Anton A. Sanderfoot

Increases in the Number of SNARE Genes Parallels the Rise of Multicellularity among the Green Plants

The green plant lineage is the second major multicellular expansion among the eukaryotes, arising from unicellular ancestors to produce the incredible diversity of morphologies and habitats observed today. In the unicellular ancestors, secretion of material through the endomembrane system was the major mechanism for interacting and shaping the external environment. In a multicellular organism, the external environment can be made of other cells, some of which may have vastly different developmental fates, or be part of different tissues or organs. In this context, a given cell must find ways to organize its secretory pathway at a level beyond that of the unicellular ancestor. Recently, sequence information from many green plants have become available, allowing an examination of the genomes for the machinery involved in the secretory pathway. In this work, the SNARE proteins of several green plants have been identified. While little increase in gene number was seen in the SNAREs of the early secretory system, many new SNARE genes and gene families have appeared in the multicellular green plants with respect to the unicellular plants, suggesting that this increase in the number of SNARE genes may have some relation to the rise of multicellularity in green plants.

Cooperation in Viral Movement: The Geminivirus BL1 Movement Protein Interacts with BR1 and Redirects It from the Nucleus to the Cell Periphery.

For plant viruses to systemically infect a host requires the active participation of viral-encoded movement proteins. It has been suggested that BL1 and BR1, the two movement proteins encoded by the bipartite geminivirus squash leaf curl virus (SqLCV), act cooperatively to facilitate movement of the viral single-stranded DNA genome from its site of replication in the nucleus to the cell periphery and across the cell wall to adjacent uninfected cells. To better understand the mechanism of SqLCV movement, we investigated the ability of BL1 and BR1 to interact specifically with each other using transient expression assays in insect cells and Nicotiana tabacum cv Xanthi protoplasts. In this study, we showed that when individually expressed, BL1 is localized to the periphery and BR1 to nuclei in both cell systems. However, when coexpressed in either cell type, BL1 relocalized BR1 from the nucleus to the cell periphery. This interaction was found to be specific for BL1 and BR1, because BL1 did not relocalize the SqLCV nuclear-localized AL2 or coat protein. In addition, mutations in BL1 known to affect viral infectivity and pathogenicity were found to be defective in either their subcellular localization or their ability to relocalize BR1, and, thus, identified regions of BL1 required for correct subcellular targeting or interaction with BR1. These findings extend our model for SqLCV movement, demonstrating that BL1 and BR1 appear to interact directly with each other to facilitate movement cooperatively and that BL1 is responsible for providing directionality to movement of the viral genome.

The specificity of vesicle trafficking: coat proteins and SNAREs.

Proteins that are destined for the secretory system usually begin their journey at the endoplasmic reticulum (ER), where proteins are translocated across the ER membrane into the lumen, before being selectively removed from the ER and packaged as cargo into transport vesicles bound for the stacks of

The geminivirus BR1 movement protein binds single-stranded DNA and localizes to the cell nucleus.

Plant viruses encode movement proteins that are essential for infection of the host but are not required for viral replication or encapsidation. Squash leaf curl virus (SqLCV), a bipartite geminivirus with a single-stranded DNA genome, encodes two movement proteins, BR1 and BL1, that have been implicated in separate functions in viral movement. To further elucidate these functions, we have investigated the nucleic acid binding properties and cellular localization of BR1 and BL1. In this study, we showed that BR1 binds strongly to single-stranded nucleic acids, with a higher affinity for single-stranded DNA than RNA, and is localized to the nucleus of SqLCV-infected plant cells. In contrast, BL1 binds only weakly to single-stranded nucleic acids and not at all to double-stranded DNA. The nuclear localization of BR1 and the previously demonstrated plasma membrane localization of BL1 were also observed when these proteins were expressed from baculovirus vectors in Spodoptera frugiperda insect cells. The biochemical properties and cellular locations of BR1 and BL1 suggest a model for SqLCV movement whereby BR1 is involved in the shuttling of the genome in and/or out of the nucleus and BL1 acts at the plasma membrane/cell wall to facilitate viral movement across cell boundaries.

The Secretory System of Arabidopsis

Abstract Over the past few years, a vast amount of research has illuminated the workings of the secretory system of eukaryotic cells. The bulk of this work has been focused on the yeast Saccharomyces cerevisiae, or on mammalian cells. At a superficial level, plants are typical eukaryotes with respect to the operation of the secretory system; however, important differences emerge in the function and appearance of endomembrane organelles. In particular, the plant secretory system has specialized in several ways to support the synthesis of many components of the complex cell wall, and specialized kinds of vacuole have taken on a protein storage role—a role that is intended to support the growing seedling, but has been co-opted to support human life in the seeds of many crop plants. In the past, most research on the plant secretory system has been guided by results in mammalian or fungal systems but recently plants have begun to stand on their own as models for understanding complex trafficking events within the eukaryotic endomembrane system.

