Functional characterization of Nicotiana benthamiana chromomethylase Nb CMT

Functional characterization of Nicotiana benthamiana chromomethylase Nb. CMT 3 -2 gene in defense to To. LCTWV 圓葉菸草甲基化轉移酶 Nb. CMT 3 -2 基因 在防禦臺灣蕃茄捲葉病毒中之功能分析 報告者：魏雋瑀 S 0124010 指導教授：傅士峯教授 報告日期： 2016. 04. 22

2 • Light intensity • Gene mutation • Aberrant gene expression • Temperature • Various pathogens and insects (Kinoshita and Seki 2014)

3 DNA methylation M Methyl group M G C T G A C T G C G A C T G A C M M S CH 2 NH 2 C 3 H 5 NH 2 COOH CH 3 M O N C 5 N 6 H 4 5 4 SAM 6 3 N enzyme CH 3 1 5 2 4 N H cytosine O 6 C 3 H 5 NH 2 COOH N S 1 3 N H 2 O 5 -methylated cytosine CH 2 O N SAH C 5 N 6 H 4

DNA methyltransferases (DNMTs) M C T G A C T G M G A C T G A C DNA methyltransferases Methyl group ？ 4 M C T G A C M M

DNA methyltransferases (DNMTs) 5 MET：Methyltransferase C-terminal part NLS RFTS BAH Catalytic domain DRM：Domians-rearranged methyltransferase N-terminal part C-terminal part UBA UBA Catalytic domain CMT：Chromomethylase C-terminal part N-terminal part BAH ← unique to plants CD Catalytic domain (Meyer 2011, Vanyushin and Ashapkin 2011)

CMT family 6 The CMT 1 coding sequence is disrupted. CMT 1 CMT 3 CMT 2 (Henikoff and Comai 1998, Jones et al. 2001)

7 CMT 2 相關前人研究 Oryza sativa 水稻 Rice cytosine DNA methyltransferases – gene expression profiling during reproductive development and abiotic stress (Sharma and Mohan Singh 2009) c. DNA microarray Os. CMT 2 Seedling + Cold stress (CS) Hypothesis: Seedling CS SS CMT 2 involved in some abiotic stress responses. Os. CMT 2 1 1. 80 1. 28 Seedling + Salt stress (SS) DS 0. 86 Seedling + Dehydration stress (DS) Relative gene expression Low High (Sharma and Mohan Singh 2009)

8 CMT family The CMT 1 coding sequence is disrupted. CMT 1 Hypothesis: CMT 2 involved in some abiotic stress responses. CMT 3 CMT 2 (Henikoff and Comai 1998, Bartee et al. 2001)

N-terminal part BAH CMT 3 C-terminal part CD Catalytic domain Bromo-adjacent homology domain Chromo domain 讓 CMT 3 能穩定的附著於 DNA 上 Catalytic domain H 3 K 9 me 2 CN G H 3：Histone 3 K 9：第 9個胺基酸 Lysine me 2 ：上面接有兩個甲基 9 Catalytic domain 催化甲基轉移到胞�� 上 CMT 3 G CN H 3 CNG ( N： A、C、T ) (Patel and Jacobsen et al. 2012, Jacobsen and Cao 2002)

11 CMT family The CMT 1 coding sequence is disrupted. CMT 1 CMT 3 regulates the development of plant. Hypothesis: CMT 2 involved in some abiotic stress responses. CMT 3 CMT 2 (Henikoff and Comai 1998, Bartee et al. 2001)

12 CMT family M M 植物 DNA CMT 1 Methyl group G C T G A C T G C G A C T G A C M M M CMT 3 CMT 2 (Henikoff and Comai 1998, Bartee et al. 2001)

13 Interaction between Geminiviruses and CMT 3 Arabidopsis thaliana 阿拉伯芥 & Beet curly top virus (BCTV) 甜菜曲頂病毒 Viral Genome Methylation as an Epigenetic Defense against Geminiviruses (Raja et al. 2008) CMT 3 M M C T G A C M 植物 DNA CMT 3 Attack Defense M CMT 3 Geminiviruses (Raja et al. 2008)

14 Interaction between Geminiviruses and CMT 3 Arabidopsis thaliana 阿拉伯芥 & Beet curly top virus (BCTV) 甜菜曲頂病毒 Viral Genome Methylation as an Epigenetic Defense against Geminiviruses (Raja et al. 2008) Attack Defense CMT 3 M 植物 DNA M M M G C T G A C T G C G A C T G A C M M M Geminiviral DNA (Raja et al. 2008)

15 Interaction between Geminiviruses and CMT 3 Arabidopsis thaliana 阿拉伯芥 & Beet curly top virus (BCTV) 甜菜曲頂病毒 Viral Genome Methylation as an Epigenetic Defense against Geminiviruses (Raja et al. 2008) + wild-type symptoms ++ moderately enhanced +++ severe ++++ extensive stunting Beet curly top virus (BCTV) WT WT cmt 3 - + + (Raja et al. 2008)

16 Interaction between Geminiviruses and CMT 3 Nicotiana benthamiana 圓葉菸草 Relative transcript levels (%) 利用雙生病毒感染植物，會使植物體內 Nb. CMT 3 的表現量受到影響 (Negrete et al. 2013) 100 RT-q. PCR 50 0 Nb. MET 1 Nb. CMT 3 TYS AC TYM Geminiviruses TG M (Negrete et al. 2013)

17 Interaction between Geminiviruses and CMT 3 ？ Geminivirus Plant Hypothesis: CMT 3 會藉由進行 DNA methylation 參與植物對 Geminivirus 的防禦機制 (Raja et al. 2008; Negrete et al. 2013)

18 研究目的 CMT 3 Geminivirus Defense Plant ？ Hypothesis: CMT 3 會藉由進行 DNA methylation 參與植物對 Geminivirus 的防禦機制 M 植物 DNA M M M G C T G A C T G C G A C T G A C M M M Geminiviral DNA

19 Experimental Procedures

Nicotiana benthamiana 20 Kingdom Plantae 植物界 Phylum Magnoliophyta 木蘭植物門 Class Magnoliopsida 木蘭綱 Order Solanales 茄目 Family Solanaceae 茄科 Genus Nicotiana 菸草屬 Nicotiana benthamiana 圓葉菸草 • 茄科植物中的模式植物 • 可被多種病原感染，廣用於病毒學領域

Nb. CMT 3 & Nb. CMT 3 -2 Nb. CMT 3 (740 aa) BAH CD Catalytic domain Nb. CMT 3 -2 (901 aa) NLS Nicotiana benthamiana 圓葉菸草 BAH CD Catalytic domain 讓 CMT 3 能穩定的附著於 DNA 上 21 實驗室學長姐 (Hou et al. 2014) 實驗室學長姐 (Lin et al. 2015) 催化甲基轉移到胞�� 上 (Patel and Jacobsen et al. 2012, Jacobsen and Cao 2002)

Nb. CMT 3 & Nb. CMT 3 -2 Nb. CMT 3 (740 aa) BAH CD Catalytic domain Nb. CMT 3 -2 (896 aa) NLS BAH CD Catalytic domain 23 實驗室學長姐 (Hou et al. 2014) 實驗室學長姐 (Lin et al. 2015) Nicotiana benthamiana 圓葉菸草 (Patel and Jacobsen et al. 2012, Jacobsen and Cao 2002)

24 To. LCTWV (Tomato leaf curl Taiwan virus) Viruses 病毒 Order ss. DNA 單鏈 DNA 病毒 Family Geminiviridae 雙生病毒科 Genus Begomovirus 豆類金黃嵌紋病毒屬 Tomato leaf curl Taiwan virus 台灣番茄捲葉病毒 病徵： ① 植株嚴重矮化 ② 葉片明顯捲曲 ③ 葉緣及中肋附近區域明顯 黃化現象 ④ 花枯萎 ⑤ 罹病植株結果率低 (MI Reyes 2013, 台南農改場) To. LCTWVWT To. LCTWV

25 Framework ① Nb. CMT 3 -2 是否參與植物 與病毒間的交互作用 Nb. CMT 3 -2 To. LCTWV Defense ？ ？ Nicotiana benthamiana 圓葉菸草 Nb. CMT 3 -2 overexpression Nb. CMT 3 -2 knockdown ② 基因靜默 G Nb. CMT 3 -2 進行功能分析 C M M M C T G A C T G G C T G A C C G A C T G A M M ④ 分離 Nb. CMT 3 -2 作用之目標基因 G ③ 大量表現 Nb. CMT 3 -2 C 進行功能分析 M

26 Framework Nb. CMT 3 -2 ① Nb. CMT 3 -2 是否參與植物 與病毒間的交互作用 To. LCTWV Nicotiana benthamiana 圓葉菸草

預期結果 Real-Time PCR Relative m. RNA expression level Nb. CMT 3 -2 Con To. LCTWV 27

28 Framework Nb. CMT 3 -2 Defense Nicotiana benthamiana 圓葉菸草 ？ Nb. CMT 3 -2 knockdown ② 基因靜默 Nb. CMT 3 -2 進行功能分析 Nb. CMT 3 -2 overexpression To. LCTWV

VIGS (Virus-induced gene silencing) 煙草脆裂病毒 (Tobacco rattle virus, TRV) Recombinant plasmid 30 TRV 35 S promoter NOS terminator p. TRV 1 ：帶有負責病毒複製與運送相關蛋白的病毒基因體 Movement Protein 35 S promoter LB 2 x 35 S Nopaline Synthase terminator Rd. Rp MP Rz NOSt RB RNA-dependent RNA polymerase self-cleaving ribozyme p. TRV 2 ：帶有外殼蛋白病毒基因體及多種限制內切酶切位 Coat Protein LB 2 x 35 S CP Nb. CMT 3 -2 MCS Rz NOSt RB Multiple Cloning Site (Liu et al. , 2002)

31 VIGS Recombinant plasmid 35 S promoter TRV 2 TRV 1 NOS terminator 35 S promoter Electroporation Agrobacterium inoculation Nicotiana benthamiana 圓葉菸草 NOS terminator

32 TRV 1 VIGS TRV 2 ：負責病毒複製與運送病毒 RNA-dependent RNA polymerase RISC (RNA-induced silencing complex) ss. RNA RISC Rd. RP ds. RNA Dicer si. RNA 植物m. RNA

預期結果 Real-Time PCR Relative m. RNA expression level Nb. CMT 3 -2 TRV 2 -GUS TRV 2 -Nb. CMT 3 -2 (Con) 34

37 預期結果 To. LCTWV TRV 2 -GUS (Con) TRV 2 -Nb. CMT 3 -2 - - + +

39 預期結果 Real-Time PCR Relative DNA expression level To. LCTWV Coat Protein To. LCTWV TRV 2 -GUS TRV 2 -Nb. CMT 3 -2 (Con) + + -

40 Framework Nb. CMT 3 -2 Defense Nicotiana benthamiana 圓葉菸草 To. LCTWV ？ Nb. CMT 3 -2 knockdown Nb. CMT 3 -2 overexpression ③ 大量表現 Nb. CMT 3 -2 進行功能分析

建立 Nb. CMT 3 -2 大量表現轉殖株 Gateway Recombination (TOPO-D) Recombinant plasmid p. ENTR/D-TOPO vector 42 Nb. CMT 3 -2 p 35 S T 35 S p. JVC 52 LB Kan p 35 S att. R 1 Nb. CMT 3 -2 ccd. B att. R 2 HA-tag Electroporation Agrobacterium T 35 S RB

45 Nb. CMT 3 -2 轉植株基因與蛋白質表現分析 Ox-Ve Ox-Nb. CMT 3 -2 -HA DNA transgene Nb. CMT 3 -2 -HA m. RNA expression Nb. CMT 3 -2 -HA Protein expression

46 實驗結果 1 Primer sets bps Nb. CMT 3 -2 C HA 450 Nb. CMT 3 -2 FL HA 2648 522 2284 2706 2733 (nt) 5’ Nb. CMT 3 -2 open reading frame HA 3’ Genomic DNA PCR of transgene Transgenic OX-Nb. CMT 3 -2 -FL-HA WT #7 #9 #10 <Nb. CMT 3 -2 C HA> (450 bps) <Nb. CMT 3 -2 FL HA> (2648 bps) <EF 1> (125 bps)

47 Nb. CMT 3 -2 轉植株基因與蛋白質表現分析 Ox-Ve Ox-Nb. CMT 3 -2 -HA DNA transgene Nb. CMT 3 -2 -HA m. RNA expression Nb. CMT 3 -2 -HA Protein expression

48 預期結果 Real-Time PCR 1 bps 114 2284 2706 2733 (nt) Nb. CMT 3 -2 open reading frame Nb. CMT 3 -2 -HA Relative m. RNA expression level Primer sets Nb. CMT 3 -2 5’ 522 Ox-Ve Ox-Nb. CMT 3 -2 -HA (Con) HA 3’

49 Nb. CMT 3 -2 轉植株基因與蛋白質表現分析 Ox-Ve Ox-Nb. CMT 3 -2 -HA DNA transgene Nb. CMT 3 -2 -HA m. RNA expression Nb. CMT 3 -2 -HA Protein expression

50 預期結果 Western blot Anti-HA Ox-Ve Ox-Nb. CMT 3 -2 -HA (102 k. Da) Rubisco (50 k. Da)

53 預期結果 To. LCTWV Ox-Ve (Con) Ox-Nb. CMT 3 -2 -HA - - + +

55 預期結果 Real-Time PCR Relative DNA expression level To. LCTWV Coat Protein To. LCTWV Ox-Ve (Con) Ox-Nb. CMT 3 -2 -HA Ox-Ve (Con) - - + Ox-Nb. CMT 3 -2 -HA +

56 Framework Nb. CMT 3 -2 To. LCTWV Defense Nicotiana benthamiana 圓葉菸草 ？ M M G C T G A C T G C G A C T G A C M M M ④ 分離 Nb. CMT 3 -2 作用之目標基因

分離 Nb. CMT 3 -2 作用之目標基因 Ox-Nb. CMT 3 -2 -HA To. LCTWV - Ox-Nb. CMT 3 -2 -HA + 57

Ch. IP-seq (Chromatin Immunoprecipitation-seq) od b i t An te o r P y in C T G A C T G A C 58

分離 Nb. CMT 3 -2 作用之目標基因 Nb. CMT 3 -2 -HA 1. Add Formalin 2. Extract total DNA Fractionate chromatin by sonicating Sequencing ATGCCTACGTG Nb. CMT 3 -2 -HA Add antibodies to immunoprecipitate target protein Map to plant genome or viral genome Unlink protein; Purify DNA 59

60 預期結果 Ox-Nb. CMT 3 -2 -HA To. LCTWV Target gene retrotransposon Ox-Nb. CMT 3 -2 -HA + retrotransposon X gene (Tompa et al. 2002)

目前進度 61 Nb. CMT 3 -2 Defense Nicotiana benthamiana 圓葉菸草 To. LCTWV ？ Nb. CMT 3 -2 knockdown Nb. CMT 3 -2 overexpression ③ 大量表現 Nb. CMT 3 -2 進行功能分析

62 Thank You

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64 Supplement

CMT 1 相關前人研究 65 Oryza sativa 水稻 CHARACTERIZATION OF RICE oscmt 1 MUTANT (Pham Quoc Tuan 2014) DNA methylation is a biochemical process that plays a very important role in development of plant and animal. In rice (Oryza sativa japonica) genome, there are 10 genes that contain the highly conserved methylase catalytic domain. Os. CMT 1 belongs to Chromo Methyltransferase (CMT) group, a plant-specific Methyltransferase (MTases). This group is known to control methylation at CHG and CHH sites. In this study, we selected a mutant line of Os. CMT 1 that had the insertion of Tos 17 transposable element at the last intron. Through callus culture, the mutant line oscmt 1 showed the abnormal seed phenotype with altered at aleurone layer (糊粉層) of opaque mature seed. In wild-type, Os. CMT 1 expresses only in early stage of flowering.

CMT 1 相關前人研究 66 Oryza sativa 水稻 CHARACTERIZATION OF RICE oscmt 1 MUTANT (Pham Quoc Tuan 2014) The expression of mature m. RNA of Os. CMT 1 in both homozygous and heterozygous mutants was lower level than that of wild type indicating the knockdown effect of the mutants. Using Rice. Array. Net (RAN), a correlation network with Os. CMT 1, 149 genes were co-expressed with Os. CMT 1 under the condition (ǀr-valueǀ ≥ 0. 65, depth = 1). Expression microarray of mature seeds showed that 2, 809/4, 349 genes were up/down-regulated by the decrease of Os. CMT 1 expression in mutant. Gene Ontology analysis of microarray data highlighted the repression of DNA metabolic process, membrane lipid biosynthesis and the activation of regulation of gibberellic acid (GA)-mediated signaling. The combination of RAN analysis and microarray data, 14 genes of Os. CMT 1 were predicted as target of Os. CMT 1.

CMT 1 相關前人研究 Oryza sativa 水稻 CHARACTERIZATION OF RICE oscmt 1 MUTANT (Pham Quoc Tuan 2014) 67

c. DNA microarray Protonema 發光 → 基因有表現 不發光 → 基因沒有表現 (Campbell chapter 20 68

N-terminal part CMT 3 C-terminal part CD BAH Bromo-adjacent homology domain Catalytic domain Chromo domain 讓 CMT 3 能穩定的附著於 DNA 上 CMT 3 69 Catalytic domain 催化甲基轉移到胞�� 上 CNG ( N： A、C、T ) (Patel and Jacobsen et al. 2012, Jacobsen and Cao 2002)

70 CMT 3 Catalytic domain H 3 K 9 me 2 C N G H 3：Histone 3 K 9：第 9個胺基酸 Lysine me 2 ：上面接有兩個甲基 G N C H 3 (Patel and Jacobsen et al. 2012)

71 Interaction between Geminiviruses and CMT 3 Arabidopsis thaliana 阿拉伯芥 & Beet curly top virus (BCTV) 甜菜曲頂病毒 Viral Genome Methylation as an Epigenetic Defense against Geminiviruses (Raja et al. 2008)

建立 Nb. CMT 3 -2 大量表現轉殖株 Gateway Recombination (TOPO-D) p. ENTR/D-TOPO vector Recombinant plasmid Nb. CMT 3 -2 p 35 S 2706 bp 利用酵素送入 TOPO-D (p. JVC 52) Kan 72 T 35 S

p. JCV 52 HA-tag LB Kan p 35 S att. R 1 ccd. B att. R 2 T 35 S RB