Phospholipids are present in extracellular fluids of imbibing sunflower seeds and are modulated by hormonal treatments

Phospholipids are well known messengers involved in developmental and stress responses mediating intracellular signalling. It has been hypothesized that phospholipids exist which could participate in intercellular communication events through the apoplast of sunflower (Helianthus annuus) seeds. Here it is shown that extracellular washing fluids (EWFs) obtained from seeds imbibed for 2 h contain diverse phospholipids. Lipid profiling by electrospray ionization tandem mass spectrometry revealed that the EWFs have a particular composition, with phosphatidic acid (PA) and phosphatidylinositol (PI) being the major phospholipids. These profiles are clearly distinct from those of seed extract (SE), and comparative SDS-PAGE of EWF and SE, followed by intracellular and plasma membrane marker analyses, allowed a significant contamination of the EWF to be discarded. Treatment of the seeds with 100 microM jasmonic acid (JA) induces changes in the profile of EWF phospholipids, leading to a decrease in PI content, while the accumulation of phosphatidylinositol 4-phosphate (PI4P) and specific PA species is observed. On the other hand, the EWF from seeds subjected to 50 microM abscisic acid (ABA) treatment exhibit an increase in PA and phosphatidylglycerol levels. To our knowledge, this is the first report on the existence of phospholipids as extracellular components of seeds. Moreover, the modulation of PA, PI, and PI4P levels by hormonal treatments further suggests their contribution to intercellular communication in planta.     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Vesicular fractions of sunflower apoplastic fluids are associated with potential exosome marker proteins

Based on the presence of phospholipids in the extracellular fluids (EFs) of sunflower seeds, we have hypothesized on the existence of vesicles in the apoplastic compartment of plants. Ultracentrifugation of sunflower EF allowed the isolation of particles of 50-200 nm with apparent membrane organization. A small GTPase Rab was putatively identified in this vesicular fraction. Since Rab proteins are involved in vesicular traffic and their presence in exosomes from animal fluids has been demonstrated, evidence presented here supports the existence of exosome-like vesicles in apoplastic fluids of sunflower. Their putative contribution to intercellular communication in plants is discussed.     

A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B

The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.     

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.     

The vascular plant‐pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

The mechanism of colonization of intercellular spaces by the soil‐borne and vascular plant‐pathogenic bacterium Ralstonia solanacearum strain OE1‐1 after invasion into host plants remains unclear. To analyse the behaviour of OE1‐1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1‐1 were observed under a scanning electron microscope. OE1‐1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1‐1 cells produced mushroom‐type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom‐type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1‐1. Mutation of lecM encoding a lectin, RS‐IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom‐type biofilms and virulence on tomato plants. Together, our findings indicate that OE1‐1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS‐IIL may contribute to biofilm formation by OE1‐1, which is required for OE1‐1 virulence.     

Proteomic analysis of tomato (Lycopersicon esculentum) pollen

In flowering plants, pollen grains are produced in the anther and released to the external environment with the primary function of delivering sperm cells to the female gametophyte. This study was conducted to identify proteins in tomato pollen and to analyse their roles in relation to pollen function. Tomato is an important crop which is grown worldwide and is an excellent experimental system. Proteins were extracted from pollen, separated by two-dimensional gel electrophoresis (2-DE), and identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and peptide mass fingerprinting. Of the 960 spots observed on Colloidal Coomassie Blue (CCB)-stained 2-DE gels, 190 were selected for analysis. Of these, 158 spots, representing 133 distinct proteins, were identified by searching the NCBInr and Expressed Sequence Tag databases. The identified proteins were classified based on designated functions and the majority included those involved in defence mechanisms, energy conversions, protein synthesis and processing, cytoskeleton formation, Ca(2+) signalling, and as allergens. A number of proteins in tomato pollen were similar to those reported in the pollen of other species; however, several additional proteins with roles in defence mechanisms, metabolic processes, and hormone signalling were identified. The potential roles of the identified proteins in the survival strategy of the small, independent, two-celled pollen grain of tomato, and subsequently in pollen germination and tube growth are discussed.     

Regulation of Leaf Shape in the Solanifolia Mutant of Tomato (Lycopersicon esculentum) by Plant Growth Substances

The solanifolia mutant (sf/sf) of tomato (Lycopersicon esculentum)produces leaves consisting of leaflets with entire margins,unlike the lobed leaflets of normal plants. Normal plants treatedwith gibberellic acid (GA₃) produced leaves with entire marginswhereas mutant plants exposed to 2-chloroethyl-trimethyl ammoniumchloride (CCC)—an inhibitor of gibberellin biosynthesis—producedlobing of leaflets. The leaf area of the mutant was significantlygreater than that of the normal, but was not significantly differentfrom GA₃-treated normal leaves. Similarly, in CCC-treated mutantleaves the leaf area was not different from that of normal untreatedleaves. These observations suggest that the sf/sf mutation affectsthe leaf shape through its effect on endogenous gibberellinsand/or inhibitory substances. Leaf shape, Lycopersicon esculentum, plant growth substances, tomato     

Age-related resistance in Arabidopsis is a developmentally regulated defense response to Pseudomonas syringae

Age-related resistance (ARR) has been observed in a number of plant species; however, little is known about the biochemical or molecular mechanisms involved in this response. Arabidopsis becomes more resistant, or less susceptible, to virulent Pseudomonas syringae (pv tomato or maculicola) as plants mature (in planta bacterial growth reduction of 10- to 100-fold). An ARR-like response also was observed in response to certain environmental conditions that accelerate Arabidopsis development. ARR occurs in the Arabidopsis mutants pad3-1, eds7-1, npr1-1, and etr1-4, suggesting that ARR is a distinct defense response, unlike the induced systemic resistance or systemic acquired resistance responses. However, three salicylic acid (SA) accumulation-deficient plant lines, NahG, sid1, and sid2, did not exhibit ARR. A heat-stable antibacterial activity was detected in intercellular washing fluids in response to Pst inoculation in wild-type ARR-competent plants but not in NAHG: These data suggest that the ability to accumulate SA is necessary for the ARR response and that SA may act as a signal for the production of the ARR-associated antimicrobial compound(s) and/or it may possess direct antibacterial activity against P. syringae.     

Sunflower (Helianthus annuus L.) Pathogenesis-Related Proteins (Induction by Aspirin (Acetylsalicylic Acid) and Characterization)

Sunflower leaf discs floated on a solution containing aspirin (acetylsalicylic acid) produced a set of new proteins extractable at pH 5.2 and excreted into the intercellular space. More than 80% of the proteins found in the intercellular fluids of induced leaf discs have been identified as pathogenesis-related (PR) proteins by their immunological relationship with tobacco PR proteins. Members of the four major classes of PR proteins have been characterized. Sunflower PR proteins of type 1 (PR1) and of type 3 (PR3) were found to have acidic isoelectric points, whereas the induced PR protein of type 2 (PR2) had a basic isoelectric point. Members of the type 5 PR proteins (PR5), known in tobacco as thaumatin-like proteins, showed a more complex pattern. Multiple sunflower PR5 isomers of similar molecular weight but of different isoelectric points were excreted from the cells in response to the aspirin treatment. PR2 and PR3 proteins were found at very low basal levels in untreated leaves, whereas PR1 and PR5 proteins could not be detected at all in the same extracts. Glucanase and chitinase activities were always associated with PR2 and PR3 proteins in partially purified sunflower extracts. All of these data indicate that, in response to aspirin treatment, sunflower plants produce a complete set of PR proteins characterized by an apparently exclusively extracellular localization.     

Negative regulation of <Emphasis Type="Italic">KNOX</Emphasis> expression in tomato leaves

Leaves of seed plants can be described as simple, where the leaf blade is entire, or dissected, where the blade is divided into distinct leaflets. Both simple and dissected leaves are initiated at the flanks of a pluripotent structure termed the shoot apical meristem (SAM). In simple-leafed species, expression of class I KNOTTED1-like homeobox (KNOX) proteins is confined to the meristem while in many dissected leaf plants, including tomato, KNOX expression persists in leaf primordia. Elevation of KNOX expression in tomato leaves can result in increased leaflet number, indicating that tight regulation of KNOX expression may help define the degree of leaf dissection in this species. To test this hypothesis and understand the mechanisms controlling leaf dissection in tomato, we studied the clausa (clau) and tripinnate (tp) mutants both of which condition increased leaflet number phenotypes. We show that TRIPINNATE and CLAUSA act together, to restrict the expression level and domain of the KNOX genes Tkn1 and LeT6/Tkn2 during tomato leaf development. Because loss of CLAU or TP activity results in increased KNOX expression predominantly on the adaxial (upper) leaf domain, our observations indicate that CLAU and TP may participate in a domain-specific KNOX repressive system that delimits the ability of the tomato leaf to generate leaflets.     

Pathogen elicitor-induced changes in the maize extracellular matrix proteome

The extracellular matrix is a vital compartment in plants with a prominent role in defence against pathogen attack. Using a maize cell suspension culture system and pathogen elicitors, responses to pathogen attack that are localised to the extracellular matrix were examined by a proteomic approach. Elicitor treatment of cell cultures induced a rapid change in the phosphorylation status of extracellular peroxidases, the apparent disappearance of a putative extracellular beta-N-acetylglucosamonidase, and accumulation of a secreted putative xylanase inhibitor protein. Onset of the defence response was attended by an accumulation of glyceraldehyde-3-phosphate dehydrogenase and a fragment of a putative heat shock protein. Several distinct spots of both proteins, which preferentially accumulated in cell wall protein fractions, were identified. These three novel observations, viz. (i) secretion of a new class of putative enzyme inhibitor, (ii) the apparent recruitment of classical cytosolic proteins into the cell wall and (ii) the change in phosphorylation status of extracellular matrix proteins, suggest that the extracellular matrix plays a complex role in defence. We discuss the role of the extracellular matrix in signal modulation during pathogen-induced defence responses.     

Glycolipid-anchored form of CD4 increases intercellular adhesion but is unable to enhance T cell activation.

CD4 functions to enhance T cell activation by increasing intercellular adhesion and/or by transduction of an intracellular signal. To study the role of human CD4 in T cell activation we have used a murine T cell hybridoma, By 155.16, which produces IL-2 when stimulated by HLA-DR-bearing cells. Previously, we have shown that expression of human CD4 by this hybridoma enhances its ability to produce IL-2 in response to HLA-DR-bearing cells. Furthermore, deletion of the majority of the cytoplasmic domain renders CD4 less efficient at enhancing IL-2 production. We describe studies of a glycolipid-anchored mutant of the CD4 molecule, CD4PI. This mutant is composed of the entire extracellular domain of CD4 anchored to the outlet leaflet of the membrane via a covalent bond to glycosylphosphatidylinositol and, therefore, has no transmembrane or cytoplasmic domains. When expressed in By155.16, CD4PI shows no defect in its ability to increase intercellular adhesion but is unable to augment IL-2 production. These results clearly demonstrate that CD4 enhances T cell activation by mechanisms other than increasing intercellular adhesion.     

Transport and secretion in plant-microbe interactions

Microbial elicitors and effectors, as well as plant receptors and defence compounds, traffic at the interface of plants and microbes in pathogenic or mutualistic interactions. Net exocytosis appears to be required for surface enlargement of plasma membrane during accommodation of microbes in intact plant cells. By contrast, ligand-induced endocytosis of surface receptors operates in basal defence. The first layer of plant defence appears to depend on polarized transport of small molecules and on local secretion of defence proteins. In return, pathogen effectors target plasma membrane bound and intracellular proteins to inhibit extracellular host defences.     

Degradation of tomato pathogenesis-related proteins by an endogenous 37-kDa aspartyl endoproteinase

As a response to the stress induced by different afflicting agents, tomato plants (Lycopersicon esculentum) produce the so-called 'pathogenesis-related' proteins. Here we report the degradation of some of these proteins by a constitutive endoproteolytic activity that co-distributes with pathogenesis-related proteins in the intercellular spaces of tomato leaves infected with citrus exocortis viroid. This endoproteinase was purified, showing a pH optimum of 2.5-3.5, a Mr of 37,000 and selective inhibition by pepstatin. In crude homogenates, the enzyme does not seem to degrade other cellular proteins. This specificity indicates that the proteinase might be involved in the extracellular degradative pathway of pathogenesis-related proteins and in the regulation of their biological function.