Progress 08/15/06 to 08/14/10
Outputs
OUTPUTS: Our previous results implicated a novel pathogen-induced structure, named the Biotrophic Interfacial Complex (BIC, originally called the Effector Secretion Body), in delivery of fungal effectors across the plant membrane that encases blast biotrophic invasive hyphae (IH) inside the plant cell. Focusing on known blast effectors in the AVR-Pita and PWL families, we transformed strains of Magnaporthe oryzae to secrete chimeric fluorescent effector proteins and visualized secretion and translocation of these fluorescent effectors as the fungus invaded rice leaf sheath cells. Specifically, (1) we tested various fluorescent proteins and techniques to optimize imaging of the fungus invading living rice leaf epidermal cells; (2) we used confocal and fluorescence microscopy to visualize BIC development during fungal invasion; (3) in collaboration with the Czymmek laboratory in Delaware, we performed correlative light microscopy/TEM using chemical fixation techniques for ultrastructural analysis of BICs; (4) we constructed fungal transformants expressing chimeric fluorescent biotrophy-associated secreted (BAS) proteins and determined fluorescent BAS protein secretion/localization patterns during rice cell invasion; (5) we performed fluorescence recovery after photobleaching (FRAP) analysis to determine if effectors are continuously secreted into BICs; (6) we developed a rice cell plasmolysis assay for enhanced visualization of translocated effectors into the rice cytoplasm; (7) we enhanced the sensitivity of the host translocation assay by engineering a nuclear localization signal into the chimeric effectors, thereby concentrating them in the rice nucleus; (8) we performed quantitative microscopic analysis and documented the correlation between BIC accumulation and translocation into the rice cytoplasm; (9) we compared effector and BAS protein localization patterns in compatible (susceptible) and incompatible (resistant) interactions, and (10) in collaboration with the Lee laboratory in Korea, we tested involvement of ER-mediated secretion in BIC accumulation using fungal mutants lacking the ER chaperone LHS1. In addition to publications, this research has been disseminated in 40 invited presentaions and numerous posters at national and international scientific meetings and universities. Results obtained with this funding were highlighted in The Plant Cell (K. Farquharson, "Magnaporthe Effectors May Pave the Way for Hyphal Invasion", 22: 996, 2010), and they were highlighted for the general scientific community in Nature Reviews Microbiology (S. Molloy, "Magnaporthe Effectors on the Move", 8: 466-467, 2010). Two of the Plant Cell articles from this project (Mosquera et al., 2009 and Khang et al., 2010) were rated as "Must Read" by the Faculty of 1000. Our work was featured on the cover of the journal Current Opinion in Plant Biology (August 2010) in which we contributed a review on Magnaporthe effectors. PARTICIPANTS: Individuals at KSU: Barbara Valent (PI), Chang-Hyun Khang (Post-doctoral Fellow), Martha C. Giraldo (Graduate Student); Melinda Dalby (Research Technician); Britni Samuelson (Undergraduate researcher); Mihwa Yi (Post-doctoral Fellow). Collaborators: Kirk Czymmek (University of Delaware); Seogchan Kang (The Pennsylvania State University); Yong Hwan Lee (Seoul National University, Korea). TARGET AUDIENCES: Target audiences for this foundational research project are the scientific community studying plant diseases, the broader scientific community, and the general public with a need to better understand the impact of plant diseases on global food production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Our findings provide critical new detail on how the blast fungus invades rice cells and causes rice blast disease. Our results characterize BIC structure and dynamics and support the hypothesis that BICs are the staging area for translocation of effectors into the rice cytoplasm. Specifically, (1) BICs first occur at the tips of the filamentous primary hyphae that enter the host cell, and they are left behind beside the first differentiated IH cell as the fungus proliferates elsewhere. FRAP analysis showed that fluorescent effectors continuously accumulate in BICs, and that BICs are surrounded by rice cytoplasmic accumulations with dynamic connections to the original penetration site. (2) Correlative LM/TEM suggested that BICs are an interfacial aggregation of lamellar membranes and diverse vesicles. However, plant and fungal membranes were poorly preserved by the chemical fixation techniques used, and we postponed further TEM analysis until correlative techniques are developed using high-pressure freezing/freeze substitution for improved membrane preservation. (3) We characterized additional BIC-localized BAS proteins as well as a nonBIC-localized putative interfacial matrix protein, BAS4, which is retained in the membrane compartment surrounding successful IH. We routinely assess membrane compartment integrity and IH health using fungal transformants that secrete fluorescent BAS4 protein in addition to fluorescent effectors. (4) Effectors and BAS proteins that accumulate in BICs are translocated to the cytoplasm of invaded rice cells, but the nonBIC-localized BAS4 protein is not. (5) Fully fluorescent BICs are characteristic of the compatible (susceptible) interaction, but not of the incompatible interaction in which invaded rice cells undergo hypersensitive death. M. oryzae mutants lacking the LHS1 gene (luminal heat shock protein seventy) were severely impaired in effector avirulence function and fluorescent effector accumulation in BICs. (6) Fluorescent effectors that reached the cytoplasm of the invaded cell moved ahead of the fungus into neighboring plant cells, presumably to prepare them before fungal entry. Properties associated with this cell-to-cell movement of effectors, protein size-limitations and plant cell type differences, suggest that the effectors move through plasmodesmata. Understanding and blocking this movement would provide another mechanism for controlling disease development. (7) Our sensitive nuclear-targeting host translocation assay will facilitate discovery of motifs that control effector secretion, translocation and cell-to-cell movement using a mutagenesis approach. The assay will allow us to screen the many secreted proteins encoded in the fungal genome for putative effectors that are delivered into plant cells. Additionally, identification of effectors that work inside living plant cells will identify AVR gene candidates for triggering hypersensitive resistance through intracellular resistance genes. Understanding fungal effector secretion systems, and identifying the secreted effectors will provide new targets for protecting agricultural crops from disease.
Publications
- Mosquera, G., M.C. Giraldo, C.H. Khang, S. Coughlan, and B. Valent. 2009. Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as biotrophy-associated secreted proteins in rice blast disease. The Plant Cell, 21:1273-1290. (Rated F1000 Biology Factor 6, Must Read)
- Khang, C.H., R. Berruyer, M.C. Giraldo, P. Kankanala, S.-Y. Park, K. Czymmek, S. Kang and B. Valent, 2010. Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement. The Plant Cell, 22:1388-1403. Valent, B. and C.H. Khang. 2010. Recent advances in rice blast effector research. Current Opinion in Plant Biology 13: 434-441. (Featured on the cover of the journal)
- Valent, B., C.H. Khang, M.C. Giraldo, M. Yi and M. Dalby. 2010. Towards understanding effector secretion, translocation and cell-to-cell trafficking during biotrophic invasion by the rice blast fungus. In: 2011 Proceedings of the US-Japan Seminar on Genome-Enabled Integration of Research in Plant-Pathogen Systems. (T. Wolpert, T. Shiraishi, eds.) APS Press, St. Paul, Minnesota. In press.
- Khang, C.H. and B. Valent. 2010. Magnaporthe oryzae. In: Cellular and Molecular Biology of Filamentous Fungi. (edited by K.A. Borkovich and D.J. Ebbole) ASM Press. Washington D.C., pp 593-606.
- Yi, M., M.-H. Chi, C.H. Khang, S.-Y. Park, S., Kang, B. Valent and Y.-H. Lee. 2009. The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. The Plant Cell, 21:681-695. (Highlighted in The Plant Cell 21:366, 2009)
- Kankanala, P., G. Mosquera, C.H. Khang, G. Valdovinos-Ponce and B. Valent. 2009. Cellular and molecular analyses of biotrophic invasion by the rice blast fungus. In: Advances in Genetics, Genomics and Control of Rice Blast Disease. (edited by G.L. Wang and B. Valent) Springer Science and Business Media, New York, New York, pp 83-91.
- Advances in Genetics, Genomics and Control of Rice Blast Disease. 2009. Edited by G.L. Wang and B. Valent. Springer Science and Business Media, New York, New York. 464 pp.
