Vitaly H. Citovsky Current Research Projects

Associate Professor
Dept. Biochemistry and Cell Biology

Ph.D., Hebrew University, Jerusalem
Postdoctoral research, University of California, Berkeley

My laboratory is investigating macromolecular transport between and within cells during host-pathogen interactions. Intercellular traffic is studied on the example of cell-to-cell and systemic movement of plant viruses and post transcriptional gene silencing (PTGS) signals whereas intracellular transport is examined using nuclear import of Agrobacterium T-DNA and nucleo-cytoplasmic shuttling of geminiviruses during the infection process.

Because pathogenic microorganisms often adapt existing cellular machinery for their own needs, plant viruses and Agrobacterium likely employ host cell pathways for intercellular and nuclear transport, representing convenient model systems to study these events. Thus, our research not only helps to better understand plant-pathogen interactions but also sheds new light on molecular mechanisms of such general biological processes as transport of nucleic acids and proteins across cell boundaries and through nuclear pores. Below, I summarize our major findings in these areas of research.

I. Viral cell-to-cell and systemic movement and development of viral disease
Communication and molecular transport between plant cells often occur through specialized intercellular connections, the plasmodesmata, which represent functional analogs (paralogs) of animal gap junctions. Plasmodesmata are also utilized by plant viruses for cell-to-cell movement. Following infection, plant viruses, the "pirates of plasmodesmata", spread from cell to cell until they reach the plant vasculature. Having crossed the boundary between non-vascular and vascular tissues, the viruses move to other parts of the plant, resulting in systemic infection and development of viral disease. Both cell-to-cell and systemic spread of plant viruses represent the first major research focus of my laboratory.

A. Viral Cell-to-Cell Movement and Its Regulation. Cell-to-cell movement of plant viruses is mediated by a unique class of biological molecules known to specifically and dramatically alter plasmodesmal function: viral cell-to-cell movement proteins (MPs). The best characterized MP is that of tobacco mosaic virus (TMV) which mediates cell-to-cell spread of the viral genomic RNA. TMV MP interacts with plasmodesmata to increase their size exclusion limit; we have also shown that MP cooperatively binds viral RNA and shapes it into unfolded cell-to-cell transport complexes which can be translocated through the enlarged plasmodesmal channels. Recently, we used MP as a molecular tool to study cellular proteins involved in plasmodesmal transport. We have isolated a host cell protein which specifically interacts MP in vivo and in vitro. Microsequencing of the purified protein identified it as a pectin methyl esterase (PME) that binds MP and, thus, may function as a specific MP receptor during viral cell-to-cell movement. Supporting this idea, our results demonstrate that, in addition to TMV MP, PME is recognized by MPs of turnip vein clearing virus (TVCV) and cauliflower mosaic virus (CaMV). The use of amino acid deletion mutants of TMV MP identified its domain necessary and sufficient for association with PME. Deletion of the PME-binding region resulted in inactivation of TMV cell-to-cell movement.

Recent studies indicate that although MP is present within plasmodesmata of all infected cells, it increases the plasmodesmal permeability only at the leading edge of the expanding infection site. Thus, MP activity within cells behind the leading infection edge must be negatively regulated to prevent its continuous interference with the host plant intercellular communication. The molecular mechanism by which such regulation occurs is unknown. We addressed this question by demonstrating that MP is phosphorylated in vivo at its carboxy terminus. Mimicking MP phosphorylation by negatively charged amino acid substitutions severely disturbs MP ability to interact with plasmodesmata and to promote viral cell-to-cell movement, suggesting that phosphorylation acts as a negative regulator of plasmodesmal transport. Interestingly, this regulatory effect on plasmodesmal permeability as well as the level of MP phosphorylation are host dependent and may contribute to the differential susceptibility of various host plants to TMV.

B. Viral Systemic Spread. Another strategy to study plant virus movement is to isolate host mutants defective for viral spread. To achieve this, we have developed a genetic assay utilizing infection of Arabidopsis thaliana, a plant of choice for genetic experiments with turnip vein clearing tobamovirus (TVCV). Using this approach, we have identified an novel type of mutants (vsm1, virus systemic movement) in which the invading virus spreads locally within the inoculated leaf but is unable to exit this tissue and move systemically. In the absence of systemic infection, vsm1 plants do not show symptoms of viral disease. Genetic segregation indicated that the vsm1 phenotype is caused by a single recessive gene. Characterization of the vsm1 plants represents the first step in genetic dissection of viral systemic movement.

To further study the systemic spread of plant viruses, we have developed specific inhibitors of this process. Exposure of tobacco plants to non-toxic concentrations of heavy metal cadmium was demonstrated to completely block viral disease caused by TVCV. Cadmium-mediated viral protection was due to inhibition of the systemic movement of the virus, i.e., its spread from the inoculated into uninoculated leaves. Treatment of plants with cadmium had no effect on viral replication and local movement within the inoculated leaf. Furthermore, higher, toxic levels of cadmium did not produce this inhibitory effect on viral movement, allowing the systemic spread of TVCV and development of the viral disease. These observations suggest that cadmium-induced viral protection requires a metabolically-active healthy plant. Thus, non-toxic levels of cadmium may trigger the production of cellular factors which interfere with the viral systemic movement.

Using confocal immunofluorescence microscopy, we demonstrated that, in the infected plants exposed to non-toxic levels of cadmium, TVCV virions entered the plant vascular tissue but were unable to exit it into the non-inoculated, systemic organs. This is in contrast to vsm1 plants in which viral systemic spread was blocked at the entry into the vasculature. These results suggest that viral entry into and exit from the vascular cells occur by different mechanisms.

Because viruses often adapt existing cellular machinery for their own needs, they likely employ an endogenous pathway for systemic transport. Indeed, recent evidence indicates that nucleic acids travel throughout the plant to induce post transcriptional gene silencing (PTGS), a fascinating phenomenon potentially involved in regulation of plant gene expression as well as in defense response to pathogen attack. Our studies show that non-toxic concentrations of cadmium, which inhibit viral systemic movement, also block PTGS, suggesting that plant viruses and PTGS signals utilize a common molecular pathway for systemic transport.

C. Development of Viral Disease. In addition to viral movement, my laboratory studies a still poorly understood role of host factors in development of plant viral diseases. to this end, we are isolating Arabidopsis mutants with alterations in the disease symptoms caused by TVCV. So far, we have identified one such mutant, vid1 (virus-induced dwarf), which is indistinguishable from the wild-type plants when healthy but develops a severely dwarfed phenotype with a loss of apical dominance following viral infection. Genetic segregation showed that the this phenotype is caused by a recessive mutation in a single gene. The effect of vid1 mutation was reversed by exogenous application of a plant hormone auxin. Systemic viral infection is thought to interfere with the host plant intercellular transport; we propose that the vid1 mutation may also affect this transport process. Combination of the mutation and viral infection may disrupt transport of developmental regulators, such as hormones, resulting in the vid1 phenotype. The study of vid1 plants may help to understand the involvement of hormonal responses in formation of plant viral diseases.

II. Nuclear transport of Agrobacterium T-DNA and viral genomes
Nucleo-cytoplasmic shuttling of macromolecules is a basic biological process central to the regulation of gene expression which underlies all aspects of development, morphogenesis, and signaling pathways in eukaryotic organisms. Furthermore, nuclear transport of proteins and protein-nucleic acid complexes is an essential step in many host-pathogen interactions such as viral and bacterial infection. These processes represent the second major research focus of my laboratory.

A. Novel Genetic Assay for Nuclear Transport. To facilitate detection of protein traffic into and out of the cell nucleus, we have developed a simple genetic assay to identify active nuclear import (NLS) and export targeting signals (NES) based on their function within yeast cells. In this system, the bacterial LexA protein was modified (mLexA) to abolish its intrinsic nuclear targeting activity and fused to the activation domain of the yeast Gal4 protein (Gal4AD) in the absence or presence of the SV40 large T-antigen NLS. In the nuclear import assay, if a protein of interest fused to the mLexA-Gal4AD hybrid contains a functional NLS, the fusion product will enter the yeast cell nucleus and activate the expression of reporter genes. In the nuclear export assay, if a protein of interest fused to the mLexA-SV40 NLS-Gal4AD hybrid contains a functional NES, the fusion product localized to the cell nucleus will exit into the cytoplasm, decreasing the reporter gene expression levels. Our results indicate that this assay may be applicable as a general method to identify and quantitatively analyze functional NLS and NES as well as specifically select for proteins containing these signals.

B. Nuclear Import of T-DNA During Genetic Transformation of Plants by Agrobacterium. Genetic modification of plant cells by Agrobacterium tumefaciens is the only known natural example of DNA transport between kingdoms. In this process, a single-stranded copy (T-strand) of bacterial transferred DNA (T-DNA) is imported into the host cell nucleus and integrated into its genome, eliciting neoplastic growths on the host plant. We utilize Agrobacterium infection as an experimental system to investigate the molecular mechanisms by which nucleic acid molecules are transported into the cell nucleus. Our studies have identified two Agrobacterium proteins, VirD2 and VirE2, which associate with the T-strand and target it to and through the nuclear pore. VirD2 is covalently attached to the 5' end of the T-strand molecule whereas VirE2 cooperatively coats the rest of the single-stranded (ss) DNA, forming the transport complex (T-complex).

To better understand the structure of the T-complex, we used scanning transmission electron microscopy (STEM) to analyze VirE2-ssDNA complexes formed in vitro. This analysis suggested that VirE2 packages ssDNA into semi-rigid, hollow cylindrical filaments with a telephone cord-like coiled structure. The outer diameter of these complexes is too large to enter the nucleus by diffusion but is within the size exclusion limits of the active nuclear import.

We then showed that the active nuclear uptake of the T-complexes is likely mediated by both VirD2 and VirE2 proteins. Importantly, while VirD2 localizes to the cell nucleus both in plant and animal systems, the nuclear targeting activity of VirE2 is plant-specific.

C. Host Cell Factors Involved in T-DNA Nuclear Import. Currently, we are identifying and characterizing host plant proteins that interact with Agrobacterium VirD2 and VirE2. First, we have cloned an Arabidopsis gene coding for an NLS-binding protein which is directly involved in VirD2 nuclear import. This protein, designated AtKAPa, specifically binds VirD2 in vivo and in vitro. VirD2-AtKAPa interaction is absolutely dependent on the carboxy terminal NLS sequence of VirD2. The deduced amino acid sequence of AtKAPa is homologous to yeast and animal NLS receptors belonging to the karyopherin a family. Indeed, AtKAPa efficiently rescues a yeast mutant defective for nuclear import. Furthermore, AtKAPa specifically mediates transport of VirD2 into the nuclei of permeabilized yeast cells.

Next, we embarked on identification of cellular factors that recognize VirE2 and mediate its nuclear uptake. Isolation of such proteins is especially interesting because VirE2 nuclear import is plant-specific. Using a yeast two-hybrid assay, we identified two Arabidopsis genes whose protein products, designated VirE2 interacting proteins (VIP) 1 and 2, specifically bind VirE2. Amino acid sequence analysis of the predicted protein encoded by the VIP1 cDNA revealed a certain homology to plant but not animal or yeast proteins containing a bZIP motif. Using the described above genetic assay, VIP1 was shown to localize to the yeast cell nucleus. When VirE2 and VIP1 were co-expressed in yeast cells, VirE2, which alone remains cytoplasmic in yeast and animal cells, was also redirected into the cell nucleus. In addition, VIP1 promoted VirE2 nuclear import in mammalian cells. Thus, in yeast and animal systems, VIP1 likely facilitates the plant-specific nuclear import of VirE2. Furthermore, using antisense expression of VIP1 in transgenic tobacco plants, we showed that VIP1 was required for VirE2 nuclear import and Agrobacterium-induced tumor formation in tobacco plants, participating in early stages of T-DNA expression.

Amino acid sequence analysis of the second VirE2 interactor, VIP2, identified homology to the Rga protein of Drosophila, proposed to mediate interaction between chromatin proteins and the transcriptional complex. Unlike VIP1, VIP2 was unable to direct VirE2 into the yeast cell nucleus. However, VIP2 and VIP1 interacted with each other in the two-hybrid system. In uninfected cells, VIP1 and VIP2 may be involved in transcription, associating with the chromosomal DNA either directly or through other components of transcription complexes. Thus, it is tempting to speculate that VIP1, VIP2 and VirE2 may function in a multiprotein complex which performs a dual function: it first facilitates nuclear targeting of VirE2 and then mediates intranuclear transport of VirE2 and its cognate T-strand to chromosomal regions where the host DNA is more exposed and, thus, better suitable for T-DNA integration.

D. Nuclear Import and Export of Viral Genomes. In our laboratory, infection by the tomato yellow leaf curl geminivirus (TYLCV), is a major pathogen of tomato plants around the world, is used as a model system to examine nucleo-cytoplasmic shuttling of proteins and nucleic acids. Unlike most other geminiviruses which divide their genome between two ssDNA molecules, TYLCV contains only one genomic ssDNA encapsulated by the viral capsid protein (CP). The mechanism by which TYLCV genomes enter the host cell nucleus for replication and transcription and then exit it for cell-to-cell spread is unknown.

In an international collaboration, we have shown that TYLCV CP carries a functional NLS and is likely involved in nuclear targeting of the viral genomic DNA molecules. Furthermore, our experiments using the genetic assay for protein nuclear export (see above) identified a functional NES within CP, suggesting that this protein also plays a role in exporting TYLCV genomes from the host cell nucleus. Thus, CP may represent the first plant viral structural protein implicated in both nuclear import and export of the viral genomes.

source: http://www.sunysb.edu/biochem/BIOCHEM/facultypages/citovsky/ 11feb01