Progress 10/01/12 to 09/30/17
Outputs
Target Audience:Basic research in microbiology and plant biology. Target audience is faculty, research scientists, graduate and undergraduate students, and postdoctoral fellows. Changes/Problems: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. Our very recent data mentioned above, will lead to the design of new strategies to develop antibiotics that can kill bacteria that utilized LD-type transpeptidases. Besides Agrobacterium, several notable human bacterial pathogens (such as Mycococcus tuberculosis) utilize LD-type transpeptidases. 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. What opportunities for training and professional development has the project provided?A former graduate student, Jacob Brunkard, is now working as a postdoctoral scholar (at the PGEC) and he is preparing several manuscripts related to PD research mentioned above. A postdoctoral scholar, Romain Grangeon, has been active in our Agrobacterium research during this last year of AES research, and we have submitted one manuscript on his work, and are currently writing up another manuscript on a different related topic for which he will be co-author. I have accepted a graduate student from Ecuador who will arrive in November 2017, and a postdoc from China who will arrive in December 2017. How have the results been disseminated to communities of interest?Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) opened up her lab to 200 undergraduate students for one hour tours in March 2017. This allows students to see first-hand how research in molecular and cell biology is carried out. What do you plan to do during the next reporting period to accomplish the goals?Nothing has changed from our planned experiments.
Impacts
What was accomplished under these goals?
We address research in two areas. I summarize the last 5 years to put our current research into perspective. At the end, I describe one contribution to plant development and evolution. 1.Agrobacterium: For 40 years, we studied different aspects of the genetic transformation of plant cells by Agrobacterium. Before 2012, we analyzed the molecular composition and localization of the bacterial channel utilized for DNA export. Eleven essential proteins form the type IV secretion system (T4SS). Many animal pathogens use homologous T4SS to export toxins into their respective hosts to cause disease. Agrobacterium T4SS serves as the paradigm. Our reseach over the last 5 years shifted. As ~15 T4SS insert around the entire circumference of the bacterial cell, we wondered how so many channels might be constructed in the context of the bacterial cell cycle. We used the high-resolution microscopes at the CNR CBI and observed that Agrobacterium grows by unipolar growth from one pole versus uniform elongation along the length of the bacterial cell that occurs in model bacteria such as E.coil. We made GFP fusions to the Agrobacterium homologs of well-studied bacterial cell cycle proteins (actin homolog FtsA and tubulin homolog FtsZ). Remarkably, FtsA and FtsZ localize to the growing poles during most of the cell cycle, and then migrate to the midcell just prior to cell division. Thus, Agrobacterium has evolved to usurp proteins previously known only to facilitate cell. division [Zupan, Cameron, Anderson-Furgeson, and Zambryski. PNAS 110, 9060-9065 (2013)] Bacterial growth requires synthesis of cross-linked peptidoglycan (PG) chains essential for cell shape and vitality. We surveyed the genome of Agrobacterium to identify homologs of (~70) well known E.coli PG synthesis components, and found 50% were absent. While most divisome homologs were present, almost no proteins used for lateral growth in E.coli were present. Common D,D-type transpeptidases (DDTs) were lacking in Agrobacterium but its genome is enriched with 14 LD-type tranpeptidases (LDTs). One of these LDTs localizes to the growing pole during polar growth, and is a good candidate for directing polar PG synthesis. [Cameron, Anderson-Furgeson, Zupan, Zik, and Zambryski. mBio 5, e01210-14. (2014) doi:10.1128/mBio.01219-14]. We contributed a review article on this relatively understudied mechanism of bacterial growth. [Cameron, Zupan, and Zambryski. Trends in Microbiology 23, 347-353 (2015)] To identify factors that might regulate polar growth in Agrobacterium we searched for proteins known to exhibit polar localization in other bacteria. Caulobacter crescentus (Cc) exhibits polar asymmetry, dependent on two proteins PopZCc, and PodJCc. Agrobacterium specific PopZAt was found exclusively at the growing pole. PodJAt is initially at the old pole but then also accumulates at the growth pole as the cell cycle progresses suggesting that PodJAt may mediate the transition of the growth pole to an old pole. Thus, PopZAt is a marker for growth pole identity while PodJAt identifies the old pole. [Grangeon, Zupan, Anderson-Furgeson, and Zambryski. PNAS 112: 11666-11671 (2015)] To characterize polar localizing proteins, we created a deletion (del) of podJAt . delpodJAt cells display ectopic growth poles (branching) and growth poles that fail to transition to an old pole. Thus, PodJAt is a negative regulator of polar growth, and PodJAt impacts cell division, likely because the growth pole cannot transition to an old pole. [Anderson-Furgeson, Zupan, Grangeon, and Zambryski. J. Bacteriol. 198, 1883-1891 (2016)] We also created a deletion of popZAt and found that delpopZAt causes a dramatic phenotype with numerous ectopic bulges of growth and the cell cycle time is increased 4-fold. This work was submitted for publication in mBio in Fall 2017 and will be described in our AES report next year. We are characterizing another polar factor called GROWTH POLE RING (GPR) as GFP fusion to GPR exhibits a ring at the growth pole with striking 6-fold symmetry. We predict that GPR is an essential organizing center for polar growth. We have created genetic deletions along this very long protein (2115 aa) to determine its essential features. We will be writing up these data for publication shortly. As PG synthesis is the primary target of antibiotics we study antibiotics that affect DDTs versus LDTs and have confirmed that Agrobacterium PG synthesis and growth are very sensitive to antibiotics that target LDTs. These results will be described in detail in next year's AES report. 2. Plasmodesmata (PD): Plasmodesmata (PD) are elongated channels that traverse the thick plant cell wall and facilitate intercellular communication. My lab developed a genetic screen to identify mutants that might affect PD structure and/or function. As PD are absolutely essential to plant life, we hypothesized that mutants in PD structure or function would manifest first as defects during embryo development. Such mutants could be scored for PD function during embryogenesis, negating the need to propagate viable plants. Embryo defective lines are stably propagated as heterozygotes and 25 % embryo defective homozygous mutant embryos are detected segregating in seedpods. Our work for 10 years previous to 2012, described the identification and characterization of two mutants with an increased ability to traffic fluorescent tracers called increased size exclusion limit (ise), ise1 and ise2; both mutants also exhibited increased production of PD. Surprisingly, each mutant occurs within nuclear genes encoding essential mitochondrial or chloroplast targeted RNA helicases. Comparative transcription analyses revealed over 60 nuclear genes essential for chloroplast function were reduced in both mutants, underscoring the dominant role of the chloroplast in cell and PD function. These results lead to a paradigm shift in our thinking about regulation of PD, i.e., PD regulation does not only occur "locally at" PD but is dependent on overall cellular homeostatic regulation "distant" from PD. We dubbed this new pathway, organelle-nuclear-plasmodesmata signaling (ONPS). As a consequence of these novel data we were invited to contribute several review articles in prominent journals: a) Brunkard, Runkel, and Zambryski. Curr. Op. Plant Biol. 16, 614-620 (2013), b) Brunkard, Runkel, and Zambryski. COPB 35, 13--20 (2015), and c) Brunkard, and Zambryski. COPB 35, 76-83 (2017). As chloroplast homeostasis regulates PD, we studied chloroplast extensions, called stromules, that are very dynamic, extending and retracting within seconds. Stromules form in response to light-sensitive redox signals within the chloroplast. Stromules increase during the day or after treatment with chemicals that produce reactive oxygen species specifically in the chloroplast. Silencing expression of the chloroplast NADPH-dependent thioredoxin reductase, also increased chloroplast stromule frequency. Finally, isolated chloroplasts produced stromules after extraction from the cytoplasm, suggesting that chloroplast-associated factors are sufficient to generate stromules. [Brunkard, Runkel, and Zambryski. PNAS 112, 10044-10049 (2015)] Jake Brunkard and myself just submitted a manuscript that further documents the chloroplast-PD connection: PD transport is strongly regulated by light and the circadian clock. These data will be described in next year's AES report. 3. Plant development and evolution. We also read articles outside our area of research, and were puzzled by an article in Science proposing a new model of evolution of plant transcription factors without gene duplication. We published a technical comment refuting this claim [Brunkard, Runkel, and Zambryski. Science 347, 621a (2015)].
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Brunkard, J.O., and Zambryski, P.C. Plasmodesmata enable multicellularlity: new insights into their evolution, biogenesis, and functions in development and immunity. Curr. Op. Plant Biol. 35, 76-83 (2017).
|
Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Target Audience: Basic research in microbiology and plant biology. Target audience is faculty, research scientists, graduate and undergraduate students, and postdoctoral fellows. Products: Anderson-Furgeson, J., Zupan, J.R., Grangeon, R., and Zambryski, P.C. Loss of PodJ in Agrobacterium tumefaciens leads to ectopic polar growth, branching, and reduced cell division. J. Bacteriol. 198, 1883-1891 (2016) Brunkard, J.O., Runkel, A.M., and Zambryski, P.C.Visualizing stromule frequency with fluorescent micrsocopy. J. Vis. Exp. 117. E54692 (2016) Other Products: none Accomplishments: First field: 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 plant cells. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by numerous animal pathogens to transport toxins into host cells to cause disease. Twelve proteins, VirB1-VirB11 and VirD4 form this channel, and we demonstrated that these proteins localize in a helical pattern around the perimeter of the bacterial cell. These results challenged existing results suggesting one or few T4S systems (S). Our results support a model where Agrobacterium attaches along its length to susceptible plant cells; the numerous T4SSs then efficiently facilitate transport of DNA and proteins into the plant cell. The next question is aimed at understanding how these multiple T4SSs are inserted through the bacterial cell wall. To answer this question we embarked on studies to characterize Agrobacterium cell division. Remarkably, Agrobacterium grows from a single pole in contrast to the generalized elongation along the entire cell length utilized by well-studied bacterial species. Further, Agrobacterium has usurped proteins normally only utilized for cell division at the midcell, FtsA and FtsZ, to mediate such polar growth. In further contrast from well studied bacterial model systems (like E.coli) Agrobacterium utilizes LD-type transpeptidases (versus the more common DD-type transpeptidases) to synthesize its peptidoglycan cell wall, especially during unipolar growth from a single pole of the bacterial cell. In 2015 we described the cell cycle specific localization patterns of two proteins, PopZ and PodJ. PopZ marks the growth pole during polar growth, disappears from the growth pole just before cell division, and then reappears at the new growth poles at the site of septation. PodJ marks the non-growing old pole during the early part of the cell cycle, and then it also marks the new pole late in the cell cycle. PodJ may facilitate the transition of the growth pole to a non-growing (old) pole. Our most recent publication (noted above) analyzed the phenotype of cells with a deletion of the gene encoding PodJ. Lack of PodJ leads to the formation of ectopic growth poles, branched cells and reduced cell division. Thus, PodJ is an essential protein for the Agrobacterium specific cell cycle, and the dramatic phenotypes observed likely occur because the growth pole cannot transition to an old pole in the absence of PodJ. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. PD are exquisitely sensitive to inter- and intracellular signaling pathways, and PD are absolutely essential. We study PD mediated cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased (called increased size exclusion limit 1 and 2) or decreased PD transport (called decreased size exclusion limit1) during embryogenesis. Identification of the genes interrupted by these mutants reveals gene products essential for mitochondrial or chloroplasts RNA processing or proteins that facilitate protein-protein complex formation. While previous studies in many labs have focused entirely on identifying proteins that localize at PD, our results shift the paradigm as they reveal that proteins essential for overall cellular homeostasis and function dramatically affect cell-to-cell transport via PD. We are preparing a manuscript describing another gene with the "ise" phenotype; amazingly the gene affects a gene encoding TOR that mediates several essential signaling pathways required for sensing nutrient availability and response to stress. A second manuscript under preparation will describe how PD transport is regulated by the circadian clock. Second field: Two graduate students were involved in this research, James Anderson-Furgeson, and Anne Runkel. Both students completed their Ph.D. 2016, and both are employed as scientists in research companies in the bay area. James is working at Taxa Biotechnologies in San Francisco, and Anne is working at Mendel Biological Solutions in Hayward. A former graduate student, Jacob Brunkard, is now working as a postdoctoral scholar (at the PGEC) and he is preparing the several manuscripts related to PD research mentioned above. Third field: Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) opened up her lab to 200 undergraduate students for one hour tours in March 2015. This allows students to see first hand how research in molecular and cell biology is carried out. Fourth field: Nothing has changed from our planned experiments. Changes/Problems: None 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. Our very recent data mentioned above, will lead to the design of new strategies to develop antibiotics that can kill bacteria that utilized LD-type transpeptidases. Besides Agrobacterium, several notable human bacterial pathogens (such as Mycococcus tuberculosis) utilize LD-type transpeptidases. 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. Changes/Problems:None What opportunities for training and professional development has the project provided?Two graduate students were involved in this research, James Anderson-Furgeson, and Anne Runkel. Both students completed their Ph.D. 2016, and both are employed as scientists in research companies in the bay area. James is working at Taxa Biotechnologies in San Francisco, and Anne is working at Mendel Biological Solutions in Hayward. A former graduate student, Jacob Brunkard, is now working as a postdoctoral scholar (at the PGEC) and he is preparing the several manuscripts related to PD research mentioned above. How have the results been disseminated to communities of interest?Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) opened up her lab to 200 undergraduate students for one hour tours in March 2015. This allows students to see first hand how research in molecular and cell biology is carried out. What do you plan to do during the next reporting period to accomplish the goals?Nothing has changed from our planned experiments.
Impacts
What was accomplished under these goals?
(1) Agrobacterium: We aim to uncover the structure and function of the trans-membrane channel used by Agrobacterium to transfer DNA and proteins to plant cells. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by numerous animal pathogens to transport toxins into host cells to cause disease. Twelve proteins, VirB1-VirB11 and VirD4 form this channel, and we demonstrated that these proteins localize in a helical pattern around the perimeter of the bacterial cell. These results challenged existing results suggesting one or few T4S systems (S). Our results support a model where Agrobacterium attaches along its length to susceptible plant cells; the numerous T4SSs then efficiently facilitate transport of DNA and proteins into the plant cell. The next question is aimed at understanding how these multiple T4SSs are inserted through the bacterial cell wall. To answer this question we embarked on studies to characterize Agrobacterium cell division. Remarkably, Agrobacterium grows from a single pole in contrast to the generalized elongation along the entire cell length utilized by well-studied bacterial species. Further, Agrobacterium has usurped proteins normally only utilized for cell division at the midcell, FtsA and FtsZ, to mediate such polar growth. In further contrast from well studied bacterial model systems (like E.coli) Agrobacterium utilizes LD-type transpeptidases (versus the more common DD-type transpeptidases) to synthesize its peptidoglycan cell wall, especially during unipolar growth from a single pole of the bacterial cell. In 2015 we described the cell cycle specific localization patterns of two proteins, PopZ and PodJ. PopZ marks the growth pole during polar growth, disappears from the growth pole just before cell division, and then reappears at the new growth poles at the site of septation. PodJ marks the non-growing old pole during the early part of the cell cycle, and then it also marks the new pole late in the cell cycle. PodJ may facilitate the transition of the growth pole to a non-growing (old) pole. Our most recent publication (noted above) analyzed the phenotype of cells with a deletion of the gene encoding PodJ. Lack of PodJ leads to the formation of ectopic growth poles, branched cells and reduced cell division. Thus, PodJ is an essential protein for the Agrobacterium specific cell cycle, and the dramatic phenotypes observed likely occur because the growth pole cannot transition to an old pole in the absence of PodJ. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. PD are exquisitely sensitive to inter- and intracellular signaling pathways, and PD are absolutely essential. We study PD mediated cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased (called increased size exclusion limit 1 and 2) or decreased PD transport (called decreased size exclusion limit1) during embryogenesis. Identification of the genes interrupted by these mutants reveals gene products essential for mitochondrial or chloroplasts RNA processing or proteins that facilitate protein-protein complex formation. While previous studies in many labs have focused entirely on identifying proteins that localize at PD, our results shift the paradigm as they reveal that proteins essential for overall cellular homeostasis and function dramatically affect cell-to-cell transport via PD. We are preparing a manuscript describing another gene with the "ise" phenotype; amazingly the gene affects a gene encoding TOR that mediates several essential signaling pathways required for sensing nutrient availability and response to stress. A second manuscript under preparation will describe how PD transport is regulated by the circadian clock.
Publications
|
Progress 10/01/14 to 09/30/15
Outputs
Target Audience:Basic research in microbiology and plant biology. Target audience is faculty, research scientists, graduate and undergraduate students, and postdoctoral fellows. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Three graduate students were involved in this research, Jacob Brunkard, James Anderson-Furgeson, and Anne Runkel. Jacob Brunkard completed his Ph.D in August 2015, and he is currently pursuing postdoctoral studies at the USDA Plant Gene Expression Center in Albany, CA. How have the results been disseminated to communities of interest?Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) opened up her lab to 180 undergraduate students for one hour tours in March 2015. This allows students to see first hand how research in molecular and cell biology is carried out. What do you plan to do during the next reporting period to accomplish the goals?Nothing has changed from our planned experiments.
Impacts
What was accomplished under these goals?
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 plant cells. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by numerous animal pathogens to transport toxins into host cells to cause disease. Twelve proteins, VirB1-VirB11 and VirD4 form this channel, and we demonstrated that these proteins localize in a helical pattern around the perimeter of the bacterial cell. These results challenged existing results suggesting one or few T4S systems (S). Our results support a model where Agrobacterium attaches along its length to susceptible plant cells; the numerous T4SSs then efficiently facilitate transport of DNA and proteins into the plant cell. The next question is aimed at understanding how these multiple T4SSs are inserted through the bacterial cell wall. To answer this question we embarked on studies to characterize Agrobacterium cell division. Remarkably, Agrobacterium grows from a single pole in contrast to the generalized elongation along the entire cell length utilized by well-studied bacterial species. Further, Agrobacterium has usurped proteins normally only utilized for cell division at the midcell, FtsA and FtsZ, to mediate such polar growth. In further contrast from well studied bacterial model systems (like E.coli) Agrobacterium utilizes LD-type transpeptidases (versus the more common DD-type transpeptidases) to synthesize its peptidoglycan cell wall, especially during unipolar growth from a single pole of the bacterial cell. Our most recent publication (noted above) describes the cell cycle specific localization patterns of two proteins, PopZ and PodJ. PopZ marks the growth pole during polar growth, disappears from the growth pole just before cell division, and then reappears at the new growth poles at the site of septation. PodJ marks the non-growing old pole during the early part of the cell cycle, and then it also marks the new pole late in the cell cycle. PodJ may facilitate the transition of the growth pole to a non-growing (old) pole. We also wrote a review article (noted above) summarizes what is known about the polar mode of growth in bacteria. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. PD are exquisitely sensitive to inter- and intracellular signaling pathways, and PD are absolutely essential. We study PD mediated cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased (called increased size exclusion limit 1 and 2) or decreased PD transport (called decreased size exclusion limit1) during embryogenesis. Identification of the genes interrupted by these mutants reveals gene products essential for mitochondrial or chloroplasts RNA processing or proteins that facilitate protein-protein complex formation. While previous studies in many labs have focused entirely on identifying proteins that localize at PD, our results shift the paradigm as they reveal that proteins essential for overall cellular homeostasis and function dramatically affect cell-to-cell transport via PD. To further characterize how chloroplasts might regulate PD transport, we embarked on a study to characterize chloroplast extensions called stromules. Stromules are very thin (~ 200 nm in diameter) and they can extend 10 um or more out to the periphery of the cell where PD reside. We published a report (noted above) demonstrating that stromules form and extend in response to internal chloroplast specific redox signals, and that isolated chloroplasts form stromues. These studies underscore the autonomy of chloroplasts. We also published a review on PD mediated cell-to-cell transport (noted above). Finally we published a commentary in the journal Science, that evaluates the conclusions of recent publication in that journal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Brunkard, J.O., Runkel, A.M., and Zambryski, P.C. Comments on "A promiscuous intermediate underlies the evolution of LEAFY DNA binding specifity". Science 347, 621a (2015)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Brunkard, J.O., Runkel, A.M., and Zambryski, P.C. The cytosol must flow: Intercellular transport through plasmodesmata. Curr. Op. Cell Biol. 35, 13--20 (2015)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Cameron, T.A., Zupan, J.R., and Zambryski, P.C. The essential features and modes of bacterial polar growth. Trends in Microbiology 23, 347-353 (2015)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Brunkard, J.O., Runkel, A.M., and Zambryski, P.C. Chloroplasts extend stromules independently and in response to internal redox signals. Proc. Natl. Acad. Sci. USA. 112, 10044-10049 (2015)
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Grangeon, R., Zupan, J.R., Anderson-Furgeson, J., and Zambryski, P.C. ����PopZ identifies the new pole, and PodJ identifies the old pole during polar growth in Agrobacterium tumefaciens. Proc. Natl. Aad. Sci. USA 112, 11666-71 (2015)
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: Basic research in microbiology and plant biology. Target audience is academic, including faculty, research scientists, graduate and undergraduate students, and postdoctoral fellows. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four graduate students were involved in this research, Jacob Brunkard, Todd Cameron, James Anderson-Furgeson, and Anne Runkel. In addition, two undergraduates, Mary Ahern, and Lauren Kivlen joined our lab. Todd Cameron graduated in December 2014 and is currently pursuing postdoctoral studies at the University of Texas in Houston. How have the results been disseminated to communities of interest? Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) has given public presentations to the bay area community at Science Café (organized by Science@Cal) in Fall 2013. The PI also participated in a two-day event (February 2014) open to the Berkeley public at large called "Science and Art at Cal (organized by Science@Cal) where scientists (including the PI) presented their research in an artistic manner to stimulate interest in science in the community at large. What do you plan to do during the next reporting period to accomplish the goals? Nothing has changed from our planned experiments.
