探討 sodium butyrate 在病毒防治之利用與機轉

國立臺灣大學生物資源暨農學院 植物病理與微生物學研究所

碩士論文

Graduate Institute of Plant Pathology and Microbiology College of Bioresources and Agriculture

National Taiwan University Master Thesis

探討 sodium butyrate 在病毒防治之利用與機轉 Investigation of the effect of sodium butyrate on plant

virus prevention and its mechanism

李佩穎 Pei-ying Li

指導教授：葉信宏 博士、張雅君 博士

Advisors：Hsin-Hung Yeh, Ph. D., Ya-Chun Chang, Ph. D.

中華民國 103 年 7 月

July 2014

国立童湾大学(碩)博士学位論文

口試委員今春定書

探討sodimb巾y血e在病毒防治之利用興機構

Investigation of the effect of sodium butyrate on

plmt Vims prevention md its mechanism

本論文係李侃穎君(RO1633006)在団正室湾大学植物 病理興微生物学研究所完成之碩(悼)士学位論文･於民団 103年07月22日承下列考試委員審査通過及口試及格･特

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黎三二∴一三∴ 弗ﾂ ｩ駝" kﾂ

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(指等教授)

一一〇一 寸 亦謦

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∴∴ し 梯y? U ﾒ tH6ｨ ｲ ! (ﾄｲ kﾂ

ii

致謝

時光匆匆，碩士的兩年生活有如白駒過隙，感謝在這兩年的期間幫助過我的 所有人，讓我可以如期完成這本碩士論文。

回首碩士求學的歷程，首先要感謝我的指導教授 葉信宏教授這幾年來的指導，

不管是大學時修習的專題討論還是碩士生涯，老師在忙碌的教學工作之餘，也不 忘給予學生充分的教導及教誨，除了科學上的討論與邏輯思考的訓練之外，於人 生課題上也獲益良多，讓學生能由各個不同的角度去看每一件事。也感謝 張雅君 教授、 柯文雄教授與 洪挺軒教授細心、嚴謹的批改學生的論文，並於口試時提 供能延伸探討的觀點，使學生的論文能漸趨完善，受用不盡，特此感謝。另外感 謝實驗室學長姊正恩、政彥、管彤、張立、賀雄、睿哲、伊翎、子毓、丹彤對實 驗方法上的指導和經驗傳承，也感謝碩士兩年一同進退的同學柏荃，在我實驗發 生問題時的第一線協助和鼓勵，也很感謝光森和盈如在實驗與口試時的幫忙，實 驗室的生活因你們而能精彩充實。也感謝洪傳揚老師實驗室的易整同學對我於細 胞染色上的指導，讓我可以迅速了解實驗的流程。

由衷感謝無時無刻關心我的父母與姊姊，回首走過的每一步都有您們的關愛 和教誨，因為您們在身後的支持才能有現在的我，謝謝您們。還有書齊特地到台 北來陪伴我度過碩士的時光，除了陪我加班到深夜，也不斷的鼓勵與激勵我，讓 我能繼續前進。也謝謝巧娟在實驗上的協助及日常生活上的督促及提醒。謝謝在 實驗上、生活中幫助過我的所有人，有你們的幫助與鼓勵，我才能完成這篇論文，

僅將這篇論文獻給你們，致上我最大的謝意。

iii

中文摘要

病毒於感染植物之後，常造成植株無法復原的傷害，而穩定有效的病毒防治策略 仍有待發展。Histone deacetylases (HDACs) 在細胞中負責調控 histone 修飾行為

，藉以影響基因的表現。過去研究顯示 Brome mosaic virus, Tomato bushy stunt virus 此兩種 RNA 病毒在 HDACs 基因功能缺失的酵母菌中，其病毒的累積會受到影 響而下降，且 Histone deacetylase inhibitors (HDACi) 更被發現在 HCV replicon cell OR6 上對 C 型肝炎病毒的複製有顯著的抑制效果，但這類物質的作用卻缺乏 植物病毒方面的探討，因此本實驗選用 sodium butyrate (HDACi 中的一種) 在易 感病圓葉菸草進行菸草嵌紋病毒抗性測試。實驗結果顯示 sodium butyrate 雖無法 阻止病毒系統性感染植株，但可延遲病株的病程發展並減緩外部病徵，且在抗性 蛋白的時程表現上，有測得水楊酸抗性途徑之標記基因 PR-1a 的生成，推測其 可能的調控與水楊酸路徑相關，且其在系統葉上的表現量較處理葉為多，但相反 地水楊酸處理的植株，其處理葉上的 PR-1a 其表現量比系統葉來得多，顯示 sodium butyrate 誘導系統性抗性較水楊酸為佳，但局部抗性誘導以水楊酸為佳，

因此將水楊酸和 sodium butyrate 合併塗抹於葉片上，實驗結果顯示，合併使用其 抗性有加成效果。另外在活性氧物質的測定上，1 mM 的 sodium butyrate 單獨使 用處理也會造成系統葉上過氧化氫的累積。因此 sodium butyrate 的誘導系統性抗 病能力應為未來可應用之方法，期為植物病毒病害防治提供新的方向。

關鍵字：histone deacetylases (HDACs)、histone deacetylase inhibitors (HDACi)、

sodium butyrate、菸草嵌紋病毒、圓葉菸草

iv

Abstract

Viruses cause serious damage to crops; however, effective plant antiviral disease

managements remain largely to be explored. Histone deacetylases (HDACs) are genes

involved in histone regulation, and modulate the expression of genes. Previous studies

demonstrated that in histone deacetylases (HDACs) single-gene-knockout yeasts the

accumulation of Brome mosaic virus and Tomato bushy stunt virus decreased, and

application of histone deacetylase inhibitors (HDACi) to HCV replicon cell OR6

showed suppressive effect on Hepatitis C virus. However, whether HDACi can be

applied for plant viral disease management remained to be resolved. In this study, we

first treated sodium butyrate (one kind of HDACi) to Nicotiana benthamiana, a

susceptible host, to Tobacco mosaic virus (TMV). The data indicated that plants

pretreated with sodium butyrate showed more vigorously growth and delayed in

symptom expression than untreated plants after TMV inoculation. In addition, our

RT-PCR revealed that the application of sodium butyrate induced the expression of

