Yedidya Gafni

Interaction with host SGS3 is required for suppression of RNA silencing by tomato yellow leaf curl virus V2 protein

The V2 protein of tomato yellow leaf curl geminivirus (TYLCV) functions as an RNA-silencing suppressor that counteracts the innate immune response of the host plant. The host-cell target of V2, however, remains unknown. Here we show that V2 interacts directly with SlSGS3, the tomato homolog of the Arabidopsis SGS3 protein (AtSGS3), which is known to be involved in the RNA-silencing pathway. SlSGS3 genetically complemented an AtSGS3 mutation and restored RNA silencing, indicating that SlSGS3 is indeed a functional homolog of AtSGS3. A point mutant of V2 that is unable to bind SlSGS3 also lost its ability to suppress RNA silencing, suggesting a correlation between the V2–SlSGS3 interaction in planta and the suppressor activity of V2.

Effects of Calreticulin on Viral Cell-to-Cell Movement

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.

A genetic system for detection of protein nuclear import and export

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

The capsid protein of tomato yellow leaf curl virus binds cooperatively to single-stranded DNA

The capsid protein (CP) of tomato yellow leaf curl virus (TYLCV) is the only known component of the virus coat. Here, we identify TYLCV CP as a single-stranded (ss) DNA binding protein. Purified TYLCV CP bound ssDNA in a highly cooperative and sequence-nonspecific fashion. TYLCV CP-ssDNA complexes were resistant to nucleolytic digestion and remained stable at relatively high salt concentrations. Because TYLCV CP is known to contain an active nuclear targeting signal, we propose that its association with the viral genomic ssDNA mediates TYLCV entry into the host cell nucleus during the infection process.

Indole-3-acetic acid biosynthetic pathways in Erwinia herbicola in relation to pathogenicity on Gypsophila paniculata☆

Pathogenic strains of Erwinia herbicola incite crown and root galls in the flowering ornamental gypsophila. Both pathogenic and non-pathogenic strains of the bacterium readily produce indole-3-acetic acid in culture. Two pathways for biosynthesis of indole-3-acetic acid were identified in E. herbicola: (1) the indole-3-acetamide route occurs via the following reactions: l-tryptophan → indole-3-acetamide → indole-3-acetic acid, and (2) the indole-3-pyruvate route involves the following reactions: l-tryptophan → indole-3-pyruvate → indole-3-acetaldehyde → indole-3-acetic acid. Production of indole-3-ethanol was also linked to the latter pathway. Evidence for the existence of the two pathways was based on: (a) chemical identification of the respective indole intermediates by thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectroscopy; (b) production of indole-3-acetic acid by bacterial cells treated with the various indole intermediates; and (c) incorporation of 3-14C-l-tryptophan into the indole intermediates of the two pathways. In contrast to the indole-3-pyruvate pathway which was detected in all the pathogenic and non-pathogenic strains examined, the indole-3-acetamide pathway was detected only in the pathogenic strains of E. herbicola. The possible relationship between the indole-3-acetamide pathway and gall formation by E. herbicola is discussed.

Tomato yellow leaf curl virus, the intracellular dynamics of a plant DNA virus

UNLABELLED SUMMARY Tomato yellow leaf curl virus is a geminivirus, transmitted by whitefly (Bemisia tabaci) and causing the most destructive disease of tomato throughout the Mediterranean region, the Middle East and the tropical regions of Africa and Central America. Affected plants produce either no fruits or a few small fruits. Since it is an ssDNA virus which replicates in the host cell nucleus, the molecular mechanisms involved in the viral nuclear import have been the focus of our studies in recent years and results as well as prospects will be discussed. TAXONOMY Tomato yellow leaf curl virus (TYLCV) is a ssDNA plant virus, a member of the family Geminiviridae, of the genus Begomovirus. Physical properties: TYLCV, like all members of Geminiviridae, has geminate (twinned) particles, 18-20 nm in diameter, 30 nm long, apparently consisting of two incomplete T = 1 icosahedra joined together in a structure with 22 pentameric capsomers and 110 identical protein subunits (Fig. 1). Disease symptoms: Symptoms become visible in tomato in approximately 2-3 weeks after infection (Fig. 2). Leaf symptoms include chlorotic margins, small leaves that are cupped, thick and rubbery. The majority (up to 90%) of flowers abscise after infection, and therefore few fruits are produced. In Israel and elsewhere, weeds bridge the gap as potential perennial host and source of the virus between tomato growing seasons. Disease control: Control of TYLCV is currently based on insecticide treatments and/or physical barriers against the insect vector (Bemisia tabaci), and on tomato breeding programs based on introgression of resistance or tolerance from wild species to cultivated tomato. Useful website:

Localizing protein–protein interactions by bimolecular fluorescence complementation in planta

The application of novel assays for basic cell research is tightly linked to the development of easy-to-use and versatile tools and protocols for implementing such technologies for a wide range of applications and model species. The bimolecular fluorescence complementation (BiFC) assay is one such novel method for which tools and protocols for its application in plant cell research are still being developed. BiFC is a powerful tool which enables not only detection, but also visualization and subcellular localization of protein-protein interactions in living cells. Here we describe the application of BiFC in plant cells while focusing on the use of our versatile set of vectors which were specifically designed to facilitate the transformation, expression and imaging of protein-protein interactions in various plant species. We discuss the considerations of using our system in various plant model systems, the use of single versus multiple expression cassettes, the application of our vectors using various transformation methods and the use of internal fluorescent markers which can assist in signal localization and easy data acquisition in living cells.

Proteasomal degradation in plant-pathogen interactions

The ubiquitin/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant-pathogen interactions. The specific functions of proteasomal degradation in plant-pathogen interactions are diverse, and do not always benefit the host plant. Although in some cases, proteasomal degradation serves as an effective barrier to help plants ward off pathogens, in others, it is used by the pathogen to enhance the infection process. This review discusses the different roles of the ubiquitin/26S proteasome pathway during interactions of plants with pathogenic viruses, bacteria, and fungi.

Nuclear import and export of plant virus proteins and genomes

SUMMARY Nuclear import and export are crucial processes for any eukaryotic cell, as they govern substrate exchange between the nucleus and the cytoplasm. Proteins involved in the nuclear transport network are generally conserved among eukaryotes, from yeast and fungi to animals and plants. Various pathogens, including some plant viruses, need to enter the host nucleus to gain access to its replication machinery or to integrate their DNA into the host genome; the newly replicated viral genomes then need to exit the nucleus to spread between host cells. To gain the ability to enter and exit the nucleus, these pathogens encode proteins that recognize cellular nuclear transport receptors and utilize the hosts nuclear import and export pathways. Here, we review and discuss our current knowledge about the molecular mechanisms by which plant viruses find their way into and out of the host cell nucleus.

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid ofAgrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2–5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation,L. chilense LA1969 andL. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.