Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non–cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A–sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.

The multicellular nature of plants requires that cells should communicate in order to coordinate essential functions. This is achieved in part by molecular flux through pores in the cell wall, called plasmodesmata. We describe the proteomic analysis of plasmodesmata purified from the walls of Arabidopsis suspension cells. Isolated plasmodesmata were seen as membrane-rich structures largely devoid of immunoreactive markers for the plasma membrane, endoplasmic reticulum and cytoplasmic components. Using nano-liquid chromatography and an Orbitrap ion-trap tandem mass spectrometer, 1341 proteins were identified. We refer to this list as the plasmodesmata- or PD-proteome. Relative to other cell wall proteomes, the PD-proteome is depleted in wall proteins and enriched for membrane proteins, but still has a significant number (35%) of putative cytoplasmic contaminants, probably reflecting the sensitivity of the proteomic detection system. To validate the PD-proteome we searched for known plasmodesmal proteins and used molecular and cell biological techniques to identify novel putative plasmodesmal proteins from a small subset of candidates. The PD-proteome contained known plasmodesmal proteins and some inferred plasmodesmal proteins, based upon sequence or functional homology with examples identified in different plant systems. Many of these had a membrane association reflecting the membranous nature of isolated structures. Exploiting this connection we analysed a sample of the abundant receptor-like class of membrane proteins and a small random selection of other membrane proteins for their ability to target plasmodesmata as fluorescently-tagged fusion proteins. From 15 candidates we identified three receptor-like kinases, a tetraspanin and a protein of unknown function as novel potential plasmodesmal proteins. Together with published work, these data suggest that the membranous elements in plasmodesmata may be rich in receptor-like functions, and they validate the content of the PD-proteome as a valuable resource for the further uncovering of the structure and function of plasmodesmata as key components in cell-to-cell communication in plants.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

In many plant species, an exposure to a sublethal temperature triggers an adaptative response called acclimation. This response involves an extensive molecular reprogramming that allows the plant to further survive to an otherwise lethal increase of temperature. A related response is also launched under an abrupt and lethal heat stress that, in this case, is unable to successfully promote thermotolerance and therefore ends up in plant death. Although these molecular programmes are expected to have common players, the overlapping degree and the specific regulators of each process are currently unknown.We have carried out a high-throughput comparative proteomics analysis during acclimation and during the early stages of the plant response to a severe heat stress that lead Arabidopsis seedlings either to survival or death. This analysis dissects these responses, unravels the common players and identifies the specific proteins associated with these different fates. Thermotolerance assays of mutants in genes with an uncharacterized role in heat stress demonstrate the relevance of this study to uncover both positive and negative heat regulators and pinpoint a pivotal role of JR1 and BAG6 in heat tolerance.

HSP70-HSP90 organizing protein (HOP) is a well-studied family of cytosolic cochaperones. However, the possible role of HOP during the endoplasmic reticulum (ER) stress response and the identity of its interactors within the ER were not previously addressed in any eukaryote. We have demonstrated that Arabidopsis HOP3, whose function was not studied before, interacts in vivo with cytosolic HSP90 and HSP70, and, unexpectedly, with binding immunoglobulin protein (BiP), a HSP70 ER-resident protein. Although BiP lacks the domain described in other eukaryotes for HOP-HSP70 binding, it interacts with HOP3 through a non-canonical association to its nucleotide binding domain. Consistent with this interaction with BiP, HOP3 is partially localized at the ER. Moreover, HOP3 is induced both at transcript and protein levels by unfolded protein response (UPR) inducer agents by a mechanism dependent on inositol-requiring enzyme 1 (IRE1). Importantly, hop3 loss-of-function mutants show a reduction in pollen germination and a hypersensitive phenotype in the presence of ER stress inducer agents, a phenotype that is reverted by the addition of the chemical chaperone tauroursodeoxycholic acid (TUDCA). All these data demonstrate, for the first time in any eukaryote, a main role of HOP as an important regulator of the ER stress response, a process intimately linked in plants to important specific developmental programs and to environmental stress sensing and response.

During compatible virus infections, plants respond by reprogramming gene expression and metabolite content. While gene expression studies are profuse, our knowledge of the metabolic changes that occur in the presence of the virus is limited. Here, we combine gene expression and metabolite profiling in Arabidopsis (Arabidopsis thaliana) infected with Tobacco rattle virus (TRV) in order to investigate the influence of primary metabolism on virus infection. Our results revealed that primary metabolism is reconfigured in many ways during TRV infection, as reflected by significant changes in the levels of sugars and amino acids. Multivariate data analysis revealed that these alterations were particularly conspicuous at the time points of maximal accumulation of TRV, although infection time was the dominant source of variance during the process. Furthermore, TRV caused changes in lipid and fatty acid composition in infected leaves. We found that several Arabidopsis mutants deficient in branched-chain amino acid catabolism or fatty acid metabolism possessed altered susceptibility to TRV. Finally, we showed that increments in the putrescine content in TRV-infected plants correlated with enhanced tolerance to freezing stress in TRV-infected plants and that impairment of putrescine biosynthesis promoted virus multiplication. Our results thus provide an interesting overview for a better understanding of the relationship between primary metabolism and virus infection.

HSP70-HSP90 organizing protein (HOP) is a family of cytosolic cochaperones whose molecular role in thermotolerance is quite unknown in eukaryotes and unexplored in plants. In this article, we describe that the three members of the AtHOP family display a different induction pattern under heat, being HOP3 highly regulated during the challenge and the attenuation period. Despite HOP3 is the most heat-regulated member, the analysis of the hop1 hop2 hop3 triple mutant demonstrates that the three HOP proteins act redundantly to promote long-term acquired thermotolerance in Arabidopsis. HOPs interact strongly with HSP90 and part of the bulk of HOPs shuttles from the cytoplasm to the nuclei and to cytoplasmic foci during the challenge. RNAseq analyses demonstrate that, although the expression of the Hsf targets is not generally affected, the transcriptional response to heat is drastically altered during the acclimation period in the hop1 hop2 hop3 triple mutant. This mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under heat, which highlights the additional role of HOP in protein quality control. These data reveal that HOP family is involved in different aspects of the response to heat, affecting the plant capacity to acclimate to high temperatures for long periods.

Virus infections in plants cause changes in host gene expression that are common to other environmental stresses. In this work, we found extensive overlap in the transcriptional responses between Arabidopsis thaliana plants infected with Tobacco rattle virus (TRV) and plants undergoing senescence. This is exemplified by the up-regulation during infection of several senescence-associated Dark-inducible (DIN) genes, including AtDIN1 (Senescence 1, SEN1), AtDIN6 (Asparagine synthetase 1, AtASN1) and AtDIN11. DIN1, DIN6 and DIN11 homologues were also activated in Nicotiana benthamiana in response to TRV and Potato virus X (PVX) infection. Reduced TRV levels in RNA interference (RNAi) lines targeting AtDIN11 indicate that DIN11 is an important modulator of susceptibility to TRV in Arabidopsis. Furthermore, low accumulation of TRV in Arabidopsis protoplasts from RNAi lines suggests that AtDIN11 supports virus multiplication in this species. The effect of DIN6 on virus accumulation was negligible in Arabidopsis, perhaps as a result of gene or functional redundancy. However, TRV-induced silencing of NbASN, the DIN6 homologue in N. benthamiana, compromises TRV and PVX accumulation in systemically infected leaves. Interestingly, NbASN inactivation correlates with the appearance of morphological defects in infected leaves. We found that DIN6 and DIN11 regulate virus multiplication in a step prior to the activation of plant defence responses. We hypothesize on the possible roles of DIN6 and DIN11 during virus infection.