- Khang, C.-H., S.-Y. Park, Y.-H. Lee, B. Valent and S. Kang. 2008. Genome organization and evolution of the AVR-Pita avirulence gene family in the Magnaporthe grisea species complex. Molecular Plant-Microbe Interactions, 21:658-670.
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: In order to cause disease, biotrophic and hemibiotrophic fungi deliver effector proteins across plant membrane into the cytoplasm of living host cells in order to block host defense responses and control host cellular processes needed for disease development. Our goal is to understand the mechanisms and signaling motifs required for effecter delivery by the agronomically important rice blast fungus. Our previous results have implicated a novel pathogen-induced structure, which we named the Biotrophic Interfacial Complex (BIC), in delivery of fungal effectors across the plant extra-invasive-hyphal membrane (EIHM) that encases blast biotrophic invasive hyphae (IH) inside the plant cell. Much of our effort in the last year has focused on Objective 3, which is to demonstrate that a secreted effector protein reaches the plant cytoplasm or a plant organelle, and identify the mRNA or protein signals that are required for this localization. We have now clearly shown that the effector:fluorescent protein that accumulates in BICs is also observed in the cytoplasm of the invaded host cell. The blast effector protein PWL2, when fused to different fluorescent proteins EGFP, mRFP and tdTomato, is delivered across the EIHM into the cytoplasm of live rice epidermal cells. Since breakage of the EIHM surrounding the IH would spill fluorescent effectors into the cytoplasm without crossing of host membrane, we assess EIHM integrity in individual infection sites using fungal transformants that secrete both fluorescent effector protein and fluorescent BAS4 matrix protein. For example, BAS4:EGFP uniformly outlines IH surrounded by intact EIHM. In rice cells invaded by IH with uniform BAS4:EGFP outlining, PWL2:mRFP and not BAS4:EGFP was observed in the cytoplasm of the invaded host cell, showing that the effector and not the matrix protein was delivered to the host cytoplasm. Surprisingly, at these infection sites, the PWL2:mRFP had moved ahead of the fungus into neighboring cells surrounding the invaded cell. This intriguing result suggests that the pathogen sends its effectors ahead to prepare adjoining cells before entering them. We have shown that we can more clearly visualize PWL2:mRFP accumulation in the rice cell by adding a nuclear localization motif to the chimeric effector proteins. This results in concentrated bright fluorescence of the translocated effector protein in the host nucleus. We have now developed a robust assay for translocation of blast effector:fluorescent proteins which will facilitates a mutagenesis approach to identify the critical effector sequences required for both BIC localization and translocation into the host cytoplasm. Our results are still consistent with the hypothesis that effectors undergo targeted secretion compared to non-effector fungal proteins, and that specific signal sequences mediate this secretion. PARTICIPANTS: Chang-Hyun Khang, Martha Giraldo, Melinda Dalby, Barbara Valent TARGET AUDIENCES: The current target audiences are scientists working on molecular plant-microbe interactions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Biotrophic and hemi-biotrophic fungi that initially invade living plant cells cause many significant diseases of US crops. These fungi were believed to deliver effector proteins across plant cell membranes into the live plant cells in order to block the plant's defenses, control plant metabolism, and cause disease. However, this critical disease process has not been not understood for any fungal plant pathogen. A significant outcome of this project is our direct demonstration that fluorescently labeled effectors from the rice blast fungus accumulate in a specialized structure, the biotrophic interfacial complex, at the host-pathogen interface and that these effectors are delivered across host membrane into the cytoplasm of live plant cells. By optimizing the fluorescent reporter system, we have developed a unique, robust assay for effector translocation into plant cells. This translocation assay will now allow discovery of specific motifs that target effector secretion using a mutagenesis approach. The assay will also allow us to sort among the many secreted proteins encoded in the fungal genome for those that are effectors delivered into plant cells. A second outcome of our research is our demonstration that fluorescent effectors move ahead of the fungus into plant cells that are not yet invaded, presumably to prepare them before the fungus enters them. This significant discovery opens questions as to how effectors are able to move on their own into new plant cells. Blocking this movement would provide another mechanism for controlling disease development. Additionally, identification of fungal effectors that work inside living plant cells will identify key strategies the fungus uses during biotrophic plant invasion and will identify AVR gene candidates for triggering hypersensitive resistance through intracellular resistance genes. Understanding effector secretion systems used by a fungus to control living plant cells, and identifying effectors that mediate this control will provide new targets for protecting agricultural crops from disease. This research addresses CSREES goal 3: Enhance protection and safety of the Nation's agriculture and food supply.
Publications
- Mosquera, G., M. Giraldo, C.H. Khang, S. Coughlan, and B. Valent. 2009. Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as biotrophy-associated secreted proteins in rice blast disease. The Plant Cell, 21:1273-1290. (Rated F1000 Biology Factor 6, Must Read)
- Yi, M., M.-H. Chi, C.H. Khang, S.-Y. Park, S., Kang, B. Valent and Y.-H. Lee. 2009. The ER chaperone LHS1 is involved in asexual development and rice infection by the blast fungus Magnaporthe oryzae. The Plant Cell, 21:681-695.
- Khang, C.H. and B. Valent. 2010. Magnaporthe oryzae. In: Cellular and Molecular Biology of Filamentous Fungi. (edited by K.A. Borkovich and D.J. Ebbole) ASM Press. Washington D.C., pp 593-606.
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Progress 08/15/07 to 08/14/08
Outputs
OUTPUTS: The goals of this project are to understand how the hemi-biotrophic rice blast fungus delivers effector proteins across the rice plasma membrane into the cytoplasm of living rice cells in order to promote biotrophic invasion leading to disease. Previously, we discovered a novel pathogen-induced structure (original name Effector Secretion Bodies, renamed Biotrophic Interfacial Complexes (BIC)) in the interfacial zone between the fungal cell wall and the plant extra-invasive-hyphal membrane (EIHM) that encases biotrophic invasive hyphae (IH) inside the plant cell. Because chimeric effector:green fluorescent proteins secreted by IH accumulate preferentially in BICs, we hypothesized that these structures function to deliver fungal proteins across the EIHM into the host cytoplasm. This proposal has the following objectives: structurally characterize BICs using confocal microscopy, transmission electron microscopy and immunocytochemistry; identify additional biotrophic hyphal proteins that are secreted into BICs; demonstrate that a secreted effector protein reaches the plant cytoplasm or a plant organelle, and identify the mRNA or protein signals that are required for this localization. We have shown that each BIC develops in two-steps that correlate with differentiation of the first filamentous hypha that enters the cell into the pseudohyphal IH that colonize the cell. Each time the fungus entered a living rice cell, the BIC was first observed at the tip of the initial filamentous hypha, but then moved off the tip as the fungus switched to its IH growth-form. Fluorescence preferentially accumulated in BICs and around IH-differentiation junctions, but not around the IH that continued to grow in the cell. We have demonstrated that BICs are an interfacial aggregation of lamellar membranes and diverse vesicles, and that they are associated with FM4-64-staining endocytotic membranes and dynamically-shifting cytoplasm in the host cell. We have expanded our analyses of secretion of putative blast effectors identified by microarray analysis. To clearly demonstrate differential secretion patterns for these proteins, we have produced fungal transformants that simultaneously secrete different fluorescent reporter proteins. Specifically, we produced transformed fungal strains that express a known blast effector labeled with one fluorescent reporter along with newly identified IH-specific proteins fused to a different fluorescent reporter. This analysis clearly demonstrated that some but not all of these putative effectors preferentially localize to BICs. One IH-specific protein appears to be an extra-invasive-hyphal matrix protein with uniform distribution inside the EIHM surrounding the entire IH. This protein has become a valuable tool for assessing EIHM integrity around particular IH. In significant progress toward objective 3, we have now defined a system to demonstrate translocation of effector:fluorescent proteins across the EIHM into the plant cytoplasm. Our results are still consistent with the hypothesis that effectors undergo targeted secretion compared to non-effector fungal proteins, and that specific signal sequences may mediate this secretion. PARTICIPANTS: Chang-Hyun Khang, Martha Giraldo, Melinda Dalby, Barbara Valent TARGET AUDIENCES: The current target audiences are scientists working on molecular plant-microbe interactions. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project addresses a fundamental question of how fungi that impact agricultural productivity colonize living plant cells. Biotrophic and hemi-biotrophic fungi must deliver effector proteins across plant cell membranes into living plant cells in order to block the plant's defenses, control plant metabolism, and cause disease. This process is not understood for any fungal plant pathogen. The longer term goal of this research is to understand the molecular mechanisms of effector secretion and to develop assays or structural paradigms for identifying effectors from the M. oryzae proteome. Discovery of specific motifs that target secretion of effectors will allow us to use the available fungal genome sequence to identify a complete set of effectors from a fungal plant pathogen. Identification of fungal effectors that work inside living plant cells will identify key strategies the fungus uses during biotrophic plant invasion and will identify AVR gene candidates for triggering hypersensitive resistance through intracellular resistance genes. Understanding effector secretion systems used by a fungus to control living plant cells, and identifying effectors that mediate this control will provide new targets for protecting agricultural crops from disease. This research addresses CSREES goal 3: Enhance protection and safety of the Nation's agriculture and food supply.
Publications
- Kankanala, P., G. Mosquera, C.H. Khang, G. Valdovinos-Ponce and B. Valent. 2008. Cellular and molecular analyses of biotrophic invasion by the rice blast fungus. In: Advances in Genetics, Genomics and Control of Rice Blast Disease. (edited by G.L. Wang and B. Valent) Springer Science and Business Media, New York, New York, pp 83-91.
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Progress 08/15/06 to 08/14/07
Outputs
OUTPUTS: Biotrophic or hemibiotrophic fungi that cause major crop diseases must deliver effector proteins across plant cell membranes into living plant cells in order to block the plant's defenses, control plant metabolism, and cause disease. This process is not understood for any fungal plant pathogen. My laboratory discovered a novel structure (Originally named Effector Secretion Bodies, renamed Blast Interfacial Complexes or BIC) in the interfacial zone between the fungal cell wall and the plant extra-invasive hyphal membrane (EIHM) that encases the fungus inside the plant cell. We hypothesized that BIC represent specialized structures for delivering fungal proteins across the EIHM into the plant cytoplasm. We have made progress on our first objective to structurally characterize BIC. Live cell confocal microscopy has defined the dynamic nature of the plant cytoplasm and plant membrane connections surrounding BIC. Correlative light and electron microscopy suggest that BICs are
complex membrane-rich and vesicle-rich structures. As progress toward our second objective, we have fused the GFP reporter gene to 4 novel secreted proteins that we identified as specifically expressed by the fungus during biotrophic invasion. Two of these show BIC-localized secretion patterns and 2 show different secretion patterns. As progress toward objective 3, we have demonstrated that these different secretion patterns occur depending on whether or not effector promoters and/or signal peptide sequences are used to express and secrete GFP-fusion proteins inside the plant cell. These results are consistent with our hypothesis that effectors are secreted through a specialized secretion system compared to non-effector fungal proteins, and that effector genes/proteins must have specific signal sequences to mediate this secretion.
PARTICIPANTS: Chang-Hyun Khang, Martha Giraldo, Melinda Dalby, Barbara Valent
TARGET AUDIENCES: Agricultural Scientists
Impacts
This project addresses the fundamental question of how fungi that impact agricultural productivity colonize living plant cells. The longer term goal of this research is to understand the molecular mechanisms of effector secretion and to develop assays or structural paradigms for identifying effectors from the M. grisea proteome. Discovery of specific motifs that target secretion of effectors will allow us to use the available fungal genome sequence to identify a complete set of effectors from a fungal plant pathogen. Identification of fungal effectors that work inside living plant cells will identify key strategies the fungus uses during biotrophic plant invasion and will identify AVR gene candidates for triggering hypersensitive resistance through intracellular resistance genes. Understanding effector secretion systems used by a fungus to control living plant cells, and identifying effectors that mediate this control will provide new targets for protecting
agricultural crops from disease. This research addresses CSREES goal 3: Enhance protection and safety of the Nation's agriculture and food supply.
Publications
- No publications reported this period
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