The co-chaperone HOP participates in TIR1 stabilisation and in auxin response in plants.

HOP (HSP70-HSP90 organising protein) is a conserved family of co-chaperones well known in mammals for its role in the folding of signalling proteins associated with development. In plants, HOP proteins have been involved in the response to multiple stresses, but their role in plant development remains elusive. Herein, we describe that the members of the HOP family participate in different aspects of plant development as well as in the response to warm temperatures through the regulation of auxin signalling. Arabidopsis hop1 hop2 hop3 triple mutant shows different auxin-related phenotypes and a reduced auxin sensitivity. HOP interacts with TIR1 auxin coreceptor in vivo. Furthermore, TIR1 accumulation and auxin transcriptional response are reduced in the hop1 hop2 hop3 triple mutant, suggesting that HOP's function in auxin signalling is related, at least, to TIR1 interaction and stabilisation. Interestingly, HOP proteins form part of the same complexes as SGT1b (a different HSP90 co-chaperone) and these co-chaperones synergistically cooperate in auxin signalling. This study provides relevant data about the role of HOP in auxin regulation in plants and uncovers that both co-chaperones, SGT1b and HOP, cooperate in the stabilisation of common targets involved in plant development.

HOP family plays a major role in long-term acquired thermotolerance in Arabidopsis.

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.

HOP3, a member of the HOP family in Arabidopsis, interacts with BiP and plays a major role in the ER stress response.

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.

Tobacco rattle virus 16K silencing suppressor binds ARGONAUTE 4 and inhibits formation of RNA silencing complexes.

The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus Tobravirus, is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an Agrobacterium-mediated transient assay in Nicotiana benthamiana to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with de novo formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.

Dissecting the proteome dynamics of the early heat stress response leading to plant survival or death in Arabidopsis.

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.

Virus-induced alterations in primary metabolism modulate susceptibility to Tobacco rattle virus in Arabidopsis.

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.

HOP co-chaperones contribute to GA signaling by promoting the accumulation of the F-box protein SNE in Arabidopsis.

Gibberellins (GAs) play important roles in multiple developmental processes and in plant response to the environment. Within the GA pathway, a central regulatory step relies on GA-dependent degradation of the DELLA transcriptional regulators. Nevertheless, the relevance of the stability of other key proteins in this pathway, such as SLY1 and SNE (the F-box proteins involved in DELLA degradation), remains unknown. Here, we take advantage of mutants in the HSP70-HSP90 organizing protein (HOP) co-chaperones and reveal that these proteins contribute to the accumulation of SNE in Arabidopsis. Indeed, HOP proteins, along with HSP90 and HSP70, interact in vivo with SNE, and SNE accumulation is significantly reduced in the hop mutants. Concomitantly, greater accumulation of the DELLA protein RGA is observed in these plants. In agreement with these molecular phenotypes, hop mutants show a hypersensitive response to the GA inhibitor paclobutrazol and display a partial response to the ectopic addition of GA when GA-regulated processes are assayed. These mutants also display different phenotypes associated with alterations in the GA pathway, such as reduced germination rate, delayed bolting, and reduced hypocotyl elongation in response to warm temperatures. Remarkably, ectopic overexpression of SNE reverts the delay in germination and the thermally dependent hypocotyl elongation defect of the hop1 hop2 hop3 mutant, revealing that SNE accumulation is the key aspect of the hop mutant phenotypes. Together, these data reveal a pivotal role for HOP in SNE accumulation and GA signaling.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

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.

Specific targeting of a plasmodesmal protein affecting cell-to-cell communication.

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 helper-component protease transmission factor of tobacco etch potyvirus binds specifically to an aphid ribosomal protein homologous to the laminin receptor precursor.

Potyviruses are plant pathogens transmitted by aphids in a non-persistent manner. During transmission, the virus-encoded factor helper-component protease (HCPro) is presumed to act as a molecular bridge, mediating the reversible retention of virions to uncharacterized binding sites in the vector mouthparts. Whilst the predicted interaction between HCPro and the coat protein (CP) of virions has been confirmed experimentally, the characterization of putative HCPro-specific receptors in aphids has remained elusive, with the exception of a report that described binding of HCPro of zucchini yellow mosaic virus to several cuticle proteins. To identify other aphid components that could play a role during transmission, this study used purified HCPro of tobacco etch virus (TEV) in far-Western blotting assays as bait to select interactors among proteins extracted from aphid heads. With this approach, new HCPro-interacting proteins were found, and several were identified after mass spectrometry analysis and searches in databases dedicated to aphid sequences. Among these interactors, a ribosomal protein S2 (RPS2) was chosen for further investigation due to its homology with the laminin receptor precursor, known to act as the receptor of several viruses. The specific interaction between RPS2 and TEV HCPro was confirmed after cloning and heterologous expression of the corresponding Myzus persicae gene. The possible involvement of RPS2 in the transmission process was further suggested by testing a variant of HCPro that was non-functional for transmission due to a mutation in the conserved KITC motif (EITC variant). This variant retained its ability to bind CP but failed to interact with RPS2.

HOP3 a new regulator of the ER stress response in Arabidopsis with possible implications in plant development and response to biotic and abiotic stresses.

HOPs (heat shock protein 70 (HSP70)-heat shock protein 90 (HSP90) organizing proteins) are a highly conserved family of cytosolic cochaperones. In a recent study we showed that HOP3, a member of the HOP family in Arabidopsis, plays an essential role during endoplasmic reticulum (ER) stress in plants. Interestingly, we also demonstrated that AtHOP3 interacts with binding immunoglobulin protein (BiP), a major ER-resident chaperone. All these data suggest that HOP3 could assist BiP in protein folding in the ER. These findings open the exciting possibility that HOP3, through its role in the alleviation of ER stress, could play an important function during different developmental processes and in response to different biotic and abiotic stresses.

Activation of senescence-associated Dark-inducible (DIN) genes during infection contributes to enhanced susceptibility to plant viruses.

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.

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Arabidopsis plasmodesmal proteome.

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.

Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system.

Potyviruses are non-persistently transmitted by aphid vectors with the assistance of a viral accessory factor known as helper component (HC-Pro), a multifunctional protein that is also involved in many other essential processes during the virus infection cycle. A transient Agrobacterium-mediated expression system was used to produce Plum pox virus (PPV) HC-Pro in Nicotiana benthamiana leaves from constructs that incorporated the 5' region of the genome, yielding high levels of HC-Pro in agroinfiltrated leaves. The expressed PPV HC-Pro was able to assist aphid transmission of purified virus particles in a sequential feeding assay, and to complement transmission-defective variants of the virus. Also, HC-Pro of a second potyvirus, Tobacco etch virus (TEV), was expressed and found to be functional for aphid transmission. These results show that this transient system can be useful for production of functionally active HC-Pro in potyviruses, and the possible uses of this approach to study the mechanism of transmission are discussed.