Impacts
What was accomplished under these goals?
(1) Agrobacterium: We aim to uncover the structure and function of the trans-membrane channel used by Agrobacterium to transfer DNA and proteins to plant cells. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by numerous animal pathogens to transport toxins into host cells to cause disease. Twelve proteins, VirB1-VirB11 and VirD4 form this channel, and we demonstrated that these proteins localize in a helical pattern around the perimeter of the bacterial cell. These results challenged existing results suggesting one or few T4S systems (S). Our results support a model where Agrobacterium attaches along its length to susceptible plant cells; the numerous T4SSs then efficiently facilitate transport of DNA and proteins into the plant cell. Mathematical modeling and quantitative assessment revealed that T4SSs are arranged in a regular periodic fashion with spacing of 0.5 uM between T4SS foci. In the last year, we aimed to understand how these multiple T4SSs are inserted through the bacterial cell wall, and embarked on studies to characterize Agrobacterium cell division. Remarkably, Agrobacterium grows from a single pole in contrast to the generalized elongation along the entire cell length utilized by well-studied bacterial species. Further, Agrobacterium has usurped proteins normally only utilized for cell division at the midcell, FtsA and FtsZ, to mediate such polar growth. Recently we have found that Agrobacterium utilizes LD-type transpeptidases (versus the more common DD-type transpeptidases) to synthesize its peptidoglycan cell wall, especially during unipolar growth from a single pole of the bacterial cell. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. PD are exquisitely sensitive to inter- and intracellular signaling pathways, and PD are absolutely essential. We study PD mediated cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased (called increased size exclusion limit 1 and 2) or decreased PD transport (called decreased size exclusion limit1) during embryogenesis. Identification of the genes interrupted by these mutants reveals gene products essential for mitochondrial or chloroplasts RNA processing or proteins that facilitate protein-protein complex formation. While previous studies in many labs have focused entirely on identifying proteins that localize at PD, our results shift the paradigm as they reveal that proteins essential for overall cellular homeostasis and function dramatically affect cell-to-cell transport via PD. During the past year we were invited to contribute several review articles.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Cameron, T.A., Anderson-Furgeson, J., Zupan, J.R., Zik, J, and Zambryski, P.C.
Peptidoglycan synthesis machinery in Agrobacterium during unipolar growth and cell division. mBio 5, e01210-14. (2014) doi:10.1128/mBio.01219-14
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Brunkard, J.O., Runkel, A.M., and Zambryski, P.C. Cell-cell signaling in plants: Cross-talk among chloroplasts, mitochondria, and plasmodesmata. The Biochemist 56, 11-15 (2014)
- Type:
Book Chapters
Status:
Published
Year Published:
2014
Citation:
Brunkard, J.O., Burch-Smith, T.M., Runkel, A.M., and Zambryski, P.C. Investigating plasmodesmata genetics with virus-induced gene silencing and Agrobacterium-mediated GFP movement assay. Meth. Mol. Bio. 1217: 185-198 (2015) in "Plasmodesmata: Methods and Protocols", Methods in Molecular Biology, vol 1217. Springer, Humana Press. M.Heinlein, ed.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: Basic research scientists in microbiology and plant biology. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Four graduate students were involved in this research, Jacob Brunkard, Todd Cameron, James Anderson-Furgeson, and Anne Runkel. In addition, two undergraduates, Mary Ahern, and Lauren Kivlen joined our lab. How have the results been disseminated to communities of interest? Our research results have been disseminated in scientific journals. In addition, the PI (Professor Patricia Zambryski) has given public presentations to the bay area community, such as “Dinner with a Scientist” (held at the Oakland Zoo in Spring 2013) and Science Café (organized by Science@Cal) in Fall 2013. What do you plan to do during the next reporting period to accomplish the goals? We will continue our research in the above described areas.
Impacts
What was accomplished under these goals?
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 plant cells. This channel is the paradigm for type IV secretion (T4S), and its significance is underscored by its utilization also by numerous animal pathogens to transport toxins into host cells to cause disease. Twelve proteins, VirB1-VirB11 and VirD4 form this channel, and we demonstrated that these proteins localize in a helical pattern around the perimeter of the bacterial cell. These results challenged existing results suggesting one or few T4S systems (S). Our results support a model where Agrobacterium attaches along its length to susceptible plant cells; the numerous T4SSs then efficiently facilitate transport of DNA and proteins into the plant cell. Mathematical modeling and quantitative assessment revealed that T4SSs are arranged in a regular periodic fashion with spacing of 0.5 uM between T4SS foci. In the last year, we initiated new studies to understand how these multiple T4SSs are inserted through the bacterial cell wall, and embarked on experiments to characterize Agrobacterium cell division. Remarkably, Agrobacterium grows from a single pole in contrast to the generalized elongation along the entire cell length utilized by well-studied bacterial species. Further, Agrobacterium has usurped proteins normally only utilized for cell division at the midcell, FtsA and FtsZ, to mediate such polar growth. (2) Plasmodesmata (PD): Plants evolved membrane lined cytoplasmic bridges, called PD, to span cell walls, linking the cytoplasm between adjacent cells. PD are exquisitely sensitive to inter- and intracellular signaling pathways, and PD are absolutely essential. We study PD mediated cell-to-cell movement during embryogenesis in the model plant Arabidopsis. We developed a genetic screen for mutants with increased (called increased size exclusion limit 1 and 2) or decreased PD transport (called decreased size exclusion limit1) during embryogenesis. Identification of the genes interrupted by these mutants reveals gene products essential for mitochondrial or chloroplasts RNA processing or proteins that facilitate protein-protein complex formation. While previous studies in many labs have focused entirely on identifying proteins that localize at PD, our results shift the paradigm as they reveal that proteins essential for overall cellular homeostasis and function dramatically affect cell-to-cell transport via PD. During the past year we were invited to contribute several review articles.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
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)
Xu, M., Cho, E, Burch-Smith, T.M., and Zambryski, P. Plasmodesmata formation and transport function are reduced in decreased size exclusion limit 1 during embryogenesis in Arabidopsis. Proc. Natl. Acad. Sci. USA, 109, 5098-5103 (2012)
Burch-Smith, T.M., and Zambryski, P. Plasmodesmata paradigm shift: regulation from without versus within. Ann. Rev. Plant Biol. 63, 239-260 (2012)
Cameron, T.A., Roper, M., and Zambryski, P. Quantitative image analysis and modeling indicate the Agrobacterium tumefaciens virulence type IV secretion system is organized in a periodic pattern of foci. PLoS ONE 7, e42219 (2012)
Cameron, T.A., and Zambryski, P. Disarming bacterial type IV secretion. Chem.
Biol. 19, 934-936 (2012)
Zupan, J.R., Cameron, T.A., Anderson-Furgeson, J., and Zambryski, P. Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA, 110, 9060-9065 (2013)
Zambryski, P. Fundamental discoveries and simple recombination between circular plasmid DNAs led to widespread use of Agrobacterium tumefaciens as a generalized vector for plant genetic engineering. Int. J Dev Biol. 57, 449-452
(2013)
Brunkard, J.O., Runkel, A.M., and Zambryski, P.C. Plasmodesmata dynamics are coordinated by intracellular signaling pathways. Curr. Op. Plant Biol. 16, 614-620
(2013)
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