Disruption of Individual Members of Arabidopsis Syntaxin Gene Families Indicates Each Has Essential Functions

Syntaxins are a large group of proteins found in all eukaryotes involved in the fusion of transport vesicles to target membranes. Twenty-four syntaxins grouped into 10 gene families are found in the model plant Arabidopsis thaliana, each group containing one to five paralogous members. The Arabidopsis SYP2 and SYP4 gene families contain three members each that share 60 to 80% protein sequence identity. Gene disruptions of the yeast (Saccharomyces cerevisiae) orthologs of the SYP2 and SYP4 gene families (Pep12p and Tlg2p, respectively) indicate that these syntaxins are not essential for growth in yeast. However, we have isolated and characterized gene disruptions in two genes from each family, finding that disruption of individual syntaxins from these families is lethal in the male gametophyte of Arabidopsis. Complementation of the syp21-1 gene disruption with its cognate transgene indicated that the lethality is linked to the loss of the single syntaxin gene. Thus, it is clear that each syntaxin in the SYP2 and SYP4 families serves an essential nonredundant function.

NPSN11 Is a Cell Plate-Associated SNARE Protein That Interacts with the Syntaxin KNOLLE

SNAREs are important components of the vesicle trafficking machinery in eukaryotic cells. In plants, SNAREs have been found to play a variety of roles in the development and physiology of the whole organism. Here, we describe the identification and characterization of a novel plant-specific SNARE, NPSN11, a member of a closely related small gene family in Arabidopsis. NSPN11 is highly expressed in actively dividing cells. In a subcellular fractionation experiment, NSPN11 cofractionates with the cytokinesis-specific syntaxin, KNOLLE, which is required for the formation of the cell plate. By immunofluorescence microscopy, NSPN11 was localized to the cell plate in dividing cells. Consistent with the localization studies, NSPN11 was found to interact with KNOLLE. Our results suggest that NPSN11 is another component of the membrane trafficking and fusion machinery involved in cell plate formation.

Intercellular and Intracellular Trafficking: What We Can Learn from Geminivirus Movement

Bipartite geminiviruses such as squash leaf curl virus encode two movement proteins, BL1 and BR1, essential for virus movement. This reflects the nuclear replication of the viral single-stranded (ss)DNA genome, requiring that one movement protein (BR1) enters the nucleus and binds the viral genome, following which it interacts with the second movement protein (BL1) to facilitate transport of the viral genome to adjacent uninfected cells. Thus, geminiviruses offer the opportunity to investigate nuclear import and export in plant cells, as well as mechanisms for transporting macromolecules across the plant cell wall. The phloem-limitation of squash leaf curl virus also allows us to investigate virus movement within phloem and how a phloem-limited virus invades susceptible host plants.

The localization of AtPEP12, a syntaxin homolog, during different stages of plant development

The Iuminal binding protein BiP, a resident of the endoplasmic reticulum (ER), is a member of a wide class of protein termed molecular chaperone. It is structurally and functionally related to cytosolii HSP70 but differing in the presence of a signal peptide and an ER retention signal (K/HDEL). In tobacco, BiP is encoded by a multigene family and at least out member is able to complement a yeast mutant indicating to a same functional role (Denecke J., Grjldman M. H., Demolder .I., Seurick J. and Botterman J. (1991), Plonf Ceil 3, 1025-1035). Although BiP is present in detectable amount under normal conditions, it can be induced by a variety of stress (e.g. tunicamycin) rest&g in mtiokled secretory proteins (Denecke J. and Vi&e A. (1995). Methods in C&l Biol. 24, 335-348). Biochemical evidence shows its implication in the process of protein folding due to its ability to bind to polypeptides in the ER lumen (Pedrazzini E. and Vitale A. (1996), Plant Physid. Biochem. 34, 207-216) but the role of BiP in tbc quality contra1 by the ER retention of malfolded proreins is not clearly established. We were interested to alter expression of this molecular chaperone to provide more information about its function in secretion and tmhsport of proteins in plants. We set up a.functional assay for BiP based on the comparison of transient protein synthesis in the cytosol (GUS) and on the rough ER (barley n-amylase) after electroporation of tobiT protoplasrs in presence of corresponding genes. Co-nansfection experiments were carried out using both 1) a plasmid containing GUS and a-amylase genei and 2) a plasmid containing BiP construct. A positive effect of transient overexpression of BiP on the synthesis of a-amylase under ER stress has been observed. Then, we have generated transgenic tobacco plants exhibiting different levels of BiP or expressing BiP mutants which will be used as &ls to better understand Sip regul&on, protein synthesis and proteins transport in plants.