Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: We address fundamental questions in two areas in the last 5 years and have 14 publications to report. (1) Agrobacterium: We aim to uncover the structure and function of the trans-membrane channel used by Agrobacterium to transfer DNA and proteins to the plant cell. This channel is the model system for investigation of type IV secretion systems (T4SS), and the significance of this research is underscored further by the fact T4SS are utilized by numerous animal pathogens to transport toxins into their host cells. Twelve proteins form this channel. Our major findings were to determine the localization pattern of the T4SS. Earlier reports from other labs suggested a polar localization; these reports used over-produced proteins and poor resolution microscopy. In contrast, we used high resolution fluorescence deconvolution microscopy to show that nine T4SS components (B1,B2,B4,B5,B7,B8,B9,B10,B11) expressed at their native levels localize as multiple foci around the entire perimeter of the bacterial cell. We further showed that bacteria attach laterally, not polarly, to plant cells. We propose that multiple T4SS increase the probability of bacteria making effective contact with the plant cell during DNA and protein transfer. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells, and enabling the transport of micro- and macromolecules (such as RNA and protein). We study PD during embryogenesis in the model plant Arabidopsis. We previously identified mutants with increased PD transport called increased size exclusion limit (ise)1 and ise2. In the last 5 years we identified their genes; ISE1 and ISE2 encode two distinct RNA helicases that localize to mitochondria or chloroplasts, respectively. As RNA helicases are essential for every aspect of RNA synthesis and processing, their loss would reduce RNA levels leading to organelle dysfunction. To provide additional support that both mutants affect PD, we preformed a quantitative ultrastructural study, and found that both mutants increase de novo PD formation. How might loss of mitochondria or chloroplast function lead to increased PD formation and function We analysed which genes were reduced in expression in both mutants; 40 percent (over 350 genes) of the commonly affected nuclear genes encode essential chloroplast products. The data further imply extensive signaling between mitochondria and chloroplasts. Thus, we propose that chloroplasts are major regulators of cell-to-cell transport in plants. We demonstrated that organelle redox state affects PD mediated cell-to-cell transport; oxidized mitochondria leads to increased PD transport, while oxidized chloroplasts overrides this effect and leads to decreased PD transport. These data reiterate that organelle homeostasis signficantly impacts PD function and the dominant role of the chloroplast. Remarkably, expression of several genes involved in cell wall modification were increased up to 50-fold in ise1 and ise2. As de novo PD formation requires insertion into existing cell walls, we propose that cell wall modification is a prerequisite for PD formation. PARTICIPANTS: Julieta Aguilar, graduate student. Jacob Brunkard, graduate student. Todd Cameron, graduate student. Euna Cho, graduate student. Solomon Stonebloom, graduate student. Dr. Tessa Burch-Smith, postdoc. Dr. Ken Kobayashi, postdoc. Dr. Insoon Kim, postdoc. Dr. Min Xu, postdoc. Dr. John Zupan, Staff research associate. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Research on Agrobacterium will lead to improved methods for plant genetic transformation, and to the design of strategies to block pathogenesis in animal disease. Research on plasmodesmata (PD) mediated intercellular communication in plants will lead to better understanding of innate mechanisms of communication in plants utilized to coordinate developmental programs, as well as to strategies to decrease such communication, for example, to block the spread of viral pathogens that usurp PD channels during infection. Our recent findings, demonstrating that chloroplast homeostasis is a critical regulator of plant cell-to-cell transport via PD, will drive our future research to investigate the mechanism(s) of signaling between PD and chloroplasts. As chloroplasts are the sites of photosynthesis, this latter research will provide insights into the regulation of photosynthesis and thereby impact efforts to design synthetic photosynthetic systems and/or improve photosynthesis in crops to efficiently provide food and biofuels for our growing population.
Publications
- Aguilar, J., Cameron, T.A., Zupan, J.R., and Zambryski, P. Membrane and core periplasmic Agrobacterium tumefaciens virulence type IV secretion system components localize to multiple sites around the bacterial perimenter during lateral attachment to plant cells. mBio 2(6):00218-11 (2011)
- Burch-Smith, T.M., Brunkard, J., Choi, Y.G., and Zambryski, P. Organelle-nucleus crosstalk regulates plant intercellular communication via plasmodesmata. PNAS Plus 108: E1451-1460 (2011)
- Burch-Smith. T.M., Cui, Ya, and Zambryski, P. Reduced levels of class 1 reversibly glycosylated polypeptide increase intercellular transport via plasmodesmata. Plant Signal Behav 7:62-67(2012)
- Stonebloom, S., Brunkard, J., Cheung, A., Jiang, K., Feldman, L., and Zambryski, P. Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata. Plant Physiol.158, 190-199 (2012)
- Zambryski, P.C., Xu, M., Stonebloom, S., and Burch-Smith, T.Embryogenesis as a model system to dissect the genetic and developmental regulation of cell-to-cell transport via plasmodesmata. In Short and Long Distance Signaling. Kragler, F. and Hulskamp, M., eds. Springer, Adv. Plant Biology Vol. 3, pages 45-60 (2012)
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Progress 01/31/11 to 02/01/11
Outputs
OUTPUTS: We address fundamental questions in two areas. (1) Agrobacterium: We aim to uncover the structure and function of the trans-membrane channel used by Agrobacterium to transfer DNA and proteins to the plant cell. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by animal pathogens to transport toxins into host cells. Twelve proteins, VirB1-VirB11 and VirD4 form this channel. Last January we published that two components (VirB8 and VirD4) and three protein substrates (VirD2, VirE2 and VirF) of the T4S system (S) localize in a helical pattern around the perimeter of the bacterial cell. Proteins were detected by their fusion to green fluorescent protein (GFP) and observation by deconvolution fluorescence microscopy. In the last year we optimized detection of native proteins by immuno-fluorescence microscopy and found eight additional T4S proteins, spanning the inner membrane, periplasm and outer membrane also form multiple foci around the circumference of the bacterial cell. Such multiple T4SS likely increase the probability of bacteria making an effective contact with the host plant cell during DNA and protein transfer. We are preparing this work for publication. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. We study cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased PD transport during embryogenesis, and found two mutants, increased size exclusion limit (ise)1 and ise2. A quantitative study of PD formation in ise1, ise2 versus wildtype embryos reveals that ise mutants cause the synthesis of new PD. Thus the increased transport in these mutants is a direct consequence of more PD. Our new data challenge existing dogma on the structure and function of PD in immature tissues, and we wrote an extensive review summarizing our findings in the context of published works. Finally, during our PD studies using enhancer traps expressing GFP in different cell types, we identified a new gene, ORGAN BOUNDARY1 (OBO1), and recently published a report characterizing this gene. OBO1 is expressed at the junction between the shoot apical meristem and lateral organs, and likely plays a role in meristem maintenance and organogenesis. PARTICIPANTS: Julieta Aguilar, graduate student Todd Cameron, graduate student Solomon Stonebloom, graduate student Euna Cho (former graduate student) Tessa Burch-Smith, postdoc Min Xu, postdoc John Zupan, SRA TARGET AUDIENCES: The target audience is members of the scientific community. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research on Agrobacterium will lead to improved methods for plant genetic transformation, and to the design of strategies to block pathogenesis in animal disease. Research on intercellular communication in plants will lead to better understanding of innate mechanisms of communication in plants utilized to coordinate developmental programs, as well as to strategies to decrease such communication, for example, to block the spread of viral pathogens that usurp intercellular plasmodesmata channels during infection.
Publications
- Burch-Smith, T., and Zambryski, P. Loss of INCREASED SIZE EXCLUSION LIMIT(ISE)1 or ISE2 increases the formation of secondary plasmodesmata. Curr. Biol. 20, 989-993 (2010)
- Burch-Smith, T., Stonebloom, S. Xu, M., and Zambryski, P. Plasmodesmata during development: re-examination of the importance of primary ad secondary plasmodesmata structure versus function. Protoplasma 248, 61-74 (2011)
- Cho, E., and Zambryski, P. ORGAN BOUNDARY1 defines a gene expressed at the junction between the shoot apical meristem and lateral organs. Proc. Natl. Acad. Sci. USA 108, 2154-2159 (2011)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: We addressed fundamental questions in two areas: plant transformation by Agrobacterium and intercellular movement between plant cells via plasmodesmata. (1) Agrobacterium: Our long-term goals aim to uncover the structure and function of the trans-membrane channel used by Agrobacterium to transfer DNA from the bacterial cell to the plant cell. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by the fact that T4S is utilized also by animal pathogens to transport toxins into host cells. Twelve major protein components, VirB1-VirB11 and VirD4 form this channel. Our previous studies lead to a topological model for how the 12 proteins are assembled into a membrane-spanning channel. In the last year we have been using high-resolution (deconvolution) fluorescence microscopy to determine the localization of the T4S system. The results suggest that the T4SS localizes in a helical pattern around the perimeter of the bacterial cell. Such multiple T4SS likely increase the probability of the bacteria making an effective contact with the host plant cell during DNA transfer. This work has just been accepted for publication. (2) Plasmodesmata (PD): Plants have evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. We specifically study cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We have developed a genetic screen for mutants that have altered PD function during embryogenesis. These mutants display increased movement of fluorescent tracers. One mutation, called increased size exclusion limit (ise) 1 has recently been identified to reside in a gene that is essential for mitochondrial function (see Stonebloom et al, 2009). ISE1 is an RNA helicase that localizes to mitochondria. The results of these studies have been disseminated by presentation at national and international meetings, as well as by publication in scientific journals (see below). PARTICIPANTS: Patricia Zambryski (PI) John Zupan (SRA) Julieta Aguilar (grad student) Todd Cameron (grad student) Solomon Stonebloom (grad student) Emilie Rennie (grad student) Tessa Burch-Smith (postdoc) Min Xu (postdoc) TARGET AUDIENCES: Scientific community at UCB, USA, and Internationally PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The research on Agrobacterium will lead to improved methods for plant genetic transformation, and to the design of strategies to block pathogenesis in animal disease. Research on intercellular communication in plants will lead to better understanding of innate mechanisms of communication in plants utilized to coordinate developmental programs, as well as to strategies to decrease such communication, for example, to block the spread of viral pathogens that usurp intercellular plasmodesmata channels during infection.
Publications
- Stonebloom, S., Burch-Smith,T., Kim, I., Meinke, D., Mindrinos, M., and Zambryski, P. Loss of the plant mitochondrial DEAD-box RNA helicase ISE1 leads to defective mitochondria and increased intercellular transport via plasmodesmata. Proc. Natl. Acad. Sci. USA 106, 17229-34 (2009)
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