Visualization of Transiently Expressed mRNA in Plants Using MS2.

RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP).

Citrus Psorosis Virus Movement Protein Contains an Aspartic Protease Required for Autocleavage and the Formation of Tubule-Like Structures at Plasmodesmata.

Plant virus cell-to-cell movement is an essential step in viral infections. This process is facilitated by specific virus-encoded movement proteins (MPs), which manipulate the cell wall channels between neighboring cells known as plasmodesmata (PD). Citrus psorosis virus (CPsV) infection in sweet orange involves the formation of tubule-like structures within PD, suggesting that CPsV belongs to "tubule-forming" viruses that encode MPs able to assemble a hollow tubule extending between cells to allow virus movement.

Crosstalk between PTGS and TGS pathways in natural antiviral immunity and disease recovery.

Virus-induced diseases cause severe damage to cultivated plants, resulting in crop losses. Certain plant-virus interactions allow disease recovery at later stages of infection and have the potential to reveal important molecular targets for achieving disease control. Although recovery is known to involve antiviral RNA silencing, the specific components of the many plant RNA silencing pathways required for recovery are not known.

Bioinformatic and mutational analysis of ophiovirus movement proteins, belonging to the 30K superfamily.

Ophioviridae is a family of segmented, negative-sense, single-stranded RNA plant viruses. We showed that their cell-to-cell movement protein (MP) is an isolated member of the 30K MP superfamily with a unique structural organization. All 30K MPs share a core domain that contains a nearly-invariant signature aspartate.

Experimental virus evolution reveals a role of plant microtubule dynamics and TORTIFOLIA1/SPIRAL2 in RNA trafficking.

The cytoskeleton is a dynamic network composed of filamentous polymers and regulatory proteins that provide a flexible structural scaffold to the cell and plays a fundamental role in developmental processes. Mutations that alter the spatial orientation of the cortical microtubule (MT) array of plants are known to cause important changes in the pattern of cell wall synthesis and developmental phenotypes; however, the consequences of such alterations on other MT-network-associated functions in the cytoplasm are not known. In vivo observations suggested a role of cortical MTs in the formation and movement of Tobacco mosaic virus (TMV) RNA complexes along the endoplasmic reticulum (ER).

Cortical microtubule-associated ER sites: organization centers of cell polarity and communication.

Anisotropic cell growth and the ability of plant cells to communicate within and across the borders of cellular and supracellular domains depends on the ability of the cells to dynamically establish polarized networks able to deliver structural and informational macromolecules to distinct cellular sites. Studies of organelle movements and transport of endogenous and viral proteins suggest that organelle and macromolecular trafficking pathways involve transient or stable interactions with cortical microtubule-associated endoplasmic reticulum sites (C-MERs). The observations suggest that C-MERs may function as cortical hubs that organize cargo exchange between organelles and allow the recruitment, assembly, and subsequently site-specific delivery of macromolecular complexes.

Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein.

Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV), members of the Ophioviridae family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution.

Citrus psorosis and Mirafiori lettuce big-vein ophiovirus coat proteins localize to the cytoplasm and self interact in vivo.

Citrus psorosis (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV) belong to the family Ophioviridae, plant viruses with filamentous nucleocapsids and segmented genomes of negative polarity, causing the worldwide distributed citrus psorosis and lettuce big-vein diseases, respectively. To gain insight into the replication cycle of these viruses, the subcellular localization of the viral coat proteins (CP) was studied. Immunoblot analysis of fractionated extracts derived from natural and experimental infected hosts indicated that the CP of CPsV occurs in the soluble cytoplasmic fraction.

Malachite green derivatives for two-photon RNA detection.

The design, preparation and characterisation of a library of malachite green (MG) derivatives for two-photon RNA labelling is described. Some of these MG derivatives exhibit an increased affinity for an MG-aptamer, as well as improved two-photon sensitivity when compared to the classical malachite green chloride. The underlying mechanisms and potential benefits for in vivo RNA visualisation are discussed.

Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein-RNA silencing.

The lack of naturally occurring resistance to Citrus psorosis virus (CPsV) has demanded exploitation of a transgenic approach for the development of CPsV-resistant sweet orange plants. Transgenic sweet orange plants producing intron-hairpin RNA transcripts (ihpRNA) corresponding to viral cp, 54K or 24K genes were generated and analyzed at the molecular and phenotypic levels. Two independent CPsV challenge assays demonstrated that expression of ihpRNA derived from the cp gene (ihpCP) provided a high level of virus resistance, while those derived from 54K and 24K genes (ihp54K and ihp24K) provided partial or no resistance.

Differential resistance to Citrus psorosis virus in transgenic Nicotiana benthamiana plants expressing hairpin RNA derived from the coat protein and 54K protein genes.

Citrus psorosis virus (CPsV), genus Ophiovirus, family Ophioviridae, is the causal agent of a serious disease affecting citrus trees in many countries. The viral genome consists of three ssRNAs of negative polarity. Post-transcriptional gene silencing (PTGS), a mechanism of plant defence against viruses, can be induced by transgenic expression of virus-derived sequences encoding hairpin RNAs.

Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus.

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks.