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Background: Wounded leaves of Arabidopsis thaliana produce reactive oxygen species (ROS) within minutes after wounding and become resistant to the pathogenic fungus Botrytis cinerea at a local level. This fast response of the plants to the wound is called wound-induced resistance (WIR). However the molecular mechanisms of this response and the signal cascade between the wound and ROS production are still largely unknown. Calcium is a conserved signal and it is involved in many abiotic stress responses in plants, furthermore, calcium pathways act very fast.Results: The results of this study show that leaves treated with calcium channels inhibitors (verapamil) or calcium chelators (oxalate and EGTA) are impaired in ROS production. Moreover, leaves treated with verapamil, EGTA or oxalate were more susceptible to B. cinerea after wounding. The intracellular measurements of calcium changes indicated quick but transient calcium dynamics taking place few seconds after wounding in cells neighbouring the wound site. This change in the cytosolic calcium was followed in the same region by a more stable ROS burst.Conclusions; These data further extend our knowledge on the connection between wounding, calcium influx and ROS production. Moreover they provide for the first time the evidence that, following wounding, calcium changes precede a burst in ROS in the same location.

Background: in a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound–induced resistance to B. cinerea.Results: in this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves.Conclusion: Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H2O2 and O2 2, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H2O2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

[EN] Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H 2O 2 and O 2 -, are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H 2O 2 was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defens

This study provides an explanation for the strong resistance to B. cinerea observed in wounded plants or plants with cuticular defects. We have observed that a production of ROS and a permeable cuticle is common to all these situations. ROS, that include hydrogen peroxide, are known inducers of resistance and can also act directly against the invading fungus. Degradation of the cuticle by exposure to cutinase also results in the production of ROS and resistance. These observations lead to a model where the cuticle plays a central role as a barrier against water-soluble elicitors from the surface. Under normal circumstances, the cuticle does not allow the passage of elicitors and no responses are induced. Under conditions where the cuticular barrier is broken, ROS and resistance are induced. This illustrates why plants that are in fact permanently exposed to potential elicitors do not constantly induce immune responses: this only takes place once the cuticle has been permeabilized, for example after an infection with a pathogen. This study also demonstrates how a cuticle-degrading pathogen avoids the generation of ROS by producing an effector that interferes with ROS production. Removal of this effector restores both ROS and resistance.

Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G0) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85–Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1GTP, which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1GDP, caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 β-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.

Background: Wounded leaves of Arabidopsis thaliana produce reactive oxygen species (ROS) within minutes after wounding and become resistant to the pathogenic fungus Botrytis cinerea at a local level. This fast response of the plants to the wound is called wound-induced resistance (WIR). However the molecular mechanisms of this response and the signal cascade between the wound and ROS production are still largely unknown. Calcium is a conserved signal and it is involved in many abiotic stress responses in plants, furthermore, calcium pathways act very fast.Results: The results of this study show that leaves treated with calcium channels inhibitors (verapamil) or calcium chelators (oxalate and EGTA) are impaired in ROS production. Moreover, leaves treated with verapamil, EGTA or oxalate were more susceptible to B. cinerea after wounding. The intracellular measurements of calcium changes indicated quick but transient calcium dynamics taking place few seconds after wounding in cells neighbouring the wound site. This change in the cytosolic calcium was followed in the same region by a more stable ROS burst.Conclusions; These data further extend our knowledge on the connection between wounding, calcium influx and ROS production. Moreover they provide for the first time the evidence that, following wounding, calcium changes precede a burst in ROS in the same location.

This study provides an explanation for the strong resistance to B. cinerea observed in wounded plants or plants with cuticular defects. We have observed that a production of ROS and a permeable cuticle is common to all these situations. ROS, that include hydrogen peroxide, are known inducers of resistance and can also act directly against the invading fungus. Degradation of the cuticle by exposure to cutinase also results in the production of ROS and resistance. These observations lead to a model where the cuticle plays a central role as a barrier against water-soluble elicitors from the surface. Under normal circumstances, the cuticle does not allow the passage of elicitors and no responses are induced. Under conditions where the cuticular barrier is broken, ROS and resistance are induced. This illustrates why plants that are in fact permanently exposed to potential elicitors do not constantly induce immune responses: this only takes place once the cuticle has been permeabilized, for example after an infection with a pathogen. This study also demonstrates how a cuticle-degrading pathogen avoids the generation of ROS by producing an effector that interferes with ROS production. Removal of this effector restores both ROS and resistance.

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N-terminal and large natively unfolded C-terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two-hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N-terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C-terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.

Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G0) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85–Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1GTP, which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1GDP, caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

Background: in a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea, the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound–induced resistance to B. cinerea.Results: in this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves.Conclusion: Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea.