Rep and V2 Suppress Post-Transcriptional Gene Silencing via Distinct Modes of Action.

(BCTIV) is a yield-limiting geminivirus belonging to the genus. The genome organization of BCTIV is unique such that the complementary strand of BCTIV resembles , whereas the virion strand organization is similar to the genus. Geminiviruses are known to avoid the plant defense system by suppressing the RNA interference mechanisms both at the transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS) levels.

Identification of the Actin-Binding Region and Binding to Host Plant Apple Actin of Immunodominant Transmembrane Protein of ' Phytoplasma mali'.

' Phytoplasma mali' ('. P. mali') has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins.

Small RNA-seq dataset of wild type and 16C leaves sprayed with naked dsRNA using the high-pressure spraying technique.

Double-stranded RNA (dsRNA) applications have emerged as promising alternatives to chemical plant pesticides. It has been proposed that the protective effect of dsRNA is mediated by the RNA interference (RNAi) mechanism. Small RNAs (sRNAs) are one of the landmarks of RNAi mechanisms.

Beyond Destabilizing Activity of SAP11-like Effector of Phytoplasma mali Strain PM19.

It was shown that the SAP11 effector of different Phytoplasma can destabilize some TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTORs (TCPs), resulting in plant phenotypes such as witches' broom and crinkled leaves. Some SAP11 exclusively localize in the nucleus, while the others localize in the cytoplasm and the nucleus. The SAP11-like effector of Phytoplasma mali strain PM19 (SAP11) localizes in both compartments of plant cells.

High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing.

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA.

The Effect of the Anticipated Nuclear Localization Sequence of ' Phytoplasma mali' SAP11-like Protein on Localization of the Protein and Destabilization of TCP Transcription Factor.

SAP11 is an effector protein that has been identified in various phytoplasma species. It localizes in the plant nucleus and can bind and destabilize TEOSINE BRANCHES/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors. Although SAP11 of different phytoplasma species share similar activities, their protein sequences differ greatly.

High-Pressure-Sprayed Double Stranded RNA Does Not Induce RNA Interference of a Reporter Gene.

In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs).

Bottom-up assembly of a bilayer structure of icosahedral viral nanoparticles.

The development of 2D and 3D structures on the nanoscale containing viral nanoparticles (VNPs) as interesting nanobuilding blocks has come into focus for a bottom-up approach as an alternative to the top-down approach in nanobiotechnology. Our research has focused on the plant Tomato Bushy Stunt Virus (TBSV). In a previous study, we reported the impact of the pH value on the 2D assembly of viral monolayers.

' Phytoplasma mali' Genome Encodes a Protein that Functions as an E3 Ubiquitin Ligase and Could Inhibit Plant Basal Defense.

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation ' Phytoplasma mali' and its host plant.

Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs.

In this study, we show that aberrant pre-mRNAs from non-spliced and non-polyadenylated intron-containing transgenes are channelled to the RNA silencing pathway. In plants, improperly processed transcripts are called aberrant RNAs (ab-RNAs) and are eliminated by either RNA silencing or RNA decay mechanisms. Ab-RNAs transcribed from intronless genes are copied by RNA-directed RNA polymerases (RDRs) into double-stranded RNAs which are subsequently cleaved by DICER-LIKE endonucleases into small RNAs (sRNAs).

Delivery of Hairpin RNAs and Small RNAs Into Woody and Herbaceous Plants by Trunk Injection and Petiole Absorption.

Since its discovery, RNA interference has been widely used in crop protection. Recently, transgene-free procedures that were based on exogenous application of RNA molecules having the capacity to trigger RNAi have been reported. Yet, efficient delivery of such RNA molecules to plants and particularly to trees poses major technical challenges.

Electrostatic conditions define the 2D self-assembly of tomato bushy stunt viruses on solid surfaces.

Plant viruses which are self-assembled on a substrate are interesting building blocks in nanobiotechnology, in particular, for the creation of 2D ordered structures. In this article, the self-assembly of different genetically modified types of the tomato bushy stunt virus spin-coated on pristine silicon was investigated by scanning force and scanning electron microscopy. Amino acid side chains were integrated in the capsids of the viruses by extending the coat protein with different charged amino acid clusters (tetra-aspartate-hexa-histidine, hexa-aspartate, or tetra-arginine-tags).

A simple method to estimate the isoelectric point of modified Tomato bushy stunt virus (TBSV) particles.

We present a simple method to estimate the isoelectric point (pI) of Tomato Bushy Stunt particles. We demonstrate that the combination of agarose gels with different pH buffers can be used to electrophorese the virus particles and their migration patterns can be compared. This method allows us to estimate the pI of the virus particles (wild type, wt, and genetically modified particles) and to monitor the effect of the pI of modified peptide side chains of the viral capsid subunit on the pI of the whole virus particle.

Identification of Highly Specific scFvs against Total Adiponectin for Diagnostic Purposes.

Adiponectin is one of the most abundant adipokines secreted from adipose tissue. It acts as an endogenous insulin sensitizer and plasma concentrations are inversely correlated with obesity and metabolic syndrome. A decrease in plasma adiponectin levels normally indicates increased hormonal activity of the visceral lipid tissue, which is associated with decreased insulin sensitivity.

Reverse Genetics and High Throughput Sequencing Methodologies for Plant Functional Genomics.

In the post-genomic era, increasingly sophisticated genetic tools are being developed with the long-term goal of understanding how the coordinated activity of genes gives rise to a complex organism. With the advent of the next generation sequencing associated with effective computational approaches, wide variety of plant species have been fully sequenced giving a wealth of data sequence information on structure and organization of plant genomes. Since thousands of gene sequences are already known, recently developed functional genomics approaches provide powerful tools to analyze plant gene functions through various gene manipulation technologies.

Induction of Silencing in Plants by High-Pressure Spraying of In vitro-Synthesized Small RNAs.

In this report, we describe a method for the delivery of small interfering RNAs (siRNAs) into plant cells. In vitro synthesized siRNAs that were designed to target the coding region of a GREEN FLUORESCENT PROTEIN (GFP) transgene were applied by various methods onto GFP-expressing transgenic Nicotiana benthamiana plants to trigger RNA silencing. In contrast to mere siRNA applications, including spraying, syringe injection, and infiltration of siRNAs that all failed to induce RNA silencing, high pressure spraying of siRNAs resulted in efficient local and systemic silencing of the GFP transgene, with comparable efficiency as was achieved with biolistic siRNA introduction.

Immunodominant membrane proteins of phytoplasmas.

Phytoplasmas are plant-pathogenic, phloem-colonizing, cell wall-less microorganisms that are primarily dependent on insect transmission for their spread and survival. The life cycle of phytoplasmas involves replication in insects and host plants. Until recently, phytoplasmas have resisted all attempts at cultivation in cell-free media, making these pathogens poorly characterized on a physiological and biochemical basis.

RNA-directed DNA methylation efficiency depends on trigger and target sequence identity.

RNA-directed DNA methylation (RdDM) in plants has been extensively studied, but the RNA molecules guiding the RdDM machinery to their targets are still to be characterized. It is unclear whether these molecules require full complementarity with their target. In this study, we have generated Nicotiana tabacum (Nt) plants carrying an infectious tomato apical stunt viroid (TASVd) transgene (Nt-TASVd) and a non-infectious potato spindle tuber viroid (PSTVd) transgene (Nt-SB2).

Functional Analysis of Cotton Leaf Curl Kokhran Virus/Cotton Leaf Curl Multan Betasatellite RNA Silencing Suppressors.

In South Asia, Cotton leaf curl disease (CLCuD) is caused by a complex of phylogenetically-related begomovirus species and a specific betasatellite, Cotton leaf curl Multan betasatellite (CLCuMuB). The post-transcriptional gene silencing (PTGS) suppression activities of the transcriptional activator protein (TrAP), C4, V2 and βC1 proteins encoded by Cotton leaf curl Kokhran virus (CLCuKoV)/CLCuMuB were assessed in Nicotiana benthamiana. A variable degree of local silencing suppression was observed for each viral protein tested, with V2 protein exhibiting the strongest suppression activity and only the C4 protein preventing the spread of systemic silencing.

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS.

Three gene products of a begomovirus-betasatellite complex restore expression of a transcriptionally silenced green fluorescent protein transgene in Nicotiana benthamiana.

Single-stranded DNA geminiviruses replicate via double-stranded DNA intermediates forming mini-chromosomes that are targets for transcriptional gene silencing (TGS) in plants. The ability of the cotton leaf curl Kokhran virus (CLCuKoV)-cotton leaf curl Multan betasatellite (CLCuMuB) proteins, replication-associated protein (Rep), transcriptional activator protein (TrAP), C4, V2 and βC1, to suppress TGS was investigated by using the Nicotiana benthamiana line 16-TGS (16-TGS) harbouring a transcriptionally silenced green fluorescent protein (GFP) transgene. Inoculation of 16-TGS plants with a recombinant potato virus X vector carrying Rep, TrAP or βC1 resulted in re-expression of GFP.

An endogene-resembling transgene is resistant to DNA methylation and systemic silencing.

In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)).

Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus.

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III-like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants.

An endogene-resembling transgene delays the onset of silencing and limits siRNA accumulation.

In plants, transgenes are generally more sensitive against RNA silencing than endogenes are. In this study, we generated a transgene that structurally mimicks an endogene. It is composed of endogenous promoter, 5'-UTR, introns, 3'-UTR and terminator elements.

Transgenerational maintenance of transgene body CG but not CHG and CHH methylation.

In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny.