ϣࢉᛏિᑗᏉጤႝݚપϯ

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

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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)

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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^TM II 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

20

4.11. ႝႝऀϾᙯࠠբҔ (Electroporation Transformation)

ஒയҺಒझᆶऊ 50 ng ፦ᡏ DNA షӝࡕǴܫΕ٣ӃܭӇ΢Ⴃհޑ cuvette ύǴ

21

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

23

๺঒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

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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

25

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

26

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

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ಃ

ಃ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

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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

29

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܄

30

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

31

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

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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

33

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

34

ಃ

ಃѤക ૸ፕ

ੰ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

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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

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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

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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

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೏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

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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

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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

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et al., 2009)Ǵ٠යఈёૈᙖԜࣴᔕԖշܭբނჹלଯࢥΚߙ࢏ੰ๵๵ਲ਼Ӓ্ϐੰ

্ٛݯ฼ౣǶ

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

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在文檔中 效應蛋白RSp0213與PopP3在青枯病菌致病力之功能研究 (頁 27-0)