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With the discovery that pattern-triggered immunity (PTI) is active against virus infection in plants less than a decade ago, we began to understand that antiviral immunity goes far beyond RNA silencing and resistance gene-mediated immunity and is much more complex than previously thought. Since then, receptor kinases, signaling components and outputs, and viral suppressors of PTI were discovered and double-stranded RNAs as well as possibly other viral nucleic acids identified as candidates for viral pathogen-associated molecular patterns (PAMPs) in plants. Here, we summarize recent progress in PAMP-triggered antiviral immunity in plants and discuss possible crosstalk between dsRNA-triggered defense pathways.

International audience; Pattern-triggered immunity (PTI) is a plant defense response that relies on the perception of conserved microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs, respectively). Recently, it has been recognized that PTI restricts virus infection in plants; however, the nature of the viral or infection-induced PTI elicitors and the underlying signaling pathways are still unknown. As double-stranded RNAs (dsRNAs) are conserved molecular patterns associated with virus replication, we applied dsRNAs or synthetic dsRNA analogs to Arabidopsis thaliana and investigated PTI responses. We show that invitro-generated dsRNAs, dsRNAs purified from virus-infected plants and the dsRNA analog polyinosinic-polycytidylic acid (poly(I:C)) induce typical PTI responses dependent on the co-receptor SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1), but independent of dicer-like (DCL) proteins in Arabidopsis. Moreover, dsRNA treatment of Arabidopsis induces SERK1-dependent antiviral resistance. Screening of Arabidopsis wild accessions demonstrates natural variability in dsRNA sensitivity. Our findings suggest that dsRNAs represent genuine PAMPs in plants, which induce a signaling cascade involving SERK1 and a specific dsRNA receptor. The dependence of dsRNA-mediated PTI on SERK1, but not on DCLs, implies that dsRNA-mediated PTI involves membrane-associated processes and operates independently of RNA silencing. dsRNA sensitivity may represent a useful trait to increase antiviral resistance in cultivated plants.

Summary RNA silencing and antiviral pattern‐triggered immunity (PTI) both rely on recognition of double‐stranded (ds)RNAs as defence‐inducing signals. While dsRNA recognition by dicer‐like proteins during antiviral RNA silencing is thoroughly investigated, the molecular mechanisms involved in dsRNA perception leading to antiviral PTI are just about to be untangled. Parallels to antimicrobial PTI thereby only partially facilitate our view on antiviral PTI. PTI against microbial pathogens involves plasma membrane bound receptors; however, dsRNAs produced during virus infection occur intracellularly. Hence, how dsRNA may be perceived during this immune response is still an open question. In this short review, we describe recent discoveries in PTI signalling upon sensing of microbial patterns and endogenous ‘danger’ molecules with emphasis on immune signalling‐associated subcellular trafficking processes in plants. Based on these studies, we develop different scenarios how dsRNAs could be sensed during antiviral PTI.

Nutrient transfer is a key feature of the arbuscular mycorrhizal (AM) symbiosis. Valuable mineral nutrients are transferred from the AM fungus to the plant, increasing its fitness and productivity, and, in exchange, the AM fungus receives carbohydrates as an energy source from the plant. Here, we analyzed the transcriptome of the Populus trichocarpa-Rhizophagus irregularis symbiosis using RNA-sequencing of non-mycorrhizal or mycorrhizal fine roots, with a focus on the effect of nitrogen (N) starvation. In R. irregularis, we identified 1,015 differentially expressed genes, whereby N starvation led to a general induction of gene expression. Genes of the functional classes of cell growth, membrane biogenesis and cell structural components were highly abundant. Interestingly, N starvation also led to a general induction of fungal transporters, indicating increased nutrient demand upon N starvation. In non-mycorrhizal P. trichocarpa roots, 1,341 genes were differentially expressed under N starvation. Among the 953 down-regulated genes in N starvation, most were involved in metabolic processes including amino acids, carbohydrate and inorganic ion transport, while the 342 up-regulated genes included many defense-related genes. Mycorrhization led to the up-regulation of 549 genes mainly involved in secondary metabolite biosynthesis and transport; only 24 genes were down-regulated. Mycorrhization specifically induced expression of three ammonium transporters and one phosphate transporter, independently of the N conditions, corroborating the hypothesis that these transporters are important for symbiotic nutrient exchange. In conclusion, our data establish a framework of gene expression in the two symbiotic partners under high-N and low-N conditions.

Plant viruses use plasmodesmata (PD) to spread infection between cells and systemically. Dependent on viral species, movement through PD can occur in virion or non-virion form, and requires different mechanisms for targeting and modification of the pore. These mechanisms are supported by viral movement proteins and by other virus-encoded factors that interact among themselves and with plant cellular components to facilitate virus movement in a coordinated and regulated fashion.

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T-DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, alpha-tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus-end marker GFP-EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell-to-cell movement of TMV encoding GFP-tagged movement protein (MP-GFP) is reduced in ATER2 accompanied by a reduced association of MP-GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.

We describe a simple fluorescent protein-based method to investigate interactions with a viral movement protein in living cells that relies on the in vivo re-localization of proteins in the presence of their interaction partners. We apply this method in combination with fluorescence lifetime imaging microscopy (FLIM) to demonstrate that a domain of the Tobacco mosaic virus (TMV) movement protein (MP) previously predicted to mediate protein:protein interactions is dispensable for these contacts. We suggest that this method can be generalized for analysis of other protein interactions in planta.

Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature.

The spread of Tomato yellow leaf curl virus (TYLCV) was accompanied by the formation of coat protein (CP) aggregates of increasing size in the cytoplasm and nucleus of infected tomato (Solanum lycopersicum) cells. In order to better understand the TYLCV-host interaction, we investigated the properties and the subcellular accumulation pattern of the non-structural viral protein V2. CP and V2 are the only sense-oriented genes on the virus circular single-stranded DNA genome. Similar to CP, V2 localized to cytoplasmic aggregates of increasing size and as infection progressed was also found in nuclei, where it co-localized with CP. V2 was associated with viral genomic DNA molecules, suggesting that V2 functions as a DNA shuttling protein. The formation and the 26S proteasome-mediated degradation of V2 aggregates were dependent on the integrity of the actin and microtubule cytoskeleton. We propose that the cytoskeleton-dependent formation and growth of V2 aggregates play an important role during TYLCV infection and that microtubules and actin filaments are important for the delivery of V2 to the 26S proteasome.

The plant's innate immune system detects potential biotic threats through recognition of microbe-associated molecular patterns (MAMPs) or danger-associated molecular patterns (DAMPs) by pattern recognition receptors (PRR). A central regulator of pattern-triggered immunity (PTI) is the BRI1-associated kinase 1 (BAK1), which undergoes complex formation with PRR upon ligand binding. Although viral patterns inducing PTI are well known from animal systems, nothing similar has been reported for plants. Rather, antiviral defense in plants is thought to be mediated by post-transcriptional gene silencing of viral RNA or through effector-triggered immunity, i.e., recognition of virus-specific effectors by resistance proteins. Nevertheless, infection by compatible viruses can also lead to the induction of defense gene expression, indicating that plants may also recognize viruses through PTI. Here, we show that PTI, or at least the presence of the regulator BAK1, is important for antiviral defense of Arabidopsis plants. Arabidopsis bak1 mutants show increased susceptibility to three different RNA viruses during compatible interactions. Furthermore, crude viral extracts but not purified virions induce several PTI marker responses in a BAK1-dependent manner. Overall, we conclude that BAK1-dependent PTI contributes to antiviral resistance in plants.

[EN] Background: Microarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees. Results: Upon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection. Conclusions: Our results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies., We thank Drs M.A. Perez-Amador y J. Gadea for helping in the result analysis. This work was supported by grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica for the transcriptomic analyses and from the grant 2009CL0020 from the bilateral project INIA-Chile/CSIC-Spain for the phytosanitary evaluation. MC Herranz was the recipient of a contract from the Juan de la Cierva program of the Ministerio de Educacion y Ciencia of Spain.

Plants are continuously exposed to changing environmental conditions and must, as sessile organisms, possess sophisticated acclimative mechanisms. To gain insight into systemic responses to local virus infection or wounding, we performed comparative LC-MS/MS protein profiling of distal, virus-free leaves four and five days after local inoculation of Arabidopsis thaliana plants with either Oilseed rape mosaic virus (ORMV) or inoculation buffer alone. Our study revealed biomarkers for systemic signaling in response to wounding and compatible virus infection in Arabidopsis, which should prove useful in further addressing the trigger-specific systemic response network and the elusive systemic signals. We observed responses common to ORMV and mock treatment as well as protein profile changes that are specific to local virus infection or mechanical wounding (mock treatment) alone, which provides evidence for the existence of more than one systemic signal to induce these distinct changes. Comparison of the systemic responses between time points indicated that the responses build up over time. Our data indicate stress-specific changes in proteins involved in jasmonic and abscisic acid signaling, intracellular transport, compartmentalization of enzyme activities, protein folding and synthesis, and energy and carbohydrate metabolism. In addition, a virus-triggered systemic signal appears to suppress antiviral host defense.

