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Our laboratory performs research in two distinct areas. In microbial biology we study the molecular mechanisms utilized by Agrobacterium that leads to the genetic transformation of plant cells. In plant biology we study how plant cells communicate with each other via unique plant specific intercellular structures called plasmodesmata.

 

1. Agrobacterium mediated DNA transfer to plant cells. A specific DNA segment, T-DNA, is transferred from a large bacterial plasmid across bacterial and plant cell membranes and ultimately integrates stably into the plant nuclear genome. We study Agrobacterium-specific proteins and their respective molecular mechanisms responsible for producing a DNA-protein complex capable of plant cell transformation. Current research is aimed at defining the topology of the bacterial export channel. This channel is the paradigm for type IV secretion systems (T4SS). T4SS are utilized by plant and animal pathogens to transport DNA as well as protein toxins that manipulate their respective host cells to cause disease.

A model of how the 12 proteins (VirB1-B11, VirD4) essential for T4SS form a membrane-spanning channel is presented in Figure 1. The T-DNA is transferred as a single stranded intermediate called the T-strand. In addition, 4 proteins, VirD2, VirE2, VirE3, and VirF are exported by the T4SS to plant cells. This model was predicted by genetic and bioinformatics approaches. Current efforts aim to define the topology, location and function of the T4SS. We have recently determined that the multiple T4SS localize in a periodic (potentially) helical pattern around the circumference of the bacterial cell.This localization pattern was determined by deconvolution fluorescence microscopy of GFP fusions to T4SS components and substrates. Figure 2 shows several optical sections through the bacterial cell. This localization pattern was confirmed by immuno-fluorescence microscopy of native T4SS proteins. This localization pattern likely facilitates binding of Agrobacterium to susceptible plant cells. Indeed, if one tags the T4SS with GFP, one can see Agrobacterium binding laterally along their lengths to single plant cells. (Figure 2).

 

We now are investigating what constitutive bacterial components facilitate T4SS localization. We reasoned that the T4SS may either use an existing periodic/helical scaffold for its assembly, such as the bacterial cytoskeleton, or it may take advantage of interruptions in the bacterial cell wall (peptidogylcan) that occur during bacterial cell elongation. Recent studies suggest both hypotheses are correct and converge on the specific mechanisms underlying the determination of Agrobacterium cell shape. In addition we are determining the role of the extracellular T-pilus (colored in yellow  Figure 1) in mediating plant cell contact and substrate transport.

 

Our laboratory is interested in defining the factors that regulate PD structure and function that are localized at the entrance and along the length of the channel. We have developed a genetic screen using embryo defective lines of Arabidopsis and have several candidate mutants that define genes that affect PD aperture during mid embryogenesis.

We are also studying the innate capacity of plant embryos to traffic proteins cell to cell via PD. Different sized derivatives of green fluorescent protein (GFP) were initially expressed in meristematic regions of the embryo. Subsequent movement of encoded proteins from their site of synthesis then reveals PD aperture. 1X sized GFP moves throughout all regions of the embryo. 2X sized GFP cannot move to the embryonic leaves, and 3X sized GFP cannot move to the tip of the root. Thus, different regions of the embryo contain PD with different apertures that likely regulate the movement of critical regulators of development (Figure 6).

 

We have identified the genes in two mutants with increased intercellular transport during embryogenesis, called increased size exclusion limit 1 (ise1) and ise2. ISE1 encodes a mitochondrial localized DEAD box RNA helicase, and ISE2 encodes a DEVH box RNA helicase that localizes to chloroplasts. Whole genome expression analyses reveal that nuclear genes encoding plastid specific products are overwhelmingly down regulated in both ise1 and ise2 mutants (genes down regulated in both mutants are shown in red in Figure 7A). In fact, both mutants affect plastid development and cause leaf chlorosis (Figure 7B). These data imply extensive crosstalk among mitochondria, plastids, and nuclei. Since both mutants show increased de novo production of plasmodesmata, these expression analyses further imply that organelle-nuclear signaling affects plasmodesmata formation and function. While mitochondrial-nuclear or plastid-nuclear signaling are established and known retrograde signaling pathways, we have uncovered a novel pathway, dubbed organelle-nucleus-plasmodesmata signaling (ONPS)(Figure 7C). Independent analyses of redox states in mitochondria versus plastids confirm that organelle homeostasis affects plasmodesmata transport.Ongoing research aims to uncover genes and signals that regulate ONPS and de novo plasmodesmata synthesis.

Plasmodesmata structure and function

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. In revision

Burch-Smith, T.M., Brunkard, J., Choi, Y.G., and Zambryski, P. Organelle-nucleus crosstalk regulates plant intercellular communication via plasmodesmata. PNAS Plus. In press (2011)

AAAS (American Association for the Advancement of Science) Fellow, 2010

International Francqui Chair, University of Gent, Belgium, 2009 - Francqui Foundation - 2008
Miller Research Professorship - Miller Institute for Basic Research in Science - 2004
Fellow of the American Society for Microbiology - American Society for Microbiology - 2001
Member of the National Academy of Sciences - National Academy of Sciences - 2001