Yuval Peretz

A Universal Expression/Silencing Vector in Plants

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plants genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl viruss natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper “virus,” transactivation occurs, resulting in an inducible expressing/silencing system. The systems potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.

Silencing of a single gene in tomato plants resistant to Tomato yellow leaf curl virus renders them susceptible to the virus

A reverse-genetics approach was applied to identify genes involved in Tomato yellow leaf curl virus (TYLCV) resistance, taking advantage of two tomato inbred lines from the same breeding program—one susceptible (S), one resistant (R—that used Solanum habrochaites as the source of resistance. cDNA libraries from inoculated and non-inoculated R and S plants were compared, postulating that genes preferentially expressed in the R line may be part of the network sustaining resistance to TYLCV. Further, we assumed that silencing genes located at important nodes of the network would lead to collapse of resistance. Approximately 70 different cDNAs representing genes preferentially expressed in R plants were isolated and their genes identified by comparison with public databases. A Permease I-like protein gene encoding a transmembranal transporter was further studied: it was preferentially expressed in R plants and its expression was enhanced several-fold following TYLCV inoculation. Silencing of the Permease gene of R plants using Tobacco rattle virus-induced gene silencing led to loss of resistance, expressed as development of disease symptoms typical of infected susceptible plants and accumulation of large amounts of virus. Silencing of another membrane protein gene preferentially expressed in R plants, Pectin methylesterase, previously shown to be involved in Tobacco mosaic virus translocation, did not lead to collapse of resistance of R plants. Thus, silencing of a single gene can lead to collapse of resistance, but not every gene preferentially expressed in the R line has the same effect, upon silencing, on resistance.

Tomato yellow leaf curl virus infection of a resistant tomato line with a silenced sucrose transporter gene LeHT1 results in inhibition of growth, enhanced virus spread, and necrosis

To identify genes involved in resistance of tomato to Tomato yellow leaf curlvirus (TYLCV), cDNA libraries from lines resistant (R) and susceptible (S) to the virus were compared. The hexose transporter LeHT1 was found to be expressed preferentially in R tomato plants. The role of LeHT1 in the establishment of TYLCV resistance was studied in R plants where LeHT1 has been silenced using Tobacco rattle virus-induced gene silencing (TRV VIGS). Following TYLCV inoculation, LeHT1-silenced R plants showed inhibition of growth and enhanced virus accumulation and spread. In addition, a necrotic response was observed along the stem and petioles of infected LeHT1-silenced R plants, but not on infected not-silenced R plants. This response was specific of R plants since it was absent in infected LeHT1-silenced S plants. Necrosis had several characteristics of programmed cell death (PCD): DNA from necrotic tissues presented a PCD-characteristic ladder pattern, the amount of a JNK analogue increased, and production of reactive oxygen was identified by DAB staining. A similar necrotic reaction along stem and petioles was observed in LeHT1-silenced R plants infected with the DNA virus Bean dwarf mosaic virus and the RNA viruses Cucumber mosaic virus and Tobacco mosaic virus. These results constitute the first evidence for a necrotic response backing natural resistance to TYLCV in tomato, confirming that plant defense is organized in multiple layers. They demonstrate that the hexose transporter LeHT1 is essential for the expression of natural resistance against TYLCV and its expression correlates with inhibition of virus replication and movement.

Expression of an entire bacterial operon in plants.

Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plants life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.

A novel role for Pax6 in the segmental organization of the hindbrain

Complex patterns and networks of genes coordinate rhombomeric identities, hindbrain segmentation and neuronal differentiation and are responsible for later brainstem functions. Pax6 is a highly conserved transcription factor crucial for neuronal development, yet little is known regarding its early roles during hindbrain segmentation. We show that Pax6 expression is highly dynamic in rhombomeres, suggesting an early function in the hindbrain. Utilization of multiple gain- and loss-of-function approaches in chick and mice revealed that loss of Pax6 disrupts the sharp expression borders of Krox20, Kreisler, Hoxa2, Hoxb1 and EphA and leads to their expansion into adjacent territories, whereas excess Pax6 reduces these expression domains. A mutual negative cross-talk between Pax6 and Krox20 allows these genes to be co-expressed in the hindbrain through regulation of the Krox20-repressor gene Nab1 by Pax6. Rhombomere boundaries are also distorted upon Pax6 manipulations, suggesting a mechanism by which Pax6 acts to set hindbrain segmentation. Finally, FGF signaling acts upstream of the Pax6-Krox20 network to regulate Pax6 segmental expression. This study unravels a novel role for Pax6 in the segmental organization of the early hindbrain and provides new evidence for its significance in regional organization along the central nervous system.

Only minimal regions of tomato yellow leaf curl virus (TYLCV) are required for replication, expression and movement

The IL-60 platform, consisting of a disarmed form of tomato yellow leaf curl virus (TYLCV) and auxiliary components, was previously developed as a nontransgenic universal vector system for gene expression and silencing that can express an entire operon in plants. IL-60 does not allow rolling-circle replication; hence, production of viral single-stranded (ss) DNA progeny is prevented. We used this double-stranded (ds) DNA-restricted platform (uncoupled from the dsDNA→ssDNA replication phase of progeny viral DNA) for functional genomics studies of TYLCV. We report that the noncoding 314-bp intergenic region (IR) is the only viral element required for viral dsDNA replication. None of the viral genes are required, suggesting recruitment of host factors that recognize the IR. We further show that IR-carrying reporter genes are also capable of replication but remain confined to the cells into which they were introduced. Only two sense-oriented viral genes (V1 and V2) need to be added to the IR-carrying construct for expression and movement. Hence, any IR-dsDNA construct supplemented with V1 and V2 becomes a replication-competent, mobile and expressing plant plasmid. All viral functions (replication, expression and movement) are determined by the IR and the sense-oriented genes. The complementary-oriented viral genes have auxiliary roles in the late phase of the virus “life cycle”. The previously reported involvement of some viral genes in expression and movement is therefore revised.

