效應蛋白RSp0213與PopP3在青枯病菌致病力之功能研究

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୯ҥᆵ᡼εᏢғڮࣽᏢଣ෌ނࣽᏢࣴز܌

ᅺγፕЎ

Institute of Plant Biology College of Life Science

National Taiwan University Master Thesis

ਏᔈೈқ RSp0213 ᆶ PopP3 ӧߙ࢏ੰ๵ठੰΚ ϐфૈࣴز

Roles of Effectors RSp0213 and PopP3 in Virulence of Ralstonia solanacearum

ᗫੀ๔ Tai-Hsiang Chu

ࡰᏤ௲௤Ǻᎄࣿ๩ റγ Advisor: Dr. Chiu-Ping Cheng

ύ๮҇୯΋ԭ႟ΟԃΎД July, 2014

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I

!

! !

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II

ᖴ ᖴᇞ!

!

! ! ߚதགᖴࣿ๩ԴৣޑڐշǴᡣךԖᐒ཮ୖᆶаϷֹԋࣴزᐕำޑᏢಞǴ٠๏

ϒךཱུεޑߞҺǵЍ࡭ᆶх৒ǴᖴᖴாǼΨाགᖴα၂ہ঩ቅྷ޹Դৣǵᒘᅟ㧌 Դৣǵ݅Ώ։Դৣǵቅ௴ቺԴৣǴԭԆύᜫཀኘޜঅׯךޑፕЎǴாॺޑࡌ᝼з ךᕇ੻ؼӭǴΨ๏Α೭ঁࣴزЬᚒ࣬྽ჴሞЪమཱޑว৖БӛǶ!

! ! གᖴҏఘǵӼฐǵ܃։Ǵགۺգॺ΋ޔаٰჹჴᡍ࠻ޑбрǶགᖴѯᐪǴӧ ך߃፯Εჴᡍ࠻ඌඌόӼਔǴیᕴࢂడคᏢۆࢎηӦ๏ϒ੿၈ᜢЈǴ΋ۓा۩ᅽ ഽǼགᖴຐԮǵࣦૠǵ஭޻Ǵᖴᖴգॺჴᡍ΢ޑಒЈ໺܍Ƕགᖴ੻௵Ǵ྽ךޑჴ ᡍ่݀ۘ҂೏ᇡёਔǴգࢂಃ΋ঁ๏ϒךႴᓰޑΓǴ٠Ъ௲Αךࡐӭ၀Ԗޑჴᡍ ᄊࡋϷೕჄޑཷۺǴ੿ޑࢂࡐӳޑৣഡǼགᖴआ♻ǵ݅៛Ǵ׆ఈیॺӧֹԋᅺγ ၂ྡྷࡕૈԐВפډ໺ᇥύޑӳتΓǶགᖴ϶ችǵ໳ฐǴคፕӵՖ೿ाᖴᖴیॺ೭ ٿԃٰߚதӭ٣௃΢ޑᔅԆᆶ dpwfsǴᏃᆅεৎதத཮೏եᓸ᠈࿽ǴՠךЈ္మ

ཱǴࢂیॺ΋ၡ΢ޑᔅԆᡣךૈόېൂӦوډϞϺǶགᖴঢ়ᝩکᐛۯǴӢࣁԖգ ॺޑуΕǴᡣჴᡍ࠻໒ۈр౜ό΋ኬޑݗൎǴգॺࢂךᅺΒਔӀ္ࡐख़ाޑӣᏫǴ Ψࢂᜤளၶډૌښ࣬׫ޑܻ϶Ƕགᖴ໡൛Ǵیؒ໒α߻๊ჹࢂঁ਻፦҅ۂǴᖴᖴ یٿԃٰਔόਔޑΖړᆶࢃЃլӳ϶ϩ٦Ƕགᖴڂউǵ۸ۢǵ൹҅Ǵکգॺ΋ଆ Ӟ໭ǵಠϺǵ໶ᖱ၉ޑਔӀ๊ჹࢂࣴز܌ғࢲ࣬྽ख़ाޑ೽ϩǴᖴᖴգॺ΋ၡޑ ഉՔǶགᖴℱଙǵ๋ྨǵර଻ǵች࣑ǵৎᆝǴᖴᖴӚՏΐኴӳუՔॺӧჴᡍ஝ው ϐᎩϝόבӚᅿ࣬ऊܡྣǶགᖴ଼ғ)εߡ*ǵകႠ)૛ቬᙝ*ǵ㤟ْ)औη*ǵറৱ )Kvmjf ૤ಹ*ǵᏫ௵Ǵ཮੝ձѺᆖဦ๊ჹࢂӢࣁԖ٤ΓᆖဦКӜӷӳ૶Ǵᖴᖴգ ॺӧךα၂ਔޑӚᅿᔅԆǴ೭ۛখӳ೚ӭ஥Ԗ౦த࡜ۺޑԴΓ೿ᚆ໒ΑǴ੿Ј׆

ఈգॺૈࡌҥଆឦܭգॺԾρޑჴᡍ࠻਻ݗǴӳӳуݨഽǼ!

! ! ٿԃޑਔӀ०ೳǴ೚ӭޑ៿ઢᆶఽНǴՔᒿ๱౲ӭޑᓸΚᆶ਋שǴᡣךుڅ ӦᏢಞډΑೀШޑᄊࡋکԋߏǶশ२ࣴزޑВη္ǴࡾহҬඹǵЭཪᗋൣǴ΋ၡ

΢وၸޑǵ࣮ډޑǵᏢډޑǴ੿ޑϼӭϼӭǴᇻຬၸԾρ྽߃ޑགྷႽǴ೭ҽ࿶ᐕ

๊ჹࢂᡣΓᡏ཮ుڅǶᜫεৎѤණܿՋࡕǴૈаԖज़ޑғڮញܫคज़ޑӀؽǴᜫ

΋Ϫ໩ճǵ΋ϪѳӼǶ!

!

ੀ๔ܭ 3125ǸѠчǸহ!

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III

ύ

ύЎᄔा

ߙ࢏ੰ๵ȐRalstonia solanacearumȑЇวӄౚ೚ӭբނϐठԝ๺঒ੰ্Ǵத

೷ԋӄౚբނѮε࿶ᔮཞѨǶᗧឦܭ phylotype I ޑߙ࢏ੰ๵๵ਲ਼ёਥᏵӧלགੰ

ࠔسพन΢Їଆޑठԝ౗Ǵ຾΋؁ۓကځࢥΚଯեǴՠҞ߻ᜢܭ،ۓߙ࢏ੰ๵ठ

ੰΚϐϩηᐒڋ܌ޕཱུϿǶࣁᕕှߙ࢏ੰ๵ϐठੰΚӢηǴךॺςֹ᏾ۓׇѤঁ

όӕࢥΚޑߙ࢏ੰ๵ਲ਼Ǵว౜ਏᔈೈқȐeffectorȑRSp0213 ѝӸӧύࢥΚ๵ਲ਼ Ȑӵ Pss4ȑᆶեࢥΚ๵ਲ਼Ȑӵ Pss216ȑύǴԶଯࢥΚ๵ਲ਼Ȑӵ Pss190ȑ߾คԜ effectorǶҁࣴزϐࣴزҞ኱ࣁ௖૸ RSp0213 ӧߙ࢏ੰ๵ठੰΚϷᆶ஌ЬҬϕբҔ ύϐفՅǶ่݀ว౜ӧڀԖ RSp0213 ޑύࢥΚ๵ਲ਼ Pss4 ύஒ RSp0213 কନ཮ග ଯ Pss4 ӧพनϐठੰΚǴԶӧค RSp0213 ޑଯࢥΚ๵ਲ਼ Pss190 ύεໆ߄౜Ԝ effector ୷Ӣ߾ёफ़ե Pss190 ӧพनϐठੰΚǴࡺ RSp0213 ዴჴࢂ،ۓߙ࢏ੰ๵

ठੰΚϐᜢᗖӢηǶԜѦǴӧ๨૛ယТύ߄౜ RSp0213 ཮೷ԋಒझԝΫޑ౜ຝǹ ӧพनύၸໆ߄౜ RSp0213 ҭᏤठಒझԝΫᆶ෌ਲ਼࿖ϯǴЪ཮ᇨᏤНླྀለǵΌ౎

Ϸၸ௵܄ϸᔈૻ৲໺Ꮴ࣬ᜢ୷Ӣϐ߄౜Ǵ຾ԶቚமพनჹଯࢥΚߙ࢏ੰ๵๵ਲ਼ Pss190 ϐל܄ǶќѦǴׇӈϩ݋Ⴃෳᆶ GFP ᑼӝೈқᔠෳዴᇡ RSp0213 ০ပӧ

๨ ૛ ಒ झ ጢ ΢ Ǵ Ъ Ԝ ০ ပ ੝ ܄ ࣁ ୺ Չ ځ ф ૈ ϐ Ѹ ा చ ҹ Ƕ а ΢ ่ ݀ ᡉ Ң Ǵ RSp0213 ёૈ೏พनಒझᒣᇡ٠຾ԶᇨวٛᑇᐒڋǴᏤठ֖ԖԜਏᔈೈқϐߙ࢏

ੰ๵๵ਲ਼ޑठੰΚफ़եǴԶଯࢥΚ๵ਲ਼߾Ӣค RSp0213 Զၨό཮ᇨว஌Ьϐமਗ਼ לੰᐒڋǶ! ፕЎޑಃΒ೽ϩҗܭྍԾᑳनޑ Pss190 ๵ਲ਼ڀҗཱུଯࢥΚǴਥᏵځ

୷ӢᡏׇӈᡉҢ Pss190 ੝Ԗਏᔈೈқ Pseudomonas outer protein P 3 (PopP3)Ǵࡺ

ҁࣴزԑӧ௖૸ PopP3 ӧߙ࢏ੰ๵ठੰΚϐфૈᆶځ࣬ᜢᐒڋǶҁࣴز࠻ޑӃ߻

ࣴز᛾ჴԜ T3E ཮ڙ T3SS ፓ௓Ǵࣴز่݀ҭᡉҢ୷Ӣᙯ౽߄౜ԿύࢥΚ๵ਲ਼ Pss4 ёቚமځӧพन΢ޑठੰΚǴԶஒ PopP3 ߄౜ܭ๨૛ယТคੰቻౢғՠԖճ ܭߙ࢏ੰ๵ϐғߏǴ٠௢ෳځӧ෌ނಈጕᡏޑ০ပՏ࿼ᆶځфૈԖᜢǴёૈᙖԜ ቹៜพन PTI ࡰ኱ϐ΋ޑᘰ໾ဟᆫᑗಕᑈ (callose deposition)Ǵа׭ڋ෌ނ PTI ϸ ᔈǶҁࣴزԑӧ຾΋؁᛾ჴ PopP3 ಈጕᡏ০ပՏ࿼ჹځфૈϐख़ा܄Ǵ٠ᔠෳԜ T3E ӧ୷Ӣᙯ౽߄౜ΠǴߙ࢏ੰ๵ӧพन΢ޑठੰΚ߄౜Ǵکځфૈᆶ HR ϐ࣬

ᜢ܄Ǵаឍܴ PopP3 ӵՖ׭ڋ෌ނޑ࣬ᜢٛᑇϸᔈᐒڋǶ

ᜢᗖӷǺߙ࢏ੰ๵ǵਏᔈೈқǵRSp0213ǵPopP3ǵठੰΚǵeffector-triggered immunity, effector-triggered susceptibility

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Abstract

Ralstonia solanacearum, the causal bacterium of bacteria wilt (BW), causes severe production losses on many important crops worldwide. R. solanacearum phylotype I strains confer varied degrees of virulence/aggressiveness on tomato plants;

however, genetic information on pathogen virulence-related factors is still very limited.

To uncover virulence-related factors, genomes of four R. solanacearum phylotype I strains with different virulence were sequenced. A putative effector protein RSp0213 is present in medium-virulence and low-virulence strain such as Pss4 and Pss216, but absent in high-virulnece strain such as Pss190. This study aimed to elucidate roles of RSp0213 in R. solanacearum virulence and plant-bacterium interaction. Diruption of RSp0213 in medium-virulence strain Pss4 resulted in increased bacterial virulence in tomato, while RSp0213 overexpression in high-virulence strain Pss190 leads to decreased bacterial virulence, evidencing a decisive role of RSp0213 in R.

solanacearum virulence. Additionally, transient local overexpression of RSp0213 in Nicotiana benthamiana leaves led to cell death. Virus-mediated overexpression of RSp0213 in tomato also caused cell death, reduced plant growth, increased expression of marker genes involved in defense hormones and hypersensitive-response signaling pathways, and enhanced resistance to the high-virulence strain Pss190. Furthermore, GFP-tagged RSp0213 colocalizes with cell membrane of N. benthamiana and membrane localization is required for its functions. These results suggest that RSp0213 can be recognized by host cell, leading to induced defense response and thus reduced virulence of RSp0213-containing R. solanacearum strains. The absence of this effector enables high-virulence strains escape from host recognition, leading to the high virulence. The second part of this thesis aimed to uncover a YopJ-type effector protein Pseudomonas outer protein P 3 (PopP3) unique to a high-virulence strain Pss190. Our

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previous study showed that overexpression of popP3 in Rs medium-virulence strain Pss4 led to increased bacterial virulence in tomato. Transient overexpression of popP3 in tomato suppresses pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) related callose deposition. popP3 transcripts was positively regulated by the type III secretion system (T3SS) regulator HrpG. Furthemore, This study proved the mitochondria localization of PopP3 in plant cell is critical to it function on tomato.

These results together suggest that PopP3 is a T3SS effector and contributes to the virulence of R. solanacearum by suppressing plant defense response which related to mitochondrial functions.

Keywords: Ralstonia solanacearum, effector, RSp0213, PopP3, virulence, effector- triggered immunity, effector-triggered susceptibility

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த

தҔᕭቪᆶӄӜჹྣ߄

ᕭቪ ӄӜ

a.a. Amino acid

BSA Bovine serum albumin

bp Base pair

CFU Colony formation unit DEPC Diethypyrocarbonate DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid

dNTP Deoxyribonucleotide triphosphate DPI Days post inoculation

EtBr Ethidium bromide

EtOH Ethanol

GFP Green fluorescence protein GUS β-glucuronidase reporter gene H7996 Hawaii 7996

HR Hypersensitive response

IPTG Isopropyl-beta-D-thiogalactopyranoside MES 2-(N-morpholino)-ethanesulfonic acid

Pcc Pectobacterium carotovorum subsp. carotovorum PCR Polymerase chain reaction

PEG Polyethylene glycol

Pst Pseudomonas syringae pv. tomato PVX Potato virus X

ROS Reactive oxygen species

sqRT-PCR Semi-quantitative reverse transcription PCR rpm Rotation per minutes

Rs Ralstonia solanacearum

SDS Sodium dodecyl sulfate SM1 Semi-selective medium

X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside

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VII

Ҟ Ҟᒵ

α၂ہ঩ቩۓਜ I

ᖴᇞ II

ύЎᄔा III

मЎᄔा IV

தҔᕭቪᆶӄӜჹྣ߄ VI

Ҟᒵ VII

ಃ΋ക ߻ق ... 1

1. ಃΟࠠೈқϩݜس಍ ... 1

1.1 ፓ௓ೈқ ... 1

1.2 ᄬ೷ೈқ ... 2

1.3 ਏᔈೈқ ... 2

2. ߙ࢏ੰᆶߙ࢏ੰ๵ …... 3

3. ߙ࢏ੰ๵ޑϩηठੰᐒڋ ... 4

4. ߙ࢏ੰ๵ಃΟࠠϩݜೈқ T3Es ... 4

4.1 Harpin ... 6

4.2 GALA ৎ௼ ... 6

4.3 AvrA ... 7

4.4 YopJ superfamily ... 7

5. ෌ނੰ্ٛᑇϸᔈ ... 8

5.1 ෌ނ PTI ٛᑇϸᔈ ... 8

5.2 ෌ނ ETI ٛᑇϸᔈ ... 9

5.3 ෌ނಒझጢᄬ೷ೈқୖᆶٛᑇϸᔈ ... 9

6. ੰচಒ๵ਏᔈೈқჹ෌ނϐբҔ ... 10

6.1 բҔܭಒझጢϐਏᔈೈқ ... 10

6.2 բҔܭಈጕᡏϐਏᔈೈқ ... 11

6.3 բҔܭယᆘᡏϐਏᔈೈқ ... 11

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6.4 բҔܭ៶ݰၮᒡϐਏᔈೈқ ... 12

6.5 բҔܭ MAPK ૻ৲໺ሀϐਏᔈೈқ ... 12

6.6 բҔܭಒझਡϐਏᔈೈқ ... 12

6.7 װᔐᙯ᝿ϐਏᔈೈқ ... 13

7. ߻Γࣴزᆶࣴز୏ᐒ ... 13

ಃΒക ׷਑ᆶБݤ ... 15

1. ٮ၂๵ਲ਼ǵ፦ᡏϷ๵ਲ਼୻Ꭶచҹ ... 15

2. ٮ၂෌ނ୻ػచҹ ... 15

3. ғނ಍ीϩ݋ ... 15

4. ख़ಔ፦ᡏᄬᑐᆶ popP3 ၸໆ߄౜๵ਲ਼ࡌᄬ ... 15

4.1. DNA ᛏિᑗᏉጤႝݚ ... 15

4.1.1. ѦࢉᛏિᑗᏉጤႝݚϩ݋ ... 15

4.1.2. ϣࢉᛏિᑗᏉጤႝݚપϯ ... 16

4.2. ᆫӝ䁙ೱᙹϸᔈ ... 16

4.2.1. Taq PCR (Violet) ... 16

4.2.2. Q5® Taq PCR (Invitrogen) ... 16

4.3. ፦ᡏ๧ڗ ... 17

4.3.1. εဉఎ๵፦ᡏ๧ڗ ... 17

4.3.2.ߙ࢏ੰ๵ᆶၭఎ๵፦ᡏ๧ڗ ... 17

4.4. DNA પϯ ... 18

4.5. DNA ज़ڋ䁙੃ϯНှ ... 18

4.6. DNA Тࢤௗӝ ... 18

4.7. TOPO^®፦ᡏᄬᑐ ... 18

4.8. LR ख़ಔϕඤϸᔈ ... 19

4.9. εဉఎ๵യҺಒझ዗ҶլᙯࠠբҔ ... 19

4.10. ႝऀϾᙯࠠբҔϐയҺಒझ࿼ഢ ... 19

4.10.1.ߙ࢏ੰ๵ǵၭఎ๵ ... 20

4.10.2. εဉఎ๵ ... 20

4.11. ႝऀϾᙯࠠբҔ ... 20

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IX

4.12. ߙ࢏ੰ๵ࢉՅᡏ DNA ๧ڗ ... 21

5. ߙ࢏ੰ๵ਏᔈೈқфૈࣴز ... 21

5.1. ๨૛চғ፦ᡏ๧ڗᆶೈқ০ပՏ࿼ᔠෳ ... 21

5.2. ਏᔈೈқܭ๨૛ယТಒझ০ပՏ࿼ᔠෳ ... 22

5.3. ߙ࢏ੰ๵ठੰΚෳ၂ ... 22

6. ෌ނᡏอኩ߄౜ਏᔈೈқ ᆶځфૈ܄ࣴز ... 23

6.1. ๨૛ယ೽୷Ӣอኩ߄౜ ... 23

6.2. พनอኩ܄ੰࢥᇨᏤ୷Ӣၸໆ߄౜ ... 23

6.3. ෌ނᡏอኩ߄౜୷Ӣᔠෳ ... 24

6.3.1 ෌ނ RNA ๧ڗ ... 24

6.3.2 ϸᙯᒵᆫӝ䁙ೱᙹϸᔈ ... 24

6.3.3.ъۓໆ RT-PCR ... 24

6.3.4. ջਔۓໆ Real-time PCR ... 25

ಃΟക ่݀ ... 27

I. ե/ύࢥΚ๵ਲ਼੝Ԗϐ RSp0213 ᆶ RSc3174 ӧߙ࢏ੰ๵ठੰΚϐфૈࣴز …... 27

1. RSp0213 ᆶ RSc3174 ӧςֹӄှׇϐߙ࢏ੰ๵๵ਲ਼ύϐϩթ ... 27

2. RSp0213 ک RSc3174 ჹߙ࢏ੰ๵ठੰΚϐቹៜ ... 27

3. RSp0213 ೈқӧ෌ނಒझύϐ০ပՏ࿼ ... 28

4. RSp0213 ၸໆ߄౜ჹ෌ނϐቹៜ ... 28

5. ӧพन߄౜ RSp0213 ჹੰ্ϸᔈϐቹៜ ... 29

6. ӧพन߄౜ RSp0213 ჹٛᑇϸᔈ࣬ᜢ኱ᇞ୷Ӣ߄౜ϐቹៜ………... 29

7. ،ۓ RSp0213 ೈқӧ෌ނಒझύϐ০ပՏ࿼ޑख़ा୔ࢤϩ݋…... 29

8. RSp0213 ০ပܭ෌ނಒझጢჹځфૈϐख़ा܄ ... 29

II. ଯࢥΚ๵ਲ਼੝Ԗϐਏᔈೈқ PopP3 ӧߙ࢏ੰ๵ठੰΚϐфૈࣴز …... 30

1. PopP ৎ௼ԋ঩ӧߙ࢏ੰ๵๵ਲ਼ϐϩթᆶׇӈ࣬՟܄ ... 30

2. ӧଯࢥΚ๵ਲ਼ PopP3 ϐϩթ ... 31

3. PopP2 ჹߙ࢏ੰ๵ӧพन΢ठੰΚϐቹៜ ... 31

4. PopP3 ჹੰ๵ᇨᏤϐ๨૛ၸ௵܄ϸᔈޑ׭ڋૈΚ ... 31

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X

5. ӧพन߄౜ popP3 ჹੰ্ϸᔈϐቹៜ ... 32

6. ،ۓ PopP3 ೈқӧ෌ނಒझύϐ০ပՏ࿼ޑख़ा୔ࢤϩ݋ ... 32

7. Тࢤմନࡕϐ PopP3 ჹߙ࢏ੰ๵ठੰΚϷჹพनੰ্ϸᔈϐቹៜ ... 33

ಃѤക ૸ፕ ... 34

΋ǵߙ࢏ੰ๵ RSp0213 ϐфૈࣴز ... 34

1. RSp0213 फ़եߙ࢏ੰ๵ჹพनϐठੰΚ ... 34

2. ӧ෌ނ߄౜ RSp0213 ཮ᇨᏤٛᑇϸᔈ ... 35

3. RSp0213 ০ပܭ෌ނಒझጢჹځфૈԿᜢख़ा ... 36

4. RSp0213 ᇨᏤ෌ނ ETI ϸᔈޑёૈᐒڋᆶ҂ٰ৖ఈ ... 37

Βǵߙ࢏ੰ๵ PopP3 ϐфૈࣴز ... 37

1. ଯࢥΚ๵ਲ਼ Pss190 ੝Ԗϐ PopP3 ࣁߙ࢏ੰ๵ख़ाޑठੰΚӢη ... 37

2. PopP3 ཮׭ڋ෌ނ PTI ϸᔈ ... 38

3. PopP3 ০ပܭ෌ނಈጕᡏჹځфૈԿᜢख़ा ... 39

4. PopP3 ׭ڋ෌ނ PTI ϸᔈޑёૈᐒڋᆶ҂ٰ৖ఈ ... 40

ୖԵЎ᝘ ... 42

߄ ... 50

კ ... 52

ߕ߄ ... 75

ߕკ ... 90

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ಃ

ಃ΋ക ߻ق

1. ಃΟࠠೈқϩݜس಍

ಃΟࠠೈқϩݜس಍ (type III secretion system, T3SS) ቶݱӸӧܭॠើМ഍܄

๵ Ǵ ӵ ୏ ނ ੰ চ ๵ Yersinia spp. ǵ Pseudomonas aeruginosa а Ϸ ෌ ނ ੰ চ ๵ Pseudomonas syringe ǵ Xanthomonas spp. ǵ Pectobacterium spp. ฻ (Troisfontaines and Cornelis, 2005)Ƕ྽ੰচ๵ௗ᝻ډ஌ЬਔǴջ཮ε൯ӦࢲϯಃΟࠠೈқϩݜس

಍Ǵ೸ၸԜس಍ёஒਏᔈೈқ (effector) ޔௗଌΕ੿ਡಒझύǴᙖаቹៜ஌Ьಒझ ޑ҅தғ౛ࢲ୏Ǵ຾Զׯᡂੰচ๵ޑགࢉ่݀ (Deslandes and Rivas, 2012)Ƕ

Զᜢܭ T3SS ୖᆶੰ্ϸᔈᐒڋޑਔ໔ᗺǴ߈යࣴزࡰрಃΟࠠೈқϩݜس

಍ӧགࢉ߃යᆶࡕය೿཮࡭ុ߄౜ǴᡉҢಃΟࠠϩݜس಍ӧठੰၸำύᔈࣣԖᜢ ᗖфૈ (Poueymiro and Genin, 2009; Monteiro et al., 2012; Stotz et al., 2014)Ƕ෌ނ

ੰচ๵ޑಃΟࠠೈқϩݜس಍߯җ hrp (hypersensitive response and pathogenicity)/

hrc (hypersensitive response and conserved) ୷Ӣಔ܌ᙯᒵǴ೭٤୷Ӣಔୖᆶಒ๵ӧ གੰࠔس෌ނ΢ޑठੰΚǵаϷלੰࠔس෌ނ΢ޑၸ௵܄ϸᔈ (hypersensitive response, HR) (Tang et al., 2006)Ƕੰচ๵གࢉ෌ނਔ܌ౢғϐಃΟࠠϩݜೈқǴ٩ Ᏽ஌Ь෌ނޑᒣ᛽ૈΚёᏤठٿᅿֹӄ࣬ϸޑ่݀ǺऩಃΟࠠϩݜೈқڙډ෌ނ ޑל܄ೈқ (resistant protein, R protein) ܌ᒣᇡǴ߾෌ނ཮௴୏זೲԶᐟਗ਼ޑխࣝ

ϸᔈǴхࡴౢғ callose ஒಒझᏛቚࠆǴ٠ӧ೏གࢉޑಒझϣεໆಕᑈၸ਼ϯނߦ

٬ಒझ঒Ϋ (programmed cell death, PCD)Ǵஒੰচ๵߳ज़ӧߟΕՏ࿼ǴԶԜᜪڙ ᒣ᛽ޑಃΟࠠϩݜೈқᆀࣁ avirulence protein (Avr protein)ǹ࣬ϸӦǴऩಃΟࠠϩ ݜೈқόڙډ஌Ь෌ނᒣᇡǴ߾Ԝᜪਏᔈೈқёૈԋф׭ڋ஌ЬٛᑇϸᔈǴ٬ੰ

চ๵ԋфགࢉ෌ނԶ೷ԋੰቻǴԜᜪޑಃΟࠠϩݜೈқ߾ᆀࣁ virulence protein (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ƕੰচ๵ޑಃΟࠠϩݜೈқس

಍ϐ࣬ᜢ୷Ӣ٩ྣځфૈ੝܄Ǵё୔ϩԋΟᅿೈқǺ௓ڋಃΟࠠϩݜس಍߄౜ϐ ፓ௓ೈқǵஒ੝ۓೈқଌΕ஌Ьᡏϣޑᄬ೷ೈқϷ೏ϩݜԿੰচ๵ಒझѦޑਏᔈ

ೈқǴ၁ಒ௶ॊӵΠǺ

1.1 ፓ௓ೈқ

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෌ނੰচ๵ਥᏵ hrp ᏹᕵಔ (operon) ޑ่ᄬаϷፓ௓БԄǴёஒಃΟࠠϩݜ

ೈқޑፓ௓س಍୔ϩԋٿεᜪǺಃ΋ᜪࣁ P. syringe ǵPectobacterium spp.ک Pantoea stewartiiǴԜᜪ෌ނੰচ๵ hrp ᏹᕵಔڙډᗧឦ ECF family ޑᙯᒵӢη HrpL ܌ፓ௓ǴHrpS ᆶ HrpR ҬϕբҔࡕᆶ sigma δ⁵⁴่ӝ٠ࢲϯ HrpLǴ΋ѿ HrpL ڙډࢲϯߡ཮௴୏Πෞ୷Ӣޑ߄౜ǹಃΒᜪ߾ࣁ R. solanacearum ک Xanthomonas spp.ǴԜᜪ෌ނੰচ๵ޑ hrp ᏹᕵಔёҗ AraC-like ޑࢲϯη (activator) ܌ࢲϯǴ ӧ Xanthomonas spp. ύᆀࣁ HrpXǴӧ R. solanacearum ύᆀࣁ HrpBǴٿޣࣣ཮ᒣ

᛽௴୏η஥Ԗ੝ۓׇӈޑ୷Ӣ௴୏߄౜Ǵ཮ڙ HrpG ࢲϯࡕӆࢲϯΠෞ hrp ୷Ӣ ϐᙯᒵ (Tang et al., 2006) (ߕკ΋)Ƕ

1.2 ᄬ೷ೈқ

ੰচ๵ஒਏᔈೈқଌΕ෌ނಒझޑၸำǴሡा೯ၸނ౛ࡀምǺٿቫಒ๵Ծ يޑಒझጢǵ΋ቫ两ᆫᑗ (peptidylglycan) ಒझᏛǴаϷ෌ނޑಒझጢکᠼᆢન (cellulose) ಒझᏛǶӢԜੰচ๵ว৖р΋ᅿߏଞᆅރޑᄬ೷ᆀࣁ Hrp pilusǴॄೢ

ೱௗಒ๵ᆶ෌ނಒझǴаճஒਏᔈೈқ໺ଌԿ஌Ьಒझύ (Büttner and He, 2009)Ƕ

ࣴزว౜ᗨฅόӕ٥ᅿ (subspecies) ໔ Hrp pili ޑႣෳΒભ่ᄬΜϩ࣬՟Ǵՠಔԋ ޑữ୷ለׇӈࠅৡ౦ࣗε (Weber and Koebnik, 2005)Ǵ௢ෳಒ๵Ѹ໪ᙖҗᄽϯٰ

όᘐׯᡂԜޔௗᆶ෌ނಒझௗ᝻ϐᆅރ่ᄬǴаᗉխ೏෌ނᒣ᛽Զᇨว෌ނޑٛ

ᑇϸᔈ (Büttner and He, 2009)Ƕ

1.3 ਏᔈೈқ

࿶ಃΟࠠೈқϩݜس಍܌ϩݜǵᒡଌϐೈқᆀࣁਏᔈೈқ (effector)ǴځЬा

фૈӧܭυᘋ෌ނಒझғ౛ࢲ୏ǵቹៜ෌ނಒझխࣝϸᔈǴаׯᡂੰচ๵ठੰૈ

ΚǶਏᔈೈқӧ෌ނಒझޑբҔՏ࿼΢ϩࣁٿᜪǺ΋ᜪࢂ཮೏ଌΕ෌ނಒझϣޑ

ೈқǴٯӵ hop (hrp-dependent outer protein) ୷Ӣ܌ጓ᝿ޑೈқ (Alfano et al., 2000) ǹќ΋ᜪޑਏᔈೈқբҔܭ෌ނಒझ໔ሜǴό཮຾ΕಒझϣǴനԐว౜ޣ Ԗ P. syringae ޑ HrpZ Ϸ R. solanacearum ޑ PopA (Arlat et al., 1992)Ƕӧғނၗૻ

ϩ݋΢ޑࣴزᡉҢǴคፕ෌ނ܈୏ނੰচ๵ޑਏᔈೈқǴځ N ᆄ 25 ঁữ୷ለׇ

ӈࣣ൤֖Ԗ Ser ᆶ coiled ޑ୔ୱǵ٠લЮ Leu (Arnold et al., 2009; Samudrala et al., 2009)Ǵ௢ෳ೭ኬё៻Ԕޑೈқ่ᄬԖշܭਏᔈೈқ೯ၸ੟ઞޑ Hrp pilus (Büttner

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and He, 2009)ǹԜѦǴਏᔈೈқӧϩݜၸำύǴӭኧޑਏᔈೈқѸ໪Ԗ੝ۓޑՔ ᒿೈқ (chaperone) ڐշωૈ຾ՉǴಃΟࠠϩݜೈқس಍ޑՔᒿೈқ೯தλܭ 170

ঁữ୷ለǵևለ܄ǵӧ C ᆄڀԖᚈཱུ܄ (amphipathic) ޑ α-helix ่ᄬ (Parsot et al., 2003) ǹ Ԗ ਔ Ք ᒿ ೈ қ ᗋ ת ᄽ ᆢ ࡭ ਏ ᔈ ೈ қ ᛙ ۓ ܄ ޑ ف Յ Ǵ ӵ Pectobacteria amylovora ޑՔᒿೈқ DspFǴ൩ڀԖᛙۓ၀ੰ๵ᜢᗖਏᔈೈқ DspA/E ޑфૈ

(Gaudriault et al., 2002)Ƕ

2. ߙߙ࢏ੰᆶߙ࢏ੰ๵

ߙ࢏ੰǴΞᆀಒ๵܄๺঒ੰ (bacterial wilt, BW)Ǵࢂҗߙ࢏ੰ๵ (Ralstonia solanacearum, Rs) ܌Їଆϐβᝆ໺ኞ܄ᆢᆅ״ੰ্ǶགࢉԜੰ্ޑ෌ਲ਼ಒਥፃϯ ᆭឳǵӦ΢೽ΠՏယ२ӃΠࠟǴԶࡕယТᅌԛ๺঒ǴӢ๺঒זೲࡺ෌ਲ਼ԝΫਔϝ և౜ߙᆘՅǴࡺᆀࣁȨߙ࢏ੰȩǶԜੰচ๵஌Ьጄൎ఼ᇂऊ 50 ࣽǴຬၸ 200 ᅿ෌

ނǴхࡴพनǵଭႍᖘϷ३ᑴ฻ख़ा࿶ᔮբނǴҞ߻ۘคԖਏϐϯᏢٛݯБݤǴ

ࢂШࣚ΢നᝄख़ޑ෌ނੰ্ϐ΋ (Poueymiro and Genin, 2009; Genin and Denny, 2012)Ƕ

ߙ ࢏ ੰ ๵ ࣁ ॠ ើ М ഍ ܄ (Gram-negative) ǵ ӳ ਼ (aerobic) ǵ ఎ ރ Ǵ ឦ β- proteobacteriaǴғߏޑཱུज़ྕࡋࣁ 4-40 °CǴคݤғߏܭ֖ 2%а΢ NaCl ᕉნǴӧ ଯᔼᎦ୻Ꭶ୷ΠܰౢғፃՅનǶߙ࢏ੰ๵ёߏԃӸࢲܭβᝆ܈НύǴ΋૓ރᄊΠ ёූӸܭβᝆऊ 14 ঁДǴനߏёၲ 25 ԃǴӧ 20-25 °C પНύ߾ёӸࢲߏၲ 40 ԃϐΦ (Denny, 2006)Ƕߙ࢏ੰ๵ޑ୷ӢಔӅऊ 5.8 MbǴҗ΋ঁᕉރࢉՅᡏ (3.7 Mb) аϷ΋ঁ୮ރѮ፦ᡏ (megaplasmid, 2.1 Mb) ܌ಔԋǴځύӸӧ೚ӭ mobile element Ϸ mosaic structureǴЪ୷Ӣಔύ֖ଯКٯ (66.5-68%) ޑ GC ᡵ୷ჹǶԾ 2002 ԃಃ΋ঁҗพनϩᚆԶٰϐߙ࢏ੰ๵๵ਲ਼ GMI1000 ୷Ӣಔ೏ֹ᏾ۓׇࡕ (Salanoubat et al., 2002)ǴҞ߻ӅԖ 5 ঁ๵ਲ਼ޑ୷Ӣಔֹ᏾ׇӈǴаϷ 17 ঁ๵ਲ਼ޑ

ׇӈ߃ዺ (genome draft) ೏ۓׇֹԋ (Boucher, NCBI website; Genin and Denny, 2012; Remenant et al., 2012; Cao et al., 2013; ߄΋ǵ߄Β) Ƕ

ߙ࢏ੰ๵ޑ๵س٩ྣځόӕ੝܄ёճҔΟᅿس಍຾ՉᅿϣϩဂǺਥᏵ஌Ьጄ ൎёϩԋϖᅿ racesǹ٩Ᏽჹόӕᚈᑗ/ᑗᎇϐճҔૈΚёϩԋϤঁ biovarsǹԶа DNA ׇӈϩ݋ᄽϯᜢ߯ёჄϩԋѤঁ phylotypes (Denny, 2006)Ƕphylotypes ᆶ๵

ਲ਼ޑӦ౛ٰྍεठ֍ӝǴϩձࣁ٥ࢪޑ phylotype IǴऍࢪޑ phylotype IIǴߚࢪޑ

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phylotype IIIǴаϷٰԾӑѭǵᐞεճ٥کВҁޑ phylotype IV (Fegan and Prior, 2006)ǶԜѦǴᆶߙ࢏ੰ๵ᒃጔ࣬߈ޑ΍३ᐋੰচ๵ Ralstonia syzygii ᆶ३ᑴੰচ

๵ blood disease bacterium (BDB) Ψឦܭ phylotype IV (Remenant et al., 2011)Ƕ୷ ܭ΢ॊޑፄᚇ܄ǴҞ߻දၹа R. solanacearum species complex ٰᆀڥԜဂ๵ᅿ (Genin and Denny, 2012)Ƕ

3. ߙߙ࢏ੰ๵ޑϩηठੰᐒڋ

ߙ ࢏ ੰ ๵ ག ࢉ ஌ Ь ෌ ނ ၸ ำ Μ ϩ ፄ ᚇ Ǵ ӧ ᕉ ნ ڈ ᐟ ᆶ ဂ ᡏ ག ᔈ (quorum sensing) ޑૻ৲໺ሀၡ৩ΠǴठੰӢηڙډፄᚇЪᆒஏޑፓ௓Ǵ຾Զ௓ڋߙ࢏ੰ

๵᏾ᡏޑੰচ܄৖౜ (Valls et al., 2006; Vailleau et al., 2007; Genin and Denny, 2012)Ƕߙ࢏ੰ๵ޑགࢉၸำύǴ߃යූӸܭβᝆύޑߙ࢏ੰ๵གڙډ஌Ь෌ނਥ

೽ϩݜނϐࡕǴߡ཮ڙᖿϯ܄ (chemotaxis) ᇨᏤ౽୏Կ෌ނߕ߈Ǵ׎ԋғނጢ (biofilm) ຾Զߕ๱ឺᏵ (root attachment and colonization) (Schell, 2000; Yao and Allen, 2006)Ǵӕਔ߄౜ಃΟࠠೈқϩݜس಍ (type III secretion system, T3SS) ϩݜ ਏᔈೈқǴଌΕ෌ނಒझυᘋ෌ނ҅தғ౛фૈ (Poueymiro et al., 2009)Ƕ྽ᙖҗ

෌ނୁਥғߏೀ܈໾αߟΕࡕ (Yao and Allen, 2007)Ǵߡёس಍܄གࢉ᏾ਲ਼෌ނޑ ಳ ೽ Ǵ ݮ ๱ Е ፦ ೽ (xylem) ӛ ΢ ቚ ғ ᕷ ෗ ǵ ౢ ғ ε ໆ झ Ѧ ӭ ᗐ ᡏ (exopolysaccarides, EPS) ߔ༞Ր෌ނНϩၮᒡǴᕇڗ܌ሡᎦҽޑӕਔᏤठ෌ނԝ Ϋ (Denny, 2006)Ƕ

җܭߙ࢏ੰ๵ޑठੰၸำ౐ੋቶᗡǴ࣬ᜢޑठੰӢηཱུӭǴх֖ߙ࢏ੰ๵ޑ ᖿϯ܄ᆶݚ୏ૈΚ (motility)ǵझѦӭᗐᡏޑౢғǵಃΒࠠೈқϩݜس಍ (type II secretion system, T2SS) Ϸځ܌ϩݜϐಒझᏛНှሇન (cell wall degradation enzyme) (Liu et al., 2005)ǵᆶಃΟࠠೈқϩݜس಍฻ (Peeters et al., 2013)Ƕҁࣴزޑჹຝࣁ ߙ࢏ੰ๵ޑಃΟࠠϩݜਏᔈೈқ (Type III effectors, T3Es)Ƕ

4. ߙ࢏ੰ๵ಃΟࠠϩݜೈқ T3Es

ӧߙ࢏ੰ๵ύǴӧځ 1.9-Mb ޑε፦ᡏύಃΟࠠϩݜೈқፓ௓س಍ϐ hrp ୷Ӣ ဂεऊ՞Ԗ 23 kbǴϩԋԿϿϤঁᙯᒵൂՏǴጓ᝿рຬၸ 20 ঁᆫᴏ两᜘Ǵಔԋᐉ

ၠϣѦጢޑೈқϩݜس಍ (Arlat et al., 1992)Ƕੰচ๵२Ӄ೸ၸѦጢௗڙᡏ PrhA receptor གڙ෌ނૻဦϐࡕǴஒૻ৲໺ሀ๏ϣጢೈқ PrhRǴ٬চҁᆶ PrhR ่࣬ӝ

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ޑ sigma factor PrhI ϩᚆញܫډಒझ፦྽ύǴ௴୏ prhJ ޑ߄౜ǴᙯᒵӢη PrhJ ཮

຾΋؁ࢲϯ hrpGǴHrpG ӆᇨᏤ hrpB ޑ߄౜Ǵനಖҗ HrpB ፓ௓ΠෞޑಃΟࠠϩ ݜೈқϐ߄౜ǴHrpB ཮ࢲϯ௴୏η஥Ԗ hrpII box ׇӈ TTCG-N16-TTCG ޑ୷ӢǴ

ࣴزว౜ऩׯᡂԜࢤߥӺ܄ׇӈǴ཮ቹៜډ௴୏ηڀԖ hrpII box ޑ popABCǵ hrpY ୷Ӣ߄౜ (Cunnac et al., 2004a)ǶЪ᏾ঁૻဦ໺ሀޑၡ৩೿཮ڙډठੰύኰፓ

௓Ӣη PhcA ܌ᆅ௓ (Peeters et al., 2013) (ߕკ΋)ǶԜѦǴHrpG ޑ਒سӕྍೈқ (paralogue) PrhG Ψ೏᛾ჴڀԖࢲϯ hrpB ޑфૈǴՠ prhG ѝ཮ӧ୷ҁ୻Ꭶ୷ (minimal medium) ύ೏ᇨᏤ߄౜Ǵࠅόڙډ෌ނಒझޑૻဦ܌ࢲϯ (Plener et al., 2010)ǶಃΟࠠϩݜೈқፓ௓س಍ჹܭߙ࢏ੰ๵ޑठੰߚதख़ाǴࣴزᡉҢஒߙ࢏

ੰ๵ޑҺ΋ hrp ୷ӢँᡂࡕǴ཮٬ߙ࢏ੰ๵ӧགੰ෌ނ΢ѨѐठੰΚǴӕਔΨ཮

Ѩѐӧלੰ෌ނ΢ᇨᏤၸ௵܄ϸᔈޑૈΚ (Van Gilsegem et al., 1995)Ƕ

ᒿ๱२ঁߙ࢏ੰ๵๵ਲ਼ GMI1000 ޑ୷ӢಔှׇǴ೸ၸғނၗૻϷᙯᒵᡏᏢޑ ϩ݋Ǵߙ࢏ੰ๵ޑਏᔈೈқ೏ว౜ڀԖаΠΟঁ੝܄ (Deslandes and Genin, 2014;

Kazan and Lyons, 2014)Ǻಃ΋Ǵൂ΋ߙ࢏ੰ๵๵ਲ਼ޑਏᔈೈқኧໆᇻӭܭځд෌

ނੰচ๵Ǵѳ֡΋ঁߙ࢏ੰ๵๵ਲ਼൩ڀԖ 60-80 ঁ T3Es ཮೏ଌΕ෌ނᡏ (Cunnac et al., 2004b)Ǵ࣬ၨܭ Xanthomonas spp.ᆶ P. syringae ޑ 30-45 ঁǴ೭ёૈΨࢂߙ

࢏ੰ๵஌ЬጄൎཱུቶޑচӢ (Poueymiro and Genin, 2009; Genin and Denny, 2012)Ǵ ՠ໻ԖϿኧೈқޑфૈ೏ϩ݋ǶಃΒǴߙ࢏ੰ๵Ӛ๵ਲ਼໔ᏱԖޑਏᔈೈқ۶Ԝৡ ౦܄ࡐଯǴ௢ෳࢂҗܭߙ࢏ੰ๵৒ܰ೸ၸНѳ୷Ӣᙯ౽ (horizontal gene transfer) ᕇளཥޑਏᔈೈқ (McCann and Guttman, 2008)ǴԖ٤ёૈٰԾ੿ਡಒझǴӵ஥Ԗ F-box domain ޑ GALA ೈқ (Angot et al., 2006)ǹԖ٤߾ёૈٰԾځд୏ǵ෌ނੰ

চ๵Ǵӵ TAL (transcriptional activator-like) ೈқǴځ೏ᇡࣁྍԾܭ Xanthomonas spp.ޑ TAL ೈқǴЪӕኬ೏᛾ჴ০ပӧ෌ނಒझਡ٠ڀԖӧ෌ނಒझύբࣁᙯᒵ ࢲϯӢη (transcriptional activator) ޑфૈ (Li et al., 2013)ǶಃΟǴߙ࢏ੰ๵ޑਏᔈ

ೈқ۳۳аৎ௼ (family) ޑ׎Ԅр౜Ǵӵ GALA ৎ௼ǵSKWP ৎ௼ (Poueymiro and Genin, 2009)Ǵ௢ෳߙ࢏ੰ๵ࣁΑ፾ᔈቶݱޑ஌Ь෌ނǴԶว৖р࣬՟ೈқࠅ фૈӭኬޑᄽϯ׎Ԅ (Remigi et al., 2011)Ƕ

ᜢܭߙ࢏ੰ๵ਏᔈೈқޑࣴزǴ߈ԃٰ໣ύӧағނၗૻБݤǵ܈೸ၸфૈ

܄ᑔᒧٰ൨פ҂ޕޑਏᔈೈқ (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ǵ

୤໻ԖϿኧਏᔈೈқԖ೏ుΕࣴزǴᖐٯ௶ॊӵΠǺ

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4.1 Harpin

೚ӭॠើМ഍܄෌ނੰচ๵ޑ hrp ୷Ӣဂ཮ᙯ᝿р΋ᅿᆀࣁ harpin ޑೈқǴ ၀ೈқޑӅӕ੝܄ࣁǺ዗ᛙۓ (heat-stable)ǵữ୷ለׇӈύ൤֖ glycine Ъ཮ᇨᏤ לੰ෌ނޑၸ௵܄ϸᔈ (Kanda et al., 2003)ǶPopA (Pseudomonas outer protein A) ࣁߙ࢏ੰ๵ύಃ΋ঁ೏ว౜ޑ harpin (Arlat et al., 1992)ǴԜਏᔈೈқբҔܭ෌ނ ಒझ໔ሜǴёᇨᏤ๨૛ޑၸ௵܄ϸᔈǴ٠ᆶ popB (Pseudomonas outer protein B)ǵ popC (Pseudomonas outer protein C) Ӆࣁፓ௓ಔ (operon)Ǵڙ HrpB ܌ፓ௓Ƕࣴز ว౜ݙ৔஥Ԗ popA ϐߙ࢏ੰ๵ܭ๨૛ယύǴpopA ܭъλਔϣ൩཮ଯࡋ߄౜Ǵՠ ࠅ཮ѨѐᇨᏤၸ௵܄ϸᔈޑૈΚǹԶ࡭ុ߄౜ popA ϐߙ࢏ੰ๵ӧ๨૛዆ឲ၂ᡍ ύठੰΚΠफ़Ǵՠޔௗಳ೽ݙ৔၂ᡍࠅόቹៜځठੰૈΚǶᡉҢ PopA ӧߙ࢏ੰ

๵Ԑයགࢉਔ཮ڙډ๨૛܌ᒣᇡǴ٬๨૛ӧᒣᇡډੰচ๵ΕߟਔջЇวٛᑇϸᔈ (Kanda et al., 2003)Ƕ

4.2 GALA ৎ௼

GALA ৎ௼ϐਏᔈೈқӧߙ࢏ੰ๵ύ֖ԖΎঁԋ঩Ǵϩձࣁ GALA1-7Ǵځ Ӆӕ੝܄ӧܭữ୷ለׇӈύ֖Ԗ෌ނ੝Ԗϐ leucine-rich repeats (LRR) ᆶ੿ਡғނ (eukaryote) ϐ F-box domainǹځύӧ LRR ׇӈ္Ǵ׳ڀԖߥӺ܄ GAxALA ޑׇ

ӈǴӢԶᆀࣁ GALA ৎ௼ (Angot et al., 2006)ǶΔgala1-7 ϐँᡂਲ਼ӧߓ܎դ޺ᆶพ नύ཮फ़եठੰΚ (Angot et al., 2006)ǴځύΞа GALA2ǵ3ǵ6ǵ7 ჹགࢉߓ܎դ

޺ၨࣁख़ाǴGALA3ǵ7 ߾ࣁགࢉพन܌Ѹ໪ (Remigi et al., 2011)ǹԜѦǴ GALA7 ࣁ೷ԋरጴ (Medicago truncatula) ठੰ܌όё܈લޑೈқǴЪځ F-box domain ჹܭ GALA7 ޑठੰΨΜϩᜢᗖǴ F-box ೈқ೏ᇡࣁࢂ੿ਡೈқύᆶ SKP1-like ೈқҬϕբҔϐ୔ୱǴԶ SKP1-like ೈқΞ཮ᆶ Cullin1 ೈқҬϕբҔǴ Ӆӕ׎ԋ SCF-type E3 ubiquitin ligaseǴ೷ԋೈқ፦ޑݱનϯ (ubiquitination) ᆶफ़

ှ (Angot et al., 2006)ǶӢԜǴGALA ৎ௼೏ᇡࣁ཮೸ၸυᘋ஌Ь෌ނޑݱનϯफ़

ှ೼৩Ǵѐቹៜ෌ނޑғ౛фૈǴԶόӕޑ GALA ৎ௼ԋ঩ӧόӕ෌ނ΢܌೷ԋ ޑठੰૈΚΨόӕǴ௢ෳ GALA ৎ௼ԋ঩໔ϝԖфૈৡ౦Ǵࣁޑࢂ፾ᔈቶݱޑ஌

Ь෌ނǶ

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4.3 AvrA

ߙ࢏ੰ๵ AvrA ਏᔈೈқǴ೏ว౜ࢂ๨૛ N. tabacum ᆶ N. benthamiana ύന Ьाޑᒣ᛽ჹຝǴavrA ँᡂਲ਼཮Ѩѐӧ N. tabacum ᆶ N. benthamiana ΢ᇨᏤၸ௵

܄ϸᔈޑૈΚǴځύữ୷ለಃ 32 ဦډಃ 50 ဦׇӈǴԜ 18 ঁữ୷ለࣁౢғၸ௵ϸ ᔈޑख़ा୔ୱǶҗܭ AvrA ׇӈύӸӧԖεໆޑख़ፄׇӈ (tandem repeats)Ǵ؂ঁ

ׇӈх֖ 12 ঁᡵ୷ჹ (base pairs)ǴЪ೽ҽׇӈᙯ᝿ࡕখӳՏܭಃ 32 ډ 50 ဦữ୷ ለϐ໔ǴᡉҢ೭٤ख़ፄׇӈӧᄽϯ΢നಖڙډ๨૛܌ᒣᇡ (Poueymiro et al., 2009)Ƕ

4.4 YopJ superfamily

ӧߙ࢏ੰ๵ख़ाЪ೏ుΕࣴزϐਏᔈೈқύǴхࡴ PopP ৎ௼Ǵᗧឦܭ YopJ (Yersinia outer protein J) superfamilyǴࣁಃΟࠠϩݜೈқύޑ΋ঁѮεৎ௼Ǵ നԐӧ୏ނޑဉၰࣽੰচ๵ Yersinia pseudotuberculosis ύ೏ว౜ (Monack et al., 1997)ǴԶځӕسނ (homologue) ቶݱӸӧܭӚᅿόӕޑ୏ǵ෌ނੰচ๵྽ύǴх

ࡴ୏ނੰচ๵ Salmonella spp.ޑ AvrAǹ෌ނੰচ๵ Pseudomonas spp.ޑ HopZ ৎ

௼ǴXanthomonas spp.ޑ AvrRxvǵAvrXv4ǵAvrBsTǵXopJǴPectobacterium spp.

ޑ ORFBǴR. solanacearum ޑ PopP ৎ௼ǹаϷ෌ނӅғ๵ Rhizobium spp.ޑ Y4LO (Lewis et al., 2011)Ƕ

ӧ ߙ ࢏ ੰ ๵ ύ Ԗ Ο ঁ ᗧ ឦ YopJ superfamily ޑ ਏ ᔈ ೈ қ ಍ ᆀ ࣁ PopP (Pseudomonas outer protein P)Ǵϩձࣁ PopP1ǵPopP2 Ϸ PopP3Ƕࣴزว౜ߙ࢏ੰ

๵ GMI1000 popP1::Tn ँᡂਲ਼ӧགࢉלੰ෌ނ࿖౐Ф (Petunia) ࡕ཮೷ԋ෌ނԝΫǵ གੰ෌ނߓ܎դ޺ᆶพनޑ๺঒௃׎߾཮ౣ༾ගϲǴԶচҁؒԖ PopP1 ޑߙ࢏ੰ

๵ Rd15 ๵ਲ਼Ǵӧ߄౜ PopP1 ࡕ཮Ꮴठ၀๵ਲ਼ӧচҁགੰޑ࿖౐Ф΢ࢥΚΠफ़ (Lavie et al., 2002)ǴЪаၭఎ๵อኩ߄౜ PopP1 ܭ๨૛ယύ཮ౢғၸ௵܄ϸᔈǴ ߄Ң PopP1 ڙ๨૛܌ᒣ᛽ǴᡉҢ PopP1 ӧߙ࢏ੰ๵ύࣁ avirulence protein ޑفՅ (Poueymiro et al., 2009)Ƕ

PopP2 ߾ӧߓ܎դ޺ύ೏ుΕ௖૸ǺGMI1000 ΔpopP2 ӧགࢉלੰޑߓ܎դ޺

ࠔس Nd-1 ࡕǴ཮Ꮴठ Nd-1 ޑԝΫǴЪࣴز᛾ჴ Nd-1 ύڀԖёޔௗᆶ PopP2 Ҭ ϕբҔޑ R ೈқ RRS1-RǴ၀ೈқ C ᆄڀԖӧ R ೈқύவ҂ว౜ޑ WRKY motif (Deslandes et al., 2003)ǹӕਔǴҗ RRS1-R ܌ᇨᏤޑ෌ނל܄ሡा΋ঁ cysteine ೈ қ䁙 Rd19 ٰӅӕ௴୏ǴRd19 ᆶ RRS1-R ΋ኬ೏᛾ჴᆶ PopP2 ёޔௗҬϕբҔ

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(Bernoux et al., 2008)ǶPopP2 ӧ N ᆄ 34-53 ঁữ୷ለׇӈ೏ႣෳڀԖΕਡׇӈ (nuclear localization signal, NLS)Ǵჴᡍ่݀Ψᡍ᛾ PopP2 ዴჴ཮຾Ε෌ނಒझਡ ύǴᆶ RRS1-R ڀԖ࣬ӕޑ০ပՏ࿼ (Deslandes et al., 2003)ǹќѦǴPopP2 ೏ว౜

ёԾךΌㄽϯ (auto-acetylation) ޑૈΚǴΌㄽϯՏᗺӧ K383Ǵ၀ữ୷ለՏᗺӧ YopJ superfamily ԋ঩ύߥӺӸӧǴЪ PopP2 ԾךΌㄽϯޑࢲ܄ჹܭ RRS1-R ܌ᇨ Ꮴޑ෌ނל܄ࢂߚதख़ाޑ (Tasset et al., 2010)Ƕ

5. ෌෌ނੰ্ٛᑇϸᔈ

෌ނޑխࣝϸᔈ߯җ΋঺ፄᚇޑགޕၸำᆶૻဦ໺ሀୖᆶځύǴ΋૓ԶقǴ Ҟ߻෌ނޑٛᑇϸᔈё୔ϩࣁٿεᜪǺҗੰচ๵ي΢ޑߥӺ܄ϩη pathogen- associated molecular patterns (PAMP) ܌ᇨᏤޑ PTI (PAMP-triggered immunity) ٛᑇ ϸᔈǴаϷ೸ၸלੰ R ೈқѐᒣ᛽ੰচ๵ ਏᔈೈқԶ௴୏ޑ ETI (effector- triggered immunity) ٛᑇϸᔈ (Pieterse et al., 2009)ǶԶੰচ๵ࣁΑၲԋགࢉ෌ނҞ ޑǴᄽϯр׭ڋ෌ނٛᑇϸᔈޑਏᔈೈқǴҔаυᘋ෌ނޑғ౛фૈǵ׭ڋ஌Ь ϐխࣝᆀࣁ ETS (effector-triggered susceptibility)Ƕനಖੰচ๵ᏱԖӭኬৡ౦ϯޑ ਏᔈೈқǴԶ෌ނᡏύࢂցڀԖ஑΋܄ёᒣᇡੰ๵ਏᔈೈқϐלੰ R ೈқǴ߾،

ۓΑ෌ނૈց௴୏஑΋܄ၨଯޑխࣝس಍ǵౢғၸ௵܄ϸᔈϷ PCD ฻аज़ڋੰ๵

ᘉණԶ،ۓஎЬޑלੰૈΚ (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ƕ

5.1 ෌ނ PTI ٛᑇϸᔈ

ੰচ๵ي΢ёᇨᏤ PTI ٛᑇϸᔈޑ PAMPǴ೯தࣁੰ๵ߥӺ (conserved) Ъ ό ё ܈ લ ޑ ่ ᄬ ೈ қ Ǵ ӵ ಒ ๵ ᚎ Л ೈ қ flagellin ǵ ಒ ๵ ಒ झ Ꮫ ԋ ҽ peptidoglycan (PGN) ǵ চਡғނ两᜘ۯߏӢ η (elongation factor Tu, EFTu) ฻ (Boller and Felix, 2009) Ƕ ੰ চ ๵ ௗ ᝻ ډ ෌ ނ ਔ Ǵ ෌ ނ ཮ ೸ ၸ ጢ ΢ ޑ pattern recognition receptors (PRRs) གڙډੰ๵ي΢ޑ PAMPǴ଺ࣁᑈᐟ䁙 (kinase) ޑ PRR ೈқߡ཮௴୏ᕗለϯϸᔈ (phosphorylation)Ǵ֎Їځдೈқ፦่ӝ܈ׯᡂԾ ي่ᄬញрҬϕբҔೈқǴࢲϯΠෞޑխࣝᐒڋǶ྽෌ނௗԏډੰচ๵ૻဦǴ२ Ӄಒझጢޑᚆη೯೸܄཮ӧࡐอޑਔ໔ϣวғׯᡂǴගϲಒझ፦ϣޑ້ᚆη֖ໆǴ ᇨว෌ނ਻Ͼ (stomata) ᜢഈǴᗉխ෌ނᝩុௗ᝻ੰচ๵ǹ້ᚆηӕਔΨёᇨᏤಒ झϣ ROS ޑғԋǴROS ନޔௗ೷ԋಒ๵ғӸӒ্ѦǴ׳ёࢲϯΠෞޑ MAPK

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cascades ޑૻ৲໺ሀၡ৩Ǵ຾Զ௴୏ٛᑇ୷Ӣ߄౜Ǵ܈ᘰ໾ဟᆫᑗ (callose) ғӝ ԋ୷ӢǴӧ෌ނಒझ໔ሜϣಕᑈᘰ໾ဟᆫᑗǴа߳ज़ੰচ๵ޑΕߟǶΨӢԜǴ PTI ٛᑇϸᔈࣁ෌ނჹбੰচ๵ޑ२ၰٛᑇس಍ǴځЬा੝ቻࣁᜢഈ਻Ͼǵ້ᚆ ηޑቚуǵזೲᇨᏤ ROS аϷ୴ᑈᘰ໾ဟᆫᑗ (Muthamilarasan and Prasad, 2013)Ƕ

5.2 ෌ނ ETI ٛᑇϸᔈ

Ꮓᆅ෌ނ PTI ϸᔈӧᄽϯ΢ԋфܢᏲΑε೽ϩੰচ๵ǴࠅΨԖੰচ๵ᄽϯр

࣬ᔈޑჹ฼ٰँઇ PTI ٛጕǴҭջᄽϯрբࣁ Virulence factor ޑӭᅿόӕਏᔈೈ

қٰυᘋ෌ނஎЬޑ҅தғ౛ᐒڋǵ׭ڋ஌ЬϐխࣝǶฅԶ΋ѿਏᔈೈқ೏෌ނ ޑ R ೈқ܌ᒣᇡǴٗ൩཮ᇨᏤ෌ނಃΒၰٛᑇϸᔈǴΨ൩ࢂ ETI ϸᔈǶETI ϸᔈ ନёᇨᏤಒझϣ ROS ޑғԋǴΨ཮ࢲϯΠෞޑ MAPK ૻ৲໺ሀၡ৩Ǵ຾Զ௴୏

೚ӭس಍܄ٛᑇລᅟᆾ࣬ᜢ୷Ӣޑ߄౜ǴᇨᏤхࡴНླྀለ (salicylic acid, SA) GluA Ϸ PR1a ฻࣬ᜢ኱ᇞ୷Ӣ߄౜ (Milling et al., 2011)ǵΌ౎ (ethylene, ET) PR1b1 ک Osmotin ฻ғӝԋ࣬ᜢ኱ᇞ୷Ӣޑ߄౜ (Milling et al., 2011) (ߕ߄Ύ)Ǵ٬

෌ނౢғس಍܄խࣝϸᔈ (systemic acquired resistance, SAR)Ǵ٠ᇨᏤ୔ୱ܄ޑಒ झԝΫ (programmed cell death, PCD) ฻ૻ৲໺ᏤǴаߔЗੰচ๵ᝩុΕߟ෌ނ (Deslandes and Genin, 2014; Kazan and Lyons, 2014)Ƕ

5.3 ෌ނಒझጢᄬ೷ೈқୖᆶٛᑇϸᔈ

೚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) Ǵ ٠ Ъ ӧ ೚ ӭ ࣴ ز ύ ೿ ࡰ р Α PRR ᆶ PAMP ٿ ޣ ϐ ໔ ޑ ჹ ᔈ ᜢ ߯ (Muthamilarasan and Prasad, 2013)Ǵՠჹܭ PRRs ޑ࣬ᜢڐӕբҔೈқ (association proteins)Ǵ߾ࢂӧ߈൳ԃഌុ೏ుΕࣴزǴٯӵ PRR FLS2Ǵࣁ΋ᅿ leucine-rich receptor kinaseǴӧௗڙډಒ๵ᚎЛࡕǴջሡाӧ෌ނಒझጢᆶќ΋ঁ receptor- like kinase BIK1 ӅӕբҔǴۈૈᇨᏤΠෞ࣬ᜢ PTI ໺ሀၡ৩ (Lu et al., 2010)ǶԶ ჹܭԖᜢ෌ނಒझጢୖᆶ෌ނ࣬ᜢٛᑇϸᔈޑࣴزǴନΑᚈቫᕗિޑᄬ೷ගٮΑ ނ౛܄ࡀም (physical barrier) аѦǴಒझጢ΢ޑᕗિжᖴፓ௓ҭ཮ჹಒझٛᑇϸ ᔈౢғख़ाቹៜǴӵፓ௓ಒझᕗિޑሇન phospholipaseǴջ೏᛾ჴჹܭ෌ނಒझ

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ޑ PCD ϸᔈϷٛᑇૻ৲໺Ꮴתᄽख़ाفՅ (Kim et al., 2014)ǴΞӵಒझጢ΢ޑ੝

ۓ่ᄬિใ (lipid raft)ǴӢځୖᆶፓ௓ಒझϣޑિ፦жᖴ (Bhat and Panstruga, 2005;

Lingwood and Simons, 2010)ǴЪς᛾ჴᆶᇨᏤ PCD ౢғϐ ROS ಕᑈஏϪ࣬ᜢ (Mersmann et al., 2010)Ǵࡺӧ෌ނٛᑇϸᔈޑፓ௓΢ڀԖᜢᗖفՅ (Fallahi-Sichani and Linderman, 2009)Ƕ

િใ߯җᕗિǵڰᎇᜪ (sterols) аϷ࣬ᜢ੝ਸфૈೈқ܌ᄬԋǴቶݱӸӧܭ

੿ਡғނύхࡴ෌ނಒझǶ࣬ᜢࣴزࡰрિใӧಒझጢޑᄬԋᛙۓ΢ǴаϷಒझ ϣ࣬ᜢિ፦жᖴޑፓ௓΢ڀԖԿᜢख़ाޑفՅ (Lingwood and Simons, 2010)ǴԶ ಒझޑ࣬ᜢિ፦жᖴፓ௓߾΋ޔ೏ຎࣁᆶ෌ނޑٛᑇϸᔈஏϪ࣬ᜢ (Munnik, 2001)ǶᖐٯԶقǴӧ෌ނ༾ғނҬϕբҔύǴԖ٤ੰচ๵཮ӧ෌ނಒझύញрિ

፦Нှ䁙 (lipid hydrolases) Ǵаઇᚯ஌Ьಒझޑٛᑇ่ᄬǴ٠Ъ٬எЬಒझሡा

ᙖҗಒझጢೈқ܌Їว܈ቚமޑ࣬ᜢٛᑇᐒڋфૈ഼ѨǴٰၲډߔ๊෌ނಒझౢ

ғس಍܄ٛᑇϸᔈϐҞޑ (Shah, 2005)ǶԶੰচ๵ନΑаሇનНှޑБԄઇᚯ෌

ނ ಒ झ ጢ Ѧ Ǵ ҭ ཮ ᙖ җ υ ᘋ ෌ ނ ಒ झ ޑ ਼ ϯ ᄊ ޑ િ ፦ (oxidized fatty acid) Oxylipins ޑբҔǴٰቹៜ෌ނಒझғߏᆶٛᑇϸᔈ (Vellosillo et al., 2007)Ƕ

6. ੰੰচಒ๵ਏᔈೈқӧ෌ނᡏϐբҔ

٩ᏵҞ߻ςޕϐਏᔈೈқӧ෌ނᡏբҔޑՏ࿼Ǵёεౣஒੰ๵ϐਏᔈೈқϩ ᜪǺϩձࣁբҔܭಒझጢǵಈጕᡏ (mitochondria)ǵယᆘᡏ (chloroplast)ǵ៶ݰ (vesicle) ၮᒡǵMAPK ૻ৲໺ሀǵᆶಒझਡϐਏᔈೈқ (Deslandes and Rivas, 2012;

Bohm et al., 2014)Ƕ

6.1 բҔܭಒझጢϐਏᔈೈқǺ

෌ނಒझጢ΢ڀԖ PRRs ฻ڙᡏೈқǴаϷ೚ӭᇨᏤΠෞٛᑇϸᔈϷፓ௓ᕗ

િжᖴޑᄬ೷ǴӢԜஎЬಒझጢதԋࣁੰচ๵ਏᔈೈқޑբҔҞ኱Ǵӵߓ܎դ޺

ಒझጢ΢ޑ RIN4 (RPM1-interacting protein 4)ǴRIN4 ࣁ෌ނխࣝϸᔈύॄፓ௓ೈ

қ (negative regulator)ǴPseudomonas ਏᔈೈқ AvrB ᆶ AvrRpm1 ൩೸ၸᇨᏤ RIPK (RIN4-interacting receptor-like protein kinase) ޑᕗለϯ߄౜Ǵ຾Զᕗለϯځ ҬϕբҔϐೈқ RIN4Ǵ׭ڋ PTI ϸᔈǹځ࣬ჹᔈϐ R ೈқ RPM1 Ψς೏פډ (Liu et al., 2011)ǶPseudomonas ਏᔈೈқ AvrRpt2 ߾ࣁ cysteine ೈқ䁙 (protease)Ǵ

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཮೷ԋ RIN4 ޑϩှǴа׭ڋ෌ނ PTI ϸᔈ (Afzal et al., 2011)Ǵՠځϩှޑ࿗Т ࠅΨ཮೏ R ೈқ RPS2 ܌ᒣᇡǵ௴୏ ETI ٛᑇǴࡺќ΋ P. syringae HopF2 ਏᔈೈ

қǴ཮ᆶ RIN4 ҬϕբҔǴᗉխ AvrRpt2 ϩှ RIN4 ೷ԋ ETI ޑࢲϯǶ

ќѦǴP. syringae ޑ AvrPto ᆶ AvrPtoB ΨࢂբҔӧ෌ނಒझጢ΢ޑਏᔈೈқǶ AvrPto ཮׭ڋߓ܎դ޺Ϸพन FLS2 ޑᕗለϯфૈǴߔЗΠෞޑ PTI ૻဦ໺ሀ (Xiang et al., 2008)ǹAvrPtoB ߾ࢂ཮ᆶ FLS2ǵFLS2 ϐӅௗڙᡏ (co-receptor) BAK1 (BRI1-associated kinase 1) ҬϕբҔǴ׭ڋჹಒ๵ᚎЛೈқޑགޕௗڙ (Gohre et al., 2008)ǶԶӧพनύǴAvrPto ᆶ AvrPtoB ߾཮ᆶพनಒझύޑ Pto ᐟ 䁙ҬϕբҔǴࣴز௢ෳ Pto ёૈתᄽᝡݾ่ӝ (competitive binding) ޑفՅǴ٬

AvrPto ᇻᚆ PRR ೈқǴ຾ԶᇨᏤ෌ނޑ ETI ٛᑇϸᔈ (Xiang et al., 2008)Ƕ

6.2 բҔܭಈጕᡏϐਏᔈೈқǺ

੿ਡғނಈጕᡏࢂᆅ௓ PCD ϸᔈޑύኰ (Lam et al., 2001)ǴPCD ϸᔈՔᒿޑ

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

6.3 բҔܭယᆘᡏϐਏᔈೈқǺ

෌ނੰচ๵ύ೏ว౜০ပܭယᆘᡏύޑ ਏᔈೈқǺP. syringae HopI1 ᆶ HopN1ǶHopI1 ຾Εߓ܎դ޺෌ނಒझࡕǴ཮׎ԋ΋ঁεޑፄӝᡏ (complex)Ǵځ

ೈқ፦ C ᆄᆶ Hsp70 (heat shock protein 70) ่࣬ӝǴஒಒझ፦ύණပޑ Hsp70 ໣ ύԿယᆘᡏύǴယᆘᡏࣁ SA ғӝԋ൑܌ǴHsp70 ೏ᇡࣁ཮ቹៜ SA ޑғӝԋ܈ၮ ᒡ࣬ᜢфૈ (Jelenska et al., 2010)ǶԶӧ๨૛ᆶพनύǴHopN1 ཮෧եੰচ๵܌ᇨ Ꮴޑ PCDǵROS ౢғǵᆶᘰ໾ဟᆫᑗಕᑈǹࣴزว౜ HopN1 ཮ᆶ෌ނӀس಍

(photosystem II, PSII) ϐख़ाԋ঩ PsbQ ่ӝǴ೷ԋ PsbQ ޑफ़ှ຾Զ׭ڋӀس಍ޑ ࢲ܄ǵ׭ڋ ROS ғԋǴυᘋ෌ނ҅தғ౛ࢲ୏ (Rodriguez-Herva et al., 2012)Ƕ

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6.4 բҔܭ៶ݰၮᒡϐਏᔈೈқǺ

ӧߓ܎դ޺ύǴP. syringae ϐਏᔈೈқ HopM1 ཮ಕᑈܭ෌ނಒझޑϸԄଯ୷ Мᡏᆛ๎ (trans-Golgi network ,TGN)Ǵᆶ AtMIN7 (Arabidopsis thaliana HopM1 interactor7) ่ӝǴӕਔ೷ԋځफ़ှ (Nomura et al., 2011)ǶAtMIN7 ࣁ៶ݰၮᒡၸำ ϐᜢᗖೈқǴჹܭ෌ނᘰ໾ဟᆫᑗϐಕᑈתᄽख़ाفՅǴࡺ HopM1 ཮׭ڋ෌ނ ಒझᏛ࣬ᜢϐٛᑇᐒڋ (Nomura et al., 2006)Ƕ

6.5 բҔܭ MAPK ૻ৲໺ሀϐਏᔈೈқǺ

MAPK ૻ৲໺ሀࣁ௴୏ PTI Ϸ ETI ٛᑇόё܈લޑ΋ᕉǴၸำፄᚇǵ౐ੋ೚

ӭᕗለϯೈқ䁙ୖᆶځύǴԋࣁਏᔈೈқբҔޑख़ᗺҞ኱ϐ΋ǺP. syringae ਏᔈ

ೈқ HopAI1 ڀԖ phosphothreonine lyase ޑࢲ܄Ǵ٬ளߓ܎դ޺ MPK3ǵMPK4 ᆶ MPK6 ѐᕗለϯ (dephosphorylation)Ǵՠ׭ڋ MPK4 ࢲ܄ޑ่݀཮Ꮴठ HopAI1

೏ R ೈқ SUMM2 ܌ᒣ᛽ǵ௴୏ ETI ٛᑇ (Zhang et al., 2012)ǶP. syringae ќ΋ਏ ᔈ ೈ қ HopF2 ߾ڀԖ mono-ADP-ribosyltransferase ޑࢲ܄Ǵ཮೸ၸਡᑗ୷ϯ (ribosylate) MKK5Ǵ׭ڋځᕗለϯфૈǴυᘋߓ܎դ޺ޑٛᑇϸᔈ (Wang et al., 2010)Ƕ

6.6 բҔܭಒझਡϐਏᔈೈқǺ

೚ӭੰচ๵཮೸ၸޔௗυႣ෌ނٛᑇ୷Ӣޑ߄౜ٰቹៜ෌ނϐխࣝϸᔈǴࡺ

ಒझਡதԋࣁਏᔈೈқբҔޑჹຝǴӵ TAL (transcription activator-like) ਏᔈೈқǺ TAL ਏᔈೈқࣁ DNA ่ӝೈқ (DNA binding protein)Ǵӧ Xanthomonas spp.ᆶ R.

solanacearum ύࣣӸӧǴTAL ೈқ೸ၸኳү੿ਡғނϐᙯᒵӢηǴޔௗᆶ஌Ь DNA ϐ௴୏η (promoter)่࣬ӝǴࢲϯ෌ނ୷Ӣǵᏹ௓խࣝϸᔈǶX. oryzae pv.

oryzae (Xoo)ϐ PthXo1 ཮ᇨวፓ௓ዿԯᗐᜪၮᒡޑ OsSWEET11 ߄౜Ǵஒᗐᜪᙯ ଌ๏ੰ๵٬Ҕ (Yang et al., 2006; Chen et al., 2010)ǹӧच෍ (pepper) ύǴX.

campestris pv. vesicatoria ϐ AvrBs3 ่ӝӧ UPA20 ϐ௴୏ηޑ UPA box ΢Ǵ UPA20 ࣁᙯᒵӢηǴљᙍ෌ނಒझᘉ஭ (enlargement) ፓ௓ǴלੰࠔسӸӧԖ R

ೈқ Bs3Ǵૈ୼٬෌ނౢғၸ௵܄ϸᔈ (Kay et al., 2007)Ƕ

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6.7 װᔐᙯ᝿ϐਏᔈೈқǺ

Զќ΋ᜪਏᔈೈқǴӵ P. syringe ޑ HopU1Ǵ཮ቹៜ෌ނ RNA ޑжᖴ (metabolism) ၸำǴ ࣴ زว౜ mono-ADP-ribosyltransferse ਏᔈೈқ HopU1 ཮ ribosylate ෌ނύΟঁယᆘᡏ RNA ่ӝೈқ (chloroplast RNA-binding protein, CP- RBPs)ǵٿঁ൤֖ glycine ϐ RNA ่ӝೈқ (GR-RBPs)ǴЪ཮೷ԋځჹܭ RNA ޑ

่ӝૈΚ෧১Ǵ௢ෳ HopU1 ޑሇનࢲ܄཮υᘋډ෌ނޑᙯ᝿ (Fu et al., 2007)Ƕ

7. ߻߻Γࣴزᆶࣴز୏ᐒ

ࢥΚࣁੰচ๵ठੰૈΚޑໆϯǴ߄Ңੰচ๵೷ԋ஌Ь෌ނठੰำࡋޑம১Ƕ ᜢܭᆵ᡼ߙ࢏ੰ๵ϐࢥΚϩ݋Ǵന߃ࢂҗ٥ጫύЈЦᆬ޹റγࣴزი໗ۓကԶۓ ޑ (Jaunet and Wang, 1999)ǴஒԾᆵ᡼ϩᚆԶளޑ 40 ਲ਼ߙ࢏ੰ๵๵ਲ਼Ǵௗᅿډ 6 ᅿόӕלགੰพनࠔس٠इᒵ෌ނ๺঒ރݩǴа಍ीᘜᜪрόӕ๵ਲ਼ޑࢥΚǴε ठёϩࣁϤဂǴΨёᙁൂ୔ϩࣁଯǵύǵեࢥΚΟᜪǴځၨڀж߄܄ϐ๵ਲ਼ϩձ ࣁǺଯࢥΚϐ Pss190 ᆶ Pss1308ǵύࢥΚϐ Pss4 аϷեࢥΚϐ Pss216ǶଯࢥΚ๵

ਲ਼ᆶύǵեࢥΚ๵ਲ਼ޑϩᜪ٩Ᏽࣁځӧόӕพनࠔسύ܌೷ԋϐ๺঒౗ (ߕ߄΋)Ƕ ԜѦǴ߈ԃٰΨԖࣴز೸ၸௗᅿพनǵच෍ᆶनηǴቶݱ܄Ӧϩ݋όӕ phylotype ޑߙ࢏ੰ๵ᆶځӧόӕ஌Ь΢ϐࢥΚ৖౜Ǵՠ٠҂ว౜ phylotype ᆶࢥΚ໔Ԗޔ

ௗᜢೱ (Lebeau et al., 2011)Ƕ

ᜢܭߙ࢏ੰ๵ϐࣴزӭኧ๱ख़ܭ௖૸ठੰ࣬ᜢӢηǴӵझѦӭᗐᡏǵݚ୏܄

ᆶ෌ނਥ೽ߕ๱Κ฻ǴԶᜢܭߙ࢏ੰ๵ޑࢥΚࣴزߚதϿǴ࣬ᜢၗૻલЮǶࣁΑ פ൨ёૈ،ۓߙ࢏ੰ๵ࢥΚϐংᒧ୷ӢǴҁࣴز࠻ᆶ٥ࢪጫ๼ύЈЦᆬ޹റγࣴ

زი໗Ϸځд୯Ѧࣴزი໗ӝբǴа comparative genomic analyses ฼ౣКၨόӕ ࢥΚϐߙ࢏ੰ๵๵ਲ਼ޑ୷Ӣ౦ӕǴЪᆫขܭӧੰ๵-஌Ь෌ނҬϕբҔύڀᜢᗖف ՅޑಃΟࠠೈқϩݜس಍ᒡрϐਏᔈೈқ (Type III effectors, T3Es)Ƕ

ӧᆶݤ୯ Christian Boucher ࣴز঩ (INRA-CNRS, Toulouse) ϐࣴزი໗ޑӝ բࣴزύǴճҔύࢥΚϐ type strain GMI1000 Ⴃෳϐ୷Ӣׇӈ೛ीޑ microarray

຾ՉޑКၨϩ݋่݀ว౜΋٤ύ/եࢥΚ੝Ԗϐ୷Ӣ (ߕკΒ)Ǵځύхࡴ೏Ⴃෳࣁ T3Es ޑ RSp0213 ᆶ RSc3174Ƕҁࣴز࠻ޑӃ߻ࣴز᛾ჴԜΒ T3Es ڀଯࡋߥӺ܄

