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RNA processing defects in chloroplasts were previously associated with increased plasmodesmata (PD) permeability. However, the underlying mechanisms for such association are still unknown. To provide insight into this, we silenced the expression of chloroplast-located INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) RNA helicase in Nicotiana benthamiana leaves and determined an increase in PD permeability which is caused by a reduction of PD callose deposition. Moreover, the silencing of two other nuclear genes encoding chloroplastic enzymes involved in RNA processing, RH3, and CLPR2, also increased PD permeability accompanied by reduced callose accumulation at PD. In addition, we quantified the plastidic hydrogen peroxide levels using the chloroplast-targeted fluorescent sensor, HyPer, in ISE2, RH3, and CLPR2 silenced N. benthamiana leaves. The levels of chloroplastic hydrogen peroxide were not correlated with the increased cell-to-cell movement of the marker protein GFP2X. We, therefore, propose that defects in chloroplast RNA metabolism mediate PD gating by suppressing PD callose deposition, and hydrogen peroxide levels in the organelles are not directly linked to this process., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

RNA transport and localization represent important post-transcriptional mechanisms to determine the subcellular localization of protein synthesis. Plants have the capacity to transport messenger (m)RNA molecules beyond the cell boundaries through plasmodesmata and over long distances in the phloem. RNA viruses exploit these transport pathways to disseminate their infections and represent important model systems to investigate RNA transport in plants. Here, we present an in vivo plant RNA-labeling system based on the Escherichia coli RNA-binding protein BglG. Using the detection of RNA in mobile RNA particles formed by viral movement protein (MP) as a model, we demonstrate the efficiency and specificity of mRNA detection by the BglG system as compared with MS2 and λN systems. Our observations show that MP mRNA is specifically associated with MP in mobile MP particles but hardly with MP localized at plasmodesmata. MP mRNA is clearly absent from MP accumulating along microtubules. We show that the in vivo BglG labeling of the MP particles depends on the presence of the BglG-binding stem-loop aptamers within the MP mRNA and that the aptamers enhance the coprecipitation of BglG by MP, thus demonstrating the presence of an MP:MP mRNA complex. The BglG system also allowed us to monitor the cell-to-cell transport of the MP mRNA, thus linking the observation of mobile MP mRNA granules with intercellular MP mRNA transport. Given its specificity demonstrated here, the BglG system may be widely applicable for studying mRNA transport and localization in plants., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Numerous cell surface receptors and receptor-like proteins (RLPs) undergo activation or deactivation via a transmembrane domain (TMD). A subset of plant RLPs distinctively localizes to the plasma membrane-lined pores called plasmodesmata. Those RLPs include the Arabidopsis thaliana Plasmodesmata-located protein (PDLP) 5, which is well known for its vital function regulating plasmodesmal gating and molecular movement between cells. In this study, we report that the TMD, although not a determining factor for the plasmodesmal targeting, serves essential roles for the PDLP5 function. In addition to its role for membrane anchoring, the TMD mediates PDLP5 self-interaction and carries an evolutionarily conserved motif that is essential for PDLP5 to regulate cell-to-cell movement. Computational modeling-based analyses suggest that PDLP TMDs have high propensities to dimerize. We discuss how a specific mode(s) of TMD dimerization might serve as a common mechanism for PDLP5 and other PDLP members to regulate cell-to-cell movement.

Cytoplasmic RNA granules consist of microscopic agglomerates of mRNAs and proteins and occur when the translation is reversibly and temporally halted (stress granules, SGs) or mRNAs are targeted for decapping (processing bodies, PBs). The induction of RNA granules formation by virus infection is a common feature of mammalian cells. However, plant-virus systems still remain poorly characterized. In this work, the SG marker AtUBP1b was expressed in Nicotiana benthamiana plants to decipher how the virus infection of plant cells affects SG dynamics. We found that the hypoxia-induced SG assembly was substantially inhibited in Potato virus X (PVX)-infected cells. Furthermore, we determined that the expression of PVX movement protein TGBp1 by itself, mimics the inhibitory effect of PVX on SG formation under hypoxia. Importantly, overexpression of AtUBP1b showed inhibition of the PVX spreading, whereas the overexpression of the dominant negative AtUBP1brrm enhanced PVX spreding, indicating that AtUBP1b negatively affects PVX infection. Notably, PVX infection did not inhibit the formation of processing bodies (PBs), indicating PVX has distinct effects depending on the type of RNA granule. Our results suggest that SG inhibition could be part of the virus strategy to infect the plant., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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. Consistent with this hypothesis, we show that the MP of CPsV (MP CPsV ) indeed forms tubule-like structures at PD upon transient expression in Nicotiana benthamiana leaves. Tubule formation by MP CPsV depends on its cleavage capacity, mediated by a specific aspartic protease motif present in its primary sequence. A single amino acid mutation in this motif abolishes MP CPsV cleavage, alters the subcellular localization of the protein, and negatively affects its activity in facilitating virus movement. The amino-terminal 34-kDa cleavage product (34K CPsV ), but not the 20-kDa fragment (20K CPsV ), supports virus movement. Moreover, similar to tubule-forming MPs of other viruses, MP CPsV (and also the 34K CPsV cleavage product) can homooligomerize, interact with PD-located protein 1 (PDLP1), and assemble tubule-like structures at PD by a mechanism dependent on the secretory pathway. 20K CPsV retains the protease activity and is able to cleave a cleavage-deficient MP CPsV in trans Altogether, these results demonstrate that CPsV movement depends on the autolytic cleavage of MP CPsV by an aspartic protease activity, which removes the 20K CPsV protease and thereby releases the 34K CPsV protein for PDLP1-dependent tubule formation at PD. IMPORTANCE Infection by citrus psorosis virus (CPsV) involves a self-cleaving aspartic protease activity within the viral movement protein (MP), which results in the production of two peptides, termed 34K CPsV and 20K CPsV , that carry the MP and viral protease activities, respectively. The underlying protease motif within the MP is also found in the MPs of other members of the Aspiviridae family, suggesting that protease-mediated protein processing represents a conserved mechanism of protein expression in this virus family. The results also demonstrate that CPsV and potentially other ophioviruses move by a tubule-guided mechanism. Although several viruses from different genera were shown to use this mechanism for cell-to-cell movement, our results also demonstrate that this mechanism is controlled by posttranslational protein cleavage. Moreover, given that tubule formation and virus movement could be inhibited by a mutation in the protease motif, targeting the protease activity for inactivation could represent an important approach for ophiovirus control., (Copyright © 2018 American Society for Microbiology.)

Citrus psorosis virus and Mirafiori lettuce big-vein virus are two members of the genus Ophiovirus, family Ophioviridae. So far, how these viruses can interfere in the antiviral RNA silencing pathway is not known. In this study, using a local GFP silencing assay on Nicotiana benthamiana, the 24K-25K and the movement protein (MP) of both viruses were identified as RNA silencing suppressor proteins. Upon their co-expression with GFP in N. benthamiana 16c plants, the proteins also showed to suppress systemic RNA (GFP) silencing. The MP CPsV and 24K CPsV proteins bind long (114 nucleotides) but not short-interfering (21 nt) dsRNA, and upon transgenic expression, plants showed developmental abnormalities that coincided with an altered miRNA accumulation pattern. Furthermore, both proteins were able to suppress miRNA-induced silencing of a GFP-sensor construct and the co-expression of MP CPsV and 24K CPsV exhibited a stronger effect, suggesting they act at different stages of the RNAi pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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. We examined its role in the MP of Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV). Alanine substitution of this aspartate prevented plasmodesmata accumulation of MP(MiLBVV), while MP(CPsV) was not affected. The capacity of ophiovirus MPs to increase the plasmodesmata size exclusion limit and non-cell autonomous protein feature was abolished in both mutants. To investigate the role of the signature aspartate in cell-to-cell movement, we constructed a new movement-deficient Tobacco mosaic virus vector used for trans-complementation assays. We showed that both ophiovirus MP mutants lack the cell-to-cell movement capacity, confirming that this signature aspartate is essential for viral cell-to-cell movement., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sweet orange (Citrus sinensis), one of the most important fruit crops worldwide, may suffer from disease symptoms induced by virus infections, thus resulting in dramatic economic losses. Here, we show that the infection of sweet orange plants with two isolates of Citrus psorosis virus (CPsV) expressing different symptomatology alters the accumulation of a set of endogenous microRNAs (miRNAs). Within these miRNAs, miR156, miR167 and miR171 were the most down-regulated, with almost a three-fold reduction in infected samples. This down-regulation led to a concomitant up-regulation of some of their targets, such as Squamosa promoter-binding protein-like 9 and 13, as well as Scarecrow-like 6. The processing of miRNA precursors, pre-miR156 and pre-miR171, in sweet orange seems to be affected by the virus. For instance, virus infection increases the level of unprocessed precursors, which is accompanied by a concomitant decrease in mature species accumulation. miR156a primary transcript accumulation remained unaltered, thus strongly suggesting a processing deregulation for this transcript. The co-immunoprecipitation of viral 24K protein with pre-miR156a or pre-miR171a suggests that the alteration in the processing of these precursors might be caused by a direct or indirect interaction with this particular viral protein. This result is also consistent with the nuclear localization of both miRNA precursors and the CPsV 24K protein. This study contributes to the understanding of the manner in which a virus can alter host regulatory mechanisms, particularly miRNA biogenesis and target expression., (© 2015 BSPP and John Wiley & Sons Ltd.)