Camalexin accumulation as a component of plant immunity during interactions with pathogens and beneficial microbes.

MAIN CONCLUSION: This review provides an overview on the role of camalexin in plant immunity taking into account various plant-pathogen and beneficial microbe interactions, regulation mechanisms and the contribution in basal and induced plant resistance. In a hostile environment, plants evolve complex and sophisticated defense mechanisms to counteract invading pathogens and herbivores. Several lines of evidence support the assumption that secondary metabolites like phytoalexins which are synthesized de novo, play an important role in plant defenses and contribute to pathogens' resistance in a wide variety of plant species. Phytoalexins are synthesized and accumulated in plants upon pathogen challenge, root colonization by beneficial microbes, following treatment with chemical elicitors or in response to abiotic stresses. Their protective properties against pathogens have been reported in various plant species as well as their contribution to human health. Phytoalexins are synthesized through activation of particular sets of genes encoding specific pathways. Camalexin (3'-thiazol-2'-yl-indole) is the primary phytoalexin produced by Arabidopsis thaliana after microbial infection or abiotic elicitation and an iconic representative of the indole phytoalexin family. The synthesis of camalexin is an integral part of cruciferous plant defense mechanisms. Although the pathway leading to camalexin has been largely elucidated, the regulatory networks that control the induction of its biosynthetic steps by pathogens with different lifestyles or by beneficial microbes remain mostly unknown. This review thus presents current knowledge regarding camalexin biosynthesis induction during plant-pathogen and beneficial microbe interactions as well as in response to microbial compounds and provides an overview on its regulation and interplay with signaling pathways. The contribution of camalexin to basal and induced plant resistance and its detoxification by some pathogens to overcome host resistance are also discussed.

Priming of camalexin accumulation in induced systemic resistance by beneficial bacteria against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000.

Plants harbor various beneficial microbes that modulate their innate immunity, resulting in induced systemic resistance (ISR) against a broad range of pathogens. Camalexin is an integral part of Arabidopsis innate immunity, but the contribution of its biosynthesis in ISR is poorly investigated. We focused on camalexin accumulation primed by two beneficial bacteria, Pseudomonas fluorescens and Bacillus subtilis, and its role in ISR against Botrytis cinerea and Pseudomonas syringae Pst DC3000. Our data show that colonization of Arabidopsis thaliana roots by beneficial bacteria triggers ISR against both pathogens and primes plants for enhanced accumulation of camalexin and CYP71A12 transcript in leaf tissues. Pseudomonas fluorescens induced the most efficient ISR response against B. cinerea, while B. subtilis was more efficient against Pst DC3000. Analysis of cyp71a12 and pad3 mutants revealed that loss of camalexin synthesis affected ISR mediated by both bacteria against B. cinerea. CYP71A12 and PAD3 contributed significantly to the pathogen-triggered accumulation of camalexin, but PAD3 does not seem to contribute to ISR against Pst DC3000. This indicated a significant contribution of camalexin in ISR against B. cinerea, but not always against Pst DC3000. Experiments with Arabidopsis mutants compromised in different hormonal signaling pathways highlighted that B. subtilis stimulates similar signaling pathways upon infection with both pathogens, since salicylic acid (SA), but not jasmonic acid (JA) or ethylene, is required for ISR camalexin accumulation. However, P. fluorescens-induced ISR differs depending on the pathogen; both SA and JA are required for camalexin accumulation upon B. cinerea infection, while camalexin is not necessary for priming against Pst DC3000.

Bacterial rhamnolipids and their 3-hydroxyalkanoate precursors activate Arabidopsis innate immunity through two independent mechanisms.

Plant innate immunity is activated upon perception of invasion pattern molecules by plant cell-surface immune receptors. Several bacteria of the genera Pseudomonas and Burkholderia produce rhamnolipids (RLs) from l-rhamnose and (R)-3-hydroxyalkanoate precursors (HAAs). RL and HAA secretion is required to modulate bacterial surface motility, biofilm development, and thus successful colonization of hosts. Here, we show that the lipidic secretome from the opportunistic pathogen Pseudomonas aeruginosa, mainly comprising RLs and HAAs, stimulates Arabidopsis immunity. We demonstrate that HAAs are sensed by the bulb-type lectin receptor kinase LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION/S-DOMAIN-1-29 (LORE/SD1-29), which also mediates medium-chain 3-hydroxy fatty acid (mc-3-OH-FA) perception, in the plant Arabidopsis thaliana HAA sensing induces canonical immune signaling and local resistance to plant pathogenic Pseudomonas infection. By contrast, RLs trigger an atypical immune response and resistance to Pseudomonas infection independent of LORE. Thus, the glycosyl moieties of RLs, although abolishing sensing by LORE, do not impair their ability to trigger plant defense. Moreover, our results show that the immune response triggered by RLs is affected by the sphingolipid composition of the plasma membrane. In conclusion, RLs and their precursors released by bacteria can both be perceived by plants but through distinct mechanisms.

Biosurfactants in Plant Protection Against Diseases: Rhamnolipids and Lipopeptides Case Study.

Biosurfactants are amphiphilic surface-active molecules that are produced by a variety of microorganisms including fungi and bacteria. Pseudomonas, Burkholderia, and Bacillus species are known to secrete rhamnolipids and lipopeptides that are used in a wide range of industrial applications. Recently, these compounds have been studied in a context of plant-microbe interactions. This mini-review describes the direct antimicrobial activities of these compounds against plant pathogens. We also provide the current knowledge on how rhamnolipids and lipopeptides stimulate the plant immune system leading to plant resistance to phytopathogens. Given their low toxicity, high biodegradability and ecological acceptance, we discuss the possible role of these biosurfactants as alternative strategies to reduce or even replace pesticide use in agriculture.

Bacillus subtilis and Pseudomonas fluorescens Trigger Common and Distinct Systemic Immune Responses in Arabidopsis thaliana Depending on the Pathogen Lifestyle.

Plants harbor various beneficial bacteria that modulate their innate immunity, resulting in induced systemic resistance (ISR) against various pathogens. However, the immune mechanisms underlying ISR triggered by Bacillus spp. and Pseudomonas spp. against pathogens with different lifestyles are not yet clearly elucidated. Here, we show that root drenching of Arabidopsis plants with Pseudomonas fluorescensPTA-CT2 and Bacillus subtilis PTA-271 can induce ISR against the necrotrophic fungus B. cinerea and the hemibiotrophic bacterium Pseudomonas syringae Pst DC3000. In the absence of pathogen infection, both beneficial bacteria do not induce any consistent change in systemic immune responses. However, ISR relies on priming faster and robust expression of marker genes for the salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) signaling pathways upon pathogen challenge. These responses are also associated with increased levels of SA, JA, and abscisic acid (ABA) in the leaves of bacterized plants after infection. The functional study also points at priming of the JA/ET and NPR1-dependent defenses as prioritized immune pathways in ISR induced by both beneficial bacteria against B. cinerea. However, B. subtilis-triggered ISR against Pst DC3000 is dependent on SA, JA/ET, and NPR1 pathways, whereas P. fluorescens-induced ISR requires JA/ET and NPR1 signaling pathways. The use of ABA-insensitive mutants also pointed out the crucial role of ABA signaling, but not ABA concentration, along with JA/ET signaling in primed systemic immunity by beneficial bacteria against Pst DC3000, but not against B. cinerea. These results clearly indicate that ISR is linked to priming plants for enhanced common and distinct immune pathways depending on the beneficial strain and the pathogen lifestyle.

Sphingolipids: towards an integrated view of metabolism during the plant stress response.

Plants exist in an environment of changing abiotic and biotic stresses. They have developed a complex set of strategies to respond to these stresses and over recent years it has become clear that sphingolipids are a key player in these responses. Sphingolipids are not universally present in all three domains of life. Many bacteria and archaea do not produce sphingolipids but they are ubiquitous in eukaryotes and have been intensively studied in yeast and mammals. During the last decade there has been a steadily increasing interest in plant sphingolipids. Plant sphingolipids exhibit structural differences when compared with their mammalian counterparts and it is now clear that they perform some unique functions. Sphingolipids are recognised as critical components of the plant plasma membrane and endomembrane system. Besides being important structural elements of plant membranes, their particular structure contributes to the fluidity and biophysical order. Sphingolipids are also involved in multiple cellular and regulatory processes including vesicle trafficking, plant development and defence. This review will focus on our current knowledge as to the function of sphingolipids during plant stress responses, not only as structural components of biological membranes, but also as signalling mediators.

Apoplastic invasion patterns triggering plant immunity: plasma membrane sensing at the frontline.

Plants are able to effectively cope with invading pathogens by activating an immune response based on the detection of invasion patterns (IPs) originating from the pathogen or released by the plant after infection. At a first level, this perception takes place at the plasma membrane through cell surface immune receptors and although the involvement of proteinaceous pattern recognition receptors (PRRs) is well established, increasing data are also pointing out the role of membrane lipids in the sensing of IPs. In this review, we discuss the evolution of various conceptual models describing plant immunity and present an overview of well-characterized IPs from different natures and origins. We summarize the current knowledge on how they are perceived by plants at the plasma membrane, highlighting the increasingly apparent diversity of sentinel-related systems in plants.

Synthetic Rhamnolipid Bolaforms trigger an innate immune response in Arabidopsis thaliana.

Stimulation of plant innate immunity by natural and synthetic elicitors is a promising alternative to conventional pesticides for a more sustainable agriculture. Sugar-based bolaamphiphiles are known for their biocompatibility, biodegradability and low toxicity. In this work, we show that Synthetic Rhamnolipid Bolaforms (SRBs) that have been synthesized by green chemistry trigger Arabidopsis innate immunity. Using structure-function analysis, we demonstrate that SRBs, depending on the acyl chain length, differentially activate early and late immunity-related plant defense responses and provide local increase in resistance to plant pathogenic bacteria. Our biophysical data suggest that SRBs can interact with plant biomimetic plasma membrane and open the possibility of a lipid driven process for plant-triggered immunity by SRBs.

Screening of wheat endophytes as biological control agents against Fusarium head blight using two different in vitro tests.

In order to find biological control agents (BCAs) for the management of Fusarium head blight (FHB), a major disease on wheat crops worldwide, 86 microorganisms isolated from inner tissues of wheat plants were discriminated for their ability to inhibit the growth of Fusarium graminearum and Fusarium culmorum by in vitro dual culture assays. A group of 22 strains appeared very effective to inhibit F. graminearum (inhibition of 30-51%) and they were also globally effective in controlling F. culmorum (inhibition of 15-53%). Further evaluation of a subselection of strains by screening on detached spikelets in vitro confirmed three species, namely Phoma glomerata, Aureobasidium proteae and Sarocladium kiliense, that have not yet been reported for their efficacy against Fusarium spp., indicating that looking for BCAs toward FHB among wheat endophytes proved to be promising. The efficacy of some strains turned out different between both in vitro screening approaches, raising the importance of finding the most appropriate screening approach for the search of BCAs. This study pointed out the interest of the test on detached wheat spikelets that provided information about a potential pathogenicity, the growth capacity and efficacy of the endophyte strains on the targeted plant, before testing them on whole plants.

Vacuum Infiltration-Centrifugation Method for Apoplastic Protein Extraction in Grapevine.

The apoplastic fluid moving in the extracellular space external to the plasma membrane provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Here, we describe an optimized vacuum-infiltration-centrifugation method to extract soluble proteins from apoplastic fluid of grapevine leaves. This optimized method allows recovering of the grapevine apoplastic soluble proteins suitable for mono- and bi-dimensional gel electrophoresis for further proteomic analysis in order to elucidate their physiological functions.

Spatial and Temporal Variation of Cultivable Communities of Co-occurring Endophytes and Pathogens in Wheat.

The aim of this work was to investigate the diversity of endogenous microbes from wheat (Triticum aestivum) and to study the structure of its microbial communities, with the ultimate goal to provide candidate strains for future evaluation as potential biological control agents against wheat diseases. We sampled plants from two wheat cultivars, Apache and Caphorn, showing different levels of susceptibility to Fusarium head blight, a major disease of wheat, and tested for variation in microbial diversity and assemblages depending on the host cultivar, host organ (aerial organs vs. roots) or host maturity. Fungi and bacteria were isolated using a culture dependent method. Isolates were identified using ribosomal DNA sequencing and we used diversity analysis to study the community composition of microorganisms over space and time. Results indicate great species diversity in wheat, with endophytes and pathogens co-occurring inside plant tissues. Significant differences in microbial communities were observed according to host maturity and host organs but we did not find clear differences between host cultivars. Some species isolated have not yet been reported as wheat endophytes and among all species recovered some might be good candidates as biological control agents, given their known effects toward plant pathogens.

Cold-night responses in grapevine inflorescences.

Cold nights impact grapevine flower development and fruit set. Regulation at the female meiosis stepmay be of considerable importance for further understanding on how flower reacts to cold stress. In this study, the impact of chilling temperature (0 °C overnight) on carbon metabolism was investigated in the inflorescencesof two cultivars, Pinot noir (Pinot) and Gewurztraminer (Gewurtz.). Fluctuations in photosynthetic activity and carbohydrate metabolism were monitored by analyzing gas exchanges, simultaneous photosystem I and II activities, andcarbohydrate content. Further, the expression of PEPc, PC, FNR, ISO, OXO, AGPase, amylases and invertase genes, activities of various enzymes, as well as metabolomic analysis were attained. Results showed that the chilling night has different impacts depending on cultivars. Thus, in Gewurtz., net photosynthesis (Pn) was transiently increased whereas, in Pinot, the Pn increase was persistent and concomitant with an inhibition of respiration. However, during the days following the cold night, photosynthetic activity was decreased, and the cyclic electron flow was inhibited in Gewurtz., suggesting lower efficient energy dissipation. Likewise, metabolomic analysis revealed that several metabolites contents (namely alanine, GABA, lysine and succinate)were distinctly modulated in the two cultivars. Taking together, these results reflect a photosynthetic metabolism alteration or internal CO2 conductance in Gewurtz. explaining partly why Pinot is less susceptible to cold stress.

Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis.

Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids.

Pseudomonas fluorescens PTA-CT2 Triggers Local and Systemic Immune Response Against Botrytis cinerea in Grapevine.

Although induced systemic resistance (ISR) is well-documented in the context of plant-beneficial bacteria interactions, knowledge about the local and systemic molecular and biochemical defense responses before or upon pathogen infection in grapevine is very scarce. In this study, we first investigated the capacity of grapevine plants to express immune responses at both above- and below-ground levels upon interaction with a beneficial bacterium, Pseudomonas fluorescens PTA-CT2. We then explored whether the extent of priming state could contribute to the PTA-CT2-induced ISR in Botrytis cinerea-infected leaves. Our data provide evidence that this bacterium colonized grapevine roots but not the above-ground plant parts and altered the plant phenotype that displayed multiple defense responses both locally and systemically. The grapevine roots and leaves exhibited distinct patterns of defense-related gene expression during root colonization by PTA-CT2. Roots responded faster than leaves and some responses were more strongly upregulated in roots than in leaves and vice versa for other genes. These responses appear to be associated with some induction of cell death in roots and a transient expression of HSR, a hypersensitive response-related gene in both local (roots) and systemic (leaves) tissues. However, stilbenic phytoalexin patterns followed opposite trends in roots compared with leaves but no phytoalexin was exuded during plant-bacterium interaction, suggesting that roots could play an important role in the transfer of metabolites contributing to immune response at the systemic level. Unexpectedly, in B. cinerea-infected leaves PTA-CT2-mediated ISR was accompanied in large part by a downregulation of different defense-related genes, including HSR. Only phytoalexins and glutathion-S-transferase 1 transcripts were upregulated, while the expression of anthocyanin biosynthetic genes was maintained at a higher level than the control. This suggests that decreased expression of HSR, as a marker of cell death, and activation of secondary metabolism pathways could be responsible for a reduced B. cinerea colonization capacity in bacterized plants.

Distinct regulation in inflorescence carbohydrate metabolism according to grapevine cultivars during floral development.

Carbohydrate metabolism is important in plant sexual reproduction because sugar contents are determining factors for both flower initiation and floral organ development. In woody plants, flowering represents the most energy-consuming step crucial to reproductive success. Nevertheless, in these species, the photosynthesis performed by flowers supplies the carbon required for reproduction. In grapevine (Vitis vinifera), the inflorescence has a specific status because this organ imports carbohydrates at the same time as it exports photoassimilates. In this study, fluctuations in carbohydrate metabolism were monitored by analyzing gas exchanges, photosynthetic electron transport capacity, carbohydrate contents and some activities of carbohydrate metabolism enzymes, in the inflorescences of Pinot noir and Gewurztraminer, two cultivars with a different sensitivity to coulure phenomenon. Our results showed that photosynthetic activity and carbohydrate metabolism are clearly different and differently regulated during the floral development in the two cultivars. Indeed, the regulation of the linear electron flow and the cyclic electron flow is not similar. Moreover, the regulation of PSII activity, with a higher Y(NPQ)/Y(NO) ratio in Gewurztraminer, can be correlated with the higher protection of the photosynthetic chain and consequently with the higher yield under optimal conditions of this cultivar. At least, our results showed a higher photosynthetic activity and a better protection of PSI in Pinot noir during the floral development.

Drought stress tolerance in grapevine involves activation of polyamine oxidation contributing to improved immune response and low susceptibility to Botrytis cinerea.

Environmental factors including drought stress may modulate plant immune responses and resistance to pathogens. However, the relationship between mechanisms of drought tolerance and resistance to pathogens remained unknown. In this study, the effects of drought stress on polyamine (PA) homeostasis and immune responses were investigated in two grapevine genotypes differing in their drought tolerance; Chardonnay (CHR), as sensitive and Meski (MSK), as tolerant. Under drought conditions, MSK plants showed the lowest leaf water loss and reduction of photosynthetic efficiency, and expressed a lower level of NCED2, a gene involved in abscisic acid biosynthesis, compared with CHR plants. The improved drought tolerance in MSK was also coincident with the highest change in free PAs and up-regulation of the genes encoding arginine decarboxylase (ADC), copper amine-oxidase (CuAO), and PA-oxidases (PAO) and their corresponding enzyme activities. MSK plants also accumulated the highest level of amino acids, including Arg, Glu, Gln, Pro, and GABA, emphasizing the participation of PA-related amino acid homeostasis in drought tolerance. Importantly, drought-tolerant plants also exhibited enhanced phytoalexin accumulation and up-regulation of PR genes, especially PR-2 and Chit4c, compared with the sensitive plants. This is consistent with a lower susceptibility of MSK than CHR to Botrytis cinerea. Data suggest a possible connection between water stress tolerance and immune response in grapevine. Pharmacological experiments revealed that under drought conditions CuAO and PAO pathways were involved in the regulation of photosynthetic efficiency, and also of immune response and resistance of grapevine to a subsequent pathogen attack. These results open new views to improve our understanding of crosstalk between drought tolerance mechanisms and immune response.

Uncovering plant-pathogen crosstalk through apoplastic proteomic studies.

Plant pathogens have evolved by developing different strategies to infect their host, which in turn have elaborated immune responses to counter the pathogen invasion. The apoplast, including the cell wall and extracellular space outside the plasma membrane, is one of the first compartments where pathogen-host interaction occurs. The plant cell wall is composed of a complex network of polysaccharides polymers and glycoproteins and serves as a natural physical barrier against pathogen invasion. The apoplastic fluid, circulating through the cell wall and intercellular spaces, provides a means for delivering molecules and facilitating intercellular communications. Some plant-pathogen interactions lead to plant cell wall degradation allowing pathogens to penetrate into the cells. In turn, the plant immune system recognizes microbial- or damage-associated molecular patterns (MAMPs or DAMPs) and initiates a set of basal immune responses, including the strengthening of the plant cell wall. The establishment of defense requires the regulation of a wide variety of proteins that are involved at different levels, from receptor perception of the pathogen via signaling mechanisms to the strengthening of the cell wall or degradation of the pathogen itself. A fine regulation of apoplastic proteins is therefore essential for rapid and effective pathogen perception and for maintaining cell wall integrity. This review aims to provide insight into analyses using proteomic approaches of the apoplast to highlight the modulation of the apoplastic protein patterns during pathogen infection and to unravel the key players involved in plant-pathogen interaction.

Elicitors as alternative strategy to pesticides in grapevine? Current knowledge on their mode of action from controlled conditions to vineyard.

Development and optimisation of alternative strategies to reduce the use of classic chemical inputs for protection against diseases in vineyard is becoming a necessity. Among these strategies, one of the most promising consists in the stimulation and/or potentiation of the grapevine defence responses by the means of elicitors. Elicitors are highly diverse molecules both in nature and origins. This review aims at providing an overview of the current knowledge on these molecules and will highlight their potential efficacy from the laboratory in controlled conditions to vineyards. Recent findings and concepts (especially on plant innate immunity) and the new terminology (microbe-associated molecular patterns, effectors, etc.) are also discussed in this context. Other objectives of this review are to highlight the difficulty of transferring elicitors use and results from the controlled conditions to the vineyard, to determine their practical and effective use in viticulture and to propose ideas for improving their efficacy in non-controlled conditions.

Grapevine NAC1 transcription factor as a convergent node in developmental processes, abiotic stresses, and necrotrophic/biotrophic pathogen tolerance.

Transcription factors of the NAC family are known to be involved in various developmental processes and in response to environmental stresses. Whereas NAC genes have been widely studied in response to abiotic stresses, little is known about their role in response to biotic stresses, especially in crops. Here, the first characterization of a Vitis vinifera L. NAC member, named VvNAC1, and involved in organ development and defence towards pathogens is reported. Expression profile analysis of VvNAC1 showed that its expression is closely associated with later stages of leaf, flower, and berry development, suggesting a role in plant senescence. Moreover, VvNAC1 expression is stimulated in Botrytis cinerea- or microbe-associated molecular pattern (MAMP)-infected berries or leaves. Furthermore, cold, wounding, and defence-related hormones such as salicylic acid, methyl jasmonate, ethylene, and abscisic acid are all able to induce VvNAC1 expression in grapevine leaves. VvNAC1-overexpressing Arabidopsis plants exhibit enhanced tolerance to osmotic, salt, and cold stresses and to B. cinerea and Hyaloperonospora arabidopsidis pathogens. These plants present a modified pattern of defence gene markers (AtPR-1, AtPDF1.2, and AtVSP1) after stress application, suggesting that VvNAC1 is an important regulatory component of the plant signalling defence cascade. Collectively, these results provide evidence that VvNAC1 could represent a node of convergence regulating grapevine development and stress responses, including defence against necrotrophic and biotrophic pathogens.

Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes.

BACKGROUND: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. RESULTS: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. CONCLUSIONS: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function.