The nonstructural NSm protein of tomato spotted wilt tospovirus (TSWV) represents a putative viral movement protein involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures. To study the molecular basis of NSm function, we expressed the protein in Escherichia coli and investigated protein-protein and protein-RNA interactions of NSm protein in vitro. NSm specifically interacts with TSWV N protein and binds single-stranded RNA in a sequence-nonspecific manner. Using NSm as a bait in a yeast two-hybrid screen, we identified two homologous NSm-binding proteins of the DnaJ family from Nicotiana tabacum and Arabidopsis thaliana.

The nucleocapsid protein (N) of tomato spotted wilt tospovirus (TSWV) plays a central role in the viral life cycle. With the aid of the yeast two-hybrid system and surface plasmon resonance analysis, homotypic interaction and multimerization of the N protein was detected. Analysis of deletion mutants identified two binding regions in the protein, located at the N terminus (amino acids 1-39) and the C terminus (amino acids 233-248), respectively, implying a ''head-to-tail'' interaction of the N terminus with the C terminus to form a multimeric chain. Further characterization of the binding domains was performed by site-directed mutagenesis. Two phenylalanines (F242 and F246) highly conserved in the N proteins within the Tospovirus genus were shown to play a crucial role in the interaction.Tomato spotted wilt tospovirus (TSWV) is the type member of the Tospovirus genus, the only plant-infecting genus of the arthropod-borne family Bunyaviridae. Tospoviruses have a very broad host range encompassing more than 650 plant species belonging to 70 families, including many crops and ornamentals. Losses to world agriculture are estimated to be more than US$1 billion per year (1).Molecular biological analysis has confirmed that TSWV is a typical member of the Bunyaviridae. The tospoviral particle consists of a core of nucleocapsids in which the genomic single-stranded RNA (ssRNA) molecules are tightly associated with nucleocapsid (N) proteins and a few copies of the L protein. These nucleocapsids are surrounded by a lipid membrane carrying two glycoproteins. TSWV has a tripartite genome consisting of three RNA species called L, M, and S, encoding structural and nonstructural proteins in a negative or ambisense orientation. Despite the fact that the TSWV genome was sequenced several years ago (2-4), knowledge of the biological function of the viral proteins is still very poor. One of the reasons for this lack of information is the limited availability of reverse genetics for Bunyaviridae; so far reverse genetics has been developed only for Bunyamvera virus (5). The TSWV L RNA encodes a large polypeptide of 2,875 amino acids proposed to be the viral RNA-dependent RNA polymerase (6). The M RNA codes for the putative movement protein NSm and for a glycoprotein precursor that is processed to the spike proteins G1 and G2 involved in thrips transmission. The S RNA encodes the nonstructural protein NSs with unknown function and the N protein, the main constituent of the TSWV nucleocapsid (1).Several functions have been established for nucleocapsid proteins of enveloped ssRNA viruses. In general they are basic proteins with nucleic acid binding capacities. Recently, Richmond et al. (7) showed that the TSWV N protein nonspecifically binds ssRNA and characterized multiple RNA binding domains. The NP protein of influenza virus (Orthomyxoviridae) binds the viral RNA, melts secondary structures, and is in this way involved in the regulation of transcription (8). Direct interaction of the influenza virus NP protein with the polyme...

SummaryIn plants, Rop/Rac GTPases have emerged as central regulators of diverse signalling pathways in plant growth and pathogen defence. When active, they interact with a wide range of downstream effectors. Using yeast two-hybrid screening we have found three previously uncharacterized receptor-like protein kinases to be Rop GTPase-interacting molecules: a cysteine-rich receptor kinase, named NCRK, and two receptor-like cytosolic kinases from the Arabidopsis RLCK-VIb family, named RBK1 and RBK2. Uniquely for Rho-family small GTPases, plant Rop GTPases were found to interact directly with the protein kinase domains. Rop4 bound NCRK preferentially in the GTP-bound conformation as determined by flow cytometric fluorescence resonance energy transfer measurements in insect cells. The kinase RBK1 did not phosphorylate Rop4 in vitro, suggesting that the protein kinases are targets for Rop signalling. Bimolecular fluorescence complementation assays demonstrated that Rop4 interacted in vivo with NCRK and RBK1 at the plant plasma membrane. In Arabidopsis protoplasts, NCRK was hyperphosphorylated and partially co-localized with the small GTPase RabF2a in endosomes. Gene expression analysis indicated that the single-copy NCRK gene was relatively upregulated in vasculature, especially in developing tracheary elements. The seven Arabidopsis RLCK-VIb genes are ubiquitously expressed in plant development, and highly so in pollen, as in case of RBK2. We show that the developmental context of RBK1 gene expression is predominantly associated with vasculature and is also locally upregulated in leaves exposed to Phytophthora infestans and Botrytis cinerea pathogens. Our data indicate the existence of cross-talk between Rop GTPases and specific receptor-like kinases through direct molecular interaction.

Background: Protein-protein interactions have decisive roles in almost all aspects of the structural and functional organization of cells. But in spite of the increasing amount of complete genome sequence data, the ability to predict protein function from sequences alone is limited. Therefore comprehensive analysis of protein-protein interactions, as derived from the yeast twohybrid mating system, will yield valuable information for functional biology on a proteomic scale.