植物-病原菌互作的分子機制課件

1、 植物-病原菌互作的分子機制生物對抗病原菌的防衛(wèi)反應策略：1. 適應性免疫途徑（The adaptive immune response) 采用抗體的形式。（只在動物中）2. 先天免疫反應（The innate immunity） 病原分子模式識別防衛(wèi)反應 寄主細胞的受體接收病原菌攜帶的分子特征 (pathogen-associated molecular patterns, PAMPs，也稱microbe- associated molecular patterns, MAMP) 激發(fā)防衛(wèi)反應。 (動物和植物）-基礎防衛(wèi)反應的主要形式 ?；钥共》磻?由抗病基因編碼的受體接收病原菌編碼的配體

2、分子（無毒性蛋白）引發(fā)主動的抗性反應。(植物！ 動物？） 主動防衛(wèi)反應機械/物理性屏障（Physical Barrier) 內(nèi)容：第一部分：植物病原微生物第二部分：植物-病原菌互作的分子機理第三部分： 植物抗病分子育種第一部分： 植物病原微生物Plant Models for Host-Pathogen InteractionsAdapted from Baker, B. et al. Science 276:726 (1997)Rice Arabidopsis不同病原菌侵染植物的?；课?，引起不同的病癥番茄有100種以上的病原菌植物病原菌（pathogens）：真菌（fungi)、細菌(ba

3、cteria)、病毒(virus)和線蟲(nematodes)病原菌: 小種(race)、菌系(strain)或分離物(isolate)寄主植物(host)：病原菌侵染的專化植物即非寄主植物（non-host）: 某個植物種對某個病原菌的所有菌系均有抗性。是植物防御潛在病原菌的主要機制，也是植物最基礎最普遍的抗病類型抗病(resistance)：植株能限制病原菌在侵染點附近、病斑不擴展或只產(chǎn)生小斑點: 非親和性反應（incompatibility)感?。╯usceptibility）病斑擴大形成典型病斑: 親和性反應（compatibility)Plant diseaseM. griseaC.

4、 fulvumB. cinereaP. infestansI 植物病原菌的侵染機理植物病原菌寄生方式 腐生（necrotroph) 活體寄生 (biotroph)半活體寄生(semibiotroph)侵染途徑分泌胞壁降解酶、毒素菌體進入寄主細胞內(nèi)先活體寄生，后腐生特征寄主組織死亡、病原菌定殖、大面積組織軟化寄主細胞一般保持成活狀態(tài)侵染早期寄主組織仍成活，而后死亡寄主范圍廣窄，一般侵染個別植物兩者之間病原真菌在植物表皮細胞內(nèi)形成吸器（Haustorium)從寄主內(nèi)吸收營養(yǎng)病原細菌定殖于寄主細胞間隙100 nmssRNAdsDNA 植物病毒可在寄主細胞內(nèi)大量增殖并通過 胞間連絲進行“cell to

5、 cell”擴散植物線蟲也形成類似于真菌的營養(yǎng)吸收器官II 病原菌侵染植物的遺傳基礎病原菌無毒性基因(Avr)和植物抗病基因(R): 基因?qū)虻幕プ鱣ene for gene interactionsGene for gene interactions between plants and pathogensAdapted from Hammond &Jones, 2000Interactions involved in toxin-dependent diseaseAdapted from Hammond &Jones, 2000Maize resistance gene Hm1 to C

6、. carbonumencodes HC-toxin reductase Adapted from Hammond &Jones, 2000從革蘭氏陰性病原菌（Pseudomonas和Xanthomonas) 中至少克隆了50個avr基因Avr蛋白一般稱為效應子（effector）： 在具有對應抗病基因的寄主上激發(fā)激發(fā)抗病反應 在沒有抗病基因的寄主上起毒性作用病原菌無毒性基因Bacterial pathogens of plants and animals use similar secretion pathways (Type III ) during pathogenesis因此Avr蛋白

7、又叫Type III 效應子: T3SS effectorButtner and He (2009) 丁香假單胞菌效應蛋白作用示意圖Block and Alfano (2011)Plants and animals share similar defense signalingConserved domains exist in receptors or key regulators of plants and animalsROS, NO, SA function similarly in both plant and animal defensesPlants may have caspa

8、se system which regulates cell death in animalsAnd moreThe bacterial Pseudomonas aeruginosa is pathogen to both plants and mice Adapted from Hammond &Jones, 2000 Plant models for animal pathogenesisPseudomonas aeruginosa infects Arabidposis thaliana roots Cellular Microbiology 7, 2005 腸球菌 Enterococc

9、us feacalis multiplies in the intercellular spaces of Arabidopsis thaliana leavesCellular Microbiology 7, 2005 金黃色葡萄球菌Staphylococcus aureus infects Arabidopsis thaliana matrixA. the polysaccharide B. Trichome colonizationC. Biofilm on the rootsD.Transgenic plants with high level of SA Cellular Micro

10、biology 7, 2005 第二部分：植物-病原菌互作的分子機理 重點：植物抗病反應及其信號轉(zhuǎn)導網(wǎng)絡植物免疫反應（Innate immunity） 1. 病原分子模式識別防衛(wèi)反應（PAMP-triggered immunity, PTI) 寄主細胞的受體接收病原菌攜帶的分子特征 (pathogen-associated molecular patterns, PAMPs，也稱microbe- associated molecular patterns, MAMP) 激發(fā)防衛(wèi)反應。這類受體稱為PRR（pattern recognition receptor)。 2.?；钥共》磻?（spec

11、ific resistance, effector-triggered immunity, ETI) 由抗病基因 (resistance genes, R) 編碼的受體R蛋白接收病原菌編碼的無毒性蛋白（Avr, type lll effector proteins,T3SS effectors ）引發(fā)?；钥共》磻?。PAMP-Triggered Immunity (PTI) PAMP的作用：病原菌的適應性與生存 已知的主要PAMPPRR Bacterial flagellin (flg22)FLS2Bacterial PAMPs EF-TuEFRXoo Ax21XA21Fungal xylan

12、aseLeEIX1/2Fungal chitinCEBiP Oomycete glucans GBPPlant Pattern-recognition receptor Zigfel (2009) 1. Plant immunity through flagellin perception by RLK FLS2 Zipfel et al. (2004) NatureLigand-induced endocytosis of FLS2 during immunity responseRobatzek et al.(2006) Genes & Dev 2. Plant immunity thro

13、ugh EF-TU perception by RLK EFRZipfel et al. (2006) CellTransgenic expression of EFR in tomato confers broad-spectrum bacterial resistanceLacombe et al. (2010)Nat Biotech 28PAMP signaling upstream of MAPKKK in Arabidopsis innate immunityPing He et al. Cell 125 (2006)II. Effector-triggered immunity(

14、ETI)遺傳基礎：基因?qū)虻幕プ?gene for-gene interactions 由抗病基因 (resistance genes, R) 編碼的受體R蛋白接收病原菌編碼的無毒性蛋白（Avr, type lll effector proteins, T3SS effectors ）引發(fā)專化性抗病反應。 丁香假單胞菌效應蛋白作用示意圖Block and Alfano (2011)Gene for gene interactions between plants and pathogensAdapted from Hammond &Jones, 2000Classes of plant di

15、sease resistance genes (2005-8)Class R genePlantPathogenAvr geneStructure123456PtoRpg1RPS2RPS5RPM1RPP13HRTR1RxGpa2RPP8SW-5HeroDm3Bs2RCY1MiPrfI2Rp1-DXa1Pi-taPi-bMla1Mla6Rp3Lr10Lr21Pm3NL6MPRPS4RPP5RPP1RPP4RPP2RRS1-RSSI4RAC1Gro1-4Cf2Cf4Cf5Cf9Hcr9-4ERPP27Xa21Hm1VemloRPW8HS1pro-1xa5Xa27RTM1RTM2TomatoBarl

16、eyArabidopsisArabidopsisArabidopsisArabidopsisArabidopsisPotatoPotatoArabidopsisArabidopsisTomatoTomatoLettucePepperArabidopsisTomatoTomatoTomatoMaizeRiceRiceRiceBarleyBarleyMaizeWheatWheatWheatTobaccoFlaxFlaxFlaxArabidopsisArabidopsisArabidopsisArabidopsisArabidopsisArabidopsisArabidopsisArabidopsi

17、sPotatoTomatoTomatoTomatoTomatoTomatoArabidopsisRiceMaizeTomatoBarleyArabidopsisSugar beetRiceRiceArabidopsisArabidopsisP. syringaeP. graminisP. syringaeP. syringaeP. syringaeP. parasiticaTCVP. infestansPotato virus XNematodeP. parasticaTospovirusNematodeB. lactucaX. campestrisCMV-YM. incognitaP. sy

18、ringaeF.oxysporumP. sorghiX. oryzaeM. griseaM. griseaB. graminisB. graminisP. sorghiP. triticinaP. triticinaB. graminisTMVM. liniM. liniM. liniP. syringaeP. parasticaP. parasticaP. parasticaP. parasticaR. solanacearumP. syringaeA. candidaNematodeC. fulvumC. fulvumC. fulvumC. fulvumC. fulvumP. parast

19、icaX. oryzaeC. carbonumVerticilliumB. graminisE. cruciferarumH. schachtiiX. oryzaeX. oryzaeTEVTEVavrPtounknownavrRpt2avrPhBavrRpm1,avrBUnkonwnCPUnknownCPUnknownavrRPP8UnknownUnknownUnknownavrBS2UkonwnUnknownavrPtoUnknownavrRp1DavrXa1avrPi-taUnknownUnknownUnknownUnknownUnknownUnknownUnknownHelicaseUn

20、knownUnknownUnknownavrRps4UnknownUnknownUnknownATR2PopP2UnknownUnknownUnknownavr2avr4avr5avr9avr4EUnknownUnknownNoneUnknownUnknownUnknownUnknownavrxa5avrXa27UnknownUnknownProtein kinaseProtein kinaseLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRLZ-NBS-LRRNBS-LRRNBS-

21、LRRNBS-LRRNBS-LRRNBS-LRRNBS-LRRNBS-LRRNBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRTIR-NBS-LRRLRR-TMLRR-TMLRR-TMLRR-TMLRR-TMLRR-TMReceptor kinaseToxin reductaseCSLRaGPCRbTM- CCNew proteinTFIIAgammaNew ProteinJacalin repeatNew pr

22、oteinHammond-Kosack and Parker: Current Opinion In Biotechnology, 2003 14:177-193 Schematic representation of domains found in plant LRR R proteinsTameling and Takken (2008) EJPPGene-for-gene抗性-小種?；钥剐裕╮ace-specific resistance）只能抵抗某些帶相同非毒性基因的病原小種，它們表現(xiàn)為經(jīng)典的顯性或隱性遺傳，并為高度抗性或免疫。在自然界，由于病原小種群體不斷變化，這種抗性很容易被病

23、原菌所克服而產(chǎn)生抗性喪失(resistance breakdown)。因而在農(nóng)業(yè)生產(chǎn)上，用這些抗病基因進行傳統(tǒng)的抗病育種的效果往往不大。Resistance gene can be bred into crop plants to control disease but this approach has only limited successAdapted from Hammond &Jones, 2000ATP/GTPCC/TIR NB LRR分子內(nèi)互作、間接設別病原菌信號。結(jié)合和水解ATP，下游因子互作？LRR-N端結(jié)合互作因子：R蛋白激活調(diào)節(jié)？LRR-C端結(jié)合病原菌無毒蛋白或上游激發(fā)

24、子：信號接收 NB-LRR抗病蛋白的結(jié)構域與可能的功能 Model for R-protein activationTakken and Tameling (2009) ScienceNB-LRR抗病蛋白往往成對起功能Eitas and Dangl (2010) Curr Opin Plant Biol 13(4)Protein kinase：蕃茄的Pto蛋白既能識別病原菌的AvrPto, 也能傳遞抗病信號（在另一個LRR蛋白Prf的協(xié)助下）；水稻XA21(LRR-kinase), 其LRR接受病原菌Avr蛋白后，kinase被激活，傳遞抗病信號。 R和Avr蛋白(分子)的互作和抗性反應激發(fā)R

25、和Avr蛋白(分子)的互作符合一般的受體-配體模式:R蛋白作為受體一方面識別病原菌Avr配體，另一方面把這個信號向下游傳遞而激活防衛(wèi)反應。R蛋白往往作為受體信號Biochemical interpretations of the gene-for-gene modelCurrent opinion Microbiology 2002Hammond-Kosack and Parker: Current Opinion In Biotechnology (2003)R protein complex and activation Current Biol (2004)R-Avr蛋白(分子) 互作引

26、發(fā)抗性反應信號概況R/Avr+其它植物蛋白 植物過敏反應(細胞死亡、氧迸發(fā)) 防衛(wèi)反應基因激活植保素積累系統(tǒng)獲得性抗性植物防衛(wèi)反應激發(fā)和放大的順序III 植物防衛(wèi)的細胞、生化和分子生物學反應植物的過敏反應和細胞死亡ROS產(chǎn)生與HR細胞死亡NO和植物抗病性植物防衛(wèi)反應的其它因子與信號分子胞壁強化和胞外活性分子植物PR（pathogenesis-related)蛋白和其它DR(defense-related)蛋白水楊酸(SA) 和苯甲酸(BA) 茉莉酸和乙烯激素病毒感染與基因沉默1. 植物的過敏反應和細胞死亡過敏反應(hypersensitive response，HR):病原菌侵染點周圍寄主細胞

27、死亡，這種死亡類似于動物細胞病理和發(fā)育過程的程序性死亡(Programmed cell death) ；機理是寄主細胞受侵后迅速合成一些有毒成分和游離基團(如O2-、NO等)，以及病原菌所產(chǎn)生的毒性成分如一些降解酶、毒素等，使細胞產(chǎn)生胞質(zhì)崩潰、核DNA斷裂、內(nèi)容物外滲；HR細胞死亡的一個直接效應是把病原菌限制在侵染點附近而不擴散。隨著這個局部過程的延續(xù)，植物產(chǎn)生系統(tǒng)的反應，從而把防衛(wèi)信號傳送到遠處的組織 。 Hypersensitive response (cell death) 是大部分抗病反應的原初、局部（local)特征HR (disease resistance) and lesion

28、-mimics HRDisease HRDiseaseHRLesion-mimicAdapted from Hammond &Jones, 2000HR過程往往伴隨活性氧（reactive oxygen species, ROS)和NO的產(chǎn)生(氧迸發(fā): oxidative burst)HR原初反應局限于侵染點的細胞Plant nematode causes similar HR as microbial pathogensResistant SusceptibleAdapted from Hammond &Jones, 2000Key regulators and signals in dis

29、ease resistance ROSHRSANPR1(NIM1)SARPR geneInductionNORar1HSR203JSGT1Reactive oxygen species, NO and SA act as signal molecules in the establishment of plant defense responsesAdapted from Hammond &Jones, 2000Plants produce ROS probably through a plasma membrane-associated NADPH oxidase similar to th

30、e system in mammalian neutrophilsGp91phoxRacReductantAntioxidantOxidant Interactions in Redox Homeostasis and SignalingFoyer & Noctor (2005) Plant Cell (17) Sensitive Control of Cellular Redox Responses by Protein Thiol StatusFoyer & Noctor (2005) Plant Cell (17)Feedback regulation of the Arabidopsi

31、s HRZhang et al. 2004, MPMI2. 植物防衛(wèi)反應的其它因子與信號分子茉莉酸（JA) 乙烯(ethylene)壬二酸 (Azelaic acid)甘油-3-磷酸 (甘油-3-磷酸)BRGA和auxin胞壁強化和胞外活性分子植物PR蛋白有報道表明 vitamin C 在植物抗病性有重要功能（Pastori et al. 2003, Plant Cell)植物防衛(wèi)反應的重要信號分子 -水楊酸(SA) 在植物與病原菌非親和互作時，SA 和BA在侵染點周圍高濃度積累；SA在植物SAR的建立中，起著關鍵的信號分子的作用，但不是移動的信號分子。COOHOHMethyl salicyl

32、ate is a critical mobile signal for SARPark et al. (2007) Science 318:116 ?Azelaic acid is components of plant SARJung et al. (2009) Science 324:89?Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plantsChanda et al. (2011) Nature Genetics植物防衛(wèi)反應的其它因子與信號分子-茉莉酸（JA)和乙烯(ethylene

33、)JA和ethylene的含量在植物遭受病原菌和昆蟲侵染時會顯著增加；兩者在親和與非親和反應中均積累，不是抗性反應的必要成分；與SA信號途徑不一樣，兩者在傷害和昆蟲侵襲所產(chǎn)生的防衛(wèi)反應中起著較為重要的作用；乙烯與JA的信號往往是重合的，兩者為蛋白酶抑制子(PI)基因、某些PR基因和幾丁質(zhì)酶基因的激活所必需。Cross-talks between hormone signaling pathways Pieterse et al. (2009)3. 植物防衛(wèi)反應的其它因子與信號分子-植物PR蛋白這些基因的誘導在非親和反應中較為快速和強烈；有些基因的激活往往被看作植物與病原菌非親和性反應的一個特征

34、，例如擬南芥的PR1, PR2 和 PR5，而PR3和PR4主要與JA、乙烯、傷害途徑有關；某些PR蛋白是一些酶類如葡聚糖酶、幾丁質(zhì)酶，可直接降解病原真菌細胞壁，抑制其生長；防衛(wèi)素（defensin）或抗菌肽（antimicrobial peptides):小分子肽類（7kDa），富含半胱氨酸，也在受病原菌侵染后積累并有直接的抗菌作用。4. 系統(tǒng)獲得性抗性(Systemic acquired resistance, SAR)SAR建立于病原菌侵染后幾小時至幾十小時；非親和性病原細菌、真菌和病毒誘導的SAR，特征是SA的參與和遠距離組織中PR基因的誘導；機械傷害或昆蟲噬食引發(fā)的SAR，特征是有J

35、A和ethylene的合成和遠距離組織中傷害反應蛋白和蛋白酶抑制子的產(chǎn)生；線蟲誘發(fā)SAR介于兩種模式之間；非病原性的根瘤菌或腐生病原菌所誘導的系統(tǒng)抗性(induced systemic resistance，ISR)，特征是有JA和乙烯的合成，遠距離組織中PR基因沒有激活。Adapted from Hammond &Jones, 2000Systemically induced immune responsesPieterse et al. (2009)Systemic defense signaling networkHammond-Kosack and Parker: Current Op

36、inion In Biotechnology (2003)Key regulators and signals in disease resistance ROSHRSANPR1(NIM1)SARPR geneInductionNORar1HSR203JSGT1* Activated NPR1Inducers of Plant Systemic Acquired Resistance Regulate NPR1 Function through Redox ChangesMou et al. 2003, Cell 113:935-944Turnover of NPR1 Plays Dual R

37、oles in Regulating Plant ImmunitySpoel et al. (2009) Cell 137：860NPR3 and NPR4 are salicylic acid receptors through degradation of NPR1Fu et al. (2012) NatureNPR3 and NPR4 mediate degradation of NPR1SA receptors control NPR1 stability to regulate SAR and ETIThe npr3 npr4 double mutant is defective i

38、n SAR and ETIFu et al. (2012) NatureRAR1 and SGT1 antagonistically control R protein stability Holt III 2005, Science 309R protein complex and activation Current Biol 2004, 14(1)IV 植物防衛(wèi)反應的整體信號轉(zhuǎn)導和關鍵基因體系 (ETI)植物防衛(wèi)反應信號體系的主要特征受病原菌侵染后，細胞從正常的代謝轉(zhuǎn)到防衛(wèi)反應的次生代謝并激活防衛(wèi)反應基因，最少只需要幾分鐘；細胞和生化上的反應包括離子通道打開，磷酸化/去磷酸化，信號分子H

39、2O2，NO ，SA，JA和ET的合成；R-Avr基因介導的防衛(wèi)反應與非寄主抗性和激發(fā)子（elicitor) 誘導的抗性在機理上是類似的 ，而在防衛(wèi)反應表達的強度、時間上有所差異；植物防衛(wèi)反應的關鍵調(diào)控因子與動物中的防衛(wèi)或免疫體系有類似性；植物防衛(wèi)反應的信號傳導與其他環(huán)境脅迫反應途徑有交互作用（cross-talking）；抗病蛋白與病原菌配體結(jié)合激發(fā)抗性后，會迅速降解，從而使HR過程限制在一個小區(qū)域。Overview of biochemical signal pathwaysand coordinating plant defense responsesHammond & Jones (2

40、000)A. brassicicolaPythium sp.P. fluorescens colonizationAvirulent pathogens(Gene-for-Gene resistance)Systemic signalP. syringaeErisyphe sp.TIR-NBS-LRRR genesLZ-NBS-LRR R genesEDSNDR1DND1Lesion-minic genesHRCPR5CamalexinResistance toA. brassicicolaCPR6SSI1PAD4CPR1SANPR1PR 1, PR2, PR5P.syringae resis

41、tancefactor, SARResiostance to P. syringae,P. parasitica, Erisyphe sp.Adapted from Current Opinion in Plant Biology 1999, 2:280-290JA, COI1, JAR1Ethylene, ETR1, EIN2 NPR1ISRPDF1,2PR3, PR4Resistance toA. brassicicola,Pythium sp.Erisyphe sp.EDSsEDRsPHXsEDS5EDR1CDR1?Key genes and signal molecules contr

42、olling defense network in ArabidopsisV. ETI與PTI之間的互作Plant cells have two defense systems that detect bacterial pathogens:Basal defense system that recognizes PAMPs、MAMPs-PTI Disease-resistance (R) proteins to recognize type lll effector proteins -ETI Then, what is the link for the two systems?PAMP s

43、ignaling upstream of MAPKKK in Arabidopsis innate immunityPing He et al. Cell 125 (2006)Interplay between PTI and ETIHe P, Shan L, Sheen J (2007) Cell Microbiol Manipulation of Host MAPK Signaling Cascades by Bacterial Type III EffectorsShan et al. (2007) Cell Host & MicrobeJones and Dangl (2006) Na

44、ture 444 Evolution and quantitative output of the plant immune systemCo-evolution of host R genes and the pathogen effector complement-“Gene-for-Gene”的形成機制Jones and Dangl (2006) Nature 444 其他一些關鍵抗病機制NHO1 is required for general resistance (non-host resistance) against Pseudomonas bacteria Lu et al.

45、(2001) Plant CellKang et al. (2003) PNAS非寄主抗性作為基礎免疫的最原初機制Loss of a Callose Synthase Results in Salicylic Acid-Dependent Disease Resistance Callose or callose synthase negativelyregulates SA-dependent resistance to powdery mildew.Nishimura et al. (2003) ScienceAutophagy Regulates Programmed Cell Deat

46、h and disease resistanceLiu et al. (2005) CellLiu et al. (2005) CellAutophagy Regulates Programmed Cell Death Involvement of Autophagy and Cathepsins in RPM1-Conditioned HR Cell DeathDaniel Hofius et al. (2009) Cell 137, 773-783A Model for the Induction of Autophagic Cell Death upon TIR-NB-LRR and/o

47、r CC-NB-LRR Immune Receptor Activation by Bacterial Avirulence FactorsDaniel Hofius et al. (2009) Cell 137, 773-783RIN4 Negatively Regulates Basal Disease Resistance and PR Gene ExpressionMackey et al. (2002) Cell VI. 病毒感染與基因沉默Mechanism for plant virus resistance: transcriptional and posttranscripti

48、onal gene silence涉及siRNAA framework of the Dicer-initiated viral immunityAliyari & Ding (2009) Immunol Reviews, 227: 176-188 Virus miRNAs target host and viral mRNAsBryan R. Cullen (2009) Nature 457VII. 抗病性與其他重要農(nóng)藝性狀的 交互作用（cross-talk）Cooper et al. PNAS 100:4945-4950植物免疫激素途徑Model for Growth Homeostasi

49、s and Defense Response Controlled by BON1Yang and Hua (2004) Plant CellCross talk between defense and development?Navarro et al. (2006) Science 312Down-regulation of auxin signaling is required for Pto DC3000 disease resistance A dual regulation mechanism for disease resistance and susceptibility through modulation of salicylic acid and auxin signaling in Arabidopsis Zhang Z et al. (2007) Plant PhysiolYang D et al. (2008) Mol PlantGA also plays a negative role in plant basal defense Stomata function in innate immunity against bacterial invasionMelotto et