluorescence around the base of the IH showed recovery after photobleaching, indicating continued secretion while the IH grew in the cell. Fluorescence for Bas150:FP displayed a dramatic funnel shaped-pattern in the appressorium. Bas150:FP also accumulated in 1st BICs, at cell wall crossing points, and in 2nd BICs in the neighboring cells. Bas150:FP showed the punctate pattern in the rice cell wall after IH crossed. Bas53, which accumulated in BICs and in the host cell wall before IH crossed, showed an interesting hybrid BFA-sensitivity pattern. Secretion into first and second BICs appeared to be insensitive to BFA treatment as reported for cytoplasmic effectors. However, secretion at the rice cell wall before crossing appeared sensitive to BFA, as seen by restricted fluorescent protein accumulation inside IH cells that were close to the cell wall. Secretion is again BFA-insensitive as the tip-BIC associated hyphae begin to grow in the newly invaded cells. Secretion of Bas150 appeared to follow the Bas53 pattern. In contrast, secretion of Bas79 by IH in first-invaded IH cells and secretion by IH into the rice cell wall were always BFA-sensitive. Secretion of Bas83, which has not been observed in the rice cell wall before crossing, is uniformly BFA insensitive. Transformants co-expressing Bas fusion proteins confirmed the differences in localization patterns. Differential expression and localization patterns for the six effector proteins associated with cell wall crossing points suggest each has a distinct role during biotrophic rice cell invasion. (Work of Mihwa Yi and Pierre Migeon with Dr. Jung-Youn Lee and graduate student Xu Wang) Objective 3: Determine if the fungus manipulates callose deposition at PD and how this impacts hyphal cell-to-cell movement. The callose staining dye, aniline blue, did not work to identify plasmodesmata-localized callose in our system. Therefore, we searched for M. oryzae homologs of fungal effectors that are known to impact callose deposition in host plants. Specifically, we identified the M. oryzae homolog of the Fusarium graminearum lipase effector FGL1, which contributes to fungal virulence by releasing free fatty acids that inhibit innate immunity-related callose during infection of wheat heads (Blumke et al Plant Physiology 2014). The M. oryzae lipase-like effector is up-regulated during plant penetration and biotrophic development. The lipase-like effector is focally secreted from the appressorial penetration pore into the appressorial O-ring and penetration pores before host invasion. Additionally, the lipase-like effector accumulates in plant cell wall crossing points during cell-to-cell colonization by the fungus. This accumulation of the lipase-like effector at crossing points was observed during invasion of the 4th consecutive rice cells following initial successful colonization of the 1st cell by the fungus. These results suggest that infection structure-specific expression of this lipase-like effector supports appressorial functionality and fungal cell-to-cell colonization. Functional analyses are underway. (Work of Ely Oliveira Garcia) Objective 4: Compare the PD size exclusion limit for movement of fluorescent reporters and fluorescent effectors in invaded and noninvaded rice cells. Biolistic transient expression assays suggest that the fungus does impact the size exclusion limit (SEL) of rice plasmodesmata after infection. Specifically, we used particle bombardment transient expression of mCherry (28.8 kDa) and double-mCherry (57.6 kDa) in rice sheath epidermal cells and observed cell-to-cell movement of the fluorescent proteins. In uninoculated cells, mCherry moves into neighboring rice cells, but double mCherry generally does not move from the bombarded cell. However, double mCherry moves rapidly into neighboring rice cells in infected tissue. Therefore, it appears that fungal infection enlarges the SEL of plasmodesmata. (Work of Ely Oliveira Garcia) (5) Perform detailed functional analyses of putative fungal movement proteins. We are using an RNAi strategy for functional analysis of single and multiple effectors that localize at the cell wall crossing points. A silencing vector has been constructed using the BAS1 promoter, which