ғނ಍ीϩ - 效應蛋白RSp0213與PopP3在青枯病菌致病力之功能研究

ҁࣴزа SAS ಍ी೬ᡏ຾Չ಍ीϩ݋Ƕ܌ԖჴᡍϐኧᏵ࿶಍ी Student’s t-test ϩ݋Ǻ^ɀ߄Ңڀᡉ๱ৡ౦ (p<0.05) Ǵ^ɀɀ߄Ңڀཱུᡉ๱ৡ౦ (p<0.01)Ƕ

4. ख़ಔ፦ᡏᄬᑐᆶ୷Ӣၸໆ߄౜๵ਲ਼ࡌᄬ

4.1. DNA ᛏિᑗᏉጤႝݚ (Agarose Gel Electrophoresis) 4.1.1. ѦࢉᛏિᑗᏉጤႝݚϩ݋Ǻ

٩Ᏽటϩ݋ϐ DNA Тࢤελᆀڗ 0.8-1.2%ό฻ϐᛏિᑗᏉጤǴуΕ 0.5X TBE ጗ፂన (Tris-borate-EDTA buffer)Ǵ༾ݢу዗Կֹӄྋှࡕ᠗ኳǴࡑᏉڰϐࡕ ܭ 0.5X TBE ጗ፂనύа 100V/30 ϩដ຾Չႝݚϩ݋Ǵϩ݋ֹ౥ޑᛏિᑗᏉጤǴ

੆ݰܭऊ0.5 μg/mL Ethidium Bromide (EtBr) ྋనύ຾ՉѦࢉևՅ 15 ϩដǴܭ UV ᐩΠᔠຎ DNA ТࢤՏ࿼ᆶߝࡋǶ

4.1.2. ϣࢉᛏિᑗᏉጤႝݚપϯǺ

ӵటӣԏጤᡏϣ DNAǴ߾٬ҔϣࢉᛏિᑗᏉጤǶ٩Ᏽటપϯϐ DNA Тࢤε λᆀڗ 0.8-1.2%ό฻ϐᛏિᑗᏉጤǴуΕ 0.5X TAE ጗ፂన (Tris-acetate-EDTA buffer)Ǵ༾ݢу዗ԿֹӄྋှǴัհࠅࡕ؂ 10 mL уΕ 0.1 μL ޑ 50 mg/mL EtBrǴ འϬ᠗ኳǴࡑᏉڰࡕܭ 0.5X TAE ጗ፂనύа 100V/30 ϩដ຾Չႝݚϩ݋Ǵܭ UV ᐩΠᔠຎ DNA ТࢤՏ࿼ᆶߝࡋǶӵటϩ݋ъۓໆ RT-PCRǴ߾௦΢ॊБԄᇙբ TBE ϣࢉᛏિᑗᏉጤǶ

4.2. ᆫᆫӝ䁙ೱᙹϸᔈ (Polymerase Chain Reaction, PCR) 4.2.1. Taq PCR (Violet)Ǻ

٬Ҕ੝ۓЇηଛჹ (ߕ߄Ύ) аᆫӝ䁙ೱᙹϸᔈቚ൯Ҟ኱ DNA ТࢤǴϸᔈన ύа 1 ng ࢉՅᡏ DNA բࣁኳ݈ǴуΕ 2 μL ޑ 10X Taq PCR bufferǴ1.6 μL ޑ 2.5mM dNTPǴ1 μL ޑ 10 μM forward ЇηǴ1 μL ޑ 10 μM reverse ЇηǴ2 μL DMSOǴ0.2 μL ޑ Taq DNA ᆫӝ䁙 (5 U/μL)ǴуΕค๵ѐᚆηНԿ 20μLǶషӝ֡

Ϭࡕаᆫӝ䁙ೱᙹϸᔈᏔ຾ՉϸᔈǶϸᔈྕࡋᆶਔ໔ӵΠ܌ҢǺ

Cycle# Denaturation Annealing Polymerization

1 94°C /5 ϩដ

30 94°C /30 ࣾ T°C /30 ࣾ 72°C /t ϩដ

1 72°C /5 ϩដ

TǺྕࡋ٩ᏵЇη Tm ॶǹtǺਔ໔٩Ᏽቚ൯ߏࡋǴ1kb/minǶ

4.2.2. Q5® Taq PCR (Invitrogen)Ǻ

17 Cycle# Denaturation Annealing Polymerization

1 95°C /2 ϩដ

30 95°C /12 ࣾ T°C /30 ࣾ 72°C /t ϩដ

1 72°C /3 ϩដ

TǺྕࡋ٩ᏵЇη Tm ॶǴtǺਔ໔٩Ᏽቚ൯ߏࡋǴ2kb/minǶ

࿶ PCR ᘉቚϐౢނǴڗ 2 μL ຾Չ 1% ᛏિᑗᏉጤႝݚϩ݋Ǵаዴۓ DNA ߏࡋǶ

4.3. ፦፦ᡏ๧ڗ (Plasmid Purification) 4.3.1. εဉఎ๵፦ᡏ๧ڗǺ

ճҔ Gene-SpinTM Miniprep Purification Kit (Protech technology) ຾Չεဉఎ

๵፦ᡏ๧ڗǴӆа 1 % ᛏિᑗᏉጤႝݚϩ݋ዴۓ DNA ᐚࡋǴߥӸܭ-20°C ഢҔǶ уΕ฻ᡏᑈ phenol: chloroform: isoamyl alcohol (25: 24: 1)Ǵ΢ΠϸᙯషϬǴа 13000 rpm ࠻ྕᚆЈ 10 ϩដ٬ྋనϩቫǶλЈڗр΢ቫనԿཥޑ༾ໆᚆЈᆅǴу Ε฻ᡏᑈޑ isopropanol Ϸ 1/10 ᡏᑈޑ 3M NaOAcǴܫ࿼ܭ-20°CǴ10 ϩដࡕа 13000 rpm ࠻ྕᚆЈ 10 ϩដǴѐନ΢మనǴуΕ 500 μL 75%ଚᆒؑࢱ؈ᐘނǴӆ ᚆЈ 10 ϩដࡕѐନ΢మనǴѺ໒༾ໆᚆЈᆅ΢ᇂ٬ଚᆒචว 5 ϩដǴуΕ 40 μL ค๵ѐᚆηНӣྋ፦ᡏ DNAǴ٠уΕ 2 μL RNaseA (20 μg/mL)Ǵ࿼ܭ 37ɗ 30 ϩ ដǴஒ๧ڗޑ፦ᡏ DNA а 1 % ᛏિᑗᏉጤႝݚ຾Չϩ݋ (Sambrook et al., 1989)Ƕ

4.4. DNA પપϯ (DNA Purification)

ճҔ Micro-Elute DNA clean/Extraction Kit (GeneMark) ຾ՉጤᡏપϯǴӆа 1

% ᛏિᑗᏉጤႝݚϩ݋аዴۓ DNA ᐚࡋǶ

4.5. DNA ज़ڋ䁙੃ϯНှ (DNA Digestion)

ట຾Չख़ಔ፦ᡏϐ DNAǴၩᡏᆶҞ኱ DNA Тࢤ຾Չज़ڋ䁙 (New England Biolabs) ੃ϯНှǴϸᔈనᕴᡏᑈࣁ 50 μLǹటϩ݋፦ᡏ DNA ТࢤελǴϸᔈన ᕴᡏᑈ߾ࣁ20 μLǶϸᔈనϣ৒ӵΠǺ

Component 20 μL reaction 50 μL reaction

DNA 0.5 μg 4 μg

10X NEB Buffer 2 μL 5 μL

100X BSA 0.2 μL 0.5 μL

Restriction enzymes 2-4 units 4-8 units

ddH2O ံԿᕴᡏᑈ20 μL ံԿᕴᡏᑈ50 μL

ϸᔈనషӝ֡ϬࡕǴܭሇનϸᔈྕࡋ 37°C Πϸᔈ 2-4 λਔ (੝ਸሇનϸᔈྕࡋǺ SmaI ࣁ 25°C/ SfiI ࣁ 50°C)Ƕӧᄬᑐख़ಔ፦ᡏਔǴ٩ჴᡍሡ؃ஒҞ኱ DNA у዗

65°C ܈ 80°C ࡭ ុ 20 ϩ ដ ѐ ନ ज़ ڋ 䁙 ሇ ન ࢲ ܄ Ǵ ӆ у Ε ᡵ ܄ ᕗ ለ 䁙 (calf instestinal alkaline phosphatase, CIP) (New England Biolabs) 2 units ѐନ DNA 5 ᆄ΢

ޑᕗለਥǴܭ 37°C Πϸᔈ 1 λਔǶനࡕа 1 % ᛏિᑗᏉጤႝݚϩ݋Ƕ

4.6. DNA Тࢤௗӝ (DNA Ligation)

ஒज़ڋ䁙ሇનϪപપϯࡕޑҞ኱ DNA ᆶၩᡏ DNA ϐವԸКٯፓ᏾ࣁ 5: 1 ܈ 20: 1ǴDNA ᕴᐚࡋεܭ 100 ngǴуΕ 1 μL ޑ 10X T4 ligation buffer Ϸ 0.2 μL ޑ T4 DNA ligase (400 U/μL) (New England Biolabs)Ǵаค๵ѐᚆηНံԿᕴᡏᑈ 10 μLǴ֡Ϭషӝࡕܭ࠻ྕΠ຾Չௗӝϸᔈ 16 λਔǶ

4.7. TOPO® ፦ᡏᄬᑐ (TOPO® Cloning)

ஒ PCR ᘉቚϐౢނճҔ pCR®8/GW/TOPO® TA Cloning® Kit (Invitrogen)Ǵ ஒҞ኱ DNA ᄬᑐܭ pCR®8/GW/TOPO®ၩᡏ΢Ƕஒཥᗲޑ PCR ౢނ 4.5 μL уΕ 1 μL salt solution ᆶ 0.5 μL pCR®8/GW/TOPO®ၩᡏǴ࠻ྕΠϸᔈԿϿ 1 λਔǶ

4.8. LR ख़ख़ಔϕඤϸᔈ (LR Recombination)

ճҔGateway® LR Clonase™ II Enzyme Mix (Invitrogen) ஒҞ኱ DNA аӕྍ

ख़ಔϕඤޑБԄඤԿట٬Ҕޑၩᡏ΢Ƕҁࣴز٬Ҕ pCR8®/GW/TOPO® ၩᡏ຾Չ ӕྍख़ಔϕඤǴϸᔈనಔԋӵΠǺ

LR recombination components Amount (μL) Entry clone (50-150 ng)

Destination vector (150 ng/μL) LR Clonase^TMII enzyme mix TE buffer, pH 8.0

1-8 μL 1 μL 1 μL

ံԿᕴᡏᑈ10 μL ஒϸᔈనషӝ֡ϬࡕǴ࿼ܭ࠻ྕԿϿ 1 λਔ຾ՉϸᔈǶ

4.9. εဉఎ๵യҺಒझ዗ҶլᙯࠠբҔ (Heat Shock Transformation)

ҁࣴزаεဉఎ๵ DH5α ๵ਲ਼຾Չ፦ᡏख़ಔჴᡍǶڗᕴᡏᑈ΋ъϐௗӝౢނ

4.11. ႝႝऀϾᙯࠠբҔ (Electroporation Transformation)

ஒയҺಒझᆶऊ 50 ng ፦ᡏ DNA షӝࡕǴܫΕ٣ӃܭӇ΢Ⴃհޑ cuvette ύǴ

YEP ୻Ꭶ୷ /εဉఎ๵ǺLB ୻Ꭶ୷)Ǵߙ࢏ੰ๵ᆶၭఎ๵ܭ 28°C Π୻Ꭶ 2-3 ϺǴ εဉఎ๵߾ܭ 37°C Π୻Ꭶ 16 λਔǴࡷڗൂ΋๵ပǶ

4.12. ߙߙ࢏ੰ๵ࢉՅᡏ DNA ๧ڗ (Bacterial Genomic DNA Purification)

ࡷᒧൂ΋๵ပаబу੝ۓלғનϐ 523 నᡏ୻Ꭶ୷ܭ 28°C Π 200 rpm ႖ڹ᎜ Purification and Protein Localization Assay)

ڗ 4 ຼε N. benthamiana ϐԃᇸယТǴаΘТჄрኧၰ໾αǴ࿼ܭς֖ 25 mL enzyme solution ϐ୻ᎦҝύǴenzyme solution ሡӃа 50°C Ⴃ዗ 10 ϩដࡕհࠅ Կ࠻ྕǴ๨૛ယТӧ࠻ྕΠа 50 rpm ໵ས᎜ᕏ 3 ঁλਔࡕǴа༟ጤᅀᆅ֎ڗ֖চ Plasmid Midiprep Purification Kit (GeneMark) ๧ڗϐଯᐚࡋ፦ᡏ DNA (p2GWF7.0-RSp0213-GFP ᆶಒझጢ኱૶ pm-rkǵp2GWF7.0-PopP3-GFP ᆶಈဏᡏ኱૶ pBIN-mt-rk) (Nelson et al., 2007) 30 mg Ъᡏᑈόεܭ 20 uLǴуԿཥ༟ጤᚆЈᆅǴ٠у

22 microscope ᢀჸ (modified from Treuter et al., 1993)Ƕ

5.2. ਏਏᔈೈқܭ๨૛ယТಒझ০ပՏ࿼ᔠෳ (N. benthamiana Leaves Protein Localization Assay) λਔ໵སޑ෌ނғߏጃΒԿΟϺǴаᑻӀᡉ༾᜔ Confocal microscope ᢀჸ๨૛ယ ङǶ

5.3. ߙ࢏ੰ๵ࢥΚෳ၂ (Virulence Test)

ஒߙ࢏ੰ๵ Pss4 ܈ Pss190 ഁғࠠ๵ਲ਼܈୷Ӣকନϐ๵ਲ਼ǴճҔ߄౜ၩᡏ

๺঒ǹ2 ࣁΒԿΟТယТ๺঒ǹ3 ࣁନΑഗယѦځᎩယТࣣ๺঒ǹ4 ࣁӄ೽ယТࣣ

๺঒ǹ5 ࣁ෌ਲ਼ॹҷԝΫǶ

6. ෌෌ނᡏอኩ߄౜ਏᔈೈқ୷Ӣᆶځфૈ܄ࣴز

6.1. ๨૛ယ೽୷Ӣอኩ߄౜ (Transient Overexpression in Tobacco)

ճҔ஥ԖΒϡ߄౜ၩᡏ (binary vector) pCAMBIA1300 ϐၭఎ๵ GV3101 ຾Չ

୷Ӣอኩ߄౜Ƕ-80°C ཥᗲჄ๵Ǵܭ 28°C ғߏጃ୻ᎦٿϺǴаబу kanamycin ᆶ

6.2. พनอኩ܄ੰࢥᇨᏤ୷Ӣၸໆ߄౜ (Virus-mediated Gene Overexpression) а߄౜ potato virus X (PVX) ϐΒϡၩᡏ (pSfinX) းၩਏᔈೈқ୷ӢǴӆճҔ

஥ԖՔᒿೈқ (chaperon) ߄౜፦ᡏ (pSoup) ϐၭఎ๵ MOG101 ๵ਲ਼߄౜ (Takken et al., 2000)Ƕ-80°C ཥᗲჄ๵Ǵܭ 28°C ғߏጃ୻ᎦٿϺǴаబу kanamycinǵ tetracyclin ᆶ rifampin ϐ 3 mL YEP నᡏ୻Ꭶ୷Ǵ28°C Π 200 rpm ႖ڹ᎜ᕏ୻Ꭶǹ

6.3. ෌෌ނᡏอኩ߄౜୷Ӣᔠෳ

6.3.1 ෌ނ RNA ๧ڗ (RNA Extraction from Plants)Ǻ

ԏڗаၭఎ๵อኩ୷Ӣ߄౜ٿϺϐ๨૛ယТǴ܈อኩ܄ੰࢥᇨᏤ୷Ӣၸໆ߄

౜ࡕ 14 ϺϐพनယТǴऊ 0.1 g ෌ނಔᙃǴа Total RNA Mini Kit (LabPrep^TM) ܜ ڗ෌ނ RNAǶ٠а RNase-free DNase I kit (Promega) ຾Չ DNA ੃ϯНှբҔǺڗ 5 μg ޑ RNAǴуΕ 5 μL (1U/μL) ϐ DNase ሇનǵ5 μL RNase-free DNase 10X bufferǴа DEPC-H2O ံԿനಖᡏᑈ 50 μLǴషӝ֡ϬࡕуΕ༾ໆᚆЈᆅϣǴ࿼ܭ 37 ɗ ϸ ᔈ 1.5 λ ਔ Ƕ у Ε 150 μL ϐ DEPC-H2O ᆶ 200 μL RNA Ҕ phenol:chloroform (3:1) (pH= 4)Ǵϸᙯ֡Ϭషӝ 5 ϩដǴа 13000 rpm 4°C ᚆЈ 15 ϩដǴڗ΢మనԿཥޑ 1.5 mL ༾ໆᚆЈᆅǴуΕ 20 μL ޑ 3 M ᎉለ໊ (pH 5.3) Ϸ 1 mL 100%ଚᆒ٠షӝ֡ϬǴ࿼ܭ-80°C ႖ڹᓉ࿼؈ᐘǶа 4°C 13000 rpm ᚆЈ 20 ϩដǴѐନ΢మనǴуΕ500 μL 75% RNA Ҕଚᆒమࢱ؇ᐘނǴа 13000 rpm ᚆ Ј 2 ϩដࡕѐନ΢మనǴख़ፄԜ؁ᡯ΋ԛǴᄇۭ౽ନ΢మనࡕǴܭค๵ᏹբѠϣ චวଚᆒ 2 ϩដǴа 15 μL DEPC-H2O ӣྋ RNAǴߥӸԿ-80°C ഢҔǶ

6.3.2 ϸᙯᒵᆫӝ䁙ೱᙹϸᔈ (Reverse Transcription PCR, RT-PCR)Ǻ

а Reverse Transcription System kit (Promega) ຾Չϸᙯᒵᆫӝ䁙ೱᙹϸᔈǶڗ 1 μg RNAǴа DEPC-H2O ံԿ 10.2 μLǴа 70°C у዗ 10 ϩដǴҥջ࿼ܭӇ΢ 3 ϩដǶуΕ4 μL 25 mM MgCl2ǵ2 μL 10X reverse transcription bufferǵ2 μL 10 mM dNTP mixǵ1 μL Oligo (dT)15 (0.5 μg/μL)ǵ0.4 μL recombinant RNasin ribonuclease inhibitorǵ0.4 μL Avian myeloblastosis virus (AMV) reverse transcriptase (15U/μL)Ǵ షӝ֡ϬࡕܫΕᆫӝ䁙ೱᙹϸᔈᏔύǴа 42°Cǵ1.5 λਔ຾ՉϸᔈǴϐࡕа 95°Cǵ 5 ϩដಖЗሇનϸᔈǶӝԋϐ cDNA аค๵ѐᚆηНံԿᕴᡏᑈ 80 μLǴߥӸܭ-20°C ഢҔǶ

6.3.3 ъۓໆ RT-PCR (Semi-quantitative RT-PCR, sqRT-PCR)Ǻ

ڗ 100-200 ng cDNA բࣁኳ݈ǴуΕ 2 μL ޑ 10X Taq PCR bufferǴ1.6 μL ޑ 2.5mM dNTPǴ1 μL ޑ 10 μM forward ЇηǴ1 μL ޑ 10 μM reverse ЇηǴ0.2 μL ޑ

Taq DNA ᆫӝ䁙 (5 U/μL)ǴуΕค๵ѐᚆηНԿ 50μLǶషӝ֡Ϭࡕаᆫӝ䁙ೱᙹ ϸᔈᏔ຾ՉϸᔈǶϸᔈྕࡋᆶਔ໔ӵΠ܌ҢǺ

Cycle# Denaturation Annealing Polymerization

1 94°C /5 ϩដ

6.3.4 ջਔۓໆ Real-time PCRǺ

! ҁჴᡍࣁ٬Ҕྕࡋఊࡋਡለջਔۓໆୀෳس಍ (Bio-Rad Real-Time PCR Detection SystemsǴࠠဦ BIO-RAD MyiQTM)ǴReal-time PCR ϸᔈ၂Ꮚ߾ࢂᖼວ KAPA SYBR® qPCR Kit (Universal, ABI Prism®, Bio-Rad iCycler™, or Roche LightCycler™ 480)Ƕჴᡍ߻Ѹ໪Ӄ຾Չ No Template Control (NTC) ෳ၂Ǵዴᇡࢂ

ցԖ primer dimmerǶҁࣴزϐჴᡍϸᔈᕴᡏᑈࣁ 18 μLǴჴᡍБݤӵΠǴ२Ӄ cDNA ีញࣁ 30 ng/μL ࡕڗ 8 μLǴуΕ٣Ӄଛ࿼ӳޑ cocktailǴϣ֖Ԗ 9 μL 2x KAPA SYBR^® FAST qPCR Master MixǴ0.5 μL ޑ forward primer (10 μM)Ǵ0.5 μL ޑreverse primer (10 μM)Ǵషӝ֡ϬࡕܫΕ Real-Time PCR ϸᔈᏔύ຾Չ PCR ϸ ᔈǴӆа Bio-Rad ࣴวϐ iQ™5 Optical system version 2.0 ހ೬ᡏǴ຾Չϩ݋Ƕ

Real-time PCR (qPCR)

Reaction Component Amount (μL) Template cDNA (100 ~200 ng) 8 μL

Forward primer (10 μM) 0.5 μL Reverse primer (10 μM) 0.5 μL 2x KAPA SYBR® FAST qPCR Master Mix 9 μL

Total volume 18 μL

Real-time PCR program Cycling step Temperature & time Hot start Initial denaturation 3 min at 95°C PCR Denaturation 10 sec at 95°C

40 cycles Annealing 30 sec at 55°C (ຎ primer Զۓ)

Melting curve 1 min at 95°C 1 min at 55°C

10 sec at 95°C 81 cycles

ಃ

ಃΟക ่݀

I. ե/ύࢥΚ๵ਲ਼੝Ԗϐ T3Es RSp0213 ᆶ RSc3174 ӧߙ࢏ੰ๵ठੰΚϐфૈࣴز

1. RSp0213 ᆶ RSc3174 ӧςֹӄှׇϐߙ࢏ੰ๵๵ਲ਼ύϐϩթ

ࣁ߃؁ᕕှ RSp0213 ᆶ RSc3174 ϐӸӧᆶցࢂցᆶ๵ਲ਼ࢥΚ߄౜Ԗ࣬ᜢ܄Ǵ

ࡺаҞ߻ςှׇֹ᏾܈ςԖ୷Ӣಔ૛ዺ (genome draft) ޑ 22 ਲ਼ߙ࢏ੰ๵๵ਲ਼ࣁჹ ຝǴϩ݋ RSp0213 ᆶ RSc3174 ϐӸӧ௃׎ (߄΋)Ƕ่݀ᡉҢӧϩ݋ޑ๵ਲ਼ύǴύ ࢥΚ๵ਲ਼ GMI1000 ᆶ Pss4ǵեࢥΚ๵ਲ਼ Pss216 Ϸ҂ޕࢥΚ๵ਲ਼ PSI07 ڀԖ RSp0213ǴԶ GMI1000ǵPss216 Ϸ PSI07 ๵ਲ਼ΨڀԖ RSc3174 (߄΋)Ƕҁࣴز࠻

Ӄ߻ճҔ Southern blotting ᆶ genomic DNA PCR ᔠෳ܌ள่݀Ψᕇ΋ठ่݀ (ߕ კΟ)ǴЪӧեࢥΚ๵ਲ਼ᆶ GMI1000 ύ RSp0213 ᆶ RSc3174 ϐữ୷ለׇӈ൳Яֹ

ӄ࣬ӕ (ߕკѤ)Ǵࡺ೭ٿঁ T3Es ӧύࢥΚϷեࢥΚ๵ਲ਼ύࣣڀԖଯࡋߥӺ܄Ƕ

2. RSp0213 ک RSc3174 ჹߙ࢏ੰ๵ठੰΚϐቹៜ

ࣁΑ຾΋؁ᡍ᛾ RSp0213 ᆶ RSc3174 ϐӸӧᆶ๵ਲ਼ठੰΚϐ߄౜ዴჴԖᜢೱǴ

ࡺаၸໆ߄౜ᆶ୷Ӣকନ฻ࣴز฼ౣ຾Չځфૈϩ݋Ƕ२ӃǴ೭ٿঁ T3E ୷Ӣঁ

ձᒧ෗ډ low-copy-number ϐ pUFR047 ၩᡏǴ٠ϩձᙯ౽ډচҁόڀԖ၀ T3Es ޑଯࢥΚ๵ਲ਼ Pss190 ύ߄౜ǴҔޜၩᡏ (empty vector) ᙯࠠϐ๵ਲ਼଺ࣁჹྣಔǴ

ௗ๱ෳ၂ᙯࠠ๵ਲ਼ӧלੰพनࠔس H7996 ΢ϐठੰΚ߄౜ (კ΋)Ƕ่݀ᡉҢ Pss190 ๵ਲ਼߄౜ RSp0213 ࡕځठੰΚዴჴ཮ᡉ๱Ӧफ़ե (p<0.01)Ǵՠ Pss190 ๵ਲ਼ ߄౜ RSc3174 ࡕ߾ځठੰΚᗨԖΠफ़ޑᖿ༈Ǵՠᆶޜၩᡏᙯࠠ๵ਲ਼໔٠όڀᡉ๱

ৡ౦ (p>0.05)Ƕࡺࡕុჴᡍஒа RSp0213 ࣁЬाࣴزჹຝǶ

࿶ճҔ NCBI Genome ၗ਑৤຾ՉׇӈКჹ (blastp) ว౜ӧߙ࢏ੰ๵ Pss4 ύ ٠ ค RSp0213 ϐ paralogs Ǵ ࡺ ߃ ؁ ௨ ନ Ԗ ᆶ RSp0213 ф ૈ ख़ ፄ (functional redundancy) ϐ paralogs ޑёૈ܄ǶࣁΑ຾΋؁ዴᇡ RSp0213 ჹߙ࢏ੰ๵ठੰΚޑ ख़ा܄ǴӧύࢥΚ๵ਲ਼ Pss4 ύஒ RSp0213 কନǴ٠Кၨ܌ள๵ਲ਼ᆶഁғࠠ๵ਲ਼ӧ གੰพनࠔس L390 ΢ϐठੰΚ߄౜Ƕ่݀ᡉҢஒ RSp0213 কନࡕځठੰΚܴᡉ ଯܭഁғ๵ਲ਼ (კΒ)Ǵᆕ΢่݀ǴRSp0213 ϐӸӧόճܭߙ࢏ੰ๵ϐठੰΚ߄౜Ƕ

3. RSp0213 ೈೈқӧ෌ނಒझύϐ০ပՏ࿼

ࣁΑ߃؁ᕕှ RSp0213 ຾Ε෌ނಒझࡕёૈЇଆޑբҔǴӃճҔ Softberry ᆛ ઠ (http://linux1.softberry.com/berry.phtml) ϩ݋ RSp0213 ёૈڀԖϐ functional domains ᆶӧ෌ނಒझύёૈޑ০ပՏ࿼Ƕ่݀ᡉҢ RSp0213 ޑ N ᆄ 47 ঁữ୷ለ ёૈࢂᏤӛᴏ两 (targeting peptide) Ϸૻ৲ᴏ两 (signal peptide, SP)ǴЪႣෳӧԜࢤ

ૻ৲ᴏ两ύڀԖၠጢТࢤ (transmembrane segments)Ǵ௢ෳ RSp0213 ёૈՏܭಒझ ጢ΢ (ߕკϤ)Ƕ

ࣁΑᡍ᛾ RSp0213 ӧ෌ނಒझϣϐ০ပՏ࿼Ǵࡺаᆶพनӕࣁनࣽ෌ނޑ༝

ယ๨૛ (Nicotiana benthamiana) ଺ࣁჴᡍ׷਑຾Չ RSp0213 ϐۓՏϩ݋Ƕ่݀ᡉ Ң RSp0213::GFP ӧ๨૛চғ፦ᡏ (protoplasts) ύϐ০ပՏ࿼ᆶಒझጢ኱૶ೈқ (membrane marker) PIP2A::mcherry ϐϩթ΋ठ (კΟ)ǴԶЪǴӧ຾ՉቹႽ᠄კೀ

౛ࡕ (fold over) ёᢀჸډ RSp0213::GFP և౜ಈރ่ᄬϩթǴ௢ෳ RSp0213 ᔈࢂ

০ပӧ෌ނಒझጢ΢ϐೈқ፦໣ဂ (protein cluster) ԶևᗺރϩթǶќѦǴаၭఎ

๵ ᇶ շ ݙ ৔ ݤ ஒ RSp0213::GFP ߄ ౜ ܭ N. benthamiana ယ ೽ ࡕ Ψ ว ౜ RSp0213::GFP ϐ০ပՏ࿼ᆶ PIP2A::mcherry ϩթ΋ठ (კѤ)ǴᡉҢ RSp0213 ᔈ

ࢂ০ပӧ෌ނಒझጢ΢Ƕ

4. RSp0213 ၸໆ߄౜ჹ෌ނϐቹៜ

ࣁΑᕕှ RSp0213 ჹ෌ނϐёૈբҔǴࡺอኩ߄౜ஒჹྣಔᆶ 35S::RSp0213 ᏤΕ๨૛ᆶพनǴ٠ᢀჸ෌ނϐϸᔈǶӧ N. benthamiana ᆶ N. tabacum W38 ယТ

΢ճҔၭఎ๵ᇶշݙ৔ݤၸໆ߄౜ RSp0213Ǵ่݀ᡉҢǴӧ N. benthamiana ယТ

΢߄౜ RSp0213 ࡕ 48 λਔϣջ཮ЇଆܴᡉޑಒझԝΫ (programmed cell death, PCD) (კϖ A)ǴԶޜၩᡏჹྣಔೀ౛߾҂೷ԋቹៜǹӧ N. tabacum W38 ယ೽߄

౜ RSp0213 ёᢀჸډಒझԝΫ౜ຝ (კϖ BǴѰΒკ)Ǵՠӧ߄౜ RSp0213 ޑచҹ Πӕਔೀ౛ߙ࢏ੰ๵ँᡂਲ਼ Pss4 hrpG^-߾཮ౢғቃਗ਼ޑ PCD ϸᔈ (კϖ BǴѓΒ კ)Ƕ

аੰࢥᇨᏤ୷Ӣ߄౜ (Virus-mediated gene overexpression, VMGO) ฼ౣǴ٬Ҕ PVX (Potato virus X) ୷ Ӣ ߄ ౜ ၩ ᡏ Ǵ ӧ ל ੰ พ न ࠔ س H7996 س ಍ ܄ ߄ ౜ 35S::RSp0213 ࡕ (კϤ)ǴΨӕኬ཮ӧพनယ೽ЇଆܴᡉಒझԝΫ (კΎ A)Ǵ٠ᢀ

ჸ ډ ೷ ԋ พ न ෌ ਲ਼ ғߏ ڙ ׭ Զ ᡂ ࿖ λ (კΎ B)ǴᡉҢพनёૈڀԖёᒣᇡ RSp0213 ϐᐒڋǶ

5. ӧӧพन߄౜ RSp0213 ჹੰ্ϸᔈϐቹៜ

ࣁΑᕕှ߄౜ RSp0213 ࢂց཮ቹៜพनϐߙ࢏ੰϸᔈǴճҔ PVX ୷Ӣ߄౜

ၩᡏǴӧלੰࠔسพन H7996 س಍܄߄౜ 35S::RSp0213 ࡕǴаβᝆ዆ឲБԄௗᅿ ଯࢥΚߙ࢏ੰ๵ਲ਼ Pss190Ǵ٠ᢀჸੰำว৖Ƕ่݀ᡉҢس಍܄߄౜ RSp0213 ཮ཱུ

ᡉ๱Ӧफ़ե Pss190 ೷ԋϐพन๺঒௃׎ (p<0.01) (კΖ)Ƕ

6. ӧพन߄౜ RSp0213 ჹٛᑇϸᔈ࣬ᜢ኱ᇞ୷Ӣ (marker genes) ߄౜ϐቹៜ җ΢ॊჴᡍёޕǴ߄౜ RSp0213 ཮ቹៜพनϐߙ࢏ੰϸᔈǴࣁΑ຾΋؁ᙶమ

ੰ্ٛᑇૻ৲໺ᏤӵՖڙቹៜǴࡺᔠෳس಍܄ၸໆ߄౜ RSp0213 ϐלੰพनࠔس H7996 ύس಍܄ٛᑇ಻ᅟᆾᆶಒझԝΫ࣬ᜢࡰ኱܄኱ᇞ୷Ӣϐ߄౜ (ߕ߄Ύ)Ƕ่

݀ᡉҢǴᆶ߄౜ gfp ϐ௓ڋಔ࣬КǴ߄౜ RSp0213 ཮ε൯ࡋӦᇨᏤ SAǵET Ϸಒ झ ԝ Ϋ (PCD) ฻ૻ৲ ໺ Ꮴ ϐ ኱ᇞ ୷ Ӣޑ߄ ౜ ( კ ΐ Aǵ C ǵD) Ǵ Զ फ ಹ ለ (jasmonic acid, JA) ӝԋϐ࣬ᜢ኱ᇞ୷Ӣޑ߄౜߾ڙډ׭ڋ (კΐ B)ǶᡉҢӧพन ߄౜ RSp0213 ཮ᇨᏤ෌ނޑٛᑇϸᔈ୷Ӣϐ߄౜Ƕ

7. ،ۓ RSp0213 ೈқӧ෌ނಒझύϐ০ပՏ࿼ޑख़ा୔ࢤϩ݋

ࣁΑᔠᡍ RSp0213 ޑ N ᆄ 47 ঁữ୷ለᄬԋϐૻ৲ᴏ两 (SP) ჹܭځ০ပܭ෌

ނಒझጢϐѸा܄Ǵࡺ຾ՉТࢤմନ (კΜ A)Ǵ٠ᢀჸ RSp0213::GFP ӧ N.

benthamiana protoplast ᆶယ೽ϐϩթǶ่݀ᡉҢӄߏ RSp0213::GFP ϐϩթᆶಒझ ጢ኱૶ೈқ PIP2A::mcherry ΋ठǴԶմନ N ᆄૻ৲ᴏ两 21 ܈ 47 ঁữ୷ለϐ RSp0213::GFPǴځϩթ߾ᆶ free GFP ΋ठ (კΜ BǵკΜ΋)Ƕࡺ RSp0213 ޑ N ᆄ 47 ঁữ୷ለᄬԋϐૻ৲ᴏ两ӧनࣽ෌ނಒझϐಒझጢ০ပՏ࿼ዴჴ࣬྽ख़ाǴЪ Тࢤմନ N ᆄനڀߥӺ܄ޑ 21 ঁữ୷ለϐ RSp0213 ջѨѐځ০ပӧ෌ނಒझϐ ಒझጢՏ࿼Ƕ

8. RSp0213 ০ပܭ෌ނಒझጢჹځфૈϐख़ा܄

ࣁΑឍܴ RSp0213 Տܭ෌ނಒझጢჹځфૈࢂցڀख़ा܄Ǵࡺа VMGO ஒ ӄߏᆶТࢤմନϐ 35S::RSp0213 ӧלੰพनࠔس H7996 س಍܄Ӧ߄౜Ǵ٠ᔠෳพ नϐғߏᆶߙ࢏ੰϸᔈࢂցׯᡂǶ่݀ᡉҢӧ߄౜ӄߏϐ RSp0213 ӧพनယ೽೷ PopP3ǴЪឦܭӧੰচ๵ቶݱӸӧޑ YopJ superfamily ԋ঩ (Lewis et al., 2011)Ƕߙ

࢏ੰ๵ GMI1000 ڀԖΟঁឦܭ YopJ superfamily ޑԋ঩Ǵϩձࣁ PopP1ǵPopP2 ᆶ PopP3Ǵځύ PopP1 ᆶ PopP2 ڀ Avr protein ੝܄ (Lavie et al., 2002; Deslandes et al., 2003)Ǵՠ PopP3 ߾фૈ҂ޕǶҁࣴزӃ߃؁ϩ݋ YopJ superfamily ԋ঩ϐӸ ӧᆶցࢂցёૈᆶߙ࢏ੰ๵ठੰΚ߄౜ԖᜢᖄǴЪᕕှ၀ৎ௼ԋ঩໔ϐׇӈ࣬՟

܄ᆶёૈϐфૈߥӺ܄Ƕ

२ӃǴаҞ߻ςֹӄှׇޑ 22 ਲ਼ߙ࢏ੰ๵๵ਲ਼ࣁჹຝǴϩ݋ PopP ৎ௼ԋ঩

ϐӸӧ௃׎ (߄Β)Ƕ่݀ว౜ӧ Pss190ǵPss1308ǵGMI1000ǵPss216ǵFJAT_91ǵ FJAT_1458 ǵ Po82 Ϸ SD54 ๵ ਲ਼ ڀ Ԗ PopP1 ǹ Pss1308 ǵ GMI1000 ǵ Pss216 ǵ FQY_40ǵFJAT_91ǵFJAT_1458ǵPo82ǵK60ǵP673ǵCMR15 Ϸ SD54 ๵ਲ਼ڀԖ

PopP2ǹPss190ǵGMI1000 Ϸ Y45 ๵ਲ਼ڀԖ PopP3Ǵՠ GMI1000 ޑ popP3 Ԗ΋ၢ

៌ηකΕځύǶҗܭନ PopP3 ϐѦޑځд PopPs ৎ௼ԋ঩ϩණܭӚߙ࢏ੰ๵๵ਲ਼ ύǴคݤᘜયѬॺޑӸӧᆶߙ࢏ੰ๵๵ਲ਼੝܄ϐᜢᖄ܄Ƕ

аߙ࢏ੰ๵ GMI1000 ๵ਲ਼ޑ PopP1ǵPopP2 ᆶ Pss190 ๵ਲ਼ޑ PopP3 ຾Չữ

୷ለׇӈٿٿКჹǴ่݀ᡉҢӄߏׇӈ࣬ӕࡋ (identity)ǺPopP1 ᆶ PopP2 ࣁ 11.6%ǴPopP1 ᆶ PopP3 ࣁ 15%ǴPopP2 ᆶ PopP3 ࣁ 11.7%ǹԶሇન໽ϯ୔ޑׇӈ

࣬ӕࡋ߾ࢂǺPopP1 ᆶ PopP2 ࣁ 30%ǴPopP1 ᆶ PopP3 ࣁ 33%ǴPopP2 ᆶ PopP3 ࣁ 26% (ߕკΎ)ǶΟޣϐ໔คፕӄߏ܈ሇન໽ϯ୔ޑữ୷ለׇӈ೿ৡ౦ࣗεǴ௢

ෳ YopJ superfamily ޑԋ঩ӧߙ࢏ੰ๵ύΨ೚ӚԖځфૈǶ

຾΋؁Ǵӧ NCBI Genome ၗ਑৤ڀԖ PopP3 ޑ܌Ԗߙ࢏ੰ๵๵ਲ਼ (х֖୷Ӣ ಔ҂ૈֹӄှׇޑߙ࢏ੰ๵๵ਲ਼) ύǴ12 ਲ਼ߙ࢏ੰ๵๵ਲ਼ (х֖ Pss190) ڀԖ PopP3ǴԶჹ PopP3 ೈқ፦ϐׇӈ຾ՉׇӈКჹ߾ว౜ PopP3 ׇӈӧӚ๵ਲ਼ύڀ ԖଯࡋߥӺ܄ (ߕკΖ)Ƕ

2. ӧӧଯࢥΚ๵ਲ਼ PopP3 ϐϩթ

ࣁΑᕕှଯࢥΚ Pss190 ๵ਲ਼܌੝Ԗޑ PopP3 ӧځдଯࢥΚ๵ਲ਼ύޑϩթ௃׎Ǵ

߻Γࣴزᒧ᏷ 3 ਲ਼ phylotype I ޑଯࢥΚ๵ਲ਼ Pss158ǵPss365ǵPss749ǴаϷ 14 ਲ਼ phylotype II ޑଯࢥΚ๵ਲ਼ Pss525ǵPss526ǵPss1327ǵPss1351ǵPss1361ǵPss1370ǵ Pss1475ǵPss1482ǵPss1569ǵPss1586ǵPss1696ǵPss1697ǵPss1703ǵPss1710Ǵ

຾ՉࢉՅᡏ PCR ϩ݋Ƕ่݀ว౜ phylotype I ύନচҁςޕڀԖ PopP3 ϐ Pss190 ᆶ GMI1000 (GMI1000 ࣁύࢥΚ๵ਲ਼ǴӧԜբࣁჴᡍ҅௓ڋಔ) ѦǴۘԖ Pss365 ڀԖ PopP3 (ߕკΐ)ǴԶ phylotype II ߾҂ว౜ԖҺՖ๵ਲ਼ڀԖ PopP3ǶᡉҢѝԖ ϿኧଯࢥΚ๵ਲ਼ڀԖ PopP3ǴځόӸӧܭ܌ԖଯࢥΚ๵ਲ਼྽ύǶ

3. PopP2 ჹߙ࢏ੰ๵ӧพन΢ठੰΚϐቹៜ

ߙ࢏ੰ๵ଯࢥΚ๵ਲ਼ Pss190 ٠όڀԖӧߓ܎դ޺΢ࣁ Avr protein ੝܄ϐ PopP2 (Deslandes et al., 2003)Ǵՠ PopP2 ჹܭߙ࢏ੰ๵ӧพन΢ϐठੰΚ߄౜ޑቹ ៜۘ҂ޕǶࡺஒ popP2 ୷Ӣᒧ෗ډ pUFR047 ၩᡏ٠ᙯΕډ Pss190 ߄౜Ǵ࿶ෳ၂ ᙯࠠ๵ਲ਼ӧלੰพनࠔس H7996 ΢ϐठੰΚ߄౜ࡕว౜Ǵεໆ߄౜ popP2 ཮फ़ե ଯࢥΚ๵ਲ਼ Pss190 ӧלੰࠔسพनϐठੰΚ߄౜ (კΜϖ)Ƕ

ࣁΑ௖૸ PopP3 ຾Ε෌ނಒझࡕǴځфૈ܄୔ࢤ (functional domain) ჹ PopP3 ӧ෌ނಒझύϐ০ပՏ࿼ޑख़ा܄ǴךॺӃа Softberry ᆛઠϩ݋ PopP3 ӧ෌ނಒ झύёૈޑ০ပՏ࿼Ǵ่݀ว౜ PopP3 നԖёૈՏܭಈጕᡏ (mitochondria) ΢ǹ

຾΋؁а iPSORT ᆛઠ (http://ipsort.hgc.jp/) ϩ݋ PopP3 ࢂց஥Ԗૻ৲ᴏ两 (signal

peptide)Ǵว౜ PopP3 ޑ N ᆄ 30 ঁữ୷ለᄬԋΑಈጕᡏᏤӛᴏ两 (mitochondria targeting peptide, MT) Ϸૻ৲ᴏ两 (SP)ǴӕਔႣෳ PopP3 ຾ΕಈጕᡏࡕϪନᴏ两 Տ࿼ (mitochondria cleavage site) ࣁ N ᆄ߻ 10 ঁữ୷ለ (კΜΐ A)Ƕ

ԶࣁΑᡍ᛾ PopP3 ޑ N ᆄ 30 ঁữ୷ለᄬԋϐಈጕᡏᏤӛᴏ两ჹܭځ০ပܭ

෌ނಈጕᡏϐѸा܄ǴךॺஒಈጕᡏᏤӛᴏ两Тࢤմନ (sequence-truncated) ᆶӄ ߏϐ PopP3::GFP ϩձճҔ߄౜ၩᡏ p2GWF7.0ǴᙯΕ N. benthamiana চғ፦ᡏ຾

Չอኩ߄౜Ǵ่݀ᡉҢ୤Ԗ߄౜ӄߏϐ PopP3::GFP ӧ๨૛চғ፦ᡏύև౜Տ࿼

ᆶಈጕᡏ኱૶ (mitochondria marker) ScCOX4::RFP ϩթ΋ठǴԶ߄౜մନಈጕᡏ Ꮴӛᴏ两ϐ PopP3::GFP ӧ๨૛চғ፦ᡏύև౜Տ࿼߾ᆶ߄౜ free GFP ޑޜၩᡏ

௓ڋಔ࣬ӕ (კΜΐ B)Ƕ

7. ТТࢤմନࡕϐ PopP3 ჹߙ࢏ੰ๵ठੰΚϷჹพनੰ্ϸᔈϐቹៜ

຾΋؁ǴࣁΑᡍ᛾ PopP3 ϐಈጕᡏ০ပՏ࿼ჹځӧพनϐфૈࢂցڀख़ा܄Ǵ а VMGO ஒӄߏᆶТࢤմନ MT ϐ 35S::popP3 ӧགੰพनࠔس L390 س಍܄Ӧ߄

౜Ǵ٠ᔠෳพनϐߙ࢏ੰϸᔈࢂցׯᡂǶ่݀ᡉҢ߄౜ӄߏϐ PopP3 ቚமพनӧ Pss4 ೀ౛Πޑ๺঒௃׎ (კΒΜ)ǴԶ߄౜ಈጕᡏᏤӛᴏ两մନࡕϐ PopP3 ߾Ѩѐ ೭ᅿቹៜǴᆶ߄౜ GFP ϐ௓ڋಔคᡉ๱ৡ౦ (p>0.05)Ƕ

ќѦǴךॺΨஒӄߏᆶТࢤմନ MT ϐ popP3 ୷Ӣᒧ෗ډ pUFR047 ၩᡏǴ٠ ϩձᙯ౽߄౜ԿচҁόڀԖ၀ T3E ޑύࢥΚ Pss4 ᒪ໺ङඳ๵ਲ਼ύǴෳ၂ӧགੰ

ࠔسพन L390 ΢ϐठੰΚ߄౜Ǵ٠аޜၩᡏ (empty vector) ᙯࠠ๵ਲ਼଺ࣁ௓ڋಔǶ

่݀ҭว౜Ǵ୤Ԗӧ Pss4 ߄౜ӄߏϐ PopP3 ཮ቚம၀๵ਲ਼ϐठੰΚ߄౜ǴԶ߄౜

ಈጕᡏᏤӛᴏ两 (MT) մନࡕϐ PopP3 ߾Ѩѐ೭ᅿቹៜ (კΒΜ΋)ǶᡉҢ PopP3 N ᆄϐಈጕᡏૻ৲ᴏ两ᆶ෌ނಈጕᡏϐ০ပ੝܄ǴჹԜ T3E ӧ෌ނύϐфૈཱུࣁ ख़ाǶ

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ੰচ๵གࢉ෌ނਔଌΕϐਏᔈೈқǴ٩Ᏽ஌Ь෌ނޑᒣ᛽ૈΚёᏤठόӕޑ

่݀ (Alfano and Collmer, 2004)Ǻऩ T3E ೏෌ނޑ R protein ᒣᇡǴ෌ނ཮௴୏ז ೲԶᐟਗ਼ޑխࣝϸᔈǴ೭ᜪޑਏᔈೈқߡёᆀࣁ avirulence (Avr) proteinǹ࣬ϸӦǴ ऩਏᔈೈқ҂೏஌Ь෌ނᒣᇡǴ߾ёૈԋф׭ڋ஌Ьٛᑇϸᔈ܈υᘋஎЬғ౛ф

ૈǴаճੰচ๵ԋфགࢉ෌ނԶ೷ԋੰ্Ǵ೭ᜪޑਏᔈೈқߡёᆀࣁ virulence protein (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ƕ

΋ǵߙ࢏ੰ๵ RSp0213 ϐфૈࣴز

1. RSp0213 फ़եߙ࢏ੰ๵ჹพनϐठੰΚ

ߙ࢏ੰ๵གࢉ஌Ь෌ނၸำፄᚇǴӧᕉნڈᐟᆶဂᡏགᔈ (quorum sensing) ޑૻ৲໺ሀၡ৩ΠǴठੰӢηڙډፄᚇЪᆒஏޑፓ௓ (Valls et al., 2006; Vailleau et al., 2007; Genin and Denny, 2012)Ǵ٠߄౜ಃΟࠠೈқϩݜس಍ (T3SS) ϩݜਏᔈೈ

қǴଌΕ෌ނಒझׯᡂஎЬੰ্ϸᔈ (Poueymiro et al., 2009)ǴՠҞ߻ᜢܭߙ࢏ੰ

๵ޑࢥΚࣴزߚதϿǴ࣬ᜢၗૻલЮǶߙ࢏ੰ๵ RSp0213 ϐ߄౜ڙ T3SS ܌ፓ௓

(Cunnac et al., 2004a) (ߕკϖ)Ǵࡺ௢ෳ RSp0213 ᔈࢂಃΟࠠϩݜೈқ (T3E)Ǵՠ

҂ٰۘሡ຾΋؁ዴᇡځ೏ϩݜϐ੝܄Ƕҁࣴز຾΋؁ว౜ RSp0213 ޑӸӧё٬ଯ ࢥΚϐߙ࢏ੰ๵ӧพन΢ޑठੰΚΠफ़ (კ΋)ǴԶஒ RSp0213 ԾύࢥΚ๵ਲ਼ Pss4 ύকନ߾ёගଯੰ๵ӧพनϐठੰΚ (კΒ)Ǵ᛾ჴ RSp0213 ࣁቹៜߙ࢏ੰ๵ठੰ

ΚϐᜢᗖӢηǶ߻ΓࣴزΨว౜Ǵӧচҁόڀ PopP1 ޑߙ࢏ੰ๵๵ਲ਼ Rd15 ύ߄

౜ PopP1 ཮Ꮴठ၀๵ਲ਼ӧགੰ஌Ь࿖౐Ф΢ठੰΚΠफ़ǴЪᇡࣁ PopP1 ӧߙ࢏ੰ

๵ύࣁ avirulence protein ޑفՅ (Lavie et al., 2002)Ƕࡺךॺ௢ෳǺύ/եࢥΚ๵ਲ਼

੝Ԗϐ RSp0213 ᙖҗ T3SS ೏ଌΕ෌ނಒझࡕǴёૈ཮೏෌ނٛᑇس಍ᒣᇡԶᇨ Ꮴ෌ނౢғלੰϸᔈǴ٬ੰচ๵ޑठੰૈΚڙډ׭ڋԶև౜ठੰΚၨեޑ߄౜ࠠǴ Զ phylotype I ߙ࢏ੰ๵ଯࢥΚ๵ਲ਼ӢόڀԖ RSp0213Ǵளаխܭ೏෌ނᒣᇡ຾Զ ԖଯठੰΚǶќѦǴҗܭ߈ԃϩᚆளϐ phylotype I ߙ࢏ੰ๵ଯࢥΚ๵ਲ਼٠όڀԖ RSp0213Ǵࡺךॺ௢ෳӧߙ࢏ੰ๵ޑ๵ᅿᄽϯၸำύǴRSp0213 ᔈឦܭӅ೯Ъၨ

ࣁԐයޑ effectorǴԶ஌Ь෌ނΨς࿶ᄽϯр࣬ᜢޑٛᑇᐒڋୀෳϐǴ٠ளаԋф

ჹלϐ (ፎـаΠ૸ፕ)ǶԜѦǴ߈යࣴزࡰрߙ࢏ੰ๵ϐ T3SS ӧੰ๵གࢉ߃ය ᆶࡕය೿཮࡭ុ߄౜ǴᡉҢ T3SS ӧԜ๵ϐس಍܄ठੰၸำύᔈࣣԖཱུᜢᗖфૈ

(Poueymiro and Genin, 2009; Monteiro et al., 2012; Stotz et al., 2014)ǹ RSp0213 ࣁߙ

࢏ੰ๵ठੰΚ࣬ᜢӢηǴՠӧߙ࢏ੰ๵ठੰΚ΢ڀԖᜢᗖفՅޑ RSp0213 زഖࢂ

benthamiana ᆶ N. tabacum W38 ယТݙΕ஥Ԗ RSp0213 ߄౜ၩᡏϐၭఎ๵ 72 λਔ ࡕࣣ཮ӧݙΕಔᙃЇଆֽ೽ޑಒझԝΫϸᔈ (კϖ)ǴЪӧ N. tabacum W38 ယ೽ೀ พनٛᑇس಍ᒣᇡԶЇว෌ނלੰϸᔈ (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ǵࡺӧ N. benthamianaǵN. tabacum Ϸพनύᔈ၀೿Ԗёаᒣᇡ RSp0213 ϐӅӕჹᔈלੰೈқ፦ǶԜѦǴ߈ԃࣴزᡉҢพनߙ࢏ੰϐלੰᐒڋᆶ ࢲϯ SA ᆶ ET ૻ৲໺ᏤϷ፾྽ፓ௓ࢲϯ਼ϩη (reactive oxygen speciesǴᙁᆀ ROS) ಕᑈ֖ໆ৲৲࣬ᜢ (Mersmann et al., 2010; Coll et al., 2011)ǴΨᆶҁࣴز܌ள

ϐ่݀΋ठǶ҂ٰᔈ຾΋؁ཛྷ൨ᒣᇡ RSp0213 ϐբҔೈқǴаճ຾΋؁ᕕှनࣽ

෌ނჹלߙ࢏ੰ๵ϐϩηᐒڋǶ

3. RSp0213 ০০ပܭ෌ނಒझጢჹځфૈԿᜢख़ा

ੰচ๵ϐਏᔈೈқ຾Ε஌Ьಒझࡕ཮౽Տډ੝ۓϐझᏔа୺ՉځբҔǴ٠٩ ځфૈٰቹៜΠෞϐૻ৲໺ሀၡ৩ (Poueymiro and Genin, 2009; Deslandes and Rivas, 2012)Ƕҁࣴزว౜ RSp0213 ޑ N ᆄڀԖ targeting peptide ᆶ signal peptide (SP)Ǵ࿶ᆶ NCBI Genome ၗ਑৤ύ຾Չ blastp ׇӈКჹǴว౜хࡴ PSI07 Ϸ R229

฻ΐਲ਼ߙ࢏ੰ๵ਲ਼ޑ putative T3E N ᆄࣣڀԖԜ SP Тࢤ (E value < 10^-5)ǴЪႣෳ

ӧԜࢤૻ৲ᴏ两ύёૈڀԖၠጢТࢤ (transmembrane segments) (ߕკϤ)Ƕჴᡍ่

݀ᡉҢ RSp0213 ০ပܭ෌ނಒझጢ΢ǴЪև౜ᗺރϩѲ (კΟǵѤ)ǴԶځ N ᆄന ڀߥӺ܄ޑ 21 ঁữ୷ለࣁԜ০ပ੝܄ϐѸሡТࢤ (კΜǵკΜ΋)ǴЪჹځӧพन

΢ᇨᏤלੰϸᔈϐ੝܄Ԗ،ۓ܄ޑቹៜ (კΜΒǵΜΟǵΜѤ)Ǵ׳ᡉҢԜ SP Т ࢤёૈჹځѬ T3Es ০ပ੝܄ࣣڀख़ा܄ǴӢԜ҂ٰёаԜ SP Тࢤௗӝ GFP ೈ қᢀჸ০ပՏ࿼Ǵᡍ᛾ځዴϪфૈǶᆕӝ΢ॊჴᡍ่݀Ǵךॺ௢ෳ RSp0213 ᔈࢂ

ሡा০ပӧ෌ނಒझጢ΢٠׎ԋೈқ፦໣ဂ (protein cluster) Бૈ৖౜ځਏᔈǴԶ ӧᄽϯޑၸำύǴ෌ނҭςว৖р੝ۓϐᒣᇡೈқᆶಒझጢ࣬ᜢٛᑇس಍Ǵᒣ᛽

RSp0213 ٠ᇨวٛᑇϸᔈǶ

೚ӭࣴزࣣࡰр෌ނಒझጢᆶځ΢ protein complexes ჹܭੰ্ٛᑇϸᔈתᄽ ᜢᗖفՅ (Poueymiro and Genin, 2009; Bohm et al., 2014)ǴٯӵǺᒣᇡੰ๵ PAMPs ϐ PRRs ᆶ೚ӭᒣᇡੰ๵ਏᔈೈқϐ R proteins ࣣՏܭ෌ނಒझጢ (Bohm et al., 2014)ǹќѦǴಒझጢ΢ޑᕗિжᖴፓ௓ҭ཮ჹಒझٛᑇϸᔈౢғख़ाቹៜǴӵፓ

௓ಒझᕗિޑሇન phospholipaseǴջ೏᛾ჴჹܭ෌ނಒझޑ PCD ϸᔈϷٛᑇૻ৲

໺Ꮴתᄽख़ाفՅ (Kim et al., 2014)ǴΞӵಒझጢ΢ޑ੝ۓ่ᄬિใ (lipid raft)Ǵ Ӣځୖᆶፓ௓ಒझϣޑિ፦жᖴ (Lingwood and Simons, 2010)ǴЪς᛾ჴᆶᇨᏤ PCD ౢғϐ ROS ಕᑈஏϪ࣬ᜢ (Mersmann et al., 2010)Ǵࡺӧ෌ނٛᑇϸᔈޑፓ

௓΢ڀԖᜢᗖفՅ (Fallahi-Sichani and Linderman, 2009)ǶԜѦǴᜢܭਏᔈೈқբ Ҕܭ෌ނಒझጢ΢ςԖ೚ӭਢٯǴٯӵ Pseudomonas syringae pv. tomato DC3000 ޑ T3E AvrPto ᆶ AvrPtoB ջࣁբҔӧ෌ނಒझጢ΢ޑਏᔈೈқǹAvrPto ཮׭ڋߓ

܎դ޺ FLS2 ޑᕗለϯфૈ (Xiang et al., 2008)ǴߔЗΠෞޑ PTI ૻဦ໺ሀǹ

AvrPtoB ߾ࢂ཮ᆶ FLS2 Ϸ FLS2 ϐӅௗڙᡏ (co-receptor) BAK1 (BRI1-associated kinase 1) ҬϕբҔǴ׭ڋჹಒ๵ᚎЛೈқޑགޕௗڙ (Gohre et al., 2008)ǹฅԶӧ พनύǴAvrPto ᆶ AvrPtoB ߾཮ᆶพनϐ Pto ᐟ䁙ҬϕբҔǴЪ Pto ёૈ཮֎Ї AvrPto ٬ځᇻᚆ PRR ೈқǴЪёૈ೸ၸᕗለϯ AvrPtoB Զ׭ڋځݱનϯࢲ܄

(Gohre et al., 2008)Ǵ຾ԶᇨᏤ෌ނޑ ETI ٛᑇϸᔈǶ҂ٰёа຾΋؁ഄڗᆶ RSp0213 ԖҬϕբҔϐ෌ނೈқဂǴаයёཛྷ൨ᒣᇡ RSp0213 ϐբҔೈқᆶځд

ૻ৲໺Ꮴ࣬ᜢೈқ፦Ǵ٠຾΋؁௖૸ځҬϕբҔᆶ࣬ᜢϩηᐒڋǶ Զ N. benthamianaǵN. tabacum Ϸพन฻नࣽ෌ނ߾ςᄽϯрёᒣᇡ RSp0213 ϐ

࣬ჹᔈբҔೈқǴܭགࢉ୔Їว PCD ϸᔈǴ٠ࢲϯٛᑇລᅟᆾ SA ᆶ ET ϐس಍

܄ૻ৲໺Ꮴၡ৩Ǵᇨวس಍܄խࣝϸᔈǴаफ़եੰচ๵ӧ෌ނᡏޑठੰૈΚǶ҂

ٰᔈ຾΋؁ཛྷ൨ᒣᇡ RSp0213 ϐբҔೈқᆶځд interacting proteinsǴаճ຾΋؁

ᕕှनࣽ෌ނჹלߙ࢏ੰ๵ϐϩηᐒڋǴڀᡏឍܴځዴϪϩηᐒڋǴБૈᙶమ RSp0213 ϐڀᡏբҔᆶ෌ނٛᑇϸᔈޑૻ৲໺Ꮴၡ৩ (Deslandes and Genin, 2014;

Kazan and Lyons, 2014)ǶќѦǴճҔ RSp0213 ᆶځբҔೈқϐᒣᇡس಍Ǵ҂ٰҭ ёஒҗቶݱੰ๵ᇨᏤ܄୷Ӣ௴୏η (Lin et al., 2014) ௓ڋ߄౜ϐ RSp0213 ᏤΕ஥

ԖԜբҔೈқϐ፾྽नࣽբނύǴว৖ڀቶݱל๵܄ϐੰ্ٛᑇ฼ౣǶ

Βǵߙ࢏ੰ๵ PopP3 ϐфૈࣴز

1. ଯ ࢥ Κ ๵ ਲ਼ Pss190 ੝ Ԗ ϐ T3E PopP3 ࣁ ߙ ࢏ ੰ ๵ ख़ ा ޑ ठ ੰ Κ Ӣ η (Virulence factor)

Нѳ୷Ӣᙯ౽ (horizontal gene transfer, HGT) ೏ຎࣁߙ࢏ੰ๵זೲᄽϯǵ፾ᔈ όӕғᄊᕉნϷளаӧӭᅿ஌Ьس಍ԋфठੰޑᜢᗖӢન (Peeter et al., 2013)Ƕ T3E ࢂߙ࢏ੰ๵ठੰޑᜢᗖӢηǴӚ๵ਲ਼໔ڀଯݔ౦ࡋǴԶ T3E ϐНѳ୷Ӣᙯ౽

೏ᇡࣁёૈࢂᏤठߙ࢏ੰ๵ठੰΚৡ౦ޑচӢǶӧߙ࢏ੰ๵ GMI1000 ๵ਲ਼ύ popP1 ൩೏ᇡࣁࢂ೸ၸ HGT ᕇளޑ୷Ӣ (Lavie et al., 2002)Ǵӧ GMI1000ǵPo82 ᆶ CMR15 Οঁ๵ਲ਼ϐ୷Ӣಔύ popP2 ೿ᆶচᏘ๵ᡏ (prophage) ϐТࢤ࣬ᎃ (Genin and Denny, 2012)ǴԶҁࣴز࠻ᔠຎςှׇߙ࢏ੰ๵๵ਲ਼Ψว౜ӧ Pss190ǵ GMI1000 Ϸ Y45 ๵ਲ਼ϐ୷Ӣಔύ popP3 ᎃ߈୷Ӣҭᆶ prophage Тࢤ࣬ᎃǴࡺך (Pss1308ǵPss749) ᛙۓǴࡺ௢ෳ PopP3 ёૈࢂߙ࢏ੰ๵߈ය࿶ HGT Զᕇளޑཥ ᑉਏᔈೈқǴΨӢԜ෌ނۘ҂ᄽϯрىаᒣ᛽ PopP3 ϐלੰ R ೈқ (ߕკΜΟǵ ΜѤ)ǴԶ٬ PopP3 ளаԋࣁߙ࢏ੰ๵ޑख़ा virulence factorǴගଯߙ࢏ੰ๵ϐठ

ੰΚ߄౜Ƕ

ߙ࢏ੰ๵ଯࢥΚ๵ਲ਼ Pss190 ٠คӧߓ܎դ޺ύ཮೏ R protein ᒣᇡޑ Avr protein PopP2 (Deslandes et al., 2003)ǴԶҁࣴزΨ᛾ჴ PopP2 ཮फ़եଯࢥΚ๵ਲ਼ Pss190 ӧלੰࠔسพनϐठੰΚ (კΜϖ)Ƕ೭٤ၗ਑ᡉҢǴӧߙ࢏ੰ๵ύǴPopP ৎ௼ԋ঩ჹܭ Pss190 ӧพन΢ϐଯठੰΚ߄౜ڀख़ाቹៜфૈǴPss190 ନΑڀԖ virulence protein PopP3 ϐѦǴΨӢࣁόڀԖӧพन΢ёૈڀ Avr protein ੝܄ϐ PopP2ǴԶ٬ Pss190 ёೕᗉพनלੰᐒڋޑᒣᇡǴ຾Զ৖౜ Pss190 ӧלੰࠔسพ न΢ϐଯठੰΚǶ ϐᘰ໾ဟᆫᗐಕᑈ (callose deposition)Ǵ٠Ъ٬พनჹ೬ᆭੰಒ๵ (Pcc) ׳ࣁགੰǴ

ՠόቹៜ flg22 ᇨᏤϐ ROS ಕᑈ (ߕკΜϤǵΜΎǵΜΖ)Ƕҗܭਏᔈೈқ׭ڋ HR ٠ߚ׭ڋ෌ނ PTI ϸᔈޑѸाచҹ (Kazan and Lyons, 2014)ǴӢԜ௢ෳ PopP3

׭ڋ PTI ϸᔈϐၡ৩ёૈբҔܭ HR ᇨᏤၡ৩ϐѦ (independently)Ǵ܈ёૈځբҔ ᐒڋՏܭ ROS ૻ৲໺ᏤϐΠෞа׭ڋᘰ໾ဟᆫᗐϐಕᑈ (Muthamilarasan and Prasad, 2013)Ǵࡺคੋܭፓ௓ ROS ಕᑈᆶ HR ϸᔈǶฅԶǴӢӃ߻ޑ ROS ಕᑈჴ

ࢂߙ࢏ੰ๵ޑख़ा virulence factorǶՠԖ፪ޑࢂǴӧלੰพनࠔس H7996 ߄౜

popP3 ࠅό཮ቹៜ H7996 ჹߙ࢏ੰ๵ Pss190 ϐੰ্ϸᔈ (კΜΖ)ǹҗܭ PopP3 ϐ

౜ຝхࡴǺಒझ፦๺ᕭǵਡࢉՅ፦ (chromatin) ᆫ໣ǵಈጕᡏᑩεǵనݰ (vacuole) ᆶယᆘᡏ (chloroplast) ڙཞ฻ (Muthamilarasan and Prasad, 2013)Ƕੰচ๵གࢉၸำ ύதᇨว੿ਡғނ PCD ϸᔈаٛᑇੰচ๵ߟࢉǴ෌ނޑ HR ϸᔈߡࢂന٫ٯηϐ

΋ǶӢԜಈጕᡏࣁੰচ๵ਏᔈೈқբҔޑख़ाՏ࿼ϐ΋ǴӃ߻ࣴز൩ว౜୏ނੰ

চ๵ޑਏᔈೈқ཮೸ၸࢲϯಒझғӸૻဦ (cell survival signals) υᘋஎЬಈጕᡏբ Ҕǵϩှ௴୏ಒझԾఠ (apoptosis) ޑૻဦೈқ (pro-apoptotic proteins)Ǵ׭ڋಒझ Ծఠаڐշੰচ๵Εߟ (Faherty and Maurelli, 2008)Ƕ

ӧ੿ਡғނಒझύǴڀԖಈጕᡏᏤӛᴏ两 (mitochondria targeting peptide, MT) ϐೈқ፦཮೏ಈጕᡏ߄य़ڙᡏ (receptor) ᒣ᛽٠ᆶځ่ӝǴೈқ፦ω཮೏ଌΕಈ ጕᡏϣǹۘ҂೏໺ᒡԿಈጕᡏϣޑೈқև౜҂ᄙ᠄ޑރᄊ (unfolded form)ǴѝԖ

྽຾ΕಈጕᡏࡕǴಈጕᡏૻ৲ᴏ两೏ϪନǴೈқω཮ӧՔᒿೈқ (chaperon) ޑᔅ շΠᄙ᠄ǵౢғфૈ (Hicks and Galán, 2013)ǶҁࣴزճҔғނၗૻϩ݋ว౜

PopP3 ޑ N ᆄ 30 ঁữ୷ለᄬԋΑ MT ᆶૻ৲ᴏ两 (SP) (კΜΐ A)ǴԶ຾΋؁ჴᡍ ΨዴᇡԜ MT Тࢤࣁ PopP3 ০ပܭ෌ނಈጕᡏϐѸाׇӈ (კΜΐ B)ǴЪჹܭ PopP3 ෧եพनჹߙ࢏ੰ๵ Pss4 ל܄ᆶቚம Pss4 ϐࢥΚڀᜢᗖфૈ (კΒΜǵΒ Μ΋)ǶҞ߻೏ว౜০ပܭ੿ਡғނಈጕᡏϐੰচ๵ਏᔈೈқཱུϿǴ୏ނੰচ๵໻

Ԗ E. coli ϐ MapǵEspF Ϸ SopA (Hicks and Galán, 2013)Ǵ෌ނੰচ๵߾໻Ԗ P.

syringe ϐ HopG1 (Hicks and Galán, 2013)ǴԶځύѝԖ E. coli Map ೏᛾ჴ཮຾Ε ಈጕᡏύǴЪ཮Ѻ໶ಈጕᡏϣѦጢޑᚆη೯೸ࡋ٠೷ԋಈጕᡏᘐ຋ґှ (Hicks and Galán, 2013)ǴԶ P. syringe ޑ HopG1 ऩ҂০ပܭ෌ނಈጕᡏߡคݤ׭ڋ෌ނ ғߏวػ (Block et al., 2010)Ƕךॺ௢ෳ PopP3 ёૈᙖҗυᘋ෌ނಈጕᡏϐၮբǴ PopP3 ϐ interacting proteinsǴаճ຾΋؁ᕕှ PopP3 υᘋพनלੰϸᔈϐڀᡏᐒ ڋǴឍܴځዴϪϩηᐒڋᆶ࣬ᜢૻ৲໺Ꮴၡ৩ (Gohre et al., 2008; Gimenez-Ibanes

et al., 2009)Ǵ٠යఈёૈᙖԜࣴᔕԖշܭբނჹלଯࢥΚߙ࢏ੰ๵๵ਲ਼Ӓ্ϐੰ

্ٛݯ฼ౣǶ

Abramovitch, R.B., Kim, Y.-J., Chen, S., Dickman, M.B., Martin, G.B. (2003).

Pseudomonas type III effector AvrPtoB induces plant disease susceptibility by inhibition of host programmed cell death. The European Molecular Biology Organization Journal 22, 60-69.

Afzal, A.J., da Cunha, L., Mackey, D. (2011). Separable fragments and membrane tethering of Arabidopsis RIN4 regulate its suppression of PAMP-triggered immunity. The Plant Cell 23, 3798-3811.

Alfano, J.R., Charkowski, A.O., Deng, W.-L., Badel, J.L., Petnicki-Ocwieja, T., Dijk, K., Collmer, A. (2000). The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants. Proceedings of the National Academy of Sciences of the United States of America 97, 4856-4861.

Alfano, J.R. and Collmer, A. (2004). Type III secretion system effector proteins:

double agents in bacterial disease and plant defense. Annual Review of Phytopathology 42, 385-414.

Angot, A., Peeters, N., Lechner, E., Vailleau, F., Baud, C., Gentzbittel, L., Sartorel, E., Genschik, P., Boucher, C., Genin, S. (2006). Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants. Proceedings of the National Academy of Sciences of the United States of America 103, 14620-14625.

Arlat, M., Gough, C.L., Zischek, C., Barberis, P.A., Trigalet, A., Boucher, C.A.

(1992). Transcriptional organization and expression of the large hrp gene cluster of Pseudomonas solanacearum. Molecular plant-microbe interactions 5, 187-193.

Arnold, R., Brandmaier, S., Kleine, F., Tischler, P., Heinz, E., Behrens, S., Niinikoski, A., Mewes, H.W., Horn, M., Rattei, T. (2009). Sequence-based prediction of type III secreted proteins. PLoS Pathogens 5, e1000376.

Bernoux, M., Timmers, T., Jauneau, A., Briere, C., de Wit, P.J., Marco, Y., Deslandes, L. (2008). RD19, an Arabidopsis cysteine protease required for RRS1-R-mediated resistance, is relocalized to the nucleus by the Ralstonia solanacearum PopP2 effector. The Plant Cell 20, 2252-2264.

Bhat, R.A., Panstruga, R. (2005). Lipid rafts in plants. Planta 223, 5-19.

Block, A., Guo, M., Li, G., Elowsky, C., Clemente, T.E., Alfano, J.R. (2010). The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development and suppresses plant innate immunity. Cellular Microbiology 12, 318-330.

Bohm, H., Albert, I., Fan, L., Reinhard, A., Nurnberger, T. (2014). Immune receptor complexes at the plant cell surface. Current Opinion in Plant Biology 20, 47-54.

Boller, T., Felix, G. (2009). A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annual Review of Plant Biology 60, 379-406.

Buttner, D., He, S.Y. (2009). Type III protein secretion in plant pathogenic bacteria.

Plant Physiology 150, 1656-1664.

Cao, Y., Tian, B., Liu, Y., Cai, L., Wang, H., Lu, N., Wang, M., Shang, S., Luo, Z., Shi, J. (2013). Genome sequencing of Ralstonia solanacearum FQY_4, isolated from a bacterial wilt nursery used for breeding crop resistance. Genome announcements 1.

Chen, F., Gao, M.J., Miao, Y.S., Yuan, Y.X., Wang, M.Y., Li, Q., Mao, B.Z., Jiang, L.W., He, Z.H. (2010). Plasma membrane localization and potential endocytosis of constitutively expressed XA21 proteins in transgenic rice.

Molecular Plant 3, 917-926.

Choi, H.W., Kim, N.H., Lee, Y.K., Hwang, B.K. (2013). The pepper extracellular xyloglucan-specific endo-b-1,4-glucanase inhibitor protein gene, CaXEGIP1, is required for plant cell death and defense responses. Plant Physiology 161, 384-396.

Coll, N.S., Epple, P., Dangl, J.L. (2011). Programmed cell death in the plant immune system. Cell Death and Differentiation 18, 1247-1256.

Cunnac, S., Boucher, C., Genin, S. (2004a). Characterization of the cis-acting regulatory element controlling HrpB-mediated activation of the type III secretion system and effector genes in Ralstonia solanacearum. Journal of Bacteriology 186, 2309-2318.

Cunnac, S., Occhialini, A., Barberis, P., Boucher, C., Genin, S. (2004b). Inventory and functional analysis of the large Hrp regulon in Ralstonia solanacearum:

identification of novel effector proteins translocated to plant host cells through the type III secretion system. Molecular Microbiology 53, 115-128.

Denny, T.P. (2006). Plant pathogenic Ralstonia species. In Plant-Associated Bacteria, S.S. Gnanamanickam, ed. (Dordrecht, The Netherlands: Springer Science), pp.

573-644.

Deslandes, L., Genin, S. (2014). Opening the Ralstonia solanacearum type III effector tool box: insights into host cell subversion mechanisms. Current Opinion in Plant Biology 20, 110-117.

Deslandes, L., Rivas, S. (2012). Catch me if you can: bacterial effectors and plant targets. Trends in Plant Science 17, 644-655.

Deslandes, L., Olivier, J., Peeters, N., Feng, D.X., Khounlotham, M., Boucher, C., Somssich, I., Genin, S., Marco, Y. (2003). Physical interaction between RRS1-R, a protein conferring resistance to bacterial wilt, and PopP2, a type III effector targeted to the plant nucleus. Proceedings of the National Academy of Sciences of the United States of America 100, 8024-8029.

Faherty, C.S., Maurelli, A.T. (2008). Staying alive: bacterial inhibition of apoptosis

during infection. Trends in Microbiology 16, 173-180.

Fallahi-Sichani, M. and Linderman, J.J. (2009). Lipid raft-mediated regulation of G-protein coupled receptor signaling by ligands which influence receptor dimerization: a computational study. PLoS One 4, e6604.

Fegan, M., and Prior, P. (2006). Diverse members of the Ralstonia solanacearum species complex cause bacterial wilts of banana. Australasian Plant Pathol. 35, 93-101.

Fu, Z.Q., Guo, M., Jeong, B.R., Tian, F., Elthon, T.E., Cerny, R.L., Staiger, D., Alfano, J.R. (2007). A type III effector ADP-ribosylates RNA-binding proteins and quells plant immunity. Nature 447, 284-288.

Gaudriault, S., Paulin, J.P., Barny, M.A. (2002). The DspB/F protein of Erwinia amylovora is a type III secretion chaperone ensuring efficient intrabacterial production of the Hrp-secreted DspA/E pathogenicity factor. Molecular Plant Pathology 3, 313-320.

Genin, S., Denny, T.P. (2012). Pathogenomics of the Ralstonia solanacearum species complex. Annual Review of Phytopathology 50, 67-89.