Ӹӧܭύեࢥٯ๵ਲ਼ǵЪڙ T3SS ፓ௓ (ߕკΟǵѤǵϖ)Ǵ٠௦Ҕ୷Ӣᙯ౽߄౜

฼ౣǴஒեࢥΚ๵ਲ਼ᐱԖޑ T3Es ᙯ౽߄౜ԿଯࢥΚ Pss190 Ϸ Pss1308 ޑᒪ໺ङ

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ඳΠ߄౜Ǵ่݀ᡉҢᙯ౽߄౜٠҂ቹៜځӧ୻Ꭶ୷΢ғߏǵ෌ނᡏϣቚғǵӧ๨

૛΢ޑၸ௵܄ϸᔈǵғނጢ׎ԋаϷݚ୏ૈΚǴՠӧ Pss190 ङඳΠޑ୷Ӣᙯ౽๵

ਲ਼ӧਥ೽ߕ๱ૈΚԖΠफ़ޑ௃׎ǴPss1308 ङඳΠ߾คቹៜ (ߕ߄Β)Ƕҁࣴزԑ ӧ຾΋؁ᔠෳӧ T3Es ୷Ӣᙯ౽߄౜ΠǴߙ࢏ੰ๵ӧพन΢ޑࢥΚ߄౜Ǵ٠Ъғ ނၗૻϩ݋ᇶշΠ (ߕკϤ)ǴᕕှԜΒ T3Es ࢂց཮ڙ෌ނᒣᇡکӵՖᇨᏤ෌ނ ޑ࣬ᜢٛᑇϸᔈᐒڋǶ

ӧᆶम୯ Exeter εᏢ Murray Grant ௲௤ࣴزი໗ޑӝբύǴଞჹόӕࢥΚޑ ߙ࢏ੰ๵๵ਲ਼ Pss190ǵPss1308ǵPss4 Ϸ Pss216 (ߕ߄Ο)Ǵךॺςֹԋځӄ୷Ӣ ಔ (х֖ࢉՅᡏ DNA ᆶ megaplasmid) ޑۓׇǴ٠Ъว౜ځύ΋ঁଯࢥΚ๵ਲ਼ Pss190 ੝Ԗޑ୷Ӣࢂ೏Ⴃෳࣁ T3E ϐ΋ޑ PopP3 (ߕკΎǵΖǵΐ)Ƕҁࣴز࠻ޑ Ӄ߻ࣴز᛾ჴԜ T3E ཮ڙ T3SS ፓ௓ (ߕკΜǵΜ΋)Ǵࣴز่݀ҭᡉҢ୷Ӣᙯ౽ ߄౜ԿύࢥΚ๵ਲ਼ Pss4 ёቚமځӧพन΢ޑࢥΚ (ߕკΜΒ)ǴԶஒ PopP3 ߄౜ܭ

๨૛ယТคੰቻౢғՠԖճܭߙ࢏ੰ๵ϐғߏ (ߕკΜΟǵΜѤǵΜϖ)Ǵ٠௢ෳ

ځӧ෌ނಈጕᡏޑ০ပՏ࿼ᆶځфૈԖᜢǴёૈᙖԜቹៜพन PTI ࡰ኱ϐ΋ޑᘰ

໾ဟᆫᑗಕᑈ (callose deposition)Ǵа׭ڋ෌ނ PTI ϸᔈ (ߕკΜϤǵΜΎǵΜΖ)Ƕ ҁࣴزԑӧ຾΋؁᛾ჴ PopP3 ಈጕᡏ০ပՏ࿼ჹځфૈϐख़ा܄Ǵ٠ᔠෳԜ T3E ӧ୷Ӣᙯ౽߄౜ΠǴߙ࢏ੰ๵ӧพन΢ޑࢥΚ߄౜Ǵکځфૈᆶ HR ϐ࣬ᜢ܄Ǵ аឍܴ PopP3 ӵՖ׭ڋ෌ނޑ࣬ᜢٛᑇϸᔈᐒڋǶ

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ಃ

ಃΒക ׷਑ᆶБݤ

1. ٮ၂๵ਲ਼ǵ፦ᡏϷ๵ਲ਼୻Ꭶచҹ

ҁࣴز܌٬Ҕϐ๵ਲ਼ᆶ፦ᡏޑ੝܄کٰྍӈܭߕ߄ѤǴ୻Ꭶ୷ଛБӈܭߕ߄ ϖǶ܌Ԗ๵ਲ਼ࣣҗ-80°C ߥӸڗрǴჄ๵ܭӝ፾ϐڰᄊ୻Ꭶ୷܈֖፾྽ᐚࡋלғ ન (ߕ߄Ϥ) ϐ୻Ꭶ୷Ǵࡷڗ๵ပࡕ຾ՉჴᡍǶεဉఎ๵ (Escherichia coli) а Luria Bertani (LB) ୻Ꭶ୷ܭ 37°C ୻Ꭶ΋Ϻǹߙ࢏ੰ๵ (R. solanacearum, Rs) а CPG ୻ Ꭶ୷ܭ 28°C ୻ᎦٿϺǹၭఎ๵а YEP ୻Ꭶ୷ܭ 28°C ୻ᎦٿϺǶ

2. ٮ၂෌ނ୻ػచҹ

ҁࣴز٬Ҕϐ෌ނࣁߙ࢏ੰלੰࠔسพन Hawaii 7996 (BW resistant, BW^R) ᆶ གੰࠔسพन L390 (BW susceptible, BW^S)Ǵҗ٥Ԁጫ๼ύЈ (Asian Vegetable Research and Development Center) Цᆬ޹Դৣ܌ගٮǶஒᅿηݰН᎜ᕏΟϺࡕǴ ኞܭ 3 मәࣧǴа 2Ǻ1 షکϐ಻ើβᆶਥ୷ܮ୻ᎦβύǴܫ࿼ܭࡡྕ 25°CǴ12 λਔӀྣǴ12 λਔ໵སޑΓπྕ࠻୻ػǴ٩ჴᡍሡ؃Կ፾྽ελࡕ຾ՉჴᡍǶ๨

૛ࠔسࣁ Nicotiana benthamiana ک Nicotiana tabacum W38Ǵҗ٥ࢪጫ๼ύЈЦᆬ

޹ԴৣගٮǴҁჴᡍ࠻ԾՉᕷ෗ǴᅿηӃаኜኞБԄኞܭ୻ᎦβǴܭΓπғߏጃ

୻ػว޵Ǵ΋ຼࡕ౽भԿ 3 मәࣧǴ୻ػܭࡡྕ 25°CǴ12 λਔӀྣǴ12 λਔ໵

སޑΓπྕ࠻Ǵය໔аᐚࡋऊ 1 g/L ϐ޸ᝊ 2 ဦࡼޥǴNicotiana benthamiana Ѥຼ

ࡕ຾ՉჴᡍǴNicotiana tabacum Ύຼࡕ຾ՉჴᡍǶ

3. ғނ಍ीϩ݋

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

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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

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٬Ҕ੝ۓЇηଛჹ (ߕ߄Ύ) аᆫӝ䁙ೱᙹϸᔈቚ൯Ҟ኱ DNA ТࢤǴϸᔈన ύа 1 ng ࢉՅᡏ DNA բࣁኳ݈ǴуΕ 10 μL ޑ 5X Q5 PCR bufferǴ4 μL ޑ 2.5mM dNTPǴ5 μL ޑ 10 μM forward ЇηǴ5 μL ޑ 10 μM reverse ЇηǴ10 μL ޑ 5X GC enhance bufferǴ0.5 μL ޑ Q5 DNA ᆫӝ䁙 (5 U/μL)ǴуΕค๵ѐᚆηНԿ 50μLǶషӝ֡Ϭࡕаᆫӝ䁙ೱᙹϸᔈᏔ຾ՉϸᔈǶϸᔈྕࡋᆶਔ໔ӵΠ܌ҢǺ 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

4.3.2.ߙ࢏ੰ๵ᆶၭఎ๵፦ᡏ๧ڗǺ

ڗ 1.5 mL ႖ڹ୻Ꭶ๵నԿ༾ໆᚆЈᆅǴа 6000 rpm ࠻ྕᚆЈ 2 ϩដǴѐନ

΢మనǶуΕ 150 μL Solution I ᎜ᕏᝌੌ؇ᐘ๵༧ǴӆуΕ 300 μL ཥᗲଛᇙޑ Solution IIǴ጗ᄌ΢Πϸᙯ 10 ԛǴᓉ࿼ܭ࠻ྕ 4 ϩដࡕуΕ 225 μL Solution IIIǴ

΢Πϸᙯ 10 ԛࡕǴа 13000 rpm ࠻ྕᚆЈ 10 ϩដǶڗ΢మనԿཥޑ༾ໆᚆЈᆅǴ 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^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

܈ TOPO® ፦ᡏᄬᑐౢނ܈ LR ख़ಔϕඤϸᔈౢނǴуΕ 100 μL ޑεဉఎ๵യҺ ಒझDH5α ๵ਲ਼ (RBC Bioscience) ύǴ࿼ܭӇ΢ 30 ϩដǴܫԿ 42°C ዗ڈᐟ 45 ࣾǴ

٬ DNA ຾Ε๵ᡏǴِೲ࿼ܭӇ΢ 2 ϩដǴуΕ 1 mL SOB నᡏ୻Ꭶ୷Ǵܭ 37°C а 200 rpm ᎜ᕏ୻Ꭶ 1 λਔǶ୻Ꭶࡕޑ๵నа 6000 rpm ᚆЈ 2 ϩដǴѐନ 800 μL ޑ΢మనǴаഭΠޑ΢మనᝌੌ๵༧Ǵஒ๵న༡ܓӧ֖Ԗ੝ۓלғનǴ܈֖Ԗ੝

ۓלғનǵIPTG (20 mg/mL) Ϸ X-Gal (20 mg/mL) ޑ LB ڰᡏ୻Ꭶ୷Ǵܭ 37 °C Π ႖ڹ୻Ꭶ 16 λਔǶ

4.10. ႝ ऀ Ͼ ᙯ ࠠ բ Ҕ ϐ യ Һ ಒ झ ࿼ ഢ (Competent Cell Preparation for Electroporation)

ҁࣴزନ፦ᡏख़ಔჴᡍѦǴߙ࢏ੰ๵ǵၭఎ๵ᆶೈқ፦߄౜Ҕϐεဉఎ๵Ǵ ځ࣬ᜢჴᡍࣣ௦ႝऀϾᙯࠠբҔ (Electroporation) ຾Չ୷Ӣׯ೷܈፦ᡏ߄౜Ƕ

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4.10.1.ߙ࢏ੰ๵ǵၭఎ๵Ǻ

җ-80°C ཥᗲჄ๵Ǵܭ 28°C ғߏጃ୻ᎦٿϺࡕǴࡷᒧൂ΋๵ပǴаబу፾྽

לғનϐӝ፾నᡏ୻Ꭶ୷ (ߙ࢏ੰ๵Ǻ523 నᡏ୻Ꭶ୷ /ၭఎ๵ǺYEP నᡏ୻Ꭶ୷) ܭ 28°C Π 200 rpm ႖ڹ᎜ᕏ୻ᎦǶڗ 50 mL బу፾྽לғનϐనᡏ୻Ꭶ୷Ǵஒ ႖ڹ୻Ꭶϐ๵నԾ O.D.600 ࣁ 0.2 ໒ۈǴܭ 28°C ᎜ᕏ୻ᎦԾ O.D.600 ࣁ 0.4-0.6Ƕ ܭ 4ɗΠа 6000 rpm ᚆЈ 3 ϩដǴѐନ΢మనࡕǴа 20 mL Ӈޑค๵ѐᚆηНख़ ཥᝌੌ๵༧Ǵ4 °C Πа 6000 rpm ᚆЈ 3 ϩដǴѐନ΢మనǴӆа 20 mL ค๵Нమ ࢱϷᚆЈ΋ԛǶѐନ΢మనǴуΕ 5 mL Ӈޑ 10 %Ғݨख़ཥᝌੌ๵༧Ǵ4 °C Πа 6000 rpm ᚆЈ 3 ϩដǶѐନ΢మనǴуΕ 1 mL Ӈޑ 10 %Ғݨख़ཥᝌੌ๵༧Ǵڗ 100 μL ᝌੌనϩးԿ༾ໆᚆЈᆅǴаనᄊේ࡚ೲհএǴߥӸܭ-80 °C ӇጃഢҔǶ

4.10.2. εဉఎ๵Ǻ

җ-80°C ཥᗲჄ๵Ǵܭ 37°C ғߏጃ୻ᎦٿϺࡕǴࡷᒧൂ΋๵ပǴа LB నᡏ

୻Ꭶ୷ܭ 37°C Π 200 rpm ႖ڹ᎜ᕏ୻ᎦǶڗ 50 mL నᡏ୻Ꭶ୷Ǵஒ႖ڹ୻Ꭶϐ

๵నԾ O.D.600 ࣁ 0.2 ໒ۈǴܭ 37°C ᎜ᕏ୻ᎦԾ O.D.600 ࣁ 0.4-0.6Ƕܭ 4 °C Πа 6000 rpm ᚆЈ 3 ϩដǴѐନ΢మనࡕǴа 20 mL Ӈޑค๵ѐᚆηНख़ཥᝌੌ๵༧Ǵ 4 °C Πа 6000 rpm ᚆЈ 3 ϩដǴѐନ΢మనǴӆа 20 mL ค๵НమࢱϷᚆЈ΋ԛǶ ѐନ΢మనǴуΕ 5 mL Ӈޑ 10 %Ғݨख़ཥᝌੌ๵༧Ǵ4 °C Πа 6000 rpm ᚆЈ 3 ϩដǶѐନ΢మనǴуΕ 1 mL Ӈޑ 10 %Ғݨख़ཥᝌੌ๵༧Ǵڗ 100 μL ᝌੌనϩ

းԿ༾ໆᚆЈᆅǴаనᄊේ࡚ೲհএǴߥӸܭ-80 °C ӇጃഢҔǶ

4.11. ႝႝऀϾᙯࠠբҔ (Electroporation Transformation)

ஒയҺಒझᆶऊ 50 ng ፦ᡏ DNA షӝࡕǴܫΕ٣ӃܭӇ΢Ⴃհޑ cuvette ύǴ

࿼ܭӇ΢ 5 ϩដǴ٬Ҕ Bio-Red MicroPulser™а 2.5 kV /2mm ຾ՉႝऀϾբҔǴ ٠ҥջуΕ 1 mL ό֖לғનϐనᡏ୻Ꭶ୷ (ߙ࢏ੰ๵Ǻ523 నᡏ୻Ꭶ୷ /ၭఎ๵Ǻ YEP నᡏ୻Ꭶ୷ /εဉఎ๵ǺSOB నᡏ୻Ꭶ୷)Ǵߙ࢏ੰ๵ᆶၭఎ๵ܭ 28°C Π 200 rpm ᎜ᕏ୻Ꭶ 2 λਔǴεဉఎ๵߾ӧ 37°C Π 200 rpm ᎜ᕏ୻Ꭶ 1 λਔǴ୻Ꭶࡕޑ

๵నа 6000 rpm ᚆЈ 2 ϩដǴѐନ 800 μL ޑ΢మనǴаഭΠޑ΢మనᝌੌ๵༧Ǵ ஒ๵న༡ܓӧ֖Ԗ੝ۓלғનϐڰᡏ୻Ꭶ୷ (ߙ࢏ੰ๵ǺCPG ୻Ꭶ୷ /ၭఎ๵Ǻ

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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᎜

ᕏ୻ᎦǴஒ๵నа 6000 rpm ࠻ྕᚆЈ 2 ϩដࡕѐନ΢మనǴуΕ 300 μL ޑ lysis bufferǴа༾ໆ֎ᆅҔΚϸᙟܜ֎ǴӆуΕ 100 μL 5M NaClǴϸᙟܜ֎ޔډషک

֡ϬǴа 13000 rpm ࠻ྕᚆЈ 10 ϩដࡕǴڗ΢మనԿཥޑ༾ໆᚆЈᆅǴуΕ฻ᡏ ᑈ chloroformǴ΢Πᙌᙯ 50 ԛԿషϬǴӆа 10000 rpm ࠻ྕᚆЈ 10 ϩដϩቫǴ λЈڗр΢ቫྋనԿཥޑ༾ໆᚆЈᆅǴуΕ฻ᡏᑈޑ isopropanol షϬǴᚆЈ 5 ϩ ដࡕѐନ΢మనǴӆа 500 μL 75%ϐଚᆒؑࢱ؈ᐘނǴ13000 rpm ࠻ྕᚆЈ 5 ϩ ដǴஒ΢మనֹӄ౽ନǴԾฅଳᔿऊ 30 ࣾࡕǴуΕ 50 μL ค๵ѐᚆηНӣྋ DNAǴӆуΕ 2 μL ޑ RNase A (20 μg/mL)Ǵ࿼ܭ 37°C 30 ϩដǴஒ๧ڗޑߙ࢏ੰ

๵ࢉՅᡏ DNA Ӹܫܭ-20°C ഢҔǶ

5. ߙ࢏ੰ๵ਏᔈೈқфૈࣴز

5.1. ๨ ૛ চ ғ ፦ ᡏ ๧ ڗ ᆶ ೈ қ ০ ပ Տ ࿼ ᔠ ෳ (N. benthamiana Protoplast 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 ғ፦ᡏϐሇનྋనаથѲၸᘠԿཥޑ༝ۭ༟ጤᚆЈᆅǶа 100 g ᚆЈ 2 ϩដǴԜ ਔ཮р౜όӕఊࡋϐচғ፦ᡏǴа༟ጤᅀᆅ֎ڗǵߥ੮ന΢ቫϐচғ፦ᡏǴ౽Կ ཥޑ༝ۭ༟ጤᚆЈᆅύǴуΕ 3 mL W5 ጗ፂనమࢱǴӆࡋᚆЈࡕѐନ΢మనǶа 3 mL W5 ጗ፂనᝌੌচғ፦ᡏǴаՈౚीኧᏔۓໆচғ፦ᡏᐚࡋǹϐࡕ 100 g ᚆ Ј 2 ϩដǴѐନ΢మనǴа K3M ྋనፓ᏾চғ፦ᡏᐚࡋࣁ 10⁶ /mLǶڗႣӃа 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

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Ε 200 uL ᝌੌϐচғ፦ᡏǴуΕ 125 uL PEG solution ࡕǴᇸቸᚆЈᆅ٬ځషӝ֡

ϬǴ٠ӧ࠻ྕΠᆒྗբҔ 45 ϩដǴуΕ 3 mL W5 ጗ፂనӣྋǴа 100 g ᚆЈ 2 ϩដǴѐନ΢మనǴӆуΕ 3 mL W5 ጗ፂనమࢱᚆЈǴख़ፄమࢱ؁ᡯٿԛǴനࡕ а 1 mL W5 ጗ፂనᝌੌচғ፦ᡏǴ౽Կ٣Ӄа 0.1% BSA ߄य़ᙟᇂೀ౛ԿϿ 30 ϩដޑϤϾዬύǴܭ΋૓ВӀᐩྣ৔୻ᎦǴ࠻ྕΠܫ࿼ 16-20 ঁλਔǶ֎ڗ 5 μL Կ ຠ Ԗ ᕉ ׎ ຠ ર ϐ ၩ ࣒ Т ΢ Ǵ ᇂ ΢ ᇂ ࣒ Т ࠾ Т ࡕ Ǵ а ᑻ Ӏ ᡉ ༾ ᜔ Confocal microscope ᢀჸ (modified from Treuter et al., 1993)Ƕ

5.2. ਏਏᔈೈқܭ๨૛ယТಒझ০ပՏ࿼ᔠෳ (N. benthamiana Leaves Protein Localization Assay)

ճҔ஥Ԗ߄౜ၩᡏ pMDC83 ϐၭఎ๵ GV3101 ຾Չ୷Ӣอኩ߄౜Ƕ-80°C ཥ ᗲჄ๵Ǵܭ 28°C ғߏጃ୻ᎦٿϺǴаబу kanamycin ᆶ rifampin ϐ YEP నᡏ୻

Ꭶ୷Ǵ28°C Π 200 rpm ႖ڹ᎜ᕏ୻ᎦǶஒ๵న 6000 rpm ᚆЈ 2 ϩដࡕѐନ΢మనǴ Ҕ 10 mL ค๵Нӣྋ๵༧Ǵӆԛ 6000 rpm ᚆЈ 2 ϩដࡕѐନ΢మనǴаค๵Нӣ ྋፓ᏾ O.D.600 ࣁ 1ǴуΕ 200 μM ޑ acetosyringone (AS)Ǵ28°C Π 200 rpm ᎜ᕏ

୻Ꭶ 2 λਔǶҁჴᡍа 4-5 ຼεޑ๨૛բࣁ׷਑Ǵݙ৔๵న߻ 1 λਔ؂ਲ਼๨૛዆

ឲऊ 20 mL НǴ٬਻ϾѺ໒Ǵϐࡕ٬Ҕ 1 mL ଞ฿வ๨૛ယङݙ৔๵నԿယ೽ಔ ᙃ྽ύǴ٠аڻ౦฽኱ຏݙ৔ጄൎǶགࢉࡕޑ๨૛࿼ܭ 25°CǴ12 λਔӀྣǴ12 λਔ໵སޑ෌ނғߏጃΒԿΟϺǴаᑻӀᡉ༾᜔ Confocal microscope ᢀჸ๨૛ယ ङǶ

5.3. ߙ࢏ੰ๵ࢥΚෳ၂ (Virulence Test)

ஒߙ࢏ੰ๵ Pss4 ܈ Pss190 ഁғࠠ๵ਲ਼܈୷Ӣকନϐ๵ਲ਼ǴճҔ߄౜ၩᡏ pUFR047 ߄౜Ҟ኱୷ӢǴҗ-80°C ཥᗲჄ๵Ǵܭ 28°C ғߏጃ୻ᎦٿϺǴа 523 న ᡏ୻Ꭶ୷܈బу gentamycin ϐ 523 నᡏ୻Ꭶ୷ܭ 28°C Π 200rpm ႖ڹ᎜ᕏ୻ᎦǴ ڗ200 μL ๵న༡ዬܭ 523 ڰᡏ୻Ꭶ୷Ǵ႖ڹ୻Ꭶࡕаค๵НࢱΠ๵నǴ٠ፓ᏾๵

నԿ O.D.600 ࣁ 0.003-0.03 (ऊ 10⁶-10⁷ CFU/mL)Ǵ ዆ឲܭΓπྕ࠻୻ػ 31-33 Ϻ ϐགੰࠔسพन L390 ෌ਲ਼Ǵ܈୻ػ 28-32 Ϻϐלੰࠔسพन H7996 ෌ਲ਼Ǵ؂΋

෌ਲ਼዆ឲ 25 mL ๵నǴܫ࿼ܭ 25°CǴ12 λਔӀྣǴ12 λਔ໵སޑ෌ނғߏጃᢀ ჸੰቻΟډѤຼǴۓයइᒵ๺঒ࡰኧ (wilting score)Ǻ 0 ࣁคੰቻǹ1 ࣁ΋ТယТ

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๺঒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 rifampin ϐ YEP నᡏ୻Ꭶ୷Ǵ28°C Π 200 rpm ႖ڹ᎜ᕏ୻ᎦǶஒ๵న 6000 rpm ᚆЈ 2 ϩដࡕѐନ΢మనǴҔ 10 mL ค๵Нӣྋ๵༧Ǵӆԛ 6000 rpm ᚆЈ 2 ϩដ ࡕѐନ΢మనǴаค๵Нӣྋፓ᏾ O.D.600 ࣁ 1ǴуΕ 200 μM ޑ acetosyringone (AS)Ǵ28°C Π 200 rpm ᎜ᕏ୻Ꭶ 2 λਔǶҁჴᡍа 4-5 ຼεޑ๨૛բࣁ׷਑Ǵݙ

৔๵న߻ 1 λਔ؂ਲ਼๨૛዆ឲऊ 20 mL НǴ٬਻ϾѺ໒Ǵϐࡕ٬Ҕ 1 mL ଞ฿வ

๨૛ယङݙ৔๵నԿယ೽ಔᙃ྽ύǴ٠аڻ౦฽኱ຏݙ৔ጄൎǶ܈ܭݙ৔ၭఎ๵

ࡕǴӆݙ৔аค๵Нӣྋፓ᏾ O.D.600 ࣁ 0.3 ϐߙ࢏ੰ๵ँᡂਲ਼ Pss4 hrpG^-Ƕགࢉ

ࡕޑ๨૛࿼ܭ 25°CǴ 12 λਔӀྣǴ12 λਔ໵སޑ෌ނғߏጃᢀჸ܈຾Չࡕុჴ ᡍǶ

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 ႖Ϻڗ 1 mL ๵నԿ 10 mL ֖࣬ӕלғનϐ YEP నᡏ୻Ꭶ୷ǴуΕ 10 mM MESǵ 20 mM acetosyringone ຾Չ႖ڹԛ୻ᎦǶஒ๵న 6000 rpm ᚆЈ 2 ϩដࡕѐନ΢మ నǴҔ 10 mL ค๵Нӣྋ๵༧Ǵӆԛ 6000 rpm ᚆЈ 2 ϩដࡕѐନ΢మనǴаค๵

Нӣྋፓ᏾ O.D.600 ࣁ 1-1.2ǴуΕ 10 mM MgCl2ǵ10 mM MESǵ200 mM acetosyringoneǴ࿼ܭ 28°C ୻Ꭶ 3 λਔǶགࢉБԄࢂӧ 12-18 Ϻεϐพनηယа 2 μL ༾ໆ֎ᆅӾ (tip) ܭယેύ໔ᇙ೷λ໾αǴҔ 1 mL ଞ฿ஒ๵నݙᅈηယǴ࿼ܭ 21°CǴ12 λਔӀྣǴ12 λਔ໵སޑ෌ނғߏጃǴ฻ࡑ຾ՉࡕុჴᡍǶ

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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

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

Cycle# Denaturation Annealing Polymerization

1 94°C /5 ϩដ

35 94°C /30 ࣾ T°C /30 ࣾ 72°C /t ϩដ TǺྕࡋ٩ᏵЇη Tm ॶǹtǺਔ໔٩Ᏽቚ൯ߏࡋǴ1kb/minǶ

ϸᔈύ໔ܭಃ 27 ঁ cycle ኩଶϸᔈǴڗр 15 μL ϸᔈనԿќ΋༾ໆᚆЈᆅഢҔǴ ഭΠޑϸᔈన߾ܫӣᆫӝ䁙ೱᙹϸᔈᏔύᝩុϸᔈǴख़ፄ΢ॊ୏բܭϸᔈ຾ՉԿ

ಃ 30ǵ35 ঁ cycleǶϐࡕஒڗрϐϸᔈనӚڗ 5μLǴаӕ΋Т 1%ϣࢉᛏિᑗᏉጤ

ႝݚ຾Չϩ݋Ƕ

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

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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