is highly expressed during biotrophic invasion, and putative mutants have been obtained. (Work of Pierre Migeon) Discoveries/Impacts:Co-localization and time course imaging showed distinct expression profiles and different micro-subcellular localization patterns for each of the 6 putative effector proteins localized at the cell wall crossing points. This implicates finely tuned regulation of their expression and localization at both temporal and spatial levels. It appears that the fungus does manipulate the plasmodesmata SEL during biotrophic invasion, which would facilitate rapid cell-to-cell movement of cytoplasmic effectors. Our results also suggest a potential role of lipases for manipulation host cell channels, plasmodesmata, by the fungus.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Oliveira-Garcia, E. and B. Valent, 2015. How eukaryotic filamentous pathogens evade plant recognition, Current Opinion in Microbiology 26: 92-101
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Progress 02/15/13 to 02/14/14
Outputs
Target Audience: Our target audience includes the professional plant pathology community and rice and wheat production specialists. We organized and hosted a workshop entitled ‘Special Symposium: Blast control - a moving target’ for the rice stakeholder audience. The workshop was presented on February 18, 2014, directly before the 35th Rice Technical Working Group Meeting in New Orleans. On December 7, 2014, we held a Wheat Blast Workshop for wheat stakeholders. The workshop was held directly before the Annual Fusarium Head Blight Meeting in St. Louis, MO, which is already attended by many interested individuals. Our audience also includes professionals involved in regulatory issues. USDA-APHIS scientists with a need to understand wheat blast disease attended the Blast Integrated Project Meeting in Minneapolis, MN, on August 9, and the Wheat Blast Workshop in St. Louis on December 7. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Objective 1. Objective Team Leader (OTL) Yulin Jia is advising postdoctoral fellow Xueyan Wang and Biological Science Lab Technicians Tracy Bianco, Michael Lin and Tiffany Sookaerm, all working on the population biology of the rice blast fungus. Co-PI Yeshi Wameshe, Univ of Arkansas (UArk) extension specialist has coordinated field observations and collection of disease specimens, with support from field technician Tebebu Gebremariam. Wameshe and Jia are working with rice extension specialists Rick Cartwright (AR), Don Groth (LA), Shane Zhou (TX), and Chris Greer (CA), all of whom have supported field collection of fungal specimens. Mia Hodges, an undergraduate student from UARK, is a student research assistant. Co-PI Guoliang Wang (OSU) and his technician Maria Bellizzi have produced and analyzed rice transgenics carrying R genes of interest. Objective 2. OTL R. Dean (NCSU) is advising graduate student Mengying Wang on the HIGS project. Co-PI J. Xu will advise graduate student Yang Li as she begins work on HIGS objectives in Year 3. Co-PI Sunghun Park (KSU), with support from technician Jung-Eun Kim Park, will produce and analyze rice transgenics. Objective 3. OTL P. Paul and Co-PI L. Madden (OSU) are advising graduate student K. McLean Mills in research on epidemiology of Lolium and Triticum pathotype strains and disease modeling and forecasting. Co-PI M. Farman (UK) is advising post-doc Baohua Wang and graduate student Lily Chen, both working on population analyses of the M. oryzae Lolium (Mol) pathotype in the US. Co-PI G. Peterson (USDA-ARS Ft. Detrick) is supported by Biological Science Technician Kelly Thomas. Co-PI K. Pedley is advising postdoc Mike Pieck on comparative genomics of M. oryzae pathotypes with the goal of developing high-specificity diagnostic tools for wheat blast. Objective 4. OTL Bockus and Co-PI Stack advised Dr. Christian Cruz when he was a graduate student supported by our previous wheat blast grant. Cruz continues as a post-doctoral researcher on this project. He competed for and won a Rotary Humanitarian Study Grant which provided additional funding during Year 2 for wheat blast research in SA. This funding provided living expenses for Dr. Cruz to work in Bolivia and Brazil to better study wheat blast disease in the field. He established working relationships with project collaborators J. M. Fernandes (Embrapa Trigo) and D. Baldelomar (ANAPO, Bolivia), as well as with wheat blast expert Prof. Dr. Alfredo Urashima at Universidade Federal de São Carlos, Brazil. Payton DeLong is an undergraduate research assistant supporting Dr. Bockus’s work on this Objective. In collaboration with Dr. Evans Lagudah, CSIRO, Australia, the team tested Avocet and Thatcher isolines containing the adult rust and mildew R genes Lr34, Lr46 or Lr67. Co-PI Trick (KSU) has advised Kerri Neugebauer (PhD student) and Hyeonju Lee (research technician) on wheat tissue culture, transformation, selection, identification and characterization of transgenic wheat lines containing the rice Piz-t resistance gene. PD Valent (KSU) with collaborator Eduard Akhunov (KSU) guided postdoc P. Kankanala who contributed GBS genotypes for wheat varieties that have varying levels of resistance to wheat blast. Objective 5. OTL DeWolf (KSU) is advising graduate student Taylor Fisher on modeling, risk evaluation and forecasting objectives and he will advise postdoc Denis Shah (Cornell) on modeling, risk evaluation and disease forecasting in 2015. P. Knight, D. Miller and B. Mills (all at Penn State) will collaborate on modeling, risk assessment and disease forecasting in 2015. Co-PI J.M. Fernandes (Embrapa Trigo, Brazil) will complete a 3-month residency at K-State early in 2015. He will continue collaborating with OTL DeWolf and Co-PI Stack when he returns to Brazil, closely coordinating with Willingthon Pavan and Jorge Bavaresco, researchers at the University of Passo Fundo, RS BR. Objective 6. With OTL Nalley (UArk) as her advisor, Anne-Céclie Delwaide completed a double M.S. degree in agricultural economics, one from the University of Arkansas and one from the University of Gent, Belgium. She administered and analyzed the European Cisgenic Survey in Europe and the U.S. Nalley and Delwaide collaborated with Dr. Wim Verbeke, Professor at the University of Gent, and a leading European Ag Economist and E.U. advisor on GMO issues. A new graduate student will be recruited to join the project in 2015. Nalley and the new student will collaborate with economists in Africa to conduct the Cisgenics Survey in selected African countries. Objective 7: OTL Stack (KSU) advised Lindsey Ashmore, a Junior in Agricultural Communications on creation of wheat blast videos and the Wheat Blast Web Site: (http://www.k-state.edu/wheatblast/). Other contributors on outreach include Co-PI Wameshe, OTL Jia, Co-PI Trick, postdoc Cruz, OTL DeWolf, Co-PI J.M. Fernandes, and PD Valent, all of whom are mentioned earlier in this section, and all of whom are contributing to achieving unique outcomes linked to Objective 7. How have the results been disseminated to communities of interest? The outreach program created through this project has impacted the lives of the undergraduate students in 2013 through a Study Abroad trip to China covering the topics of “Food Security and Food Safety” as reported in detail in last year’s report. This trip will run again, through support of this project, in May of 2015. As part of the year 2 outreach efforts, a week-long workshop on real-time PCR on M. oryzae was conducted by Tom Mitchell at the University of Puerto Rico Mayaguez in June. For the workshop, 27 attendees performed hands-on experiments on rice blast samples to test hypotheses using real-time PCR technologies. They attended daily sessions taught by Tom on plant pathology, genomics, and bioinformatics. At the end of the workshop, students gave a presentation on their results and an interpretation of their meaning and possible biological impacts. In addition to exposing a new cohort of students to these topics, two were inspired to apply to graduate school in Plant Pathology. Additionally, Tom’s graduate student Nikki Tate assisted with all aspects of workshop training, preparation, and execution in Puerto Rico. This was an experience of a lifetime for her and helped train her to be the next generation of scientists that can communicate their work effectively to the public. A press release about the workshop can be found at http://www.uprm.edu/portada/article.php?id=2941. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project members attended a BIP meeting in Minneapolis on Aug 9 and an External Scientific Advisory Board (SAB) meeting in St. Louis on Dec 6. Progress is summarized. (OBJ.1) (1.1) Cisgenic approaches to introduce cloned rice resistance (R) genes into elite US rice varieties focused on producing Pi9 marker-free transgenic rice using the dual vector system developed last year. Transformation conditions were optimized for US varieties LaGrue and Juniper. (1.2) Understanding avirulence (AVR) gene compositions in field populations will guide R gene deployment. Isolates being analyzed include 629 archival isolates (collected 1950 to 2005), and 880 isolates from 2012 to 2014. We shortened our AVR analysis pipeline, in which purified strains are assigned to lineages using Rep-PCR fingerprinting and analyzed for AVR gene composition, by isolating DNA for PCR analysis from fungus which is still in the storage filter papers (Jia et al. 2014. Crop Journal). We are focusing on AVR genes corresponding to 4 cloned rice R genes, Pi9, Pita, Pizt, and Pik. All archival isolates contain AVR-Pi9 and 83% contain AVR-Pita1. Among 480 recent isolates, 74% contain AVR-Pi9 and 65% contain AVR-Pita1. (1.3) We developed a perfect DNA molecular marker for Pi9 and are screening germplasm used by US rice breeders. (Obj. 2) The Dean and Xu labs have prepared a list of 20 target genes for the HIGS analysis, an appropriate HIGS vector, immature suspension culture cells, and actively growing callus cultures for rice transformation. (Obj. 3) (3.1) Native Lolium (MoL) isolates pose a significant risk to US wheat. The MoL strain isolated from a wheat head in Kentucky in 2011, and 2 other Kentucky isolates are 75, 57, and 81% as aggressive as Bolivian MoT strain B2 in causing head blast under controlled environment conditions. (3.2) We sequenced and assembled genomes of 25 fungal strains from rice, wheat, ryegrass and other hosts, and we developed software for BLAST searches, SNP analyses and repeat masking. We confirmed that M. oryzae isolates are distinct from M. grisea Digitaria isolates and that MoT and MoL isolates are closely related and relatively distant from other populations; and we showed that MoT isolates show a surprising degree of SNP variability relative to isolates from other hosts. (3.3) We are applying a Seq-to-SSR approach to develop diagnostics for differentiating MoT and MoL strains. Marker2 appears specific to MoT, but the final assay may require more than one marker. 20 additional unique MoT loci are being evaluated. (3.4) Experiments in Bolivia confirm that MoT is seed-borne and can be transmitted from spike to seed and from seed to seedling. MoT sporulation on basal senescent wheat leaves might provide an inoculum source in the field. New MoT isolates from Bolivia and Paraguay show a lower temperature optimum than previous isolates. (3.5) Seed treatments can provide significant wheat blast control and fungicides applied at the head phase reduced MoT seed-borne inoculum and blast intensity and increased yield. (Obj. 4) (4.1) Successful field tests were performed in Quirusillas and Okinawa in Bolivia. Some putatively-resistant cultivars identified in controlled environment studies with single isolates were susceptible in field studies, suggesting there are races in the field. Field tests of isogenic lines from Dubcovsky and Pumphrey show that the 2NS/2AS translocation from T. ventricosum, containing the Lr37, Yr17 and Sr38 rust R genes, also confers wheat blast resistance in some genetic backgrounds and to some pathogen field populations. (4.2) Soft Red Winter Wheat (SRWW) varieties assayed at OSU show a continuum of reactions to MoL isolate PL2-1 similar to HRWWs inoculated with MoT strains. Differential reactions to point and spray inoculations suggest that SRWW may possess different types of resistance to MoL-resistance to infection and resistance to spread within the spike. Thatcher, reported in the literature to contain 2 leaf blast resistance genes, is moderately susceptible to head blast and may not be a good candidate for follow-up. Avocet and Thatcher isolines containing the adult rust and mildew R genes Lr34, Lr46 or Lr67, from the CSIRO, Australia, are highly susceptible to head blast in growth chamber inoculations. A list of resistant cultivars was produced and uploaded onto the wheat blast website (http://www.k-state.edu/wheatblast/). (4.3) DNA was isolated from about 450 wheat cultivars of known reaction to blast and screened for the presence of the 2NS translocation segment using published PCR markers. The 2NS fragment was confirmed in 14 out of 22 highly resistant wheat varieties identified at KSU. Other varieties (Hatcher, Overland, RonL, TAM 112) may contain different resistance genes. Of the 175 spring wheat entries tested at ARS, 6 appeared highly resistant and 5 of these contained the 2NS fragment. We initiated Genotyping by Sequencing (GBS) studies for wheat blast resistance. (Obj. 5) Activities for rice blast modeling focused on identifying cooperators and compiling historical observations of blast outbreaks and associated weather data for rice producing regions in AR, LA, TX and CA. Quality assessment and verification of weather data are completed from AR and LA and in process for TX and CA. We calculated potential model variables from the hourly records of temp, RH and rainfall, and developed preliminary models for rice blast. We have also identified rice blast models based on research in Asia and will take advantage of these where possible. J.M. Fernandes (Embrapa, Brazil) is working at KSU for 3 months to incorporate his expertise and his current wheat blast model into the project modeling effort. (Obj.6) The European Cisgenic Survey has been administered in 5 European countries, Belgium, England, France, Holland, and Spain. A total of 3002 surveys were completed and the data was cleaned and analyzed in SAS using a survivor model. The “willingness to pay (WTP)” survey was designed to assess consumers’ attitudes towards cisgenic rice and to estimate which factors influence consumers’ willingness to accept cisgenic rice. There were important differences among countries and consumers do have a more positive attitude towards cisgenic rice than transgenic rice. Many consumers report that they need more information, which represents opportunity for education to have an impact. (Obj. 7) (7-1) Our workshop entitled: ‘Special Symposium: Blast control-a moving target’ on February 18, before the 35th Rice Technical Working Group Meeting in New Orleans had 75 rice stakeholder attendees. After the workshop, Jia and Wamishe responded to many requests for information, DNA marker protocols, and field inspections and 200 samples of blast resistant germplasm were distributed to public and private institutions from the GSOR of DB NRRC. (7-2) A draft wheat blast extension publication was made available for comment at the wheat blast stakeholders workshop Dec 7. (7.3) We had 15 attendees from private industry at a presentation entitled Science, Risks and Benefits of GMOs at a grain purchasing workshop (KSU International Grains Program) on Mar 31. (7.4) A Wheat Blast Workshop in Bolivia provided disease survey and management information to wheat producers, diagnosticians, extension specialists, breeders, research plant pathologists, and students (52 attendees). (7.5) Our Wheat Blast Stakeholders Workshop at the annual Fusarium Head Blight Meeting in St. Louis on Dec 7 attracted 64 attendees, including scientists from universities, USDA-ARS, USDA-APHIS and industry. (OBJ.8) We presented a 1-week Genomics and Bioinformatics Workshop for undergraduates, graduate students, post-docs and faculty at the University of Puerto Rico Mayaguez in June. The 27 attendees performed hands-on real-time PCR on rice blast samples, and learned about blast, fungal diseases, genomics and bioinformatics.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Jia, Y., Wamishe, Y.A., and Zhou, B. 2014. An expedited method for isolaton of DNA for PCR from Magnaporthe oryzae stored on filter paper. The Crop Journal 2: 267-271. Doi: 10.1016/j.cj.2014.06.003. NIFA support acknowledged.
- Type:
Other
Status:
Published
Year Published:
2014
Citation:
Delwaide, Anne-C�cile. 2014. European consumers attitudes towards cisgenic rice. M.S. thesis. University of Arkansas, Fayetteville, AR. NIFA support acknowledged.
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Progress 02/15/12 to 02/14/13
Outputs
Target Audience: Target audiences for this foundational research project are the scientific community studying plant diseases, teachers educating the next generation of scientists, and the general public with a need to better understand the impact of plant diseases on global food production. Changes/Problems: Dr. Mihwa Yi has left the project and she has been replaced with Graduate Student Pierre Migeon and Postdoctoral Research Associate Ely Oliveira Garcia. What opportunities for training and professional development has the project provided? Much of this research so far has been performed by Postdoctoral Research Associate Mihwa Yi. However, for family reasons, Dr. Yi moved to another position at the Noble Foundation in Ardmore, Oklahoma. Graduate Student Pierre Migeon and Postdoctoral Research Associate Ely Oliveira Garcia will begin working on this project in 2014. Research technician Melinda Dalby continues to assist in aspects of this project. Graduate Student Stuart Sprague is working with Dr. Sunghun Park (KSU) to transform rice with a fluorescent plasmodesmata marker protein. Research technician Kim Park is assisting with the rice transformations. Collaborations: We continue our collaborations with Dr. Jung-Youn Lee and her graduate student Xu Wang at the University of Delaware. Our original collaborator, Dr. Kirk Czymmek at the University of Delaware has moved to a position in the Carl Zeiss Company in New York. Now we are working with Dr. Jeffrey Caplan and Shannon Modla in the Microscopy Facility at the Delaware Biotechnology Institute (Delaware Biotechnology Institute BioImaging Center). Dr. Nicole Donofrio helped us produce biological materials for rice leaf sheath infection assays at the University of Delaware. How have the results been disseminated to communities of interest? Project results have been disseminated to academic, government, and industry researchers at professional meetings and at universities in 2012 including the 30th New Phytologist Symposium: Immunomodulation by Plant-Associated Organisms, September 16-19, Fallen Leaf Lake, California; the Department of Plant Pathology, Kobe University, August 3, Kobe, Japan; and the 15th Meeting of the International Society of Molecular Plant Microbe Interactions, July 29-August 2, Kyoto, Japan. In 2013, presentations include the North Central Division Meeting, American Phytopathological Society, Kansas State University, June 14, Manhattan, Kansas; and the 27th Fungal Genetics Conference, March 12-17, Asilomar, Pacific Grove, California. In 2013, Dr. Yi presented talks or posters at the 2013 Midwestern Universities Filamentous Fungal Symposium, University of Missouri-Kansas City School of Biological Sciences, Kansas City, Missouri, February 14; the North Central Coordinating Committee workshop (NCCC-207), Genetics and Biochemistry of Plant-Fungal Interactions, Asilomar, California, March 12; the 27th Fungal Genetics Conference, Asilomar, California, March 12-17; and at The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, May. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1a: Detailed microscopic analyses of invasive hyphal cell-to-cell movement. (1) Mihwa Yi traveled to University of Delaware to learn and perform Correlative Light and Electron Microscopy (CLEM) and advanced confocal microscopy using the Zeiss LSM780 microscope. From the results, localization of Bas53:RFP protein (labeled with monomeric red fluorescent protein) at the cell wall crossing points in both lateral (epidermal to epidermal cell) and vertical (epidermal to mesophyll cell) invasions and at the BICs was confirmed. In the CLEM studies, Bas53:RFP fluorescence also identified double rings where IH had crossed to new cells, located on each cytoplasmic side of the wall area that was crossed. The CLEM images also enabled us to quantitate association between locations where the IH crossed the plant cell wall and pit fields/plasmodesmata. In these CLEM images, rice leaf sheath pit field diameters were about 1.3µm and IH pegs that crossed the wall were about 0.53µm in diameter. IH had clearly crossed through pit fields in 17 percent of the 42 crossing sites examined, and visible plasmodesmata and the surrounding pit field cell wall appeared undamaged. An additional 43 percent of IH crossing points showed pit fields/or plasmodesmata close by, and 40 percent of imaged IH crossing points lacked visible plasmodesmata. Detailed analysis of the size range and density of rice sheath pit fields is required for accurate interpretation of these results. (Work of Mihwa Yi, with Dr. Jung-Youn Lee, and graduate student Xu Wang, Dr. Jeffrey Caplan and Shannon Modla, and Dr. Nicole Donofrio for materials support). (2) Transformation of rice plants to express the plasmodesmata marker gene encoding PDLP5 fused to yellow fluorescent protein has been initiated. (Work of graduate student Stuart Sprague, Kim Park and Dr. Sunghun Park). Objective 1b: Localization dynamics of effectors during early infection stages. (3) To compare and differentiate the expression and localization patterns for the effectors associated with IH crossing cell walls, each effector:RFP fusion protein was co-expressed with cytoplasmic green fluorescent protein (GFP), and these transformants were imaged throughout the biotrophic infection stages. Bas150:RFP showed unique funnel shaped accumulations in appressoria during penetration in addition to accumulation at cell wall crossing points, and the signal remained in later stages. In contrast, Bas79:RFP was only detectable in the later stages after IH had crossed the cell wall. That is, Bas79:RFP fluorescence was observed in BICs in 2nd-invaded cells, but not in BICs in 1st-invaded cells. Bas53:RFP showed fluorescent signals near penetration pores and penetration pegs during penetration and was also detected in the 1st BICs (in the first-invaded cells), at cell wall crossing points and in 2nd BICs in the neighboring cells. Bas83:RFP was only detectable in 1st BICs after penetration, at crossing points and in 2nd BICs. Transformants expressing both Bas53:RFP and Bas83GFP fusion proteins confirmed this difference in localization patterns. Co-transformations with known apoplastic effector Bas4:GFP either with Bas53 and Bas83 clearly differentiated the appoplastic and crossing points localization patterns because Bas4:GFP signal was almost never detectable near cell wall crossing points and its later expression pattern, IH outlining, was evenly distributed throughout the entire IH in the neighboring cells without any remaining signal at the 1st invaded cells. Previously reported Bas2 and Bas3 had localization patterns at the cell wall crossing points and in 1st and 2nd BICs. Bas2 also shows weak fluorescence around penetration pores. Differential expression and localization patterns for the six effector proteins associated with cell wall crossing points suggest each has a distinct role during biotrophic rice cell invasion. (Work of Mihwa Yi). (4) We tested maize leaf sheath as an alternative tissue for in planta infection assays. Use of maize in place of rice can provide plant materials faster after a shorter growing time, as well as providing more variety of fluorescently-labeled transgenic lines. Maize leaf sheath cells provide a superior alternative for studying the cell biology of biotrophic blast invasion compared to commonly-studied onion epidermal cells. (Work of Mihwa Yi). Objective 2: Analysis of callose deposition during cell-to-cell movement. (5) The most common and general callose staining dye, aniline blue, did not work for the rice sheath tissues we use for infection assay, either with or without fixation. More sensitive or different approaches need to be tested to resolve this objective. Meanwhile, after fixation of infected tissues, calcoflour white, which stains cellulose and chitin, showed potential cell wall component modification due to uneven distribution around the cell wall crossing points. Calcoflour white could also differentiate the points where IH had crossed the cell wall as tiny dark spots in the stained wall, due to the lack of stained cell wall components at the points where hyphae crossed. Further assays using specific antibodies to detect individual cell wall components including callose will better address potential modifications during hyphal cell-to-cell movement. (Work of Mihwa Yi, with Dr. Jung-Youn Lee and graduate student Xu Wang). Objective 3: Functional analyses of cell-to-cell movement related effector genes. (6) We continue to face challenges in generating gene replacement mutations for in planta biotrophy-specific effector candidates. Several trials using a conventional deletion knockout strategy by Agrobacterium-mediated fungal transformation (ATMT) on a recipient strain containing a KU70 mutation (mutation in the pathway for non-homologous end joining) did not provide expected deletion mutants. We are considering an RNAi strategy for functional analysis of single and multiple effectors that localize at the cell wall crossing points. (Work of Mihwa Yi). Discoveries/Impacts: Co-localization and time course imaging showed distinct expression profiles and different micro-subcellular localization patterns for each of the 6 putative effector proteins localized at the cell wall crossing points. This implicates finely tuned regulation of their expression and localization at both temporal and spatial levels. Our current data suggests that the effector accumulation occurs after and/or simultaneously with the IH crossing rather than in advance of crossing. Efforts to determine changes in callose deposition during infection development using aniline blue have not succeeded, but this may either imply that callose deposition is not critical for the infection development or that the level is too low to detect by aniline blue dye staining. However, there were clues for cell wall component modifications with calcoflour white dye staining.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yi, M. and B. Valent. 2013. Communication between filamentous pathogens and plants at the biotrophic interface. Annual Review of Phytopathology 51: 587-611.
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