The fungal elicitor cryptogein is a sterol carrier protein

AbstractCryptogein is a protein secreted by the phytopathogenic pseudo-fungus, Phytophthora cryptogea. It is a basic 10 kDa hydrophilic protein having a hydrophobic pocket and three disulfide bridges. These common features with sterol carrier proteins led us to investigate its possible sterol transfer activity using the fluorescent sterol, dehydroergosterol. The results show that cryptogein has one binding site with strong affinity for dehydroergosterol. Moreover, this protein catalyzes the transfer of sterols between phospholipidic artificial membranes. This is the first evidence for the existence of an extracellular sterol carrier protein and for a molecular activity of cryptogein. This property should contribute to an understanding of the role of cryptogein in plant-microorganism interactions

NO signaling functions in the biotic and abiotic stress responses

In the last 1990s, NO became an increasingly popular target of investigation in plants. As in mammals, NO fulfils a broad spectrum of signalling functions in pathophysiological processes in plants. Here we summarize studies published in recent years that provide novel insights into the signalling functions of NO produced by plant cells exposed to abiotic stresses and biotic stress (pathogen-derived elicitors). Particularly, we report that NO emerges as a key messenger governing the overall control of Ca2+ homeostasis. Although the precise signalling functions of NO are poorly understood, its capacity to modulate Ca2+ homeostasis provides an extraordinary and remarkable effective way of conveying information

Real-time electrochemical detection of extracellular nitric oxide in tobacco cells exposed to cryptogein, an elicitor of defence responses

It was previously reported that cryptogein, an elicitor of defence responses, induces an intracellular production of nitric oxide (NO) in tobacco. Here, the possibility was explored that cryptogein might also trigger an increase of NO extracellular content through two distinct approaches, an indirect method using the NO probe 4,5-diaminofluorescein (DAF-2) and an electrochemical method involving a chemically modified microelectrode probing free NO in biological media. While the chemical nature of DAF-2-reactive compound(s) is still uncertain, the electrochemical modified microelectrodes provide real-time evidence that cryptogein induces an increase of extracellular NO. Direct measurement of free extracellular NO might offer important new insights into its role in plants challenged by biotic stresses

Protein S-nitrosylation: specificity and identification strategies in plants

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the Biotin Switch Technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed

Nitric oxide modulates dynamic actin cytoskeleton and vesicle trafficking in a cell type-specific manner in root apices

NO is an important regulatory molecule in eukaryotes. Much of its effect is ascribed to the action of NO as a signalling molecule. However, NO can also directly modify proteins thus affecting their activities. Although the signalling functions of NO are relatively well recognized in plants, very little is known about its potential influence on the structural integrity of plant cells. In this study, the reorganization of the actin cytoskeleton, and the recycling of wall polysaccharides in plants via the endocytic pathway in the presence of NO or NO-modulating substances were analysed. The actin cytoskeleton and endocytosis in maize (Zea mays) root apices were visualized with fluorescence immunocytochemistry. The organization of the actin cytoskeleton is modulated via NO levels and the extent of such modulation is cell-type specific. In endodermis cells, actin cables change their orientation from longitudinal to oblique and cellular cross-wall domains become actin-depleted/depolymerized. The reaction is reversible and depends on the type of NO donor. Actin-dependent vesicle trafficking is also affected. This was demonstrated through the analysis of recycled wall material transported to newly-formed cell plates and BFA compartments. Therefore, it is concluded that, in plant cells, NO affects the functioning of the actin cytoskeleton and actin-dependent processes. Mechanisms for the reorganization of the actin cytoskeleton are cell-type specific, and such rearrangements might selectively impinge on the functioning of various cellular domains. Thus, the dynamic actin cytoskeleton could be considered as a downstream effector of NO signalling in cells of root apices

Early signaling events induced by elicitors of plant defenses.

Plant pathogen attacks are perceived through pathogen-issued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models

The Pseudomonas fluorescens siderophore pyoverdine weakens arabidopsis thaliana defense in favor of growth in iron-deficient conditions

Pyoverdines are siderophores synthesized by fluorescent Pseudomonas spp. Under iron-limiting conditions, these high-affinity ferric iron chelators are excreted by bacteria in the soil to acquire iron. Pyoverdines produced by beneficial Pseudomonas spp. ameliorate plant growth. Here, we investigate the physiological incidence and mode of action of pyoverdine from Pseudomonas fluorescens C7R12 on Arabidopsis (Arabidopsis thaliana) plants grown under iron-sufficient or iron-deficient conditions. Pyoverdine was provided to the medium in its iron-free structure (apo-pyoverdine), thus mimicking a situation in which it is produced by bacteria. Remarkably, apo-pyoverdine abolished the iron-deficiency phenotype and restored the growth of plants maintained in the iron-deprived medium. In contrast to a P. fluorescens C7R12 strain impaired in apo-pyoverdine production, the wild-type C7R12 reduced the accumulation of anthocyanins in plants grown in iron-deficient conditions. Under this condition, apo-pyoverdine modulated the expression of around 2,000 genes. Notably, apo-pyoverdine positively regulated the expression of genes related to development and iron acquisition/redistribution while it repressed the expression of defense-related genes. Accordingly, the growth-promoting effect of apo-pyoverdine in plants grown under iron-deficient conditions was impaired in iron-regulated transporter1 and ferric chelate reductase2 knockout mutants and was prioritized over immunity, as highlighted by an increased susceptibility to Botrytis cinerea This process was accompanied by an overexpression of the transcription factor HBI1, a key node for the cross talk between growth and immunity. This study reveals an unprecedented mode of action of pyoverdine in Arabidopsis and demonstrates that its incidence on physiological traits depends on the plant iron status

S-Nitrosoglutathione is a component of wound- and salicylic acid-induced systemic responses in Arabidopsis thaliana

S-Nitrosoglutathione (GSNO) is a bioactive, stable, and mobile reservoir of nitric oxide (NO), and an important player in defence responses to herbivory and pathogen attack in plants. It has been demonstrated previously that GSNO reductase (GSNOR) is the main enzyme responsible for the in vivo control of intracellular levels of GSNO. In this study, the role of S-nitrosothiols, in particular of GSNO, in systemic defence responses in Arabidopsis thaliana was investigated further. It was shown that GSNO levels increased rapidly and uniformly in injured Arabidopsis leaves, whereas in systemic leaves GSNO was first detected in vascular tissues and later spread over the parenchyma, suggesting that GSNO is involved in the transmission of the wound mobile signal through the vascular tissue. Moreover, GSNO accumulation was required to activate the jasmonic acid (JA)-dependent wound responses, whereas the alternative JA-independent wound-signalling pathway did not involve GSNO. Furthermore, extending previous work on the role of GSNOR in pathogenesis, it was shown that GSNO acts synergistically with salicylic acid in systemic acquired resistance activation. In conclusion, GSNOR appears to be a key regulator of systemic defence responses, in both wounding and pathogenesis

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores