Sandy Lazarowitz, PhD
Department of Plant Pathology
Research Focus
Our long term goals are to understand the mechanisms that regulate intracellular and intercellular trafficking in plants, and common plant cell responses to pathogen attack. Most of our efforts focus on the biochemical functions and cellular interactions of the two movement proteins NSP and MP encoded by the bipartite geminiviruses, using these as models to investigate nuclear shuttling and the biogenesis of channels in plant cell walls. In collaboration with Alan Collmerís lab, we are also taking a functional genomics approach to investigate the roles of bacterial avirulence (avr) proteins in pathogenesis and in inducing host resistance responses in plants. The model geminiviruses we employ are SqLCV (squash leaf curl virus), which has a broad host range in cucurbits, and the closely-related CLCV (cabbage leaf curl virus), which has a broad host range in Brassica that includes Arabidopsis. Our functional genomics studies employ the model plant pathogenic bacterium Pseudomonas syringae pv. tomato DC3000.
Plant viruses overcome the barrier of the plant cell wall by encoding movement proteins that allow the virus to move directly cell to cell without an extracellular phase. Our research has defined the functions of NSP and MP, and their cooperative interactions to move the geminivirus single strand DNA from its site of replication in the nucleus, to and across the cell wall. Using molecular genetics, biochemistry, and cell biology, we have shown that NSP is a nuclear shuttle protein that moves the viral genome between the nucleus and the cytoplasm. MP targets to the cortical endoplasmic reticulum (ER) to induce the formation of unique transwall tubules, which may be the functional analog of the desmotubule, the core structure of plasmodesmata. Thus, the virus appears to usurp the cell machinery that forms plasmodesmata to create virus-specific channels for cell-to-cell movement. MP provides directionality to virus movement, trapping NSP-genome complexes in the cytoplasm and directing these to and across the cell wall. In adjacent uninfected cells, MP releases NSP, which now targets the viral genome to and across the nuclear pore to initiate new rounds of infection. Using site-directed mutagenesis and transient expression assays, we have identified the essential functional domains in NSP and MP, including a nuclear export sequence (NES) in NSP that resembles the leucine-rich NES in HIV Rev, TFIIIa and other rapidly shuttling nuclear proteins in animal cells and yeast. The Xenopus TFIIIa NES functionally substitutes for the NSP NES, suggesting that the mechanism of nuclear export, like import, is highly conserved among plants, animals and yeast.
We are now using NSP and MPB as models to investigate the regulation of nuclear export in plant cells and the ER remodeling that leads to the formation of MP tubules and potentially transwall channels. Using yeast-based interactive screens, we have identified a number of novel Arabidopsis proteins that interact with NSP or MP. The functions of these novel proteins in nuclear export or in vesicle trafficking and fusion, specifically, and more generally in plant growth and development, is being investigated through a combination of cell-based and biochemical assays, and RNA silencing and expression studies in transgenic plants.
A broader aspect of our interest in the cellular interactions that underlie the pathogenic process is the investigation of common plant cell responses to diverse plant pathogens. Using the model of P. syringae DC3000 and its interactions with Arabidopsis and tomato, we have obtained a collection of transgenic Arabidopsis lines that have different subcellular compartments and structures labeled with GFP and are generating a similar collection of transgenic tomato lines. These plant-based tools are being used to investigate the remodeling that occurs when plant cells respond to attack by compatible or incompatible P. syringae strains, and will also be used to examine responses to geminivirus infection.