A new role of hindbrain boundaries as pools of neural stem/progenitor cells regulated by Sox2

BackgroundCompartment boundaries are an essential developmental mechanism throughout evolution, designated to act as organizing centers and to regulate and localize differently fated cells. The hindbrain serves as a fascinating example for this phenomenon as its early development is devoted to the formation of repetitive rhombomeres and their well-defined boundaries in all vertebrates. Yet, the actual role of hindbrain boundaries remains unresolved, especially in amniotes.ResultsHere, we report that hindbrain boundaries in the chick embryo consist of a subset of cells expressing the key neural stem cell (NSC) gene Sox2. These cells co-express other neural progenitor markers such as Transitin (the avian Nestin), GFAP, Pax6 and chondroitin sulfate proteoglycan. The majority of the Sox2+ cells that reside within the boundary core are slow-dividing, whereas nearer to and within rhombomeres Sox2+ cells are largely proliferating. In vivo analyses and cell tracing experiments revealed the contribution of boundary Sox2+ cells to neurons in a ventricular-to-mantle manner within the boundaries, as well as their lateral contribution to proliferating Sox2+ cells in rhombomeres. The generation of boundary-derived neurospheres from hindbrain cultures confirmed the typical NSC behavior of boundary cells as a multipotent and self-renewing Sox2+ cell population. Inhibition of Sox2 in boundaries led to enhanced and aberrant neural differentiation together with inhibition in cell-proliferation, whereas Sox2 mis-expression attenuated neurogenesis, confirming its significant function in hindbrain neuronal organization.ConclusionsData obtained in this study deciphers a novel role of hindbrain boundaries as repetitive pools of neural stem/progenitor cells, which provide proliferating progenitors and differentiating neurons in a Sox2-dependent regulation.

Silencing of ORFs C2 and C4 of Tomato Yellow Leaf Curl Virus Engenders Resistant or Tolerant Plants

The IL-60 system is a transient universal vector system for expression and silencing in plants [1]. This vector has been derived from Tomato yellow leaf curl virus (TYLCV). The viral intergenic region (IR) is a non-coding short (314 b) sequence separating the viral sense-oriented genes from the complementary-oriented genes. IR carries the viral origin of replication as well as a promoter at each end. Placing a gene segment between two IRs at opposite orientations followed by trans-activation of the construct by the plasmid IL-60-BS, caused silencing of the pertinent gene as indicated by the silencing of the endogenous gene PDS.. The viral genes C2 and C4 are implicated as having a role in viral-directed silencing suppression. The silencing of C2 and C4 intervened with the virus ability to counter-react to viral silencing by the host plant, thus engendering resistance or tolerance.

Neural stem cells deriving from chick embryonic hindbrain recapitulate hindbrain development in culture

Neural stem cells (NSCs) are self-renewing multipotent cells that line the neural-tube and generate all the nervous system. Understanding NSC biology is fundamental for neurodevelopmental research and therapy. Many studies emphasized the need to culture NSCs, which are typically purified from mammalian embryonic/adult brains. These sources are somewhat limited in terms of quantity, availability and animal ethical guidelines. Therefore, new sources are needed. The chick is a powerful system for experimental embryology which contributed enormously to neurodevelopmental concepts. Its accessibility, genetic/molecular manipulations, and homology to other vertebrates, makes it valuable for developmental biology research. Recently, we identified a population of NSCs in the chick hindbrain. It resides in rhombomere-boundaries, expresses Sox2 and generates progenitors and neurons. Here, we investigated whether these cells can recapitulate hindbrain development in culture. By developing approaches to propagate and image cells, manipulate their growth-conditions and separate them into subpopulations, we demonstrate the ordered formation of multipotent and self-renewing neurospheres that maintain regional identity and display differential stem/differentiation/proliferation properties. Live imaging revealed new cellular dynamics in the culture. Collectively, these NSC cultures reproduce major aspects of hindbrain development in-vitro, proposing the chick as a model for culturing hindbrain-NSCs that can be directly applied to other neural-tube domains and species.

A T7-driven silencing system in transgenic plants expressing T7 RNA polymerase is a nuclear process

We previously demonstrated a case of silencing in transgenic plants expressing T7 RNA polymerase in which expression of a reporter gene placed under the control of the T7 promoter was silenced. Here we demonstrate that endogenous genes can be silenced by the same system. The T7-driven silencing system does not conform to several aspects characteristic of post-transcriptional RNA silencing in plants, and this prompted an investigation into the mechanisms underlying this type of silencing. The present paper demonstrates that T7-driven silencing is a post-transcriptional process that is restricted to the nucleus. Nuclear run-on assays indicated the presence of silenced gene transcripts in both orientations. SiRNA corresponding to the silenced gene could not be traced in the cytoplasm but was found in nuclei. The silenced gene was hypermethylated. We present evidence that a tobacco RNA-dependent RNA polymerase (RdRP) is not involved in T7-mediated silencing, but indicate the involvement of a nuclear RdRP in this type of silencing.