The aim of this study was to increase understanding of amino acid utilisation and amino acid permease (aa_permease) genes in the plant pathogen Pseudomonas syringae, which inhabits the nutrient-limited environment of the plant surface and intracellular apoplast. aa_permease genes are significantly reduced in P. syringae genomes compared to non plant-pathogenic Pseudomonas. Accordingly, this work demonstrates that P. syringae can utilise a restricted number of amino acids for growth compared to non plant-pathogenic Pseudomonas. The remaining aa_permease genes in Pseudomonas syringae pv. tomato are annotated as transporting GABA, ethanolamine, proline and aromatic amino acids. Sequence analyses, phylogenetic analyses, chemotaxis assays and gene expression analyses supported the functional annotations of the GABA, ethanolamine and aromatic aa_permeases, but showed that expression of the proline permease was specifically induced by histidine, which suggests that this encodes a histidine transporter. Expression of the GABA and histidine permeases was also induced in the presence of tomato apoplast, indicating that these amino acids may be assimilated during apoplast colonisation. P. syringae pv. tomato exhibited chemotaxis towards tomato apoplast and several constituent amino acids, which may be important for invasion of the apoplast from the plant surface. Uptake of several amino acids including GABA and histidine appeared to be negatively affected in alkaline media. The aa_permeases use electrochemical potential energy to transport substrates and this mechanism may be affected by pH. The plant apoplast becomes increasingly alkaline during infection and this may select against the retention of particular transporters in P. syringae. As GABA is an abundant amino acid in the apoplast and can be used by P. syringae strains for growth, the growth of P. syringae pv. tabaci was monitored in transgenic tobacco plants with increased GABA. Although growth was similar in all plants, significantly fewer symptoms and more callose deposition was observed upon infection.

Universiẗat, Diss., 2006--Würzburg.

<p>&nbsp;Bakteriozno su&scaron;enje tre&scaron;nje (Prunus avium L.) poslednjih nekoliko godina u mladim zasadima i plantažama tre&scaron;nje predstavlja značajan problem u proizvodnji ove voćne vrste. Simptomi bolesti se ispoljavaju u vidu su&scaron;enja grana, grančica ili celih stabala, &scaron;to se uglavnom zapaža na mestima rezidbe i oko pupoljaka, sa uočljivim promena boje tkiva kore, koje puca i nastaju rak rane. U periodu od 2012 &ndash; 2015 godine izvr&scaron;en je monitoring zdravstvenog stanja tre&scaron;nje kojim je obuhvaćeno nekoliko plantaža i manjih zasada tre&scaron;nje iz vi&scaron;e lokaliteta na području AP Vojvodine i centralne Srbije (Ritopek). Mlade voćke su očigledno najugroženije, jer smo na osnovu praćenja zdravstvenog stanja u vi&scaron;e lokaliteta i zasadima različite starosti, pojavu bakterioznog su&scaron;enja u jačem ili slabijem intenzitetu, konstatovali samo u mladim zasadima (do 3 godine starosti &ndash; Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo i Kanjiža). Izolacijama na strandardne hranljive podloge, iz prikupljenih obolelih uzoraka tre&scaron;nje, kao i sa zdravih pupoljaka i listova tre&scaron;nje (epifitna populacija), dobijeni su brojni izolati bakterija P. syringae pvs. od kojih je za dalja ispitivanja odabrano 155 izolata. Identifikikacija dobijenih izolata je izvr&scaron;ena je na osnovu fenotipskih i genotipskih metoda. Na osnovu LOPAT testova izolati pripadaju Ia grupi fluorescentnih vrsta Pseudomonas syringae. Prema GATTa testovima utvrđene su dve grupe izolata u okviru vrste P. syringae: I grupa (G+A+T&ndash;Ta&ndash;) i II grupa (G&ndash;A&ndash;T+Ta+). Dodatni testovi su potvrdili GATTa testove, na osnovu kojih je zaključeno da su&scaron;enje mladih stabala tre&scaron;nje prouzrokuju dve grupe bakterije P. s. pv. syringae (I grupa) i P. s. pv. morsprunorum rasa 1 (II grupa). Među ispitivanim izolatima nije bilo odstupanja u pogledu fenotipskih karakteristika u okviru iste grupe, osim sposobnosti stvaranja siringomicina pojedinih izolata I grupe (pv. syringae). Proverom patogenosti na raznim test biljkama i biljci domaćinu utvrđene su razlike, ali i određene sličnosti između izolata I i II grupe. Jasne razlike između grupa izolata utvrđene su pri inokulaciji zelenih plodova tre&scaron;nje, vi&scaron;nje, ringlova i kru&scaron;ke, paradajza, paprike i mahuna boranije. Pri inokulaciji odvojenih listova jorgovana izolati I grupe (pv. syringae), kao i većina izolata II grupe (pv. morsprunorum rasa 1) su pozitivno reagovali, &scaron;to ukazuje na heterogenost populacije bakterije P. s. pv. morsprunorum rasa 1. Pri inokulaciji sejanaca voćnih podloga (divlja tre&scaron;nja, magriva, divlja &scaron;ljiva, divlja kru&scaron;ka) svi izolati pv. syringae su prouzrokovali karakteristične patolo&scaron;ke promene na podlogama svih voćnih vrsta, a izolati pv. morsprunorum rase 1 takođe na svim vrstama, osim na sejancima divlje &scaron;ljive. Ovi rezultati ukazuju da je &scaron;irenje bakterija moguće i putem podloga koje takođe mogu biti zaražene. Inokulacijama dvogodi&scaron;njih grančica tre&scaron;nje u periodu mirovanja zaključeno je da su svi izolati pv. syringae i morprunorum rasa 1 podjednako patogeni na svim sortama tre&scaron;nje (Burlat, Summit, Hedelfigenska i Germerzdorfska). Najveća dužina nekroze najče&scaron;će je zabeležena na sortama Burlat i Summit u kombinaciji sa izolatima I grupe (pv. syringae) u pojedinim slučajevima i sa izolatima II grupe (pv. morsprunorum rasa 1), a najmanja uglavnom kod sorti Germerzdorfska i Hedelfigenska sa izolatima II grupe (pv. morsprunorum rasa 1). Identifikacija izolata KBNS71 &ndash; 84 (Gornji Tavankut) i KBNS85 &ndash; 94 (Selenča) na bazi MLST kori&scaron;ćenjem gena gyrB, rpoD, gapA i gltA, jasno je pokazala prisustvo dva patovara P. s. pv. syringae i P. s. pv. morsprunorum rasa 1. Pri poređenju sa sojevima H &ndash; 1, V &ndash; 85, V &ndash; 88 (vi&scaron;nja) i V &ndash; 109 (tre&scaron;nja) utvrđene su značajne razlike i postojanje genetskog diverziteta populacije ovih patogena. Simultana detekcija gena syrB i syrD utvrđena je kod 70 izolata I grupe (pv. syringae), a samo SyrB kod 9 izolata iste grupe (pv. syringae). Gen za sintezu koronatina detektovan je kod svih 76 izolata II grupe (pv. morsprunorum rasa 1). Rep &ndash; PCR metodom ustanovljene su značajne razlike (58%) između I i II grupe izolata (pv. syringae i pv. morsprunorum rasa 1). Ispitivani izolati sa tre&scaron;nje u okviru pv. syringae nisu ispoljili međusobne razlike, ali se razlikuju od sojeva sa drugih lokaliteta i ranije izolovanih sa istog domaćina (V &ndash; 109 i T6), kao i od sojeva sa drugih domaćina &ndash; vi&scaron;nje (V &ndash; 85) i uljane tikve (Tk21) do 37%. Razlike među izolatima pv. morsprunorum rase 1 iznosile su manje od 5%, a 24% u odnosu na soj CFBP2119 istog patogenog varijeteta. Rep &ndash; PCR analiza ukazala je na nizak nivo heterogenosti ispitivanih izolata u okviru istog patogenog varijeteta. RAPD metoda, kori&scaron;ćenjem većeg broja prajmera, bila je uspe&scaron;nija za poređenje ispitivanih izolata od rep &ndash; PCR. Od testiranih 11 prajmera, 4 (SPH1, DJP17, DJ15, DJ16) su selektovana za dalji rad na osnovu razlika među izolatima unutar patogenih varijeteta. Kumulativna RAPD analiza pokazala je da između ispitivanih izolata pv. syringae postoje razlike do 24%, a 41% u poređenju sa sojem KFB0103, dok su kod izolata pv. morsprunorum rase 1 razlike iznosile do 15%, a 36% u odnosu na soj</p><p>4<br />CFBP2119. Dobijeni rezultati RAPD analize ukazuju da u okviru populacije obe grupe ispitivanih izolata postoji određena heterogenost, ali je genetski diverzitet izraženiji kod pv. syringae. Proučavanjem epidemilogije ovih patogena u poljskim uslovima inokulacijom jednogodi&scaron;njih grana / mladara sortama Burlat, Germerzdorfska, Hedelfigenska i Droganova žuta, zaključeno je da tre&scaron;nja u na&scaron;im agroekolo&scaron;kim uslovima ranije postaje osetljiva (oktobar) prema P. s. pv. morsprunorum rasa 1 u odnosu na pv. syringae. Prvi pozitivni rezultati pri inokulaciji sojevima pv. syringae utvrđeni su pri inokulaciji u novembru. U pogledu dužine nekroze najuspe&scaron;nije su bile novembarske inokulacije (najduže nekroze; 2,17 &ndash; 3,35 cm), uspe&scaron;ne su bile i januarske i martovske inokulacije, ali je dužina nekroze bila sve manja, respektivno. Generalno najduže nekroze su ostvarene kod sorte Burlat, a najkraće kod sorte Germerzdorfska. Sve inokulacije urađene u periodu vegetacije su bile negativne. Inokulacijama dvo &ndash; trogodi&scaron;njih grana na sorti Summit prve uspe&scaron;ne inokulacije (oba patovara) su ostvarene tek u novembru (oktobarske su bile negativne), kada je utvrđena i veća agresivnost patovara syringae. Pri inokulacijama u januaru dužina nekroze je bila manja, a martovska je bila negativna. Sve inokulacije vr&scaron;ene u periodu od bubrenja pupoljaka do opadanja li&scaron;ća takođe su bile negativne. Ispitivanjem osetljivosti sotrimenta tre&scaron;nje i pojedinih sorti vi&scaron;nje zaključeno je da su prema oba patovara (syringae i morsprunorum rasa 1) najosetljivije sorte tre&scaron;nje Katalin, Linda, Summit, New Star i Burlat, srednje osetljive su sorte vi&scaron;nje Erdi Botermo i sorte tre&scaron;nje Droganova žuta, CarmCarmen, Germerzdorfska i Rana od Noara, a slabo osetljive sorte vi&scaron;nje &Scaron;panska i Ujfeheti firto&scaron; i sorta tre&scaron;nje Rita.</p> / <p>Bacterial die back (canker) of sweet cherry (Prunus avium L.) in young orchards and sweet cherry plantations in the past few years has been a significant problem in the production of this fruit species. Symptoms of the disease were manifested in the form of drying branches, twigs or whole trees, which were mainly observed in places of pruning or around the buds, bark changes a color, cracks and cankers has formed. In the period 2012 - 2015 monitoring of the health status of sweet cherries was carried out covering several plantations and smaller orchards of sweet cherries in several localities in Vojvodina and central Serbia (Ritopek). Young fruit trees are obviously the most susceptible, based on monitoring of the health status in many localities and plantations of different ages, the occurrence of bacterial canker in a stronger or weaker intensity was found only in young plantations (up to 3 years old - Selenča, Gornji Tavankut, Donji Tavankut, Ljutovo, Mikićevo and Kanjiža). From collected diseased samples of sweet cherries, as well as healthy buds and leaves of sweet cherry (epiphytic population) isolations on standard nutrient medium, were obtained numerous isolates of P. syringae pvs. and for further investigations was selected 155 isolates. Identification of isolates was performed on the basis of phenotypic and genotypic methods. Based on LOPAT tests isolates belonging to Ia group fluorescent Pseudomonas syringae. According to GATTa tests two groups of P. syringae isolates were identified, I group (G+A+T-Ta-) and II group (G-A-T+Ta+). Additional tests confirmed the GATT tests, on the basis which it was concluded that the drying of young sweet cherry trees caused P. s. pv. syringae (I group) and P. s. pv. morsprunorum race 1 (II group). Among the tested isolates was not exceptions in phenotypic characteristics within the same group, except for the ability to produced syringomycine for some isolates of I groups (pv. syringae). In pathogenicity tests on various plants and host plant were observed differences, but also and some certain similarity between isolates of I and II groups. Clear differences between the groups of isolates were determined in the inoculations of green fruit of sweet cherry, sour cherry, cherry plum and pears, tomatoes, peppers and green bean pods. In the case of inoculation of separate lilac leaves isolates of I group (pv. syringae) and most isolates of II group (pv. morsprunorum race 1) reactions were positive, what indicating the heterogeneity of the population of P. s. pv. morsprunorum race 1. In the inoculation of fruit rootstock seedlings (wild cherry, Magriva, wild plum, wild pear) all isolates pv. syringae caused the characteristic pathological changes on the all fruit species, isolates of pv. morsprunorum race 1 also except on the seedlings of wild plum. These results suggest that the spreading of bacteria is possibly through the rootstock that can also be infected. Inoculations of two &ndash; years old branches of sweet cherry during dormancy, was concluded that all isolates pv. syringae and morprunorum race 1 were equally pathogenic in all sweet cherry cultivars (Burlat, Summit, Hedelfigen and Germersdorf). The longest length of necrosis usually was observed on the cultivars Burlat and Summit in combination with isolates of I groups (pv. syringae), in some cases with isolates of II group (pv. morsprunorum race 1), and the lowest mainly in cultivars Germersdorf and Hedelfigen with isolates of II group (pv. morsprunorum race 1). Identification of isolates KBNS71 - 84 (GornjiTavankut) and KBNS85 - 94 (Selenča) based on MLST using genes gyrB, rpoD, gapA and gltA genes clearly showed the presence of two patovars P. s. pv. syringae and P. s. pv. morsprunorum race 1. Comparison with strains H - 1, V - 85 V - 88 (sour cherry) and V - 109 (sweet cherry) showed significant differences and the existence of genetic diversity in the population of these pathogens. Simultaneous detection of syrB and syrD gene was found in 70 isolates of I group (pv. syringae) and only syrB gene in 9 isolates of the same group (pv. syringae). The gene for coronatine synthesis was detected in all 76 isolates of II group (pv. morsprunorum race 1). Rep - PCR method detected significant differences (58%) between isolates of I and II groups (pv. syringae and pv. morsprunorum race 1). The tested isolates from sweet cherry within pv. syringae did not show differences between them, but they were different from the strains from other locations and previously isolated from the same host (V - 109 and T6), as well as strains from other hosts - cherry (V - 85) and pumpkin (Tk21) to 37 %. The differences between isolates pv. morsprunorum race 1 were less than 5% and 24% compared to the same pathovar strain CFBP2119. Rep - PCR analysis indicated a low level of heterogeneity of isolates within the same pathovar. RAPD method using a large number of primers were more successful to compare isolates than rep - PCR. Among 11 tested primers, 4 (SPH1, DJP17, DJ15, DJ16) were selected for further work on the basis of the difference between isolates within same pathovar. Cumulative RAPD analysis showed up to 24% differences among tested isolates of pv. syringae and 41% compared to the strain KFB0103, while among isolates pv. morsprunorum race 1 differences were 15% and 36% compared to the strain CFBP2119. The results of RAPD analysis indicate that a certain heterogeneity<br />7<br />exists in the population of both tested groups of isolates, but genetic diversity is more pronounced among isolates of pv. syringae. Studying the epidemiology of this pathogen in field conditions, by inoculating one &ndash; year old branches / or shoots sweet cherry cultivars Burlat, Germersdorf, Hedelfigen and Droganova žuta, it was concluded that the sweet cherry in our agroecological conditions becoming sensitive (October) to P. s. pv. morsprunorum race 1 before in relation to the pv. syringae. The first positive results of inoculations with strains pv. syringae were determined in November. Regarding the length of necrosis most successful were inoculation in the November (necrosis longest; 2.17 to 3.35 cm), inoculations also were successful in the January and the March, but the length of necrosis was smaller, respectively. Generally longest necrosis were observed in the cultivar Burlat, and the shortest in cultivar Germersdorf. All inoculations carried out in the period of vegetation were negative. Inoculations of two &ndash; three &ndash; years old branches of the cultivar Summit, first successful inoculations (for both pathovar) were observed only in November (October was negative), when a greater aggressiveness of pathovar syringae were determined. In inoculations in January length of necrosis was smaller, and in March was negative. All inoculations carried out in the period from buds swelling to leaf falling were also negative. Investigation susceptibility of sweet cherry and some sour cherry cultivars was concluded that against to both pathovars (syringae and morsprunorum race 1) the most susceptible were cultivars of sweet cherry Katalin, Linda, Summit, New Star and Burlat, medium susceptible were cultivar of sour cherry Erdi Botermo and sweet cherry cultivars Droganova žuta, Carmen, Germersdorf and Rana od Noara and low susceptible cultivars of sour cherry &Scaron;panska and Ujfeheti firto&scaron; and cultivar of sweet cherry Rita.</p>

Pea aphids, Acyrthosiphon pisum, have the potential to increase reproduction as a defence against pathogens, though how frequently this occurs or how infection with live pathogens influences this response is not well understood. Here we determine the minimum infective dose of an environmentally common bacterium and possible aphid pathogen, Pseudomonas syringae, to determine the likelihood of pathogenic effects to pea aphids. Additionally, we used P. syringae infection to investigate how live pathogens may alter reproductive rates. We found that oral bacterial exposure decreased subsequent survival of aphids in a dose-dependent manner and we estimate that ingestion of less than 10 bacterial cells is sufficient to increase aphid mortality. Pathogen dose was positively related to aphid reproduction. Aphids exposed to low bacterial doses showed decreased, although statistically indistinguishable, fecundity compared to controls. Aphids exposed to high doses reproduced significantly more than low dose treatments and also more, but not significantly so, than controls. These results are consistent with previous studies suggesting that pea aphids may use fecundity compensation as a response to pathogens. Consequently, even low levels of exposure to a common plant-associated bacterium may therefore have significant effects on pea aphid survival and reproduction.

Phytohormone spielen eine zentrale Rolle in der Regelung normalen Wachstums, der Entwicklung und der Mitwirkung an Abwehrmechanismen in Pflanzen. Allgemein betrachtet können Phytohormone in zwei Klassen unterteilt werden – in solche, die in Beziehung zu Stressreaktionen stehen und in jene, die das Wachstum begünstigen. Salizylsäure, und Jasmonsäure sind in erster Linie an der Stressresonanz, Ethylen, Auxine, Cytokinine (CKs) und Gibberilline an Entwicklungsprozessen beteiligt. In den letzten Jahrzehnten wurde den Phytohormonen aus diesem Betrachtungswinkel starke Aufmerksamkeit gewidmet und heute stehen ihre wechselseitigen Beeinflussungen im Fokus. Die Tatsache, dass Pflanzenpathogene ein hormonelles Ungleichgewicht an der Wirtspflanzen-Pathogen Schnittstelle bedingen und es begleitend zu physiologischen Veränderungen kommt, wird dabei als Werkzeug für Erforschungen in Pflanzengeweben genutzt. Abgesehen von der bekannten Bedeutung, die Cytokinine für Wachstum und Entwicklung haben, sind sie bisher am meisten vernachlässigt worden und eher als Konsequenz denn als Grund von Pathogeninfektionen angesehen worden. Die Ergebnisse dieser Arbeit basieren auf der Hyphothese, dass erhöhte Gehalte an CKs die Pflanzen mit einer Resistenz gegen hemibiotrophe Pathogene ausstatten. In diesem Zusammenhang wurden transgenetische Pflanzen untersucht, in welchen das bakterielle Gen IPT überexpremiert wurde. Kontrolliert wurde die Expression durch einen pathogen-induzierbaren, einen tetracyclin-induzierbaren oder durch einen wachstumsabhängigen Promotor. Für die weitere Validierung der an den transgenetischen Pflanzen gewonnenen Ergebnisse wurden unterschiedliche Cytokinin von abgeschnittenen Tabakblätter aufgenommen. Alle transgenetischen Ansätze und exogen applizierten Cytokiningaben zeigten ähnliche verringerte Krankheitsanzeichen. Diese Art der Resistenz wurde im Weiteren mit verschiedenen zellulären, biochemischen, mikrobiellen Techniken sowie durch Signalwirkungstests fundiert. Die Gehalte von SA und JA blieben unverändert, während die Expression des Gens PR1 in Proben mit erhöhtem Cytokiningehalt stark hoch reguliert wurde. Darüber hinaus konnte eine verringerte Akkumulation von ROS in IPT exprimierenden Blättern gegenüber der entsprechende Kontrolle beobachtet wurden. Zusätzlich konnte weder ein direkter Effekt im Wachstum von P. syringae pv. tabaci noch die Präsenz von antimikrobiellen Peptiden in Cytokinin-angereicherten Extrakten festgestellt werden. Interessanterweise ist die verstärkte Akkumulation von Phytoalexinen bei erhöhtem CKs-Status der Pflanze als ein mögliches Anzeichen für die Gefährdung durch die Ausbreitung von Pathogenen belegt. Im Gegensatz dazu konnten wir keine Wachstumsverlangsamung für Sclerotinia sclerotiorum in Blättern mit erhöhten CKs-Gehalten feststellen. Neben der Wirt-Pathogen Interaktion im Hinblick auf erhöhte CK-Gehalte wurden die Auswirkungen eines modulierten Kohlenstoffhaushalts auf das Wachstum von Pathogenen untersucht. Dafür wurden zuvor generierte transgenetische Tabakpflanzen, basierend auf ein regulierbarem Invertase Enzym verwendet. Es konnte gezeigt werden, dass induzierte und nicht-induzierte Expression von CIN1 unter der Kontrolle des Tet-Promotors das Wachstum von P. syringae pv. tabaci nicht beeinflusst. Darüber hinaus zeigten Linien, welche den Invertaseinhibitor NtCIF unter Kontrolle desselben Tet-Promotors exprimieren, keine differenzielle Veränderung des Wachstums von P. syringae pv. tabaci bei induziertem und nicht-induziertem Status der Pflanze. Ähnlich waren die Resultate in der transgenetischen Tomaten-Linie Lin6::NtCIF für P.syringae pv. tomato DC 3000. Interessanterweise zeigten die Blätter von Lin6::NtCIF Tomatenpflanzen starke Symptome nach Behandlung mit Botrytis cinerea verglichen zum Wildtyp. Eine mögliche Verbindung zwischen Cytokininen und Zuckermetabolismus im Bezug auf die Wirt-Pathogen Beziehung wurde ebenfalls untersucht. Die Expression des IPT-Gens unter der Kontrolle des pathogeninduzierbaren Promotors (4xJERE::IPT) im transgenetischen Hintergrund von Tet::CIN1 ergab lokale Unterschiede in der Entwicklung der Symptom von P. syringae pv. tabaci. Bei exogen appliziertem Kinetin an abgeschnittenen Tabakblättern von Tet::CIN1 verzögerte sich ebenfalls das Wachstum von P. syringae pv. Tabaci im Vergleich zu Tet-induzierten Blättern. Diese Ergebnisse führen zu der Schlussfolgerung, dass die extrazelluläre Invertase keine essentielle Rolle in der Cytokinin-vermittelten Resistenz gegen hemibiotrophe Pathogene spielt. / Phytohormones are known for their pivotal roles in promoting normal growth and development of the plants and contributing to the mechanism of defense. Although an over simplification, however, they may be categorized as stress specific and growth promoting. SA and JA/Ethylene are implicated in stress responses while auxins, cytokinins and gibberellins are involved in developmental processes. Phytohormones from the above perspective got much attention in the last few decades; however their reciprocal role is currently in focus. It is because of the reason that plant pathogens cause overall hormonal imbalance at host pathogen interface and alter host physiology for the sake of pathogenecity. Despite their importance in growth and development, cytokinins are among the most neglected phytohormones that are usually noticed as consequence rather than a cause of pathogen infection. Results presented in this thesis are based on the hypothesis that elevated levels of CKs embody plants with resistance against hemibiotrophic pathogens. To explore a connection between the spread of P. syringae and its tobacco host, CKs over producing transgenic plants were investigated whereby bacterial IPT gene was expressed under the control of pathogen inducible, tetracycline inducible and developmentally inducible promoters. To further validate the out-come of transgenic plants, various types of cytokinins were exogenously fed to detached tobacco leaves. Mentioned transgenics and exogenous CKs feeding approaches unanimously resulted in, “more cytokinins less disease symptoms” and vice versa. This state of cytokinins mediated resistance was further substantiated with various cellular, signaling, biochemical and microbial approaches wherein levels of SA and JA remained unaffected. Conversely, PR1 gene expression was strongly up-regulated in enhanced cytokinins accumulating samples. Moreover, less accumulation of ROS was observed in IPT expressing sites of the plants as compared to their corresponding controls. Additionally, we neither noticed any direct effect of cytokinins on the growth of P. syringae pv. tabaci nor found presence of anti-microbial peptides in cytokinins enriched extracts. Interestingly, enhanced accumulation of phtyoalexins in elevated CKs status of the plant proved to be a possible gesture in jeopardizing the spread of pathogen. Contrarily, no reduction was observed in the spread of fungal necrotrophic pathogen Sclerotinia sclerotiorum when leaves of elevated CKs were inoculated. Besides host-pathogen interaction in perspective of elevated cytokinins, impact of modulated sugar status of the plant on the spread of pathogen was also investigated. For this purpose, previously generated modulated invertase enzyme tobacco transgenic plants were analyzed. We showed that repression and de-repression of CIN1 gene under the control of tetracycline inducible-promoter did not affect the growth of P. syrinage pv. tabaci in Tet::CIN1 transgenic plants. Moreover, invertase inhibitor tobacco lines expressing NtCIF gene under the control of the same promoter failed to exhibit differential pathogenic responses in induced and non induced status of the plant. Similar was the case of tomato transgenic plants expressing NtCIF gene under the control of invertase gene Lin6 promoter in Lin6:: NtCIF plants for P.syringae pv. tomato DC 3000. Interestingly, when challenged Lin6:: NtCIF tomato plants with Botrytis cinerea, severe disease symptoms were observed on transgenic leaves as compared to control plants. To dissect a potential link between cytokinins and sugar metabolism with its effect on the growth of pathogen, invertase transgenic plants with elevated CKs were probed. When expressed exogenous IPT gene under the control of pathogen inducible promoter (4xJERE::IPT) in transgenic background of Tet::CIN1, we observed localized differences in symptom development for P.syringae pv. tabaci. Similarly, when exogenously fed with kinetin, detached leaves of Tet::CIN1 exhibited retarded growth of P.syringae pv. tabaci as compared to the tetracycline induced leaves. These results led to the conclusion that extracellular invertase may not play an essential role in cytokinins mediated disease resistance against hemibiotrophic pathogens.

1. OH-FS wurden in vitro hergestellt, um als Standardsubstanzen zur gaschromato-graphischen Identifizierung von OH-FS in Pflanzenmaterial eingesetzt zu werden. 2. Für die Untersuchung der Oxylipin-Gehalte in A. thaliana wurden der virulente Pst-Stamm DC3000 sowie der avirulente Stamm avrRPM1 verwendet, um die kompatible Interaktion mit der inkompatiblen Interaktion vergleichen zu können. Die Konzentrationen der Oxylipine sowie SA wurden innerhalb einer Versuchsdauer von 60 h verfolgt. Dabei wurden PPF1 sowie 12- und 16-OH-FS, als Vertreter der nicht-enzymatisch entstandenen Oxylipine, 9- und 13-OH-FS, sowohl als enzymatisch als auch nicht-enzymatisch entstandene Oxylipine, sowie JA und deren Vorstufe OPDA als enzymatisch gebildete Phytohormone untersucht. Es wurden monophasische Konzentrationsanstiege, bei allen untersuchten Substanzen, in der kompatiblen Interaktion ermittelt, wohingegen die Konzentrationsanstiege in der inkompatiblen Interaktion biphasisch waren. In beiden Interaktionen wurden nach 48 bis 60 h Konzentrationsmaxima der freien sowie der veresterten OH-FS und PPF1 nachgewiesen, ein früher Konzentrationsanstieg nach 5 bis 10 h konnte ausschließlich in der inkompatiblen Interaktion ermittelt werden. Die gleichzeitige Akkumulation von 9-, 10-, 12-, 13, 15- und 16-OH-FS und PPF1 deutet auf eine parallel ablaufende Oxylipin-Synthese durch enzymatische, Photo-oxidative und über freie Radikale vermittelte Prozesse hin. Die Akkumulation veresterter OH-FS und PPF1 erfolgte in beiden Interaktionen 5 bis 12 h früher als die Konzentrationsanstiege der freien OH-FS und PPF1. Die Ergebnisse bestätigen die Hypothese, dass nicht-enzymatische Oxylipine in Membranen gebildet werden können und anschließend vermutlich durch eine Lipase frei gesetzt werden. In der inkompatiblen Interaktion konnte ein erstes frühes Konzentrationsmaximum von JA und OPDA nach 5 h beobachtet werden, während späte Maxima in beiden Interaktionen nach 24 bis 36 h erfolgten. Somit akkumulierten die OH-FS und PPF1 in der inkompatiblen Interaktion zeitgleich mit den Jasmonaten nach 5 h. 3. Bei einer Kälteexposition von A. thaliana bei 4°C über 2 h wurde jeweils ein 3,3-facher Konzentrationsanstieg der freien und der veresterten enzymatisch gebildeten 13-OH-FS nachgewiesen. Darüberhinaus wurde ein 4,6-facher Anstieg der enzymatisch entstandenen 9-OH-FS ermittelt. Die nicht-enzymatisch gebildeten 12- und 16-OH-FS zeigten dagegen keine signifikanten Konzentrationsanstiege über die basalen Konzentrationen hinaus. Die angewendeten Stressbedingungen bewirken demnach ausschließlich eine enzymatische Bildung von OH-FS in A. thaliana. 4. Zur Untersuchung der OH-FS-Synthese in der inkompatiblen Interaktion in Abhängigkeit von der bei der Pflanzenanzucht eingesetzten Lichtstärke wurden A. thaliana bei Licht und in Dunkelheit mit Pst avrRPM1 infiziert. Nach 10 h wurde eine 1,1- bis 3,7-fach stärkere Bildung der freien sowie eine 2,0- bis 3,4-fach stärkere Akkumulation der veresterten 9-, 10-, 12-, 13, 15- und 16-OH-FS bei den Pflanzen ermittelt, die bei Licht angezogen wurden. Die Lichtintensität, der Pflanzen während der Infektion mit Pst ausgesetzt sind, hat demnach große Bedeutung für die Entstehung enzymatisch und nicht-enzymatisch gebildeter OH-FS. Ein 4,9-facher Anstieg veresterter 15-OH-FS, ein Marker für eine photooxidative OH-FS-Entstehung, auch bei Dunkelheit widersprach der Hypothese, dass 15-OH-FS ohne Lichteinwirkung nicht gebildet werden können und deutet auf eine bisher unbekannte Licht-unabhängige Entstehung von 1O2 bzw. von 15-OH-FS hin. 5. Die Bestimmung von OH-FS in Blättern und Wurzeln von unbehandelten A. thaliana-Pflanzen ergab eine 13- bis 31-fach höhere Konzentration veresterter 9-, 10-, 12-, 13- und 16-OH-FS in den Blättern. Darüberhinaus wurde eine 111-fach höhere Konzentration von veresterten 15-OH-FS in Blättern im Vergleich zu Wurzeln nachgewiesen. 15-OH-FS wurden als selektiver Marker für eine Photo-oxidative OH-FS-Bildung durch 1O2 verwendet. Mit 0,57 µg/g TG kommt 15-OH-FS allerdings auch im Wurzelgewebe vor, was einen Hinweis darauf darstellt, dass neben einem Licht-abhängigen Hauptweg auch ein Licht-unabhängiger Entstehungsmechanismus von 15-OH-FS bzw. 1O2 existiert. Alternativ wäre es denkbar, dass ein Transport von 15-OH-FS von den Blättern in die Wurzeln stattfindet. 6. Eine Untersuchung der Gehalte an OH-FS und PPF1 in NahG-, lsd1-, atrbohD- und atrbohF-Mutanten ergab 48 h nach Infiltration von Pst avrRPM1 keine signifikanten Unterschiede im Vergleich zu den Pflanzen des jeweiligen Wildtyps Col-0 und WS. Unter den gewählten Versuchsbedingungen bewirken die genetischen Defekte der untersuchten Mutanten keine veränderte Akkumulation enzymatisch sowie nicht-enzymatisch gebildeter Oxylipine. / 1. To obtain reference-substances for the identification of OH-FA in plant material by GC-MS, OH-FA were made in vitro. 9- and 13-hydroxy-octadecadienoic acids were formed from linoleic acid, 9- and 13-hydroxy-octadecatrienoic acids from &#61537;-linolenic acid by LOX-catalized reaction. 2. In order to compare the concentrations of oxylipins in A. thaliana during compatible and incompatible interaction, a virulent Pst-strain DC3000 and an avirulent strain avrRPM1 were utilized. The concentrations of oxylipins and salicylic acid were investigated within 60 h after inoculation. F1-phytoprostanes, 12- and 16-OH-FA were used as markers for non-enzymatically formed oxylipins. Concentrations of 9- and 13-OH-FA, either enzymatically or non-enzymatically formed, were measured as well as phytohormones, jasmonic acid and its precursor 12-oxo-phytodienoic acid. Within the compatible interaction an increase of the amount of all measured compounds was observed. In contrast, during incompatible interaction the increases of all measured substances was seperated into two phases. In both interactions, free and esterified OH-FA- and PPF1-concentrations exhibited maxima after 48 to 60 h, an early increase after 5 to 10 h was exclusively found in the incompatible interaction. The concurrent accumulation of 9-, 10-, 12-, 13, 15- und 16-OH-FA und PPF1 indicates that enzymatically, Photo-oxidative, and free-radical catalyzed synthesis of oxylipins takes place simultaneously. In both interactions the accumulation of esterified OH-FA and PPF1 occurred 5 - 12 h earlier than the increase of the concentrations of free OH-FA and PPF1. These results confirm the hypothesis that oxylipins are formed non-enzymatically from lipids inside cell membranes and are subsequently released by lipases. An early increase of JA- and OPDA-concentrations after 5 h was found in the incompatible interaction, while late maxima occurred in both interactions after 24 to 36 h. Therefore, OH-FA- and PPF1-accumulation took place at the same time as the increase of jasmonates after 5 h. 3. When A. thaliana plants were chilled for 2 h at 4°C an 3,3-fold incrceased formation of both, the enzymatically formed free and esterified 13-OH-FA was detected. The amount of enzymatically formed free 9-OH-FA increased 4,6-fold. In contrast, the amount of non-enzymatically formed 12- and 16-OH-FA did not increase significantly indicating that the applied mild stress conditions triggered exclusively enzymatical OH-FA-formation. 4. In order to get information about OH-FA-formation with regard to light intensity A. thaliana was infected by Pst avrRPM1 and cultivated either in the dark or in the light. When plants were cultivated in the light after 10 h an 1,1- to 3,7- fold increased formation of free and an 2,0- to 3,4-fold increased accumulation of esterified 9-, 10-, 12-, 13-, 15- und 16-OH-FA could be detected. Therefore, the light intensity during an infection with Pst avrRPM1 is an important factor for the formation of enzymatically as well as non-enzymatically formed OH-FA in planta. The 4,9-fold increase of esterified 15-OH-FA (a marker for photooxidative formation of OH-FA) in the dark contradicted the hypothesis that its formation is impossible without the impact of light. In contrast the accumulation of 15-OH-FA in the dark points to a light-independent 1O2- and subsequent 15-OH-FA-formation by a so far unknown mechanism. 5. Determination of the concentrations of OH-FA in leaves and roots of untreated plants of A. thaliana showed 13- to 31-fold higher amounts of esterified 9-,10-, 12-, 13 und 16-OH-FA in the leaves. Moreover, the amounts of esterified 15-OH-FA exceeded in leaves by the factor 111 in comparison to roots. 15-OH-FA was used as a selective marker for Photo-oxidative formation of OH-FA by 1O2. Though, 15-OH-FA occurred to an amount of 0,57 µg/g (dry weight) in roots as well. This indicates that, apart from an primarily used light depending mechanism, a second path for the formation of 15-OH-FA respectively 1O2 exists which does not depend on light strength. An alternative explanation could be the transport of 15-OH-FA from leaves to roots. 6. Compared to the wildtype plants no significant differences in the increase of OH-FA and PPF1 could be detected in NahG-, lsd1-, atrbohD and atrbohF-mutants 48 h after infiltration of Pst avrRPM1. Under the utilized conditions the genetic defects of the analyzed mutants did not cause a modified accumulation of both, enzymatically and not enzymatically formed oxylipins.

Horizontal gene transfer (HGT) and gene loss are important processes in the evolution of prokaryotic lineages. HGT involves the movement of genetic material between distantly related species, and can facilitate adaptation when gained genes confer advantageous phenotypes to recipient lineages. However, high levels of gene gain and loss are predicted to obfuscate patterns of vertical descent and homogenise nucleotide diversity across ecological and phylogenetic boundaries. Thus, a holistic understanding of the role of genome fluctuation in the emergence and maintenance of genetically and ecologically cohesive bacterial groups remains to be fully elucidated. In this thesis, I use the plant-associated bacterium Pseudomonas syringae as a model system to investigate the impact of HGT and gene loss on evolutionary processes such as adaptation, diversification and speciation. The Gram-negative Gammaproteobacterium P. syringae is an opportunistic plant pathogen, and has been used for decades as a model system with which to study the interaction between plants and their microbial pathogens. Recently, the diversification of lineages within this species has involved a number of host jumps onto a range of woody host plant species, resulting in the emergence of diseases such as bacterial canker of kiwi and bleeding canker of the European horse chestnut. Using whole-genome sequence data and a range of comparative genomics and phylogenetics methods, I quantitatively reconstruct the history of gene gain and loss in P. syringae and show HGT to be the predominant evolutionary force in this species. Genomes of this species are under constant permutation, are subject to a highly diverse HGT genepool and show marked differences in patterns of codon usage between imported and core genes. I then generate additional genome data for 26 strains of P. syringae that are pathogenic on a range of different woody plants, and investigate the contribution from HGT to the adaptation of these strains into the woody niche. Using a method that accounts for the underlying phylogenetic relationships among P. syringae strains, I look for the correlated evolution between gained genes and the woody niche, and find that a substantial proportion of the genome is associated with this ecological niche. I then investigate the recent adapitation of P. syringae pv. aesculi onto the European horse chestnut, and show that a number of genomic events that include both homologous and non-homologous recombination are likely to have led to the evolution of this bacterium onto its host, where it has become the causal agent of the bleeding canker disease that is currently epidemic across much of northern and central Europe. Overall, this thesis is an investigation into how HGT contributes to niche adaptation in P. syringae, and aims to further our understanding of the mechanisms that underlie bacterial evolution.

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage host immune responses and physiology to favor infection. We have previously shown that P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple pathogen-associated molecular patterns (PAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms in the suppression of two branches of PAMP-activated MAP kinase cascades. In contrast to blocking MKK5 in MEKK1-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in MEKK1-MKK1/2-MPK4 cascade and plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase involved in multiple PAMP signaling. The interaction between BAK1 and HopF2 or two additional P. syringae effectors AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of HopF2, the lethality of overexpression of HopF2 in wild-type Arabidopsis transgenic plants was largely alleviated in bak1 mutant plants. Identification of BAK1 as an additional HopF2 virulence target not only explains HopF2 suppression of multiple PAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors have multiple targets in host cells.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.