Viruses use and subvert host cell mechanisms to support their replication and spread between cells, tissues and organisms. Microtubules and associated motor proteins play important roles in these processes in animal systems, and may also play a role in plants. Although transport processes in plants are mostly actin based, studies, in particular with Tobacco mosaic virus (TMV) and its movement protein (MP), indicate direct or indirect roles of microtubules in the cell-to-cell spread of infection. Detailed observations suggest that microtubules participate in the cortical anchorage of viral replication complexes, in guiding their trafficking along the endoplasmic reticulum (ER)/actin network, and also in developing the complexes into virus factories. Microtubules also play a role in the plant-to-plant transmission of Cauliflower mosaic virus (CaMV) by assisting in the development of specific virus-induced inclusions that facilitate viral uptake by aphids. The involvement of microtubules in the formation of virus factories and of other virus-induced inclusions suggests the existence of aggresomal pathways by which plant cells recruit membranes and proteins into localized macromolecular assemblies. Although studies related to the involvement of microtubules in the interaction of viruses with plants focus on specific virus models, a number of observations with other virus species suggest that microtubules may have a widespread role in viral pathogenesis.

Like many other viruses, Tobacco mosaic virus replicates in association with the endoplasmic reticulum (ER) and exploits this membrane network for intercellular spread through plasmodesmata (PD), a process depending on virus-encoded movement protein (MP). The movement process involves interactions of MP with the ER and the cytoskeleton as well as its targeting to PD. Later in the infection cycle, the MP further accumulates and localizes to ER-associated inclusions, the viral factories, and along microtubules before it is finally degraded. Although these patterns of MP accumulation have been described in great detail, the underlying mechanisms that control MP fate and function during infection are not known. Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), a conserved chaperone controlling protein fate in yeast (Saccharomyces cerevisiae) and animal cells by extracting protein substrates from membranes or complexes, as a cellular factor regulating MP accumulation patterns in plant cells. We demonstrate that Arabidopsis (Arabidopsis thaliana) CDC48 is induced upon infection, interacts with MP in ER inclusions dependent on the MP N terminus, and promotes degradation of the protein. We further provide evidence that CDC48 extracts MP from ER inclusions to the cytosol, where it subsequently accumulates on and stabilizes microtubules. We show that virus movement is impaired upon overexpression of CDC48, suggesting that CDC48 further functions in controlling virus movement by removal of MP from the ER transport pathway and by promoting interference of MP with microtubule dynamics. CDC48 acts also in response to other proteins expressed in the ER, thus suggesting a general role of CDC48 in ER membrane maintenance upon ER stress.

Plant development depends on the intercellular transport of macromolecules through gatable channels in the cell wall known as plasmodesmata (PD). Plant viruses exploit this intercellular trafficking pathway to move cell-to-cell and to cause systemic infection. Dependent on viral species, movement through PD can occur in virion or non-virion form and by different mechanisms for targeting and modification of the pore. These mechanisms are supported by viral movement proteins and by other virus-encoded factors that interact among themselves and with plant cellular components to facilitate virus movement in a coordinated and regulated fashion. This chapter provides an overview about current knowledge in virus cell-to-cell movement with the aim to extract principal mechanisms playing a role in intercellular transport.

A panel of seven SR1 tobacco mutants (ATER1 to ATER7) derived via T-DNA activation tagging and screening for resistance to a microtubule assembly inhibitor, ethyl phenyl carbamate, were used to study the role of microtubules during infection and spread of tobacco mosaic virus (TMV). In one of these lines, ATER2, alpha-tubulin is shifted from the tyrosinylated into the detyrosinated form, and the microtubule plus-end marker GFP-EB1 moves significantly slower when expressed in the background of the ATER2 mutant as compared with the SR1 wild type. The efficiency of cell-to-cell movement of TMV encoding GFP-tagged movement protein (MP-GFP) is reduced in ATER2 accompanied by a reduced association of MP-GFP with plasmodesmata. This mutant is also more tolerant to viral infection as compared with the SR1 wild type, implying that reduced microtubule dynamics confer a comparative advantage in face of TMV infection.

In plants, the enzymes for cysteine synthesis serine acetyltransferase (SAT) and O-acetylserine-(thiol)-lyase (OASTL) are present in the cytosol, plastids and mitochondria. However, it is still not clearly resolved to what extent the different compartments are involved in cysteine biosynthesis and how compartmentation influences the regulation of this biosynthetic pathway. To address these questions, we analysed Arabidopsis thaliana T-DNA insertion mutants for cytosolic and plastidic SAT isoforms. In addition, the subcellular distribution of enzyme activities and metabolite concentrations implicated in cysteine and glutathione biosynthesis were revealed by non-aqueous fractionation (NAF). We demonstrate that cytosolic SERAT1.1 and plastidic SERAT2.1 do not contribute to cysteine biosynthesis to a major extent, but may function to overcome transport limitations of O-acetylserine (OAS) from mitochondria. Substantiated by predominantly cytosolic cysteine pools, considerable amounts of sulphide and presence of OAS in the cytosol, our results suggest that the cytosol is the principal site for cysteine biosynthesis. Subcellular metabolite analysis further indicated efficient transport of cysteine, gamma-glutamylcysteine and glutathione between the compartments. With respect to regulation of cysteine biosynthesis, estimation of subcellular OAS and sulphide concentrations established that OAS is limiting for cysteine biosynthesis and that SAT is mainly present bound in the cysteine-synthase complex.

Viruses are obligate symbionts that tightly interact with their hosts to complete their life cycle. Each infected cell is confronted with the accumulation of viral products and activities that have evolved to support the replication and spread of the virus in the context of host cell functions and defense responses. Tobacco mosaic virus encodes replicase proteins and coat protein, to replicate and protect the RNA genome, and a movement protein (MP) that binds viral RNA and manipulates the size exclusion limit of plasmodesmata to facilitate the spread of the viral genomic RNA (vRNA). The MP and replicase also interfere with the cellular RNA silencing machinery that influences plant gene expression and development. Moreover, virus-infected cells stimulate the production of a systemic signal ahead of the virus front that triggers genomic recombination leading to heritable genetic changes. Thus viruses can interact with their hosts through diverse molecular interactions. Given the high mutation rate of viruses, these interactions have implications for evolutionary processes and adaptations at the virus-host interface that may contribute to eukaryotic evolution.

A better understanding of defence responses elicited during compatible plant–virus interactions is a current goal in plant pathology. We analysed defence responses during infection of Solanum tuberosum L. cv. Desiree with Potato virus X (PVX) at the transcript and metabolite level. A mostly unchanged primary metabolism reflects the compatible nature of this plant–virus interaction. Salicylic acid biosynthesis and expression of several defence genes including PR-1 and glutathione-S-transferase, which are involved in ethylene and reactive oxygen species dependent signalling, were highly up-regulated in upper-uninoculated (systemic) leaves of PVX-infected potato plants compared with mock-inoculated controls. Moreover, the β-phenylethylamine-alkaloids tyramine, octopamine, dopamine and norepinephrine were highly induced upon infection. β-phenylethylamine-alkaloids can contribute to active plant defence responses by forming hydroxycinnamic acid amides (HCAA), which are thought to increase cell wall stability by extracellular peroxidative polymerisation. Expression of tyramine-hydroxycinnamoyl transferase (THT) and apoplastic peroxidase (POD) was highly induced upon PVX infection in systemic leaves, which suggests synthesis and extracellular polymerisation of HCAA. Since cell-wall-bound ion concentrations could contribute to this process, we measured cell-wall-bound and total ion concentrations in PVX-infected and mock-inoculated leaves. The observed metabolic and transcriptional changes might represent a systemic acquired resistance response against subsequent pathogen challenge.

Cell-division-cycle protein 48 (CDC48) is an essential, conserved ATP-driven chaperone in eukaryotic cells, which functions in diverse cellular processes including the targeting of misfolded and aggregated proteins for degradation via proteasomal and aggresomal-autophagic pathways. We recently demonstrated that plant CDC48 localizes to and interacts with Tobacco mosaic virus (TMV) movement protein (MP) in ER-associated viral protein inclusions. Our data suggest that CDC48 participates in the clearance of these viral protein inclusions in an ER-assisted protein degradation (ERAD)-like mechanism. As TMV MP-inclusions formed at late infection stages resemble aggresomes, we here propose that TMV MP enters both, ERAD-like and aggresomal pathways in its host cells and that CDC48 coordinates these processes. Moreover, as viruses often exploit host pathways for replication and spread, we propose a model in which CDC48 functions in the degradation pathway of overaccumulating viral protein and also actively participates in the regulation of TMV replication and cell-to-cell movement.