PR-1a, suggested that sodium butyrate participated in the salicylic acids (SA) related

plant defense pathway. Furthermore, in contrast to SA-treated plants the expression of

PR-1a is stronger in treated leaves than systemic leaves; sodium butyrate-treated leaf

shows stronger PR-1a induction in systemic leaves. The production of ROS showed

obviously increase in the systemic leaf of sodium butyrate-treated N. benthamiana. It

v

indicated that SA may trigger stronger local defense and sodium butyrate trigger

stronger systemic defense. Thus, we applied both SA and sodium butyrate solutions on

the leaves of N. benthamiana, and enhanced resistance was observed on the mixed

solutions-treated plants. It revealed that sodium butyrate has the potential to be applied

in development of effective systemic antiviral disease managements.

Key words：histone deacetylases (HDACs), histone deacetylase inhibitors (HDACi),

sodium butyrate, TMV, Nicotiana benthamiana

vi

Contents

口試委員審定書 ... i

致謝 ... ii

中文摘要 ... iii

Abstract ... iv

Contents ... vi

Figure contents ... vii

Table contents... x

Appendix contents ... xi

Introduction ... 1

Materials and Methods ... 7

Results ... 12

Dicussion... 18

References ... 24

Figures ... 33

Tables ... 58

Appendix ... 62

vii

Figure contents

Fig. 1 Symptoms of Nicotiana benthamiana infected with freeze-dried TMV

at different concentrations five days pi………..…………...….…..…..34

Fig. 2 Disease index for measuring the symptoms of TMV-infected Nicotiana

benthamian………..………….…....…….35

Fig. 3 Phenotypes of Nicotiana benthamiana drenched with different

chemicals...………...……...…36

Fig. 4 Phenotypes of Nicotiana benthamiana drenched with different

concentrations of sodium butyrate……….………...…...…..38

Fig. 5 Phenotypes of Nicotiana benthamiana drenched with different

concentrations of sodium butyrate………....….40

Fig. 6 Phenotypes of Nicotiana benthamiana with leaves applied with

chemicals..……….……….……..….41

Fig. 7 Leaves appearance of Nicotiana benthamiana with leaves applied with

chemicals………...….42

Fig. 8 Symptoms of Nicotiana benthamiana pre-drenched with chemicals then

inoculated with TMV ten days pi………...……….…43

Fig. 9 Symptoms of Nicotiana benthamiana pretreated with chemicals on

leaves then inoculated with TMV ten days pi………...……….45

viii

Fig. 10 Symptoms of Nicotiana benthamiana pretreated with chemicals on

leaves then inoculated with TMV ten days pi………..…..47

Fig. 11 Detection of PR1a expression in different chemical-treated Nicotiana

benthamiana in 7-day time course………...48

Fig. 12 Quantification of the symptom reduction of Nicotiana benthamiana

pretreated with single or mixed chemicals and then inoculated with

TMV………..………...….49

Fig. 13 Hydrogen peroxide accumulation was detected via DAB staining in

different chemical-treated Nicotiana benthamiana one day after

treatment………..……….…...50

Fig. 14 Quantification of hydrogen peroxide accumulation in different

chemical-treated Nicotiana benthamiana via DAB staining one day after

treatment………..……….……….51

Fig. 15 Quantification of hydrogen peroxide on Nicotiana benthamiana

applied with different chemicals seven days after treatment……..…….……52

Fig. 16 Lesion numbers and whole leaf phenotype of Nicotiana glutinosa

pre-drenched with chemicals and then inoculated with TMV five days

pi………..………..…53

Fig. 17 Lesion numbers and whole leaf phenotype of Nicotiana glutinosa

ix

pretreated with chemicals and inoculated with TMV five days

pi………..………..55

Fig. 18 Phenotypes of Nicotiana tabacum Xanthi-nc treated with different

solutions four days after treatment………...56

x

Table contents

Table 1. Primers used for RT-PCR analysis in Nicotiana

benthamiana…. ………..59

Table 2. Reported concentration of chemicals used for plant

treatment……….60

Table 3. The expression pattern of PR1a derived from repeated

experiments……….…61

xi

Appendix contents

Appendix Fig. 1 Symptoms of Nicotiana benthamiana pretreated with

chemicals then inoculated with TMV-30B-GFP………..63

Appendix Fig. 2 Symptoms of Nicotiana benthamiana infected by

TMV-U1 on plants pretreated with chemicals……….…64

Appendix Table 1. The accumulation of hydrogen peroxide on leaves of

different chemical-treated Nicotiana benthamiana…..…….………….65

1

Introduction

DNA is the important molecule that encodes whole genetic information and

dominates all living organisms. DNA is ordered by highly alkaline proteins called

histones. Histones guide DNA performance by acetylation, methylation, and

phosphorylation and ubiquitination of the nucleosome in the nucleus (Pandey et al.,

2002, Fu et al., 2007, Hu et al., 2009, Allfrey et al., 1964). Among these

modifications, acetylation is one of the best well-studied regulatory modes on

post-translational modifications of histones. Histone acetylation is controlled by

histone deacetylases (HDACs) and histone acetyltransferases (HATs) (Hu et al.,

2009, Fu et al., 2007). These two antagonistic enzymes transfer acetyl groups on the

lysine residues at the N-termini of histones oppositely.

HDACs play important roles in regulating gene expression and integrating

chromatin structure. One of the major function of HDAC is to catalyze the removal

of acetyl groups from histones to down-regulate the activity of transcription, while

another antagonistic gene, HATs, function at adding them back to activate the

expression of chromatin (De Ruijter et al., 2003, Bolden et al., 2006, Giannini et al.,

2012, Carafa et al., 2013). These modulations are involved in epigenetic inheritance

2

systems. Other than directly controlling the gene expression by modifications of

chromatin, HDACs can also deacetylate over 50 nonhistone proteins such as

structural proteins, chaperones, transcription factors, chromatin-remodelling proteins,

signaling mediators and nuclear import proteins in different regulatory processes

including cell proliferation, cell migration, and cell death (De Ruijter et al., 2003,

Dokmanovic et al., 2007, Carafa et al., 2013, Nguyen et al., 2011, Marchion &

Münster, 2007).

HDAC inhibitors (HDACi) are a group of chemicals interfering with the function of HDACs and usually don’t cause cytotoxic effects on cells directly

(Dokmanovic et al., 2007, Suliman et al., 2012, De Ruijter et al., 2003, Baidyaroy et

al., 2002). These chemical compounds can be grouped in many classes according to

their structures such as aliphatic acids, cyclic peptide, benzamides, and

hydroxamates (Dokmanovic et al., 2007). HDACi now are recognized as potential

antitumor drugs because many studies have approved that single aberrant HDAC

can result in cancer development (Marchion & Münster, 2007, Licciardi et al., 2012).

The relation between HDACs and cancer has shed the light on HDACi, so it has

emerged to be a potential therapeutic therapy for cancer (Licciardi et al., 2012).

3

Diseases caused by viruses are both agriculture and healthy problem that is

affecting countless crops and people in the world. Viruses are pathogens that are

hard to be eliminated, and can be classified into 7 major groups (dsDNA, ssDNA,

dsRNA, (+)ssRNA, (-)ssRNA, ssRNA-RT and dsRNA-RT) (Baltimore, 1971).

Among them, positive-strand virus consists of most members. For a genome wide

screening of host factors involved in virus accumulation, artificial replication system

derived from two positive-strand viruses, Brome mosaic virus (BMV) and Tomato

bushy stunt virus (TBSV), have been tested on single-gene-knockout yeast mutant

library to identify host factors involved in virus accumulation. Similarly, about 100

genes involved in accumulation of BMV or TBSV have been identified in both

systems (Panavas et al., 2005, Kushner et al., 2003). HDACs are both involved in

those identified genes related to plant virus accumulation.

On the investigation between HDACs and virus infection, some experiments

have been conducted on animal virus infection. Human immunodeficiency virus

(HIV) establishes a latent infection for life-long proviral latency by recruiting

HDAC to its promoter region to prevent the expression of viral genes (Wightman et

al., 2012, Keedy et al., 2009). Recent studies used SAHA (one kind of HDACi) to

inhibit the function of HDAC have found that the latent reservoir of HIV will

4

emerge from latency (Archin et al., 2009, Richman et al., 2009, Edelstein et al.,

2009, Margolis, 2011). On another case, SAHA can suppress the replication of

Hepatitis C virus by up- or down-regulate some host genes expression (Sato et al.,

2013). Besides, another strong HDACi, Trichostatin A (TSA), can mimic the

infection of virus that activates the antiviral function of beta interferon when treated

on murine fibroblastic L929 cells (Shestakova et al., 2001), showing that HDACs

participate in animal virus infection.

HDACs are conserved among all organisms. HDACs in plants can be divided

into four groups. Within these groups, three groups primary share homology to yeast

HDACs families, RPD3/HDA1 superfamily and SIR2 family. The fourth family,

HD2, was first discovered in maize and only found in plants (Hu et al., 2009, Fu et

al., 2007, Pandey et al., 2002). Recently, Arabidopsis has been used as the modal

plant to investigate the function of plant HDAC. Researches showed that if some

HDACs (like AtHD1/HDA19, AtRPD3A and AtRPD3B) were inactivated, the

Arabidopsis would express developmental abnormalities. Among these genes,

AtHD1/HDA19 also participate in the gene expression of jasmonic acid and ethylene

signaling pathway showing that plant HDACs may play a role in plant defense

pathway (Hu et al., 2009).

5

Recent papers have shown that HDACi have the ability to inhibit the growth of

fungi. For example, sodium butyrate (one kind of HDACi) and its derivatives could

inhibit the fungal growth strongly and had a synergy effect when applied with azole

drugs on the pathogenic yeast (Nguyen et al., 2011). On the other fungal pathogen,

Fusarium verticillioides, adding TSA (one kind of HDACi) would increase the

production of mycotoxins in maize (Visentin et al., 2012).

Sodium butyrate, a short-chain fatty acid-based HDACi, acts as morphological

and biochemical modifications inducer in animal cell lines at millimolar

concentrations, and the modifications are reversible (Boto et al., 1987, Kruh, 1981).

It is produced by ruminant and commensal bacteria like Clostridium and

Lactobacillus species from human intestinal tracts (Nguyen et al., 2011). Previous

papers have reported that sodium butyrate has the ability to inhibit the function of

some HDACs (Candido et al., 1978), causing hyperacetylation of H3 and H4 on

many mammalian cell lines (Kruh, 1981, Sealy & Chalkley, 1978, Candido et al.,

1978), H4 acetylation in Nicotiana tabacum cells (Arfmann & Haase, 1981) and

down-regulation of HDAC3 in tomato cells (Kim et al., 2001). Besides, sodium

butyrate was reported as an useful enhancer of lytic Epstein-Barr Virus (EBV)

infection in some cell lines (Westphal et al., 2000, Daigle et al., 2010) causing H3

6

hyperacetylation and triggering transcriptome change in HH514-16 cells resulting

EBV lytic activation (Daigle et al., 2010).

Another sodium butyrate structure-like chemical called DL-β-aminobutyric acid (BABA) has been reported that it’s known for its ability to activate the immune

response in plants to against wide spectrum pathogens like fungi, nematodes,

bacteria and viruses. BABA has been confirmed that it can induce local and

systemic resistance defense in host plants including annual, perennial plants,

monocots and dicots (Cohen et al., 2011, Cohen, 2002). However, whether

BABA-related pathway is related to the function of HDAC remains to be resolved.

Sodium butyrate is a cost economically HDACi, and share similar structure

with the known plant defense inducing chemical, BABA. Besides, sodium butyrate

is safe and has been already used as clinical treatment on cancer therapy on human

(Suliman et al., 2012). Our initial experiment also showed that sodium butyrate

could delay the pathogenesis of TMV infection and lower down the disease index of

TMV transcript-infected plants (Appendix Fig. 1 and 2). Thus, in this thesis I try to

conduct a more thorough studies to evaluate the effect and the beneath mechanism

of sodium butyrate for antiviral purpose on plant.

7

Materials and Methods

Plants

Tobacco plants (Nicotiana benthamiana, Nicotiana glutinosa and Nicotiana

tabacum Xanthi-nc) were planted in 11-cm commercial pots with

peat/perlite/vermiculite (6:1:1 v/v/v), and grown in a growth chamber under

controlled conditions at a temperature of 25°C under fluorescent lamps (10,000 lux)

in a 16h/8h day/night cycle. After four to six weeks, plants with six to eight fully

expanded leaves were ready to use.

Inoculum

Wild-type pTMV-U1 vector (5,000 ng) (Rabindran & Dawson, 2001) was

digested by Kpn1 digestion enzyme. The linearized plasmid was in vitro-transcribed

by use of T7 MEGAscript (Ambion, Austin, TX) following the manufacturer’s

instructions.

Inoculation

TMV-infected leaves (40 g) were homogenized with liquid nitrogen by use of

pestle and mortar, and then 40 ml of 0.1 M pH 7.0 potassium phosphate buffer (KP

buffer) was added to suspend the powder of homogenized leaves. This crude sap

8

solution was freeze-dried by use of Freeze Dryer System (Christ Beta, Germany) for

one day then stored at -80°C. Freeze-dried tissue powder (0.05 g) were diluted with

1 ml of 0.1 M KP buffer for inoculum. Carborundum was dusted on the leaf surface first and 200 μl of inoculum was gently rubbed on the leaf of plants.

Treatment of chemical inducer

All chemical inducers, salicylic acid (SA) (Sigma-Aldrich, St Louis, MO,

USA), DL-β-aminobutyric acid (Sigma-Aldrich, St Louis, MO, USA) and sodium

butyrate (Sigma-Aldrich, St Louis, MO, USA), were all prepared in deionized water.

Same amount of different solutions (150 ml) were used to drench soil and 200 μl of

solutions were sprayed on the 3^rd and 4^th leaf surface of N. benthamiana and N.

tabacum Xanthi-nc or the 2^nd, 3^rd and 4^th leaf of N. glutinosa two days before TMV

inoculation.

Phenotype measurement

Height of plants was measured from the top of main branch to stem base. The

leaf width was represented by measuring the width of the largest leaf of each plant.

Total flowers and branches were all counted in our experiment.

9 RNA extraction

Leaf tissues (0.05 g) were homogenized with liquid nitrogen by use of pestle

and mortar, and mixed with 1 ml of TRIzol (Invitrogen, Carlsbad, CA, U.S.A.). The sap was transferred to an eppendorf tube and 200 μl of chloroform were added. After

well mixing, the tube was centrifuged at 12,000 x g for 15 minutes at 4°C.

Approximately 600 to 650 μl of upper phase of homogenate were transferred to a

new eppendorf tube and 500 μl of 100% isopropanol were added. The tube was

inverted several times, and kept at room temperate for 10 minutes, and centrifuged

at 12,000 x g for 10 minutes. Then, the supernatants were discarded, and 1 ml of

70% ethanol was added to wash RNA pellets. The tubes were centrifuged at 7500 x

g for 5 minutes at 4°C. The supernatants were discarded and wait until the

precipitates are dry. The pellets were dissolved in 50 μl of DEPC water.

Reverse transcription polymerase chain reaction (RT-PCR)

RNA (800 ng) were mixed with reverse primers (Table 1.) and heated at 95°C

for 5 minutes, then immediately chilled on ice. Then 1 μl of 2.5 mM dNTP, 4 μl of 5X MMLV buffer, 1 μl of M-MLV reverse transcriptase (Promega, Madison, WI,

USA) and 8 μl of DEPC H₂O were added to each PCR tube, and incubated tubes at

42°C for 45 minutes for first-strand cDNA synthesis.

10

cDNA (200 ng, about 5 μl) were used in each PCR reaction. Beside cDNA template, 1 μl of 10 μg/μl forward primer, 1 μl of 10 μg/μl reverse primer, 1 μl of 2.5

mM dNTP, 4 μl of 10X reaction buffer and 1 μl of 5 U/μl Taq polymerase (Promega)

were added to PCR tube, and added ddH2O to 50 μl. Cycle conditions were set as

94°C 5 minutes for one cycle, 94°C 30 seconds, 55°C 30 seconds and 72°C 45

seconds for 26 cycles (for actin) or 35 cycles (for PR1a), and 72°C 5 minutes for 1

cycle.

Detection of hydrogen peroxide accumulation

The hydrogen peroxide was measured by 3, 3’-Diaminobenzidine (DAB)

staining. The treated and systemic leaf of N. benthamiana were excised, and soaked

freshly in 0.1% DAB solution (pH 3.8) (Sigma-Aldrich, St Louis, MO, USA) under

vacuum for 5 minutes for 2 times, and the leaves were kept for 10 h in DAB solution

at 25°C under fluorescent lamps. After staining, leaves were transferred to 95%

ethanol at 25°C overnight to wash out chlorophyll. After treatment, the percentage of

the brown spot area (indicated the localization of hydrogen peroxide) versus total leaf

area was measured by use of Image J public domain software (National Institute of

Health, USA).

11

To quantify hydrogen peroxide by use of spectrophotometer, 0.05 g leaf sample

were collected randomly by puncher in treated leaves or in systemic leaves to ensure

there was no bias in sample collection. After grinding with liquid nitrogen by use of

pestle and mortar, sap was mixed with 3 ml sodium phosphate buffer (50 mM, pH 6.8,

with 1 mM hydroxylamine). Then the tube was centrifuged at 6,000 x g for 25

minutes at 4°C. Upper phase (2 ml) were transferred to a new eppendorf tube, and 1

ml of TiCl4 (0.1 %, v/v, dissolved in 20 % H2SO4) was added and mixed thoroughly.

The tube was centrifuged at 1,000 x g for 15 minutes at room temperature. After

centrifugation, supernatant (200 μl) were transferred to 96-well PCR plate and

spectrophotometer was used to measure the absorption at 410 nm (blank sample is

replaced by 50 mM sodium phosphate buffer), and the content of H₂O₂ (μmol g^-1) was

measured through the formula：A₄₁₀ ÷ 0.28 (K, μmol^-1 cm ^-1 ) × 1.5 (dilution ratio) ÷

0.05 g as described in previous studies (Jana & Choudhuri, 1982, 林, 1996).

12

Results

Optimization of TMV inoculum

To optimize the TMV inoculum, TMV-U1 RNA transcript was generated from

pTMV-U1 by in vitro transcription. In order to mimic the nature inoculum, the RNA

transcript was used to inoculate N. tabacum, and freeze-dried TMV-infected leaf

powder was used for further inoculation. Different concentrations of the inoculum

were rubbed to the sensitive plant, N. benthamiana (Fig. 1). The inoculum (0.5

mg/ml) was selected for further inoculation assay (Fig. 1, 10^-2 dilution), because

plants inoculated at this concentration began to show symptoms five to seven days

and died around 14 days post-inoculation. It allowed us to better record the

pathogenesis of TMV on infected N. benthamiana. Furthermore, the symptoms

caused by TMV infection was measured through disease index (Fig. 2)

Analysis the application methods and concentration of sodium butyrate that

may cause phytotoxicity

To analyze if sodium butyrate may cause phytotoxicity, different concentrations

of sodium butyrate (1 mM, 5 mM, 10 mM and 15 mM) were used to drench or spray

on N. benthamiana. Salicylic acid (SA) and DL-β-aminobutyric acid (BABA) were

used as controls. The concentration of SA and BABA used for plant treatment varied

13

between reports (Table 2.). We used 5 mM of SA and 10 mM of BABA for plant

treatment. The results indicated that chlorosis appeared on plants drenched with 10

mM and 15 mM of sodium butyrate, and also on plants drenched with 10 mM of

BABA three days after treatment (Fig. 3), but not on other plants. The height, leaf

width, flower and branch number of sodium butyrate-treated plants were all

recorded two months after treatment to evaluate the long-term effect (Fig. 4 and 5).

The results showed that the height, leaf width and flower number were all reduced

on plants treated with 10 and 15 mM of sodium butyrate (Fig. 5a-5c), and reduced

branch number also was observed on plants treated with 15 mM of sodium butyrate

(Fig. 5d).

Besides drenching, the same concentrations of sodium butyrate (1 mM, 5 mM,

10 mM and 15 mM) were sprayed on leaves. All the treated plants were healthy

looking (Fig. 6 and 7).

N. benthamiana pretreated with sodium butyrate could delay the pathogenesis

of TMV

To assay if sodium butyrate could interfere the infection of virus, sodium

butyrate was treated on plants and plants were inoculated with TMV inoculum two

days after treatment. Because previously results indicated that 10 mM and 15 mM of

14

sodium butyrate would cause phtotoxicity to plants (Fig. 3 and 4), only 1 mM and 5

mM of sodium were used for drenching. N. benthamiana pre-drenched with 1 mM

and 5 mM sodium butyrate remained to stand upright while wilting was observed on

mock treated plants five days pi (Fig. 8). Sodium butyrate (1 mM and 5mM) also

sprayed on the 3^rd and 4^th leaf two days before TMV inoculation. Results showed

that plants pretreated with 1 mM and 5 mM all showed mild symptoms and delayed

pathogenesis as compared with mock treated plants (Fig. 9). However, even 10 mM

and 15 mM of sodium butyrate did not show pytotoxicity on plants by leaf spraying

(Fig. 6 and 7). The inoculation assay indicated that plants sprayed with sodium

butyrate at 10 mM and 15 mM did not exert antiviral activity (Fig. 10).

Sodium butyrate induced the expression of PR1a in systemic leaves on treated

N. benthamiana

Because virus usually induce SA-related palnt defense pathway (Yalpani et al.,

1993, Malamy et al., 1992), we initially monitered the marker gene, PR1a, of this

pathway (Fig. 11). Three independent experiments were conducted (Table 3 and Fig.

11), and data showed that PR1a can be detected from one day after SA treatment,

and the expression is most on treated leaves but also can be detected on systematic

leaves albeit the expression amount is less than in the treated leaves (Fig. 11). In

15

contrast, PR1a can be only be detected seven days after treatment on systematic

leaves on both BABA- and sodium butyrate-treated plants (Fig. 11).

Enhanced antiviral effect of SA and sodium butyrate-treated N. benthamiana

Although both SA and sodium butyrate all induced the expression of PR1a (Fig.

11), the spacious and temporal expression of PR1a is different in SA- and sodium

butyrate-treated plants. It suggested that SA and sodium butyrate may induce

different defense mechanism. Thus, we tried to combine the use of both chemicals to

see if they can enhance the resistance, SA and sodium butyrate were combined to

treat plants. The results showed that all treated plants showed reduced disease

symptoms (Fig. 12), and SA mixed with 5 mM of sodium butyrate displayed the

most obvious reduction of disease index among all treatments (Fig. 12).

Hydrogen peroxide accumulated at the systemic leaf of 1 mM but not 5 mM

sodium butyrate-treated N. benthamiana

Because SA can induce the generation of reactive oxygen species (ROS), we

also determined if sodium butyrate can induce the ROS. ROS was analyzed one and

seven days later on plants treated with SA, sodium butyrate, BABA or inoculation of

TMV. The data indicated that ROS can be detected on systemic leaves of plants

16

sprayed with 1 mM of sodium butyrate, and also on TMV infected plants (Fig. 13

and 14). However, ROS only can be detected seven days later on TMV infected

plants but not on other treated plants (Fig. 15).

Lesion induced by TMV on N. glutinosa treated with SA, BABA and sodium

butyrate

Besides the highly susceptible N. benthamiana, we also tested if sodium

butyrate also works on the resistance host, N. glutinosa (baring N gene). Previously

report indicated that BABA can reduce the lesion number on N. tabacum Xanthi-nc

(Siegrist et al., 2000). We followed the method to treat another N gene containing

plant, N. glutinosa. The results showed that in both drenching and leaf sparing

treated plants, no obvious decrease lesion size and lesion number on all treated

plants (Fig. 16 and 17).

Local lesion induced on BABA but not sodium butyrate treated N. tabacum

Xanthi-nc

Previous report indicated that BABA can cause phtotoxicity on sensitive

tabacco, N. tabacum Xanthi-nc (Siegrist et al., 2000). To analyze if sodium butyrate

can also cause phytotoxicity on this plants, we also sprayed sodium butyrate on N.

17

tabacum Xanthi-nc. Data showed that small lesions were observed on BABA-treated

but not on sodium butyrate-treated plants (Fig. 18). (Yalpani et al., 1991, Mitter et al.,

1998, Cohen, 1994, Lazzarato et al., 2009, Caro et al., 2007)

18

Dicussion

The ideal chemical applied on plants to induce resistance should be able to

induce desirable resistance against viruses, less phytotoxicity and also cost effective

when applied for field crops. In this thesis, I demonstrate that HDACi, sodium

butyrate, can exert the antiviral activity against TMV on treated N. benthamiana.

Compare to the effect of previously reported antiviral inducer, SA and BABA, our

results indicate that the needed amount of sodium butyrate is lower and less

phytotoxicity is observed on sodium butyrate-treated plants. Besides, our initial

result also suggested that sodium butyrate induced SA-related resistance pathway.

However, the pathway is more similar to BABA- but not SA-induced pathway.

In terms of inducing desirable resistance, our data show that when the highly

susceptible host, N. benthamiana, pretreated with sodium butyrate can delay the

pathogenesis and alleviates the symptoms (Fig. 8 and 9). Although BABA- and

SA-treated N. benthamiana also show resistance after TMV infection, the

concentrations used in alleviating the symptoms was higher than sodium butyrate.

Low concentrations of chemicals can cut down the dose and decrease the risk of

phytotoxicity.

19

Interestingly, BABA has been reported that it can enhance the ability of N gene

by reducing the lesion size and number on TMV-infected N gene-carrier N. tabacum

Xanthi-nc (Siegrist et al., 2000); however, in our experiments SA-, sodium butyrate-

and BABA-treated plants cannot reduce the lesion number and size induced by

TMV on treated N. glutinosa (N gene containing plants) (Fig. 16 and 17). It suggests

that plants differ in their ability to enhance resistance. Thus, more thorough

experiments may be needed to determine the appropriate concentration and the

treated methods to induce resistance on different plants.

Phytotoxicity usually results from over-inducing some defense responses in

plants affecting the basal expression of defense reaction (Macías et al., 2004,

Ottmann et al., 2009). SA is often doubted with its high risk of causing chemical

injury. Several SA analogs, such as INA and BTH, have been developed to alleviate

the phytotoxicity (吴 & 郝, 2005, 王 & 朱, 2002). However, INA also triggers

phytotoxicity (Pertot et al., 2008). BTH have been reported to cause less

phytotoxiciy than SA and INA and has already been commercialized; however, it

still causes chemical injury on plants (Pertot et al., 2008). In our study, we find

drenching plants with sodium butyrate in high concentrations (10 mM and 15 mM)

would cause chemical injury, but the effective concentrations in triggering antiviral

20

activity (1 mM and 5 mM) are not phytotoxic (Fig. 3-5). BABA has been reported to

be less phytotoxic than SA and its analogs; however, it also causes chemical injury

on some plants at the effective concentration (Fig. 18). Our results testing sodium

butyrate on sensitive plant, N. tabacum Xanthi-nc, also indicate that effective

concentrations in triggering antiviral activity of sodium butyrate (1 mM) wouldn’t

induce lesions; however, the effective concentration of BABA in triggering antiviral

activity causes lesion on treated plants.

Our results indicate that sodium butyrate induces the expression of PR1a. Data

display that PR1a accumulated in sodium butyrate- and BABA-treated plants only in

systemic leaves seven days after treatment, but SA-treated plants express PR1a

accumulation most in treated leaf within one day reveals a differential accumulative

pattern between treatments. Therefore, our data suggest that sodium butyrate may

participate in SA-related pathway; however, the pathway is more similar to

BABA-induced pathway but different from typical SA-induced plant response.

To be noted, the resistance induction is both determined by inducer and plants.

For example, previous reported that the expression of PR1a also differed between

BABA-treated tobacco species. PR1a expressed most in the systemic leaves of N.

21

tabacum Samsun (NN) (Lazzarato et al., 2009), but appeared apparently in the

treated leaf of N. tabacum Xanthi-nc (Siegrist et al., 2000). Furthermore, the

expression time of PR1a also seemed different in varied plants. In BABA-treated

lettuce, minor expression of PR1a could only be detected three days after treatment

(Cohen et al., 2010). In N. tabacum L. cv. Ky16 (nn), PR1a was detected for two

days after BABA spraying (Cohen, 1994).

Other than PR1a, ROS accumulation is also an important response of plant

resistance. However, like the expression of PR1a, the generation of hydrogen

peroxide is also both depended on inducers and plant species. Examples such as

hydrogen peroxide is detected 48 hours after BABA treatment on N. tabacum

Xanthi-nc (Siegrist et al., 2000) and 18 hours later on LaCl₃-treated pea (Romero‐

Puertas et al., 2004). Because we cannot find the reported timing to detect

SA-induced hydrogen peroxide in N. benthamiana, we initially detected the

hydrogen peroxide on sodium butyrate-treated plants one and seven days after

treatment. The results showed that hydrogen peroxide can be detected on

TMV-infected plants both at one and seven days pi; however, in all other treated

leaves only 1 mM of sodium butyrate-treated N. benthamiana show obvious

accumulation of hydrogen peroxidein systemic leaves one day after treatment (Fig.

22

13-15). To be noted, we cannot detect hydrogen peroxidein plants treated with 5

mM sodium butyrate or SA. One possible explanation is that different plant species

and different concentrations of the same inducer may induce differential resistance

in terms of timing and location.

The cost of sodium butyrate is cheaper than BABA. Currently, we purchase

BABA and sodium butyrate both from Sigma-Aldrich. Sodium butyrate (500 g)

costs 164.5 USD; however, BABA (5 g) cost 234 USD. Besides, sodium butyrate

can be generated by ruminant and commensal bacteria like Clostridium and

Lactobacillus species from human intestinal tracts (Nguyen et al., 2011), which can

feed on polysaccharides of plant cell wall such as cellulose and pectins (Hague et al.,

1993, Cummings, 1981). Thus, it is possible to collect their major metabolite,

sodium butyrate, by culturing these anaerobic bacteria in lab, and scale up the

production from lab to commercial purpose.

In field, TMV infects plants through mechanical injuries contacted with

TMV contaminated insects, tools or plant-remaining in the soil (翟 et al., 2012).

The main disease management is to sterilize the tools, soil and kill insects by

spraying pesticide. Thus, if mixed pesticide with sodium butyrate for spraying might

23

provide additional protection to decrease the possibility of virus infection. Although

sodium butyrate offers several advantages in inducing resistance against virus

infection, the way and timing of application in field remains to be further optimized.

My initial study indicates that sodium butyrate can induce resistance on N.

benthamiana against TMV infection. Both sodium butyrate and BABA induce the

similar expression pattern of PR1a in plants (Fig. 11); however, the accumulation of

hydrogen peroxide (Fig. 13 and 14) and also the lesion inducing on N. tabacum

Xanthi-nc (Fig. 18) is different between sodium butyrate- and BABA-treated plants.

It suggests that sodium butyrate and BABA may differ in their mechanism in

inducing resistance. Thus, the more in depth study such as whole genome analysis of

histone acetylation pattern remains to be explored to fully understand the effect and

mechanism underline the sodium butyrate-induced resistance against TMV.

24

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33

Figures

34

Fig. 1 Symptoms of Nicotiana benthamiana infected with freeze-dried TMV at

different concentrations five days pi. Six weeks-old plants were inoculated with

serial dilution of TMV inoculum. Freeze-dried TMV-infected N. tabacum tissue

(0.05 g) were grinded to powder by liquid nitrogen and dissolved in 1 ml of

potassium phosphate buffer that defined as 1X stock inoculum, and diluted at ten

times increment. Photos were taken five days pi. The side view (a) top view (b)

detached inoculated leaves (c) of N. benthamiana were shown.

35

Fig. 2 Disease index for measuring the symptoms of TMV-infected Nicotiana

benthamiana. Leaves were classified into four indexes by the severity. When the

symptom took 0-25% area on leaf, it was index 1. Symptom occupied 25-50% leaf

region was index 2, and 50-75% was index 3. The infected area over 75% was

defined as index 4. Plants infected with TMV showed symptoms five to seven days

pi, and leaves of plants were excised to estimate the severity of each treatment ten

days pi.

36

Fig. 3 Phenotypes of Nicotiana benthamiana drenched with different chemicals.

Four weeks-old plants were drenched with 150 ml of deionized water (Water), 5 mM

salicylic acid (SA), 10 mM DL-β-aminobutyric acid (BABA) and different

concentrations (1 mM, 5 mM, 10 mM and 15 mM) of sodium butyrate (SBA).

Photos were taken seven days after treatment. The side view (a) top view (b) and the

phenotypes of systemic leaf (c). Notice that systemic leaves of plants drenched with

37

10 mM BABA, 10 mM and 15 mM sodium butyrate appeared chlorosis symptoms

three days after treatment.

38

Fig. 4 Phenotypes of Nicotiana benthamiana drenched with different

concentrations of sodium butyrate. Six weeks-old N. benthamiana were drenched

with 150 ml deionized water and different concentrations (1 mM, 5 mM, 10 mM and

15 mM) of sodium butyrate once every two days for two months. Photos were taken

two months after the initial treatment. Plants were drenched with deionized water (a),

39

1 mM sodium butyrate (b), 5 mM sodium butyrate (c), 10 mM sodium butyrate (d),

and 15 mM sodium butyrate (e).

40

Fig. 5 Phenotypes of Nicotiana benthamiana drenched with different

concentrations of sodium butyrate. Plants were drenched with 150 ml of different

concentrations (1 mM, 5 mM, 10 mM and 15 mM) of sodium butyrate once every

two days for two months. The statistic data of height (a) leaf width (b) flower

number (c) and branch number (d) of treated N. benthamiana were shown. Error

bars represent standard deviation. Data were collected two months after the initial treatment, and analyzed by Student’s t test. The asterisk, (*) and (**), indicates P <

0.05 and P < 0.01, respectively, as compared with deionized water-treated control

plants.

41

Fig. 6 Phenotypes of Nicotiana benthamiana with leaves applied with chemicals.

Four weeks-old N. benthamiana were sprayed with 200 μl of deionized water

(Water), 5 mM salicylic acid (SA), 10 mM DL-β-aminobutyric acid (BABA) and

different concentrations (1 mM, 5 mM, 10 mM and 15 mM) of sodium butyrate on

the above and abaxial side of 3^rd and 4^th leaf for one time. Photos were taken one

week after treatment. The side view (a) and top view (b) of treated N. benthamiana.

42

Fig. 7 Leaves appearance of Nicotiana benthamiana with leaves applied with

chemicals. Four weeks-old N. benthamiana were applied with 200 μl of deionized

water (Water), 1 mM and 5 mM sodium butyrate (SBA). The solution-treated 3^rd and

4^th leaf with their petioles labelled with orange mark, and the arrangement of leaves

from left to right was the first leaf through the treated leaves. Photos were taken one

month after the initial treatment.

43

Fig. 8 Symptoms of Nicotiana benthamiana pre-drenched with chemicals then

inoculated with TMV ten days pi. Four weeks-old plants were drenched with 150

44

ml deionized water (Mock), 1 mM sodium butyrate (SBA) and 5 mM sodium

butyrate (SBA) first, and inoculated freeze-dried TMV two days after treatment. The

uninoculated plant (Control) was indicated. Plants started to show symptoms five

days pi. Photos and quantification of the symptom reduction were performed ten

days pi. Four independent experiments were performed. Error bars represent standard deviation. Data were analyzed by Student’s t test. The asterisk, (*) and (**),

indicates P < 0.05 and P < 0.01, respectively, as compared with mock.

45

Fig. 9 Symptoms of Nicotiana benthamiana pretreated with chemicals on leaves

then inoculated with TMV ten days pi. Four weeks-old N. benthamiana were applied with 200 μl of deionized water (Mock), 10 mM DL-β-aminobutyric acid

(BABA), 1 mM sodium butyrate (SBA) and 5 mM sodium butyrate (SBA) on the 3^rd

and 4^th leaf first, and inoculated freeze-dried TMV two days after treatment. The

uninoculated plant (Control) was indicated. Plants started to show symptoms five

days pi. Photos and quantification of the symptom reduction were performed ten

46

days pi. Four independent experiments were performed. Error bars represent standard deviation. Data were analyzed by Student’s t test. The asterisk, (*) and (**),

indicates P < 0.05 and P < 0.01, respectively, as compared with mock.

47

Fig. 10 Symptoms of Nicotiana benthamiana pretreated with chemicals on

leaves then inoculated with TMV ten days pi. Four weeks-old N. benthamiana were applied with 200 μl of deionized water (Mock), 10 mM sodium butyrate (SBA)

and 15 mM sodium butyrate (SBA) on the 3^rd and 4^th leaf first, and inoculated

freeze-dried TMV two days after treatment. Plants started to show symptoms five

days pi, and photos were taken ten days pi.

48

Fig. 11 Detection of PR1a expression in different chemical-treated Nicotiana

benthamiana in 7-day time course. Plants were treated with 200 μl of deionized

water (CK), 5 mM salicylic acid (SA), 1 mM sodium butyrate (SBA), and 10 mM

DL-β-aminobutyric acid (BABA). RNA of the 3^rd treated leaf and systemic leaf were

extracted from different plants at different days, and used RT-PCR to detect the

accumulation of PR1a. Ethidium bromide was used to stain with 1.5% agarose gels

with RT-PCR products. Three independent experiments were conducted, and gel

from one experiment was shown.

49

Fig. 12 Quantification of the symptom reduction of Nicotiana benthamiana

pretreated with single or mixed chemicals and then inoculated with TMV. Four

weeks-old N. benthamiana were applied with 200 μl of deionized water (Mock), 5

mM salicylic acid (SA), 10 mM DL-β-aminobutyric acid (BABA), 1 mM sodium

butyrate (SBA), 5 mM sodium butyrate (SBA) and 5 mM salicylic acid mixed with

1 mM or 5 mM sodium butyrate (SA+1 mM SBA and SA+ 5 mM SBA). TMV was

inoculated two days after treatment. Plants started to show symptoms five days pi,

and photos were taken ten days pi. Significance of variation in disease index of

different chemical-pretreated N. benthamiana was determined by LSD test. Values

followed by the same letter are not significantly different at the 5% level, and each

value is the mean of three replications.

50 Fig. 13 Hydrogen peroxide accumulation was detected via DAB staining in different chemical-treated Nicotiana benthamiana one day rdth after treatment.The 3and 4leaf wereapplied with 200 μlofdeionizedwater(Control), 5 mM salicylic acid(SA), 10 mM DL-β-aminobutyric acid (BABA), 1 mM sodium butyrate (SBA), 5 mM sodium butyrate (SBA), 5 mM salicylic acid mixed with 1 mM or 5 -2ndrdth mM sodium butyrate (SA+1 mM SBA and SA+ 5 mM SBA) and 10X TMV inoculum. The 2, 3 and 4 leaf were cut down to stain with DAB one day after treatment. Brown spots directly indicated the localization of hydrogen peroxide.