In eukaryotic RNA-based antiviral immunity, viral double-stranded RNA is recognized as a pathogen-associated molecular pattern and processed into small interfering RNAs (siRNAs) by the host ribonuclease Dicer. After amplification by host RNA-dependent RNA polymerases in some cases, these virus-derived siRNAs guide specific antiviral immunity through RNA interference and related RNA silencing effector mechanisms. Here, I review recent studies on the features of viral siRNAs and other virus-derived small RNAs from virus-infected fungi, plants, insects, nematodes and vertebrates and discuss the innate and adaptive properties of RNA-based antiviral immunity.

RNA-based antiviral immunity

The specificity of intracellular signaling and developmental patterning in biological systems relies on selective interactions between different proteins in specific cellular compartments. The identification of such protein-protein interactions is essential for unraveling complex signaling and regulatory networks. Recently, bimolecular fluorescence complementation (BiFC) has emerged as a powerful technique for the efficient detection of protein interactions in their native subcellular localization. Here we report significant technical advances in the methodology of plant BiFC. We describe a series of versatile BiFC vector sets that are fully compatible with previously generated vectors. The new vectors enable the generation of both C-terminal and N-terminal fusion proteins and carry optimized fluorescent protein genes that considerably improve the sensitivity of BiFC. Using these vectors, we describe a multicolor BiFC (mcBiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. Application to a protein interaction network acting in calcium-mediated signal transduction revealed the concurrent interaction of the protein kinase CIPK24 with the calcium sensors CBL1 and CBL10 at the plasma membrane and tonoplast, respectively. We have also visualized by mcBiFC the simultaneous formation of CBL1/CIPK1 and CBL9/CIPK1 protein complexes at the plasma membrane. Thus, mcBiFC provides a useful new tool for exploring complex regulatory networks in plants.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2008.03612.x

Multicolor bimolecular fluorescence complementation reveals simultaneous formation of alternative CBL/CIPK complexes in planta

Protein interactions are a fundamental mechanism for the generation of biological regulatory specificity. The study of protein interactions in living cells is of particular significance because the interactions that occur in a particular cell depend on the full complement of proteins present in the cell and the external stimuli that influence the cell. Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the association between two nonfluorescent fragments of a fluorescent protein when they are brought in proximity to each other by an interaction between proteins fused to the fragments. Numerous protein interactions have been visualized using the BiFC assay in many different cell types and organisms. The BiFC assay is technically straightforward and can be performed using standard molecular biology and cell culture reagents and a regular fluorescence microscope or flow cytometer.

/pdf/bimolecular-fluorescence-complementation-bifc-analysis-as-a-1u1cwh4pwb.pdf

Bimolecular Fluorescence Complementation (BiFC) Analysis as a Probe of Protein Interactions in Living Cells

The family Geminiviridae is one of the largest and most important families of plant viruses. The small, single-stranded DNA genomes of geminiviruses encode 5-7 proteins that redirect host machineries and processes to establish a productive infection. These interactions reprogramme plant cell cycle and transcriptional controls, inhibit cell death pathways, interfere with cell signalling and protein turnover, and suppress defence pathways. This Review describes our current knowledge of how geminiviruses interact with their plant hosts and the functional consequences of these interactions.

Geminiviruses: masters at redirecting and reprogramming plant processes

A multitude of small RNAs (sRNAs, 18–25 nt in length) accumulate in plant tissues. Although heterogeneous in size, sequence, genomic distribution, biogenesis, and action, most of these molecules mediate repressive gene regulation through RNA silencing. Besides their roles in developmental patterning and maintenance of genome integrity, sRNAs are also integral components of plant responses to adverse environmental conditions, including biotic stress. Until recently, antiviral RNA silencing was considered a paradigm of the interactions linking RNA silencing to pathogens: Virus-derived sRNAs silence viral gene expression and, accordingly, viruses produce suppressor proteins that target the silencing mechanism. However, increasing evidence shows that endogenous, rather than pathogen-derived, sRNAs also have broad functions in regulating plant responses to various microbes. In turn, microbes have evolved ways to inhibit, avoid, or usurp cellular silencing pathways, thereby prompting the deployment of counter-c...

/pdf/roles-of-plant-small-rnas-in-biotic-stress-responses-1biq2dwx14.pdf

Roles of Plant Small RNAs in Biotic Stress Responses

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

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.

https://www.pnas.org/content/pnas/105/1/157.full.pdf

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

Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel

Glick E., Levy Y., Gafni Y . (2009): The viral etiology of tomato yellow leaf curl disease – a review. Plant Protect. Sci., 45: 81–97. Tomato yellow leaf curl disease (TYLCD) is one of the most devastating plant diseases in the world. As a result of its continuing rapid spread, it now afflicts more than 30 tomato growing countries in the Mediterranean basin, southern Asia, Africa, and South, Central and North America. The disease is caused by a group of viral species of the genus Begomovirus, family Geminiviridae (geminiviruses) , referred to as Tomato yellow leaf curl virus (TYLCV). These are transmitted by an insect vector, the whitefly Bemisia tabaci , classified in the family Aleyrodidae. The genome of TYLCV generally consists of a single circular single-stranded (ss) DNA molecule, with only one exception in which two components were identified. It encodes six open reading frames, only one of which codes for the coat protein (CP) that represents a building block of the viral particle. TYLCV, like all other members of the Geminiviridae, has geminate particles, apparently consisting of two incomplete T = 1 icosahedra joined together to produce a structure with 22 pentameric capsomers and 110 identical CP subunits. Close to 50 years of intensive research into TYLCV epidemics has been conducted to find solutions to the severe problem caused by this virus. To date, breeding for resistance appears to be the best approach to controlling this disease, although only partially resistant varieties are commercially available. Since the virus consists of a ssDNA that replicates in the host-cell nucleus, the molecular mechanisms involved in its nuclear import have been the focus of our studies in recent years and results, as well as prospects, are discussed in this review. In addition, we describe our recent finding of a suppressor of gene silencing encoded by one of the TYLCV-Isr genes. This paper provides an overview of the most outstanding achievements in TYLCV research that may lead to more effective control strategies.

/pdf/the-viral-etiology-of-tomato-yellow-leaf-curl-disease-a-1k1s82hi2h.pdf

Efrat Glick

Papers

Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta.

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

Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel

The Viral Etiology of Tomato Yellow Leaf Curl Disease - A Review