Gimenez-Ibanez, S., Hann, D.R., Ntoukakis, V., Petutschnig, E., Lipka, V., Rathjen, J.P. (2009). AvrPtoB targets the LysM receptor kinase CERK1 to promote bacterial virulence on plants. Current Biology 19, 423-429.

Gohre, V., Spallek, T., Haweker, H., Mersmann, S., Mentzel, T., Boller, T., de Torres, M., Mansfield, J.W., Robatzek, S. (2008). Plant pattern-recognition receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Current Biology 18, 1824-1832.

Hajdukiewicz, P., Svab, Z., Maliga, P. (1994). The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol. Biol. 25, 989–994.

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J.

Mol. Biol. 166, 557-580.

Hayward, A.C. (1991). Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annual Review of Phytopathology 29, 65-87.

Hicks, S.W., Galán, J.E. (2013). Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors. Nature reviews Microbiology 11, 316-326.

Jaunet, T.X., Wang, J.F. (1999). Variation in genotype and aggressiveness of Ralstonia solanacearum race 1 isolated from tomato in Taiwan. Phytopathology 89, 320-327.

Jelenska, J., van Hal J.A., Greenberg, J.T. (2010). Pseudomonas syringae hijacks plant stress chaperone machinery for virulence. Proceedings of the National Academy of Sciences of the United States of America 107, 13177–13182.

Jones, J.D., Dangl, J.L. (2006). The plant immune system. Nature 444, 323-329.

Kanda, A., Yasukohchi, M., Ohnishi, K., Kiba, A., Okuno, T., Hikichi, Y. (2003).

Ectopic expression of Ralstonia solanacearum effector protein PopA early in invasion results in loss of virulence. Molecular Plant-Microbe Interactions 16, 447-455.

Karimi, M., Inze, D., Depicker, A. (2002). GATEWAY vectors for

Agrobacterium-45

mediated plant transformation. Trends in Plant Science 7, 193-195.

Karimi, M., Meyer, B.D., Hilson, P. (2005). Modular cloning in plant cells. Trends in Plant Science doi: 10.1016/j.tplants.2005.01.008.

Kay, S., Hahn, S., Marois, E., Hause, G., and Bonas, U. (2007). A bacterial effector acts as a plant transcription factor and induces a cell size regulator. Science 318, 648-651.

Kazan, K., Lyons, R. (2014). Intervention of Phytohormone Pathways by Pathogen Effectors. The Plant Cell doi: 10.1105/tpc.114.125419.

Kim, D.S., Jeun, Y., Hwang, B.K. (2014). The pepper patatin-like phospholipase CaPLP1 functions in plant cell death and defense signaling. Plant Molecular Biology 84, 329-344.

Koncz, C., Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Molecular Genetics and Genomics 204, 383–396.

Lam, E., Kato, N., Lawton, M. (2001). Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848-853.

Lavie, M., Shillington, E., Eguiluz, C., Grimsley, N., Boucher, C. (2002). PopP1, a new member of the YopJ/AvrRxv family of type III effector proteins, acts as a host-specificity factor and modulates aggressiveness of Ralstonia solanacearum.

Molecular Plant-Microbe Interactions 15, 1058-1068.

Lebeau, A., Daunay, M.C., Frary, A., Palloix, A., Wang, J.F., Dintinger, J., Chiroleu, F., Wicker, E., Prior, P. (2011). Bacterial wilt resistance in tomato, pepper, and eggplant: genetic resources respond to diverse strains in the Ralstonia solanacearum species complex. Phytopathology 101, 154-165.

Lewis, J.D., Lee, A., Ma, W., Zhou, H., Guttman, D.S., Desveaux, D. (2011). The YopJ superfamily in plant-associated bacteria. Molecular plant pathology 12, 928-937.

Li, L., Atef, A., Piatek, A., Ali, Z., Piatek, M., Aouida, M., Sharakuu, A., Mahjoub, A., Wang, G., Khan, S., Fedoroff, N.V., Zhu, J.K., Mahfouz, M.M. (2013).

Characterization and DNA-Binding Specificities of Ralstonia TAL-Like Effectors. Molecular Plant doi: 10.1093/mp/sst006.

Lin, Y.-M., Shih, S.-L., Lin, W.-C., Wu, J.-W., Chen, Y.-T., Hsieh, C.-Y., Guan, L.-C., Lin, L., Cheng, C.-P. (2014). Phytoalexin biosynthesis genes are regulated and involved in plant response to Ralstonia solanacearum infection. Plant Science 224, 86-94.

Lingwood, D., Simons, K. (2010). Lipid rafts as a membrane-organizing principle.

Science doi: 10.1126/science.1174621.

Liu, J., Elmore, J.M., Lin, Z.J., Coaker, G. (2011). A receptor-like cytoplasmic kinase phosphorylates the host target RIN4, leading to the activation of a plant innate immune receptor. Cell Host and Microbe 9, 137-146.

Liu, H., Zhang, S., Schell, M.A., Denny, T.P. (2005). Pyramiding unmarked deletions in Ralstonia solanacearum shows that secreted proteins in addition to plant cell-wall-degrading enzymes contribute to virulence. Molecular Plant-Microbe Interactions 18, 1296-1305.

Lu, D., Wu, S., Gao, X., Zhang, Y., Shan, L., He, P. (2010). A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proceedings of the National Academy of Sciences of the United States of America 107, 496-501.

McCann, H.C., Guttman, D.S. (2008). Evolution of the type III secretion system and its effectors in plant-microbe interactions. The New Phytologist 177, 33-47.

Mersmann, S., Bourdais, G., Rietz, S., Robatzek, S. (2010). Ethylene signaling regulates accumulation of the FLS2 receptor and is required for the oxidative burst contributing to plant immunity. Plant Physiology 154, 391-400.

Milling, A., Babujee, L., Allen, C. (2011). Ralstonia solanacearum extracellular polysaccharide is a specific elicitor of defense responses in wilt-resistant tomato plants. PLoS One 6, e15853.

Monack, D.M., Mecsas, J., Ghori, N., Falkow, S. (1997). Yersinia signals macrophages to undergo apoptosis and YopJ is necessary for this cell death.

Proceedings of the National Academy of Sciences of the United States of America 94, 10385-10390.

Monteiro, F., Sole, M., van Dijk, I., Valls, M. (2012). A chromosomal insertion toolbox for promoter probing, mutant complementation, and pathogenicity studies in Ralstonia solanacearum. Molecular Plant-Microbe Interactions 25, 557-568.

Munnik, T. (2001). Phosphatidic acid: an emerging plant lipid second messenger.

Trends in Plant Science 5, 227-233.

Muthamilarasan, M., Prasad, M. (2013). Plant innate immunity: an updated insight into defense mechanism. Journal of Biosciences 38, 433-449.

Nelson, B.K., Cai, X., Nebenfuhr, A. (2007). A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. The Plant Journal 51, 1126-1136.

Nomura, K., Debroy, S., Lee, Y.H., Pumplin, N., Jones, J., He, S.Y. (2006). A bacterial virulence protein suppresses host innate immunity to cause plant disease. Science 313, 220-223.

Nomuraa, K., Meceya, C., Lee, Y.N., Imboden, L.A., Changc, J.H., He, S.Y. (2011).

Effector-triggered immunity blocks pathogen degradation of an immunity-associated vesicle traffic regulator in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 108, 10774–10779.

Parsot, C., Hamiaux, C., Page, A.L. (2003). The various and varying roles of specific chaperones in type III secretion systems. Current Opinion in Microbiology 6, 7-14.

Peeters, N., Guidot, A., Vailleau, F., Valls, M. (2013). Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era. Molecular Plant Pathology doi: 10.1111/mpp.12038.

Pfund, C., Tans-Kersten, J., Dunning, F.M., Alonso, J.M., Ecker, J.R., Allen, C., Bent, A.F. (2004). Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana. Molecular Plant-Microbe Interactions 17, 696-706.

Pieterse, C.M., Leon-Reyes, A., Van der Ent, S., Van Wees, S.C. (2009). Networking by small-molecule hormones in plant immunity. Nature Chemical Biology 5, 308-316.

Plener, L., Manfredi, P., Valls, M., Genin, S. (2010). PrhG, a transcriptional regulator responding to growth conditions, is involved in the control of the type III secretion system regulon in Ralstonia solanacearum. Journal of Bacteriology 192, 1011-1019.

Poueymiro, M., Cunnac, S., Barberis, P., Deslandes, L., Peeters, N., Cazale-Noel, A.C., Boucher, C., Genin, S. (2009a). Two type III secretion system effectors from Ralstonia solanacearum GMI1000 determine host-range specificity on tobacco. Molecular Plant-Microbe Interactions 22, 538-550.

Poueymiro, M. and Genin, S. (2009). Secreted proteins from Ralstonia solanacearum:

a hundred tricks to kill a plant. Current Opinion in Microbiology 12, 44-52.

Remenant, B., Babujee, L., Lajus, A., Medigue, C., Prior, P., Allen, C. (2012).

Sequencing of K60, type strain of the major plant pathogen Ralstonia solanacearum. Journal of Bacteriology 194, 2742-2743.

Remenant, B., de Cambiaire, J.C., Cellier, G., Jacobs, J.M., Mangenot, S., Barbe, V., Lajus, A., Vallenet, D., Medigue, C., Fegan, M., Allen, C., Prior, P.

(2011). Ralstonia syzygii, the Blood Disease Bacterium and some Asian R.

solanacearum strains form a single genomic species despite divergent lifestyles.

PLoS One 6, e24356.

Remigi, P., Anisimova, M., Guidot, A., Genin, S., Peeters, N. (2011). Functional diversification of the GALA type III effector family contributes to Ralstonia solanacearum adaptation on different plant hosts. The New Phytologist 192, 976-987.

Rodriguez-Herva, J.J., Gonzalez-Melendi, P., Cuartas-Lanza, R., Antunez-Lamas, M., Rio-Alvarez, I., Li, Z., Lopez-Torrejon, G., Diaz, I., Del Pozo, J.C., Chakravarthy, S., Collmer, A., Rodriguez-Palenzuela, P., Lopez-Solanilla, E. (2012). A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses. Cellular Microbiology 14, 669-681.

Salanoubat, M., Genin, S., Artiguenave, F., Gouzy, J., Mangenot, S., Arlat, M., Billault, A., Brottier, P., Camus, J.C., Cattolico, L., Chandler, M., Choisne, N., Claudel-Renard, C., Cunnac, S., Demange, N., Gaspin, C., Lavie, M., Moisan, A., Robert, C., Saurin, W., Schiex, T., Siguier, P., Thebault, P., Whalen, M., Wincker, P., Levy, M., Weissenbach, J., Boucher, C.A. (2002).

Genome sequence of the plant pathogen Ralstonia solanacearum. Nature 415, 497-502.

Samudrala, R., Heffron, F., McDermott, J.E. (2009). Accurate prediction of secreted substrates and identification of a conserved putative secretion signal for type III secretion systems. PLoS Pathogens 5, e1000375.

Schell, M.A. (2000). Control of Virulence and Pathogenicity Genes of Ralstonia

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