大阪府立大学　学術情報リポジトリ

(1)

Analysis of cyclic symptom development in

tobacco infected with Cucumber mosaic virus

著者

SUNPAPAO Anurag

内容記述

学位授与大学: Osaka Prefecture University(大阪

府立大学), 学位の種類: 博士(応用生命科学), 学

位記番号: 論生命第34号, 学位授与年月日:

2011-03-31, 指導教員: 大木理.

(2)

大阪府立大学博士（応用生命科学）学位論文

Analysis of cyclic symptom development in tobacco

infected with Cucumber mosaic virus

（キュウリモザイクウイルス感染タバコにおける周期的病徴発現機構の解析）

Anurag SUNPAPAO

2011 年

(3)

Analysis of cyclic symptom development in tobacco infected with

Cucumber mosaic virus

(キュウリモザイクウイルス感染タバコにおける周期的病徴発現機構の解析)

By

ANURAG SUNPAPAO

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of a

Doctor of Philosophy

in

Applied Life Sciences

2011

Division of Applied Life Sciences

(4)

“Most of the important things in the world have been accomplished by people

who have kept on trying when there seemed to be no hope at all.”

(5)

Declaration

I hereby declare that this thesis is my own work.

May 31, 2011 Anurag Sunpapao

(6)

ACKNOWLEDGEMENTS

All respects are due to all of those people who gave me the encouragements and extended their help to complete this course. The present study was carried out at Department of Plant Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University during years 2008-2011.

First of all, I pay cordial gratitude to my worthy, reverend supervisor Prof. Satoshi T. Ohki of Department of Plant Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University for his supervision, invaluable help, continuous guidance, constant encouragements and keen interest throughout this study. He supervised the whole works with full zeal and eager, advised critically and gave tremendous constructive comments for the preparation of the manuscript.

Next, I would like to express my deepest gratitude and immense indebtedness to Assist. Prof. Tomofumi Mochizuki of the department for his helpful, diligent, excellent guidance, stimulating suggestions and encouragements helped me in all the time of research and he also kept an eye on the progress of my works and always was available when I needed his advices.

I am literally indebted to Assoc. Prof. Matoaki Tojo of the department for the kind advices, recommendations and all supports during the period of my work.

I am grateful to Prof. Masayuki Oda and Prof. Nozomu Koizumi of the department for critical reading of the thesis and valuable comments.

I am also grateful to Dr. T. Meshi of Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, for kindly providing RNA silencing down-regulated transgenic lines.

I would also like to thank my colleagues of the laboratory for encouraging me and maintaining friendly environments during my work.

(7)

Special thanks are due to the Osaka Prefecture University, Japan, for providing me all the facilities and the conductive academic environment to complete the course.

No acknowledgment could never adequately express my obligations to my affectionate father, and sweet loving mother, who encourage and unconditionally love me forever.

Finally, I am also indebted to the Ministry of Education, Culture, Sports, Science and Technology, Government of Japan for financial support and extend throughout the whole study course in Japan. This research has been supported and funded by Japanese Government Scholarship (MEXT scholarship). I thank you all for your confidence in me.

March 31, 2011

(8)

LIST OF ORIGINAL PUBLICATIONS AND PRESENTATIONS

The thesis is based on the following articles, which are referred to, in the text, by their Arabic numerals.

Published papers

1. Sunpapao, A., Nakai, T., Dong, F., Mochizuki, T. and Ohki, S.T. (2009). The 2b protein of Cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants. Journal of General

Virology 90: 3015-3021.

2. Sunpapao, A., Mochizuki, T. and Ohki, S.T. (2011). Relationship between viral distribution in the developing leaves and symptom severity in the fully expanded leaves of tobacco plants infected with Cucumber mosaic virus. Australasian Plant Pathology (in press).

Proceedings

1. Dong, F., Sunpapao, A., Tojo, M. and Ohki, S.T. (2007) Distribution of Cucumber

mosaic virus in leaf primordia and developing leaves of infected tobacco. Japanese Journal of Phytopathology 73: 240. (Abstract)

2. Sunpapao, A., Dong, F. and Ohki, S.T. (2008) The suppressor protein of Cucumber

mosaic virus is necessary for severe systemic infection in tobacco. Japanese Journal of Phytopathology 74: 246. (Abstract)

3. Sunpapao, A., Mochizuki, T. and Ohki, S.T. (2009) Correlation between viral amount in developing leaves and symptom severity on the fully expanded leaves of Cucumber

mosaic virus-infected tobacco. Japanese Journal of Phytopathology 75: 287. (Abstract)

4. Sunpapao, A., Mochizuki, T. and Ohki, S.T. (2010) Decreased expression of dicer-like ribonuclease 2 or 4 or RNA-dependent RNA polymerase 1 alone does not affect the viral titer and symptom severity of Cucumber mosaic virus. Japanese Journal of

(9)

SUMMARY

Analysis of cyclic symptom development in tobacco infected with

Cucumber mosaic virus

The most common disease symptom when plants are systemically infected with viruses is mosaic. Characteristic of mosaic leaves consist of two areas: dark green islands and yellow-green tissues. The former contains few infectious viruses, whereas abundant CMV-infected cells occur in the latter. Several viruses including Cucumber mosaic virus (CMV) develop cyclic symptom expression in infected plants as mosaic leaves and then symptomless leaves, and again mosaic leaves. However, the mechanism of this phenomenon is not so far clarified.

There are numerous factors such as virus-encoded proteins and plant defense mechanism should be involved in the development of cyclic symptoms. For this aim, I have examined virus-plant interaction between the pathogenesis and their related molecular events in CMV-infected tobacco.

Chapter 1. Characterization of cyclic symptom expression in CMV-infected tobacco plants

Tobacco plants inoculated with the CMV Pepo strain induced cyclic mosaic symptom expression; mosaic and mottled leaves appeared first among the newly developing leaves (L7-9) above the inoculated leaf (L0), symptomless leaves then developed (L10-11), and mosaic leaves appeared again (L12-14). To clarify the formation of the cyclic symptoms by CMV, the relationships between distribution of viral coat protein (CP) or viral RNA (vRNA) in the leaf primordia (LP, 2 to 3 mm) or young developing leaves (YDLs, 1 to 2 cm) and symptom severity in the fully expanded leaves were analyzed. Large amounts of CMV were detected in most cells of the YDLs in the early infection periods, which later led to the development of mosaic and mottled leaves. Virtually no signals were detected in the

(10)

that very few and uneven CMV distribution in the symptomless leaves and mosaic leaves generated during the late infection period was determined at LP stage, while viral distribution in the early mosaic leaves was not determined at the developing stage.

Chapter 2. The 2b protein of CMV is necessary for cyclic mosaic symptom expression in tobacco

A 2b protein-defective mutant of the CMV Pepo strain (∆2b) induces only mild symptom and no cyclic symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in the cyclic symptom expression, distribution of ∆2b in the shoot apical meristem (SAM) and LP was monitored. Time-course histochemical observations showed that ∆2b was distributed in SAM at 7 day postinoculation (dpi) but could not invade SAM and quickly disappeared from SAM, whereas Pepo transiently appeared in SAM from 4 to 10 dpi. Faint ∆2b signals were detected at 14 and 21 dpi in LP, whereas dense Pepo signals were observed in LP from 4 to 18 dpi. Northern blot analysis showed that short interfering RNA (siRNA) derived from ∆2b-RNA accumulated earlier in SAM and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and ∆2b-infected plants at the late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected with Pepo enlarged faster than those infected with ∆2b, whereas accumulation of ∆2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in SAM and LP via prevention of RNA silencing and/or acceleration of the cell-to-cell movement as well as promoted cyclic mosaic pattern in infected tobacco plants.

Chapter 3. Involvement of DCL2-4 and RDR1 in cyclic mosaic symptom development of CMV-infected tobacco

In RNA silencing mechanism, RNA-dependent RNA polymerase (RDR) in plants synthesizes complementary RNA using viral RNA as the template and the double-stranded RNA is then cleaved by Dicer-like proteins (DCL). The Nicotiana tabacum genome is considered to encode four DCL and six RDR homologues. DCL1 synthesizes 18-21 nt-long microRNA, whereas DCL2, DCL3 and DCL4 produce 22 nt, 24 nt and 21 nt-long siRNA,

(11)

respectively, in the RNA silencing process. To clarify which components were involved in the viral titer change and the cyclic symptom development in CMV-infected tobacco, transgenic tobaccos in which DCL2, 3, 4, 2/4 or RDR1 down-regulated were used and amounts of vRNA in YDLs in the transgenic tobaccos were examined by Northern blot analysis. Most transgenic plants inoculated with CMV exhibited the cyclic mosaic patterns. Amounts of vRNA were changed along with the leaf position similar to those in the case of wild-type tobacco. Furthermore, expression of those RNA silencing-related genes during high and low CMV infection were not significantly different among those transgenic plants. These results indicated that the inhibition of single DCL2-4 or RDR1 and double DCL2/4 did not affect the development of the cyclic mosaic symptom by CMV.

The results obtained in this study demonstrated the mosaic leaves development at the early infection stage was not determined but that at the late infection stage was already determined in LP. The interaction between the virus-encoded counter-defense 2b protein and the RNA silencing components was a key factor determining the severe symptom expression and the cycling symptom development. Experiments on the transgenic tobaccos suggested that the RNA silencing components compensated each other. To establish virus-tolerant crops, further analysis of systemic silencing signals and host defense proteins in the virus-infected plants will be needed.

(12)

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………..………...…………i

LIST OF ORIGINAL PUBLICATIONS AND PRESENTATIONS…………....…………..iii

SUMMARY……….…………....…..vi

TABLE OF CONTENTS………..………vii

INTRODUCTION………...……….….1

Mosaic symptom development………..1

Cucumber mosaic virus………...………4

RNA silencing as a defense mechanism against plant viruses (a) Silencing defense as a plant defense mechanism.………7

(b) Silencing suppressors of plant viruses………10

AIMS……….…...13

CHAPTER I Characterization of cyclic symptom expression in CMV-infected tobacco plants 1.1 Introduction……….………15

1.2 Materials and methods………...16

1.2 Results 1.3.1 Accumulation of viral CP, viral RNAs and viral distributions in fully expanded tobacco leaves infected with CMV………21

1.3.2 Viral CP and RNAs in young developing leaves shifted along leaf positions in CMV infected tobacco………..…..24

1.3.3 Kinetics of viral distributions during leaf development in CMV-infected tobacco….26 1.4 Discussion………...28

(13)

CHAPTER II The 2b protein of CMV is necessary for cyclic mosaic symptom expression in tobacco

2.1 Introduction……….32 2.2 Materials and methods………33 2.3 Results

2.3.1 Involvement of 2b protein in efficient CMV distribution in SAM and LP…………36 2.3.2 Accumulation of vRNA and siRNA in the SAM and LP………38 2.3.3 The 2b protein affects the efficiency of cell-to-cell movement of CMV in inoculated leaves…...………...………..41 2.4 Discussion………...43

CHAPTER III Involvement of DCL2-4 and RDR1 in cyclic mosaic symptom development of CMV-infected tobacco

3.1 Introduction……….…47 3.2 Materials and methods………49 3.3 Results

3.3.1 Expression of mRNA of RNA silencing components in RNA silencing down- regulated transgenic lines………51 3.3.2 Symptom patterns in down-regulated lines infected with CMV……….…...…52 3.3.3 Change of viral RNA amounts in CMV-infected YDLs of transgenic lines…………54 3.3.4 Expression of mRNA of RNA silencing components during CMV infection………55 3.4 Discussion………...…57

GENERAL DISCUSSION………..61

(14)

Introduction

INTRODUCTION

Plant viruses cause many important plant diseases and are responsible for great losses in crop productions and quality worldwide. Although a huge variety of plant viruses have been isolated and characterized from a multitude of agronomically important plant species, we have poor countermeasures against plant virus infection. Plant viruses cannot be directly controlled by chemical application. The most effective ways of managing viruses are cultural controls and using tolerant cultivars.

The infection by plant viruses starts from infected cells and the viruses systemically spread through vascular system. Plant viruses must accomplish three main steps to complete their infection cycles: (i) replication inside the initially infected cell, (ii) cell-to-cell movement to adjacent cells in the inoculated leaf through plasmodesmata, and (iii) long-distance movement to other parts of the plant through vascular tissues. The majority of studies on plant viral symptomatology have particularly focused on cell-to-cell movement and long-distance movement. Systemic infected plants may show a range of symptoms depending on the disease but often there is leaf mosaic (Hull 2002).

Mosaic symptom development

The normal obvious effects of plant viral infection are based on the route of viral spread in the plant host. Plant viruses are transported through phloem following the source-to-sink route for photoassimilates (Leisner & Turgeon 1993). Therefore, viruses tend to accumulate

(15)

Introduction

in young (sink) tissues and virus replication is usually high there (Leisner & Turgeon 1993). Symptoms are therefore often most severe in the upper leaves of the plant. Symptoms around the initially infected cells are denoted local symptoms, whereas viruses spread to other part of plants and cause symptom, this is referred to as systemic symptom.

The most common type of systemic symptoms produced by plant virus infection is mosaic symptom (Hull 2002). Mosaic develops only in sink leaves to which the viruses spread by long-distance movement. This symptom comprises areas of the leaves showing various degrees of chlorosis together with areas that remain green and are termed ‘dark green islands’ (DGIs). The mosaic symptom are also characterized by two shades of colors involve DGIs and a pale or yellow-green tissues (YTs). In older leaves showing mosaic, numerous of small islands of DGIs usually appear against a background of YTs. The borders between DGIs and YTs are distinct or sometimes diffused.

Many researchers have been characterized the DGIs and YTs phenomenon of mosaic in several host and viruses. In most cases, for example Tobamovirus (Atkinson & Matthews 1970), Cucumovirus (Loebenstein et al. 1977), and Potyvirus (Suzuki et al. 1989), DGIs in mosaic pattern are cytologically and biochemically normal as far as has been clarified (Atkinson & Matthews 1970; Loebenstein et al. 1977; Suzuki et al. 1989). They contain low or zero amounts of infectious viruses and no detectable viral proteins or viral RNAs (Hull 2002). For instance, cells within DGIs were almost free of viral RNA and

(16)

Introduction

virus (CMV) infectivity in DGIs were very low (1-2% of YTs). Sinderlarova & Sindelar

(2004) showed that the content of viruses in YTs of tobacco leaves infected with Tomato

mosaic virus common strain was about ten times higher than those of DGIs. Honda et al.

(1974) also observed that about 90% of cells in YTs of CMV-infected leaves contained large numbers of CMV particles, while DGIs were similar to healthy. About 50% of plantlets regenerated from DGIs of tobacco leaves systemically infected with TMV were virus-free (Murashiki & Carlson 1976). Several lines of evidence showed that DGIs are resistant to superinfection with the same virus or closely related viruses, but susceptible to infection by unrelated viruses (Atkinson & Matthews 1970; Loebensteins et al. 1972; Douherty et al. 1994; Guo & Garcia 1997)

The goal of viral infection is to obligate host plants, and use host plants as material sources for multiplication and spread their progeny to other healthy parts of the plants. However, viruses do not always win over the plant hosts because symptom disappears in some leaves, they are termed symptomless leaves. This phenomenon is typified by systemic virus infection with associated symptoms followed by decrease and disappearance of symptoms in the newly young developing leaves of the plants.The entire mechanism of this phenomenon is poorly understood but may simply by an example of completely successful plant antiviral activity via RNA silencing (MacDiarmid 2005). There is an increasing realization that the gene silencing host defense system may plays a very important role in controlling the virus infection (Ratcliff et al. 1997), suggesting that DGIs might be a result of gene silencing. Recent work by Moore et al. (2001) characterized the

(17)

Introduction

DGIs were caused by posttranscriptional gene silencing (PTGS); DGIs are related phenomenon, differing in their ability to amplify or transport the silencing signals.

Cucumber mosaic virus

CMV are widespread around the world and cause serious yield loses in important crops. This virus has a wide range of hosts (Palukaitis et al. 2003) and attacks a great number of vegetable varieties, ornamentals, weeds, and other plants than other viruses. CMV affects plants by causing mosaic, mottling or discoloration and distortion of leaves, flowers and fruits. The infected plants, consequently, are reduced greatly in size and their quality is often lowered.

CMV contains three single-stranded messenger sense RNAs, 1, 2 and 3; and also contains sub-genomic RNAs, RNA 4, RNA 4A. Proteins 1a, 2a and 3a are translated from the genomic RNAs 1, 2 and 3, respectively. The 2b and capsid proteins are translated from sub-genomic messengers RNA 4A and RNA 4, respectively. The 1a and 2a proteins work as RNA-dependent RNA polymerase involved in viral replication. The CMV 3a is essential to the viral movement (Boccard & Baulcombe 1993; Canto et al. 1997; Suzuki et al. 1991). The 3a protein was able to form tubules on the surface of infected tobacco protoplasts (Canto & Palukaitis 1999b) and is also required for long-distance movement. The CMV 3b or capsid protein is the sole protein associated with viral particles and is also required for viral movement within and between plants (Boccards & Baulcombe 1993; Canto et al.

(18)

Introduction

expansa, Momordica charantia and Physalis floridana plants were determined by the

amino acid 129 of CMV coat protein (CP) (Kobori et al. 2002), CP of CMV differentially affected transmissibility by aphids (Perry et al. 1998), and that the symptom severity is distinctly affected by 2b protein (Ding et al. 1995a, Ryang et al. 2004).Recently, the CMV 2b protein has been shown to be the suppressor of PTGS (Brigneti et al. 1998; Li et al. 1999). This protein is one of the first two viral proteins that have been identified as suppressors of PTGS (Anandalakshmi et al. 1998; Brigneti et al. 1998; Kasschau & Carrington 1998).

CMV causes cyclic mosaic symptom in infected tobacco plants (Nicotiana

tabacum). Following CMV infection, the uninoculated upper leaves of tobacco plants

appear alternately mosaic and mottle, then symptomless, and finally again became mosaic: this phenomenon is typified as cyclic symptom expression (Ohki et al. 1990; Gal-On et al. 1996; Kaplan et al. 1997). This cyclic phenomenon was founded in various host infected with several viruses. For instance, in Peanut green mosaic virus infected groundnut, the leaves that showing light green area show recovery and appear normal green in three weeks after inoculation, while the young leaves on the same plant show severe symptom (Nayudu 2008). In rice varieties TN1 susceptible and Balimau Puith tolerant to rice tungro disease, co-infection with Rice tungro bacilliform virus and Rice tungro spherical virus induced symptom of yellow orange discoloration, which developed in a cyclic pattern (Sta Cruz et al. 2003). Fig. 1 shows cyclic mosaic symptom caused by CMV infection in tobacco plant at 30 days postinoculations (dpi). Characteristic mosaic leaves consist of two areas: DGIs

(19)

Introduction

and YTs, which are mentioned above. Tomoru (1967a) examined the kinetics of CMV titers in LP and showed that the amounts of CMV-O and CMV-Y in LP shifted along leaf positions of the infected tobacco plants. The author presumed that the CMV infection in LP and young developing leaves regulated symptom expression and symptom severity in the fully expanded leaves. However, the mechanism of cyclic mosaic symptom is still remained unclear.

Figure 1. Cucumber mosaic virus (CMV) caused cyclic symptoms on tobacco.

Early mosaic Late mosaic

(20)

Introduction

In the case of Tobamovirus that also cause mosaic disease in tobacco, Hosakawa et al. (1990) observed that viral-infected areas and uninfected areas had already been divided at leaf primordia (LP) stage in leaf development. The authors indicated that the expression of mosaic symptom was determined by the viral distribution in the LP of infected plants (Hosokawa et al. 1990). In expanded leaves infected by TMV, RNA silencing against the virus was established in the cells located at the margin of DGIs, which restricted the expansion of YTs and consequently defined the mosaic patterns (Hirai et al. 2008).

RNA silencing as a defense mechanism against plant RNA viruses (a) RNA silencing as a plant defense mechanism

In the case of plant defense, plant hosts also response the viral infection by defense mechanism known as RNA silencing to control viral systemic infection (Fig. 2). RNA silencing, based on sequence-specific degradation of RNA (Ding & Voinnet 2007), is a key mechanism induced in plants for resistance to diseases caused by plant viruses (Ratcliff et al. 1997; 1999).This is an adaptive defense mechanism that is triggered by double stranded RNA (dsRNA) corresponding to the invading viral RNA. Subsequently, the dsRNA is cleaved into small fragments called short interfering RNA (siRNA) by RNase III-Like enzyme such as Dicer and Dicer-Like Protein (DCL) (Voinnet et al. 2005; Molnar et al. 2005).

(21)

Introduction

RNA polymerases (RDR), and other components of the silencing machinery in plants (Henderson et al. 2006; Margis et al. 2006; Wassenegger et al. 2006; Xie et al. 2004). DCL1 processes fold-back precursors to release microRNAs (Bartel 2004). DCL3 produces 24-nucleotide (nt)-long, DNA repeat-associated siRNAs guiding heterochromatin formation (Xie et al. 2004). DCL4 generates 21-nt-long siRNAs that mediate PTGS of some endogenous genes, and of transgene mediating RNA interference (Dunoyer et al. 2005). DCL2 synthesizes stress related natural-antisense-transcript (nat)-siRNAs (Borsani et al. 2005). The dsRNA is sequentially diced by DCL4 in a phased reaction that can be carried out by DCL2 when DCL4 is genetically inactivated (Chapman & Carrington 2007). DCL2 and DCL4 also redundantly mediate dsRNA-mediated RNA interference (RNAi) used for experimental gene knockdown in plants (Dunoyer et al. 2007).

After the cleaving process, siRNA associates with Argonautes and other components as RNA-induced silencing complex (RISC) and guides the complex to complementary sequences that are to be degraded. In addition to the primary response, siRNAs can be produced through a secondary pathway that involves synthesis of dsRNA or siRNA by host encoded RDR (Wassenegger & Krczal 2006; Makeyev & Bamford 2004; Baulcombe 2007). After infection by the virus, the infected cells also induce RNA silencing signals that elicited defense mechanism in the transported tissues. RNA silencing can initiate signals that move intercellular through the plant both cell-to-cell and also

(22)

long-Introduction

expressing the corresponding transgene (Palauqui et al. 1997; Sonoda & Nishiguchi 2000).

Figure 2. RNA silencing and its inhibition. The dsRNAs are synthesized by viral-RdRp or

plant-RdRp, and are cleaved by DCLs into 21-26 nt short interfering RNAs (siRNAs). These siRNAs combine with protein complexes as RNA-induced silencing complex (RISC), then unwinding and activate RISC for target RNA cleavage, guide plant-RdRp for the secondary amplification of dsRNA, and are responsible for short- and long-distance signaling inducing RNA silencing. In the case of counter-defense, P19 of Tombusvirus blocks short RNA species. HC-Pro of Potyvirus inhibits activities of RISC. 2b of

Cucumovirus can interfere with siRNA, long-distance signals and interacts directly with

Argonaute 1, a component of RISC. P25 of Potexvirus suppresses the long-distance signaling activities. Viral RNA RdRp DCLs siRNA unwiding Sequence specific RNA degradation Long-distance movement 2b RdRp HC-Pro P19 dsRNA siRNA P25 RISC 2b 2b

(23)

Introduction

(b) Silencing suppressors of plant viruses

Many plant viruses have developed specific counter-defense mediated by viral-encoded silencing suppressors that inhibit various steps of the RNA silencing process, such as interference with local silencing (Dunoyer et al. 2002; Pfeffer et al. 2002; Takeda et al. 2002; Bucher et al. 2003; Qu et al. 2003; Thomas et al. 2003) and prevention of systemic silencing signals (Li et al. 2002; Dunoyer et al. 2002; Takeda et al. 2002, Bucher et al. 2003; Qu et al. 2003; Thomas et al. 2003). Viral suppressor proteins that inhibit the antiviral RNA silencing defense have been identified in numerous RNA and DNA viruses (Voinnet et al. 1999; Roth et al. 2004). Many viral suppressor proteins have been characterized as viral pathogenicity factors (Voinnet et al. 1999) and as determinants of systemic viral infection (Kasschau & Carrington 2001; Yelina et al. 2002; Hevelda et al. 2003; Deleris et al. 2006; Diaz-Pendon et al. 2007).

For instance, helper component proteinase (HC-Pro) of Potyvirus was the first identified suppressor of RNA silencing. The original reports demonstrated that it suppresses both transgene- and virus-induced silencing (Anandalakshmi et al. 1998; Kasschau & Carrington 1998). HC-Pro can reverse the maintenance phase of RNA silencing and re-establish transgene expression (Brigneti et al. 1998). P1/HC-Pro functions as an effective suppressor of PTGS (Kasschau & Carrington 1998). It has been found that long-distance movement and pathogenicity enhancement are related activities of the HC-Pro (Saenz et al.

(24)

Introduction

RNA silencing defense of host plants against TYLCV-Israel infection. P25, the cell-to-cell movement protein of Potato virus X, prevents the systemic spread of the silencing signals (Hamilton et al. 2002; Voinnet et al. 2000). P19 suppressors of Tombusvirus could function as silencing suppressors in a wide range of organisms, and even in mammalian cells (Dunoyer et al. 2004).

The approximately 12-kDa 2b protein encoded by all species of Cucumovirus had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of PTGS (Guo & Ding 2002; Shi et al. 2003; Wang et al. 2004;Qi et al. 2004). The 2b protein of CMV has been shown to be one such suppressor protein (Brigneti et al. 1998).Guo & Ding (2002) demonstrated that 2b restricted the long-distance spread of RNA silencing, and Goto et al. (2007) suggested that 2b interfered with the RNA silencing pathway by binding to siRNA.Furthermore, Zhang et al. (2006) indicated that 2b interacted directly with Argonaute 1, a component of the antiviral RNA-induced silencing complex. Earlier studies suggested that 2b controlled systemic viral movement, and a lack of 2b protein was reported to be associated with reduced pathogenicity (Ding et al. 1995a, b). In addition, the 2b protein of CMV strain Fny was found to influence viral movement within leaves and throughout the whole plant (Soards et al. 2002). Diaz-Pendon et al. (2007) used silencing-defective Arabidopsis ecotypes to demonstrate that the silencing suppressive

activity of 2b was required to establish systemic CMV infection and was an essential contributor to virulence.

(25)

Introduction

It can be hypothesized that interaction between RNA silencing and suppressor proteins may play an important role in ability of viral movement and express cyclic phenotype in host plants. Cyclic mosaic pattern might be the responses of plant defense mechanism against viral infection to the host plants. Since plant viral diseases have been found worldwide, and cause serious yield loses, this study is very important to approach the mechanism of plant defense mechanism operating in plants against invading viruses. To apply this pioneer knowledge for further creation of virus-tolerant crops, the studies on the interaction of RNA silencing and virus distribution on the outcome of cyclic mosaic expression were conducted.

(26)

Aims

AIMS

The formation of cyclic mosaic symptom caused by virus infection is not so far clarified. In this thesis, I have extensively elucidated the mechanism of cyclic symptom expression by CMV in tobacco, focusing particularly on the roles of plant defense mechanism and the distribution of CMV in shoot apical meristem (SAM), leaf primordia (LP), young developing leaves (YDLs) and in the fully expanded leaves. In addition, I have investigated role of the suppressor protein in viral distribution and involvement of RNA silencing components DCLs and RDRs on the infection by CMV. By using CMV wild type and a suppressor-defective CMV mutant and the RNA silencing down-regulated transgenic tobacco lines, I have specifically aimed to study the following:

1. The relation of cyclic mosaic development with CMV kinetics

2. The role of 2b protein in viral movement and cyclic symptom expression 3. The involvement of DCLs and RDRs in cyclic symptom expression

(27)

Characterization of cyclic symptom expression in CMV-infected tobacco plants

CHAPTER I

Characterization of cyclic symptom expression in

CMV-infected tobacco plants

(28)

CHAPTER I

Characterization of cyclic symptom expression in CMV-infected tobacco plants

1.1 INTRODUCTION

CMV causes mosaic disease in infected tobacco plants (Nicotiana tabacum), which is a characteristic symptom of plant viral infections. Following CMV infection, the uninoculated upper leaves of tobacco plants are alternately mosaic and mottled, then symptomless, and finally again become mosaic: this phenomenon is known as cyclic mosaic symptom expression (Ohki et al. 1990; Kaplan et al. 1997). Characteristic mosaic leaves consist of two areas: dark green islands (DGIs) and yellow-green tissues (YTs). The DGIs contain few infectious viruses, whereas abundant CMV-infected cells occur in the YTs (Loebenstein et al. 1977, Ohki et al. 1990). Ohki et al. (1990) reported that the CMV titer in the symptomless leaves was also very low (1 to 2% of the level in YTs). Thus, it is well established that the viral content is different among YTs, DGIs, and symptomless leaves.

However, which cells in the leaf primordia (LP) become DGIs and which become YTs in the fully expanded leaves are not clear. Furthermore, the relationship between CMV infection in the LP and young developing leaves (YDLs) stage and symptom severity in fully expanded leaves has not been elucidated. It is difficult to simultaneously analyze viral infections in the LP/YDLs and symptoms in fully expanded leaves using identical leaves. Therefore, the CMV coat protein (CP), viral RNAs (vRNA) and distributions in LP/YDLs in all leaf positions of inoculated tobacco plants were examined by Western and Northern blot analyses and immunohistochemical microscopy. To elucidate the relationship between CMV infections in the developing leaves and the symptoms in fully expanded leaves, the

(29)

CMV infection and distribution in the LP and YDLs were compared with symptoms in the fully expanded leaves at their corresponding leaf positions. The association of CMV kinetics in the LP/YDLs with symptom severity in the fully expanded leaves is discussed.

1.2 MATERIALS AND METHODS

Plant materials, CMV and inoculation

The Pepo strain of CMV (CMV-Pepo) was originally obtained from Cucurbita pepo (Osaki et al. 1973). The largest leaf of the five to seven-leaf-stage of the tobacco plant (Nicotiana

tabacum cv. Samsun NN) was mechanically inoculated with purified CMV (25 µg/ml) and

designated as leaf position 0 (L0). The leaves above the inoculated leaf were sequentially numbered. The inoculated plants were grown under greenhouse conditions (24 to 30ºC), and then symptoms in the fully expanded leaves were observed for a month. Twenty infected plants were selected for symptom observation.

Immunohistochemical microscopy

Procedures for the preparation of expanded leaves (8 to 9 cm), LP (2 to 3 mm) and YDLs (1 to 2 cm) tissues from inoculated tobacco plants, immunohistochemistry were conducted using method described previously (Mochizuki & Ohki 2004). The leaf tissues samples were fixed in FAA solution (50% ethanol, 5% formaline and 5% acetic acid) vacuumed to eliminate air bubbles in tissues for 10 minutes two times and kept overnight at 4ºC. The

(30)

were then to be embedded in paraffin (Paraplast-plus; Sigma, St. Louis, MO, USA). The samples with paraffin were cut 10 µm in longitudinal sections by a rotary microtome (PR-50; Yamato Kohki, Tokyo, Japan). Then, the tissues were thoroughly dried on a warming plate overnight, fixed in 100% xylene for 30 minutes two times and later brought to 50% xylene: 50% ethanol for 30 minutes two times and washed in 100% ethanol for 30 minutes two times. After that the tissues were hydrated in a gradual series of 70% ethanol for 30 minutes two times, 50% for 30 minutes two times, 30% for 30 minutes two times and then distilled water (DW) for 10 minutes. Finally, the samples were analyzed by the immuno-histochemical microscopy.

For immunohistochemistry the sections were blocked in phosphate-buffered saline (PBS) pH 7.0 containing 1% bovine serum albumin (BSA) and 0.05% Tween 20 for 30 minutes. Then, they were incubated with a mixed antibody to CMV CP diluted to 1: 1000 and an alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (IgG) (Chemicon International, Temecula, CA, USA) diluted to 1: 1000 in the phosphate buffer saline containing 0.05% Tween 20 (PBST) for overnight at 4ºC, and washed for 10 minutes three times in PBST. The sections were incubated with AP 9.5 (100 mM Tris, 100 mM NaCl, 50 mM MgCl2, pH 9.5) for 5 minutes. Then, the sections were incubated with a color

substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Sigma-Aldrich, Steinheim, Germany) and nitrotetrazolium blue chloride (Sigma-Aldrich) for 10 minutes. Afterward, a staining section were stop in stop solution (10 mM Tris-HCl, pH 7.5, 1mM EDTA) in 10 minutes and washed in DW, finally examined with a BX-50

(31)

light microscope (Olympus, Tokyo, Japan).

Western blot analysis

Procedures for protein extraction, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), blotting onto nitrocellulose membrane and detection of CMV CP were performed as described previously by Saiