Julius-von-Sachs-Institut für Biowissenschaften
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Maize seedlings contain high amounts of glucosidically bound 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). The effects of DIMBOA on the feeding behaviour and performance of two noctuids, Spodoptera exigua Hübner and S. frugiperda Smith, were compared. The question was raised whether S. frugiperda, preferring maize and other Poaceae, is better adapted to DIMBOA than S. exigua. In addition, the effects of DIMBOA on the mycelial growth of the plant pathogen Setosphaeria turcica Leonard et Suggs (causal agent of northern corn leaf blight) was assessed in vitro. DIMBOA had an antifeedant effect on S. exigua but stimulated feeding in S. frugiperda in dual-choice experiments. In a no-choice setup, larvae of S. exigua gained less biomass and had a prolonged development when feeding on an artificial diet containing DIMBOA. However, pupal weight was not significantly different between treatments. In contrast, larvae of S. frugiperda were not affected by DIMBOA. Strong detrimental effects of DIMBOA were found on the mycelial growth of the pathogen S. turcica.
During the last few years an increasing number of physiological processes in plants have been shown to be regulated by NO. NO plays important roles in growth and development, plant disease resistance, abiotic stress, and in above and underground plant organs. In recent years several enzymatic pathways and few non-enzymatic pathways were proposed for nitric oxide production in plants. The major goal of this work was to quantify NO production by plants and especially by roots, and to identify the enzymes responsible for NO production. As a major method, NO production by roots was followed through on-line measurement of NO emission into the gas phase by chemiluminescence (= direct chemiluminescence), and also by indirect chemiluminescence where trace amounts of oxidized products like NO2- and NO3- can be easily measured. Plants used were tobacco wild-type (N. tabacum cv Xanthi or cv Gatersleben), NR-free mutants grown on ammonium in order to prevent NR induction, plants grown on tungstate to inhibit synthesis of functional MoCo-enzymes, and a NO-overproducing nitrite reductase (NiR)-deficient transformant as well as barley, rice and pea. Induction of a hypersensitive response (HR) in tobacco leaves was achieved by using avirulent Pseudomonas syringae pv phaseolicola. At oxygen concentrations of <1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it was observed with Arabidopsis, barley, pea, and tobacco. Nitrite and NADH consumption by mitochondria were also measured. Anaerobic, nitrite-dependent NO emission was exclusively associated with the membrane fraction, without participation of matrix components. It was also examined whether root mitochondria and mitochondrial membranes produce nitric oxide (NO) exclusively by reduction of nitrite or also via a nitric oxide synthase (NOS),- and to what extent direct NO measurements could be falsified by NO oxidation. In addition to chemiluminescence, Diaminofluoresceins (DAF) were used as an NO indicators for comparison. In air, mitochondria apparently produced no nitrite-dependent NO, and no NOS activity was detected by direct or indirect chemiluminescence. In contrast, with DAF-2 and DAR-4M an L-arginine-dependent fluorescence increase took place. However, the response of this apparent NOS activity to inhibitors, substrates and cofactors was untypical when compared with commercial iNOS and is considered an artefact. With iNOS, about 2/3 of the NO were oxidized to (nitrite + nitrate). Mitochondria also appear to consume NO without increasing oxidation to (nitrite+ nitrate). We therefore assume formation of NO to a volatile intermediate (eventually N2O3). It was recently shown that the hypersensitive response (HR) of tobacco triggered by the fungal elicitor cryptogein occurred independent of the presence or absence of nitrate reductase (NR). One conclusion was that NR-dependent NO formation played no role in the HR. Here we present evidence that the described scenario may be specific for cryptogein. Pseudomonas syringae pv. phaseolicola was infiltrated into tobacco leaves from WT plant and from the NiR-deficient NO-overproducing clone 271, grown either on nitrate or ammonium. Lesion development as well as bacterial growth and sugar concentrations in leaves and in the leaf apoplast was monitored. Lesion development was positively and bacterial growth was negatively correlated with nitrate nutrition and eventually with NO formation. Bacterial growth was positively correlated with ammonium nutrition and apoplastic sugar concentrations. Total (free and conjugated) SA content were always drastically increased by bacterial infection, but there was no clear correlation with NO production. In the presence of cryptogein, Pseudomonas growth was drastically reduced. This shows that the assumed interdependence of bacterial growth, NO production and the HR is complex and not unifactorial.
Brassicaceae and a few related plant families are characterized by possession of the glucosinolate-myrosinase system. Glucosinolates are amino-acid derived allelochemicals which are hydrolysed upon tissue damage by myrosinase enzymes to produce various degradation products which can be toxic for generalist insects. The larvae of the crucifer-specialist Athalia rosae, the turnip sawfly, sequester glucosinolates into their haemolymph. The role of the glucosinolate-myrosinase system for the interaction of the turnip sawfly with Brassicaceae was examined in this study from two different perspectives: variation within individual plants and between plant species. The plant responses to the feeding by herbivores and the short-term effects this induction had on insect behaviour were investigated in white mustard. Furthermore, plants can use multiple defences. Hence correlations of glucosinolates and myrosinase activities with other defences and nutritional quality and their long-term effects on the development of the insects were investigated in seven different plant species.
Lokalisation, Funktion und Regulation pflanzlicher Tandem-Poren-Kaliumkanäle in Arabidopsis thaliana
(2007)
Lokalisation - Alle TPKs bis auf TPK4, der in der Plasmamembran lokalisiert ist, sind im Tonoplasten lokalisiert. - Das 14-3-3-Bindemotiv bzw. der komplette N-Terminus spielt im Gegensatz zu den tierischen TPK´s keine Rolle beim Targeting (und evtl. auch beim Assembly), da ein Austausch der N-Termini bzw. Mutationen im 14-3-3- Bindemotiv keinen Einfluss auf die subzelluläre Lokalisation hat. - Im C-Terminus ist möglicherweise ein strukturelles Motiv bzw. eine Erkennungssequenz für das Targeting in unterschiedliche Zielmembranen lokalisiert. Eventuell ist hier auch eine Assembly-Domäne für den Zusammenbau der unterschiedlichen Kanaluntereinheiten vorhanden. TPK4 - Der Kaliumkanal TPK4 wird nach Agro-Infiltration in dem pflanzlichen Expressionssystem Nicotiana benthamiana exprimiert. - TPK4 ist auch in diesem Expressionssystem in der Plasmamembran der Zelle lokalisiert. - Die Ströme, welche aus Mesophyllzellen von TPK4 infiltrierten Blättern abgeleitet wurden, gleichen denen, von TPK4 exprimierenden Oocyten von Xenopus laevis. Somit hat TPK4 in beiden Expressionssystemen die gleichen elektrophysiologischen Eigenschaften. TPK1 - TPK1 bindet über die C-terminalen EF-Hände Calcium und wird durch diese Interaktion aktiviert. - TPK1 interagiert phosphospezifisch und isotypspezifisch mit dem 14-3-3- Protein GRF6. Diese Interaktion führt zur Aktivierung des Kanals. - Die Kinasen CPK3 und CPK29, welche das 14-3-3-Bindemotiv von TPK1 phosphorylieren um eine Interaktion mit 14-3-3-Proteinen zu ermöglichen, gehören zur Familie der CDPKs - Diese Kinasen sind selbst Calcium aktiviert und aller Wahrscheinlichkeit nach unter physiologischen Bedingungen inaktiv. Erst ein Anstieg der freien Calciumkonzentration führt zur Aktivierung der Kinase in der Zelle und damit zur Aktivierung des Kanals. - Das 14-3-3-Bindemotiv ist das einzige Target der CDPK´s im N-Terminus von TPK1 - Die Phosphatase, welche das 14-3-3-Bindemotiv von TPK1 dephosphoryliert gehört zur Familie der PP2A-Proteinphosphatasen. - Es ist möglich, dass die Kinase und damit auch der Kanal durch Salzstress und durch Kaliumunterversorgung aktiviert werden und somit die Signalkaskade für die Aktivierung von TPK1 über Kinasen/14-3-3/Calcium in einen stressphysiologischen Kontext involviert ist. - tpk1.3- und cpk3.1-Verlustmutanten zeigen eine Reduktion in der Keimungsrate unter Salzstress und limitierten Kaliumangebot. Es kann über einen funktionalen Komplex bestehend aus TPK1 und TPC1 zur Aufrechterhaltung der Na+/K+-Homeostase und der elektroneutralen Aufnahme von Na+ in die Vakuole unter Salzstressbedingungen spekuliert werden.
The continuously increase in resistance of human pathogenic microorganisms to the known antibiotics leads to the necessity for searching new sources for production of new active antimicrobial compounds from different natural sources especially plants, since many plants have been found to be able to produce antimicrobial compounds as a defense phenomenon against invading microorganisms. The aim of this work is to screen cultures for production of antimicrobial activity against representative of human pathogenic microorganisms and selection the most active cell culture producing antimicrobial protein(s) which are active against these pathogenic microorganisms and also isolation ,purification of the active protein(s) and cloning of its/their genes. Ten different plant suspension cultures have been screened in presence of nine elicitors for their antimicrobial activity against five selected human pathogenic microorganisms, and it has been found that the heterotrophic cultures are more active against the tester isolates than the autotrophic ones. The intracellular fraction of the mixotrophic Arabidopsis thaliana culture elicited with salicylic acid showed the highest antimicrobial activity against the tester isolates. The presence of proteinous antimicrobial activity has been elucidated by testing the activity of ammonium sulphate precipitate against Candida maltosa. High speed centrifugation technique has been used for partial purification of the active protein. The proteinous nature of the isolated compound has been confirmed by using bioautography technique and its molecular weight could be estimated to be around 26KDa. The active protein has been purified using gel filtration, and using mass spectrometry technique, for microsequencing of the active protein, it has been found that the function of the protein is unknown and we have termed it as AtPDP1 according to Arabidopsis thaliana Plat-Domain Protein1, since it contains a plant stress domain termed PLAT domain. It has been found that a second protein from the same plant with high homology level to AtPDP1 with the same domain, we termed it as AtPDP2. Genes for AtPDP1 and AtPDP2 have been cloned in E. coli using PGEM-T easy vector. The expression of both genes have been tested using Digital Northern program, and it has been observed that both genes are induced by different pathogens, chemicals known to induce defense in plant cells and also different hormones. We tried to clone the gene for AtPDP1 in PBI121 binary vector under the control of an elicitor inducible promoter of a proteinase inhibitor gene, to test its function in plant by overexpression, but we did not succeeded. Also the work aims to cloning the different known thaumatin genes from Arabidopsis thaliana for future work which represented by testing their expression under different stimuli, since most thaumatins have antimicrobial activity and some of them are active against Candida spp..Thirteen genes of known thaumatins from Arabidopsis thaliana have been cloned in PGEM-Teasy vector in DH5-alpha cells. coli cells. The expression of the thirteen genes has been done using Digital Northern program and it has been found that different genes show different expressions under different stimuli and the expression of At1g75800 gene was the maximum under all stimuli. The minimum expression of genes was for At1g75050. The rest of thaumatin genes showed moderate expressions under different stimuli.
Oxylipins are important biological active compounds that play essential roles in defense, growth, development, and reproduction of plants and animals. Oxylipins are formed either by enzymatic pathways or radical catalyzed reaction from polyunsaturated fatty acids. Products of oxidation of arachidonic acid (C20:4) in animals by enzymatic and non-enzymatic pathways are prostaglandins and isoprostanes, respectively. In plants, radical catalyzed reaction of -linolenic acid (C18:3) forms phytoprostanes and enzymatic oxidation of this fatty acid produces OPDA and jasmonic acid. Like plants, cyanobacterial membranes contain a high ratio of polyunsaturated fatty acid, about 25% of total fatty acids. Oxylipin biosynthesis and function was studied in two model cyanobacteria, Anabaena PCC 7120 and Synechocystis PCC 6803, for the first time: 1. The filamentous cyanobaterium Anabaena PCC 7120 can naturally produce phytoprostanes type I and II as well as hydroxy fatty acids like in plants but lacks the enzymatic capacity to form jasmonates (12-oxo-phytodienoic acid and jasmonic acid) and prostaglandins. Data obtained provide the first evidence for the occurence of phytoprostanes in cyanobacteria as well as in the baterial kingdom. 2. By GC-MS analysis, the E1- and F1-phytoprostanes in Anabaena PCC 7120 were detected both in free and esterified form. Their levels are comparable with those in plants, in the range of ng/g DW. In one week old cultures, there was no evidence of PPF1 in the medium but its level accumulated up to 142 ng/l in six weeks old cultures. In contrast, PPE1 was stable over time, about 20 ng/g DW. Free cellular PPE1 was found about 4 times higher than that of PPF1, 80.5  23.6 and 24.1  10.9 ng/g DW, respectively. However, there was no significant difference in the total cellular levels of PPF1 and PPE1, ranging from 150 to about 200 ng/g DW. 3. Phytoprostanes are inducible in Anabaena. In the combination of oxidative stress (200 µM H2O2 or 10 µM CuSO4) with high light intensity (330 µE.m-2.s-1) for 8 h, levels of total cellular PPE1 and PPF1 were increased about 2 to 4 times. Interestingly, unlike in higher plants, application of oxidative stress or high light intensity alone showed no phytoprostaneous induction in this cyanobacterium. 4. When Anabaena cells were treated with phytoprostanes, Anabaena cells became remarkably resistant against subsequently applied – otherwise lethal – oxidative stress. All phytoprostanes displayed a high protective effect except for PPE1. The highest protection level was contributed by a mixture of PPA1 type I and II. After preincubation of Anabena cells with 100 µM PPA1–type I/II for 16 h followed by application of 1 mM H2O2 or 50 µM CuSO4 for 5 h, A1-phytoprostane pre-treatment protected 84.2% and 77.5% of the cells from cell death, respectively. Without oxylipins pre-treatment, about 98% of the cells were dead. Surprisingly, preincubation of Anabaena with other oxylipins derived from enzymatic pathway in plants and animals showed also an effect, however, the protection effect was low and ranged from 10 to 30%. In contrast, phytoprostanes did not protect Pseudomonas syringae and Escherichia coli from the toxicity of hydrogen peroxide. However, these bacteria do not synthesize polyunsaturated fatty acids and are therefore devoid of and not exposed to endogenously formed oxidized lipids. 5. Exogenous application of 100 µM PPF1 or 1.5 mM H2O2 for 90 min did not activate the expression of isiA in Anabaena. Oxylipins also displayed no effect on shinorine and tocopherol levels in Anabaena. However, application of 100 µM PPF1 for 6 h altered the protein expression in Anabaena. Most PPF1-modulated proteins are down-regulated and related to photosynthesis. Since oxidative stress only in combination with high light intensity increased lipid peroxidation, down-regulation of photosynthesis after recognition of oxidised lipids (phytoprostanes) may be a survival strategy of Anabaena to avoid damage by peroxidized lipids. 6. Dead plants may be the main source of (exogenous) phytoprostanes in the natural environment of Anabaena. Dry hay releases PPE1 and PPF1 (11 µg/g DW) into an aqueous environment. Anabaena is the typical cyanobacterium in paddy rice fields. After harvesting, most of uneconomical parts of rice plants are abundant on the field, which may release phytoprostanes that in turn might have an impact on cyanobacteria in the rice ecosystems. However, field research is needed to clarify this suspection. 7. A new class of oxylipins, phytoprostanes type III and IV, was identified and quantified in vitro. The two main phytoprostanes, PPE1 and PPF1 (type III and IV), can be obtained by autoxidation of -linolenic acid or Borage oil (containing 25% esterified -linolenic acid). After 12 days of autoxidation and subsequent hydrolysis, 1 g of Borage oil yielded 112.71 ± 1.93 µg of PPF1 and 3.80 ± 0.14 mg of PPE1. PPB1 and PPA1 (type III and IV) were prepared by isomerization and dehydration of PPE1 (type III and IV). The overall yield of PPB1 was 1.71 ± 0.04 mg/g oil (type III) and 2.09 ± 0.12 mg/g oil (type IV). Those of PPA1 were 8.38 ± 0.35 µg/g and 10.18 ± 0.30 µg/oil, respectively. 8. A rapid HPLC-MS/MS method for phytoprostane and phytohormone analysis has been developed. This method was applied to quantify free and esterified E1- and F1-phytoprostanes type III and IV in Synechocystis PCC 6803. The in vivo phytoprostanes type III and IV are present both in free and esterified form. The total cellular level of PPE1 type III and IV in Synechocystis is at least 2 times higher than that of PPF1. Unlike Anabaena, PPE1 and PPF1 were detectable in the medium of one week old Synechocystis cultures. Free levels of PPF1 in the medium (231.8 ± 36.2 ng/l) and in the cells (164.9 ± 15.2 ng/g DW) are lower than those of PPE1 (1003.3 ± 365.2 ng/l and 2331.0 ± 87.7 ng/g DW).
Abiotic environmental stress, as evoked by short-term exposure of greenhousegrown plants to ambient ultraviolet radiation (UV), induces chemical and morphological adaptations of plants. Responses depend on the strength of stress and differ between species and tissues of variable age. In two Brassicaceae, Sinapis alba and Nasturtium officinale, stress responses towards short-term exposure to ambient radiation including or excluding UV reveal a high phenotypic plasticity, with strong differences their chemical composition compared to plants that remained in the greenhouse. The most pronounced defensive response against UV, the accumulation of flavonoid pigments, was strongest in young UV-exposed leaves, with an increase of the more effectice flavonol quercetin on the expense of less effectice kaempferol. Glucosinolates and myrosinase enzymes showed highly species-specific responses to UV-stress. Feeding behaviour and larval performance of the oligophagous Brassicaceae specialist, Phaedon cochleariae (Chrysomelidae; Coleoptera) were poorly affected by these differently UV-exposed host plants. Effects of plant stress on larval development were restricted to a minor variation in body mass due to variable food conversion of certain larval instars, which were compensated until pupation. Moreover, larval developmental times were unaffected by UV-exposure, but varied between species and leaves of different age. For P. cochleariae, this lack of variation in larval and pupal development towards UV-altered phytochemistry may suggest a strong genetic fixation of life history traits. In combination, the high plasticity towards variable food quality may correspond to the beetles’s specialisation on a narrow range of chemically highly variable host plants. Apart from being involved in plant defence against generalist herbivores, glucosinolates may also act as recognition cues and feeding stimulants for specialist insects. In earlier studies, glucosinolates were assumed to stimulate feeding by P. cochleariae, and they were suggested to be present on outermost leaf surfaces. However, since these findings were based on crude extraction methods, the presence of feeding stimulants in epicuticular waxes of Brassicaceae was re-investigated. In our study, glucosinolates were not detectable in mechanically removed waxes in Brassica napus and N. officinale, whereas substrate concentrations in solvent leaf extracts corresponded to densities and closure of leaf surface stomata. Therefore, glucosinolates that originate from the mesophyll may have been washed out through open stomata. Neither leaf waxes, nor leaf waxes combined with sinigrin or pure sinigrin evoked feeding. Moreover, in choice tests, these leaf beetles clearly preferred to feed on de-waxed surfaces. Finally, the presence of feeding stimulants in epicuticular waxes is highly unlikely considering the physico-chemical properties of the plant cuticle. The lack of stimulants on the outermost surface corresponds to the plant’s perspective, which should avoid easily accessible feeding stimulants. Nevertheless, the role of glucosinolates for feeding stimulation of P. cochleariae remained unclear. Therefore, S. alba leaf extracts of different polarities were tested in bioassays in order to identify which chemical leaf compounds act as stimulants. In bioassay-guided fractionations of methanol extracts by semi-preparative HPLC, two distinct fractions with stimulating activity were detected, whereas other fractions were not effective. Flavonoids were identified as main component in one stimulating fractions, the second fraction mainly contained glucosinolates, including sinalbin. The combination of both fractions was significantly more stimulating than each individual fraction, indicating additive effects of at least one compound of each fraction. However, since the combined fractions were less effective compared to the original extracts, other compounds may additionally be involved in the complex composition of leaf compounds acting as feeding stimulants for P. cochleariae. Finally, fractionated extracts of UV altered plants were used to test whether the strength of feeding responses depend on different ratios of glucosinolates and flavonoids. However, since the feeding behavior of this leaf beetle was not affected, such quantitative variations were concluded to be less important. The initiation of feeding behaviour may solely depend on the presence of stimulating compounds.
NO has been described as an important component involved in the development of the hypersensitive reaction (Delledonne et.al., 1998). Furthermore, NO induces expression of a set of defence gene, such as PR-1, PAL1 and chalcone synthase (CHS), and accumulation of SA (Durner et al., 1998). In this study, transgenic plants with altered NO levels were used to study the role of NO in plant defence. Arabidopsis plants which, due to expression of a bacterial NO dioxygenase, exhibit lower levels of NO than wild-type plants, show several weakened defence response, including the oxidative burst and expression of phenylpropanoid pathway genes. By contrast, constitutive expression of a bacterial NO synthase in Arabisopsis results in increased levels of endogenous NO. However, these plants do not show constitutively activated defence responses, but suffer from increased susceptibility to various strains of P. syringae. This might indicate that a gradient in NO production rather than constitutive elevation of NO is necessary to trigger plant defence responses. Nevertheless, NO seems to be important for regulation of the oxidative state in plant cells. This function of NO is important during leaf senescence. The data of the present work indicate that NO acts as senescence-delaying factor during plant development. The molecular action of NO in plants and signalling cascades in which NO is involved as second messenger are still poorly understood. Experiments addressing the selective quantification of NO in intact plant tissue, the identification of NO-target proteins as well as the function of NO-modified biomolecules might help to understand the role of NO in plants. Non-host resistance consists of several layers of defence that include preformed compounds existing in plants before pathogen infection and induced defences which the plant activates after recognition of a pathogen. The role of inducible defences in preventing multiplication of non-adapted bacteria is not clear. Our experiments suggest that to restrict non-adapted bacterial growth, pre-formed antimicrobial compounds and an early inducible cell wall-based defence might play an important role in Arabidopsis leaves. Upon inoculation with non-adapted bacteria, we have observed early, TTSS-independent up-regulation of PAL1 and BCB, two lignin biosynthesis genes which might be involved in papilla formation or other kinds of cell wall fortification. Moreover, Arabidopsis pal1 knockout lines permit significantly higher survival of non-adapted bacteria in leaves than wild-type plants, suggesting a functional importance of PAL1 up-regulation. Although non-host bacteria, like host bacteria, induce accumulation of SA and PR gene expression in a TTSS-dependent manner, SA-dependent or JA/ET-dependent defences do not directly contribute to non-host resistance. Moreover, non-adapted bacteria activate similar defence signalling pathways as do host bacteria. However, because of varieties in effector protein composition between different non-adapted bacterial strains, the activated signalling pathways might also include different compounds. The Arabidopsis ecotype Ler 0 is more susceptible to a non-adapted strain of P. syringae than ecotype Col-0. Although differences in glucosinolate content and composition between those ecotypes exist, they are probably not a major reason for the observed difference in non-host resistance. To further understand the mechanisms underlying non-host resistance, the generation of double or triple mutants with deficits in both cell wall-based defences and SA-dependent signal cascades is necessary. Moreover, the study of genome polymorphism and composition of secondary metabolites between Ler-0 and Col-0 can shed new light into the mechanisms of non-host resistance against bacterial pathogens. Additionally, experiments addressing papilla formation and callose biosynthesis in Ler-0 and Col-0 could help to further elucidate bacterial non-host resistance. Our data indicate that localized contact of Arabidopsis leaves with non-adapted bacteria, type III secretion-defective P. syringae strains and bacterial pathogen-associated molecular patterns (PAMPs) induce systemic acquired resistance (SAR) at the whole plant level. This finding contrasts the general belief that an HR or other leaf necroses are required for SAR induction. The observed symptomless systemic response was abolished in all SAR-deficient mutants tested in this study, but was intact in the jar1 mutant, which is compromised in induction of ISR, indicating that non-host bacteria and PAMPs induce SAR in a mechanistically similar way than host bacteria. In addition, our data show that the extent of SA accumulation or PR gene expression induced at sites of virulent or avirulent P. syringae inoculation rather than the amount of tissue necroses or jasmonate accumulation determine the magnitude of SAR. The fact that systemic responses were also triggered after local treatment with type III secretion-defective P. syringae strains and bacterial PAMPs indicate that induction of SAR is TTSS-independent. Instead, recognition of general elicitors like flagellin and LPS play an important role in activation of the SAR process. To broaden the concept of PAMP-based SAR initiation, further general elicitors from bacteria and fungal pathogens should be tested for their capability to induce SAR. Screens for mutants with deficiency in SAR activation by individual PAMPs can help to identify new components involved in the SAR signalling cascade. Possible functions of PAMPs as mobile systemic signals should be tested in future experiments. By selection of candidate genes whose expression is up-regulated in Arabidopsis leaves infected with avirulent and virulent P. syringae and pathophysiological analyses of corresponding T-DNA knockout lines, FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) was identified as a key SAR regulator. SAR triggered by P. syringae is completely abolished in fmo1 mutant plants, and pathogen-induced expression of FMO1 in systemic leaves is closely correlated with the capability of different Arabidopsis lines to develop SAR. According to our findings, we have proposed that the FMO1 acts in signal amplification in non-inoculated, systemic leaves to trigger SAR. Experimental verification of the postulated potential amplification cycle underlying SAR should be tested in future experiments. The generation of transgenic lines expressing FMO1::GFP will provide useful information about the cellular localization of the FMO1 protein. Moreover, a comparative metabolomic analysis using SAR-induced wild-type, fmo1 knockout and FMO1 overexpressing lines can be used to identify substrates and reaction products of the FMO1 monooxygenase. As the single yeast FMO (yFMO) provides oxidizing equivalents at the ER for correct protein folding, expression of FMO1 in yfmo mutant yeast combined with protein activity assays might indicate whether FMO1 exhibits functional similarities with yeast FMO, e.g. in assuring proper folding of ER-targeted proteins essential for SAR establishment. Identification of further genes involved in activation of systemic resistance and biochemical characterization of the corresponding proteins can help to understand the SAR process in more detail.
Wirkmechanismen von Hefe-Elicitoren sowie die Rolle von Jasmonaten in Pflanze-Pathogen-Interaktionen
(2007)
Die Anwendung von Hefe (Saccharomyces cerevisiae) als Elicitor wurde bisher in Zellkulturen, ebenso in Sojabohne und Gerste beschrieben. Im Rahmen der vorliegenden Arbeit wurde eine mögliche Elicitorwirkung von Hefe auf A. thaliana untersucht. Das Sprühen mit autoklavierter Bäckerhefe führte zu einem Anstieg des Phytoalexins Camalexin mit einem Maximum (54 nmol/g FG) am 5. Tag nach der Behandlung mit dem Elicitor. Bei nachfolgenden Infektionen am 5. Tag nach Hefebehandlung mit Pseudomonas syringae pv. tomato DC3000 wurde eine Schutzwirkung detektiert, die beim Wildtyp Col-0 zu einer 3 bis 4fachen Verringerung des Bakterienwachstums im Vergleich zur Wasserbehandlung führte. Die Schutzwirkung setzte mit dem 5. Tag nach Hefebehandlung ein und hielt bis einschließlich dem 11. Tag an. Ein Schutz gegen Pseudomonas syringae pv. tomato DC3000 war auch systemisch in nicht mit Hefe behandelten Blättern zu detektieren. Infektionen mit Botrytis cinerea 5 Tage nach Hefebehandlung führten beim Wildtyp Col-0 zu Nekrosengröße, die nur 17 % der Nekrosengröße der mit Wasser behandelten Kontrolle betrugen. Veränderungen in der Genexpression 48 Stunden nach Hefebehandlung wurden in einer Microarray-Analyse (in Kooperation mit der GSF Neuherberg) ermittelt. Von rund 1400 Stress-responsiven Genen konnte eine Induktion von 6 Genen nachgewiesen werden. Dabei handelte es sich um Salicylsäure-abhängige Gene (Pr1, Pr2 und Pr5), Gluthation-S-Transferasen (Gst2 und Gst11) und eine UDP Glucosyltransferase. Die Erhöhung der Gene Pr1 und Pr2 deutet auf eine Aktivierung des Salicylsäure-Weges hin. Die Induktion der anderen Gene deutet auf eine Aktivierung der Detoxifizierung hin. Gene aus dem Jasmonsäure (JA)- und Ethylen-Weg wurden nicht induziert. Reprimiert wurde das Gen Asa1, das für eine JA-induzierte Antranilatsynthase kodiert. In Northernblot-Analysen wurden Gene auch zu früheren Zeitpunkten als in der Microarray-Analyse untersucht. Für die Untersuchung, welche Signalwege für die Resistenz durch Hefebehandlung verantwortlich sind, wurden verschiedene Mutanten mit den korrespondierenden Wildtypen von Arabidopsis thaliana aus dem JA-Weg (dde2, opr3 und jin1), aus dem Salicylsäure-Weg (nahG und npr1) und aus dem Camalexin-Weg (cyp79B2/B3 und pad3) mit Pseudomonas syringae pv. tomato DC3000 oder Botrytis cinerea infiziert. Nach Infektionen mit Pseudomonas syringae pv. tomato DC3000 konnte nur in den Salicylsäure-Mutanten keine erhöhte Hefe-vermittelte Resistenz festgestellt werden. Das deutet darauf hin, dass Salicylsäure für den Schutzeffekt der Hefe gegenüber Pseudomonas syringae pv. tomato DC3000 notwendig ist. Bei den getesteten Wildtypen und den Mutanten aus dem JA- und Camalexin-Weg wurden in den mit Hefe vorbehandelten Pflanzen Schutzfaktoren gegen Pseudomonas syringae pv. tomato DC3000 zwischen 2 und 5fach nachgewiesen. Bei Infektionen mit Botrytis cinerea wurde in allen getesteten Mutanten nach Hefebehandlung eine Schutzwirkung aufgezeigt (Schutzfaktoren von 3 bis 7). Das deutet darauf hin, dass weder JA, noch Salicylsäure oder Camalexin für die Schutzwirkung gegen Botrytis cinerea verantwortlich ist. Eine direkte hemmende Wirkung der Hefe auf das Wachstum des nekrotrophen Pilzes konnte durch Wachstumsversuche auf unterschiedlichen Medien ausgeschlossen werden. In Versuchen mit den Mutanten dde2 und opr3 konnte nachgewiesen werden, dass dde2, die weder 12-Oxo-Phytodiensäure noch JA bilden kann, größere Läsionen nach Botrytis cinerea Infektionen ausbildet als der Wildtyp. Größere Läsionen zeigte auch opr3, die 12-Oxo-Phytodiensäure, aber keine JA bildet, die sich aber nicht signifikant vom Wildtyp unterschieden. Daraus lässt sich schließen, dass 12-Oxo-Phytodiensäure eine wichtige Rolle für die Abwehr gegenüber dem nekrotrophen Pilz Botrytis cinerea spielt, wobei JA vermutlich zusätzlich zur Abwehr beiträgt. Infektionen mit Pseudomonas syringae pv. tomato DC3000 führten bei beiden Mutanten zu einer geringeren Symptomausprägung als in den Wildtypen. Übereinstimmend mit den makroskopisch sichtbaren Symptomen zeigte die Mutante dde2 ein mehr als 20fach geringeres Bakterienwachstum als der Wildtyp. Dieses Ergebnis deutet darauf hin, dass sich die Anwesenheit von 12-Oxo-Phytodiensäure und JA im Wildtyp negativ auf die Abwehr gegen das biotrophe Pathogen Pseudomonas syringae pv. tomato DC3000 auswirkt. In Fusarium graminearum konnte JA nachgewiesen werden. Ob es sich bei der JA um einen Pathogenitätsfaktor des Pilzes handelt, sollte durch Mutanten mit einem Defekt im Lipoxygenasegen untersucht werden. Infektionsversuche mit Lipoxygenase-Knockout-Mutanten und Stämmen mit komplementierter Lipoxygenase-Expression zeigten keine Unterschiede in der Symptomausprägung an Blüten und jungen Schoten von Arabidopsis thaliana im Vergleich zum Wildtyp-Pilz. Dieses Ergebnis deutet darauf hin, dass die Lipoxygenase in Fusarium graminearum keine Rolle in der Pathogenität gegenüber Arabidopsis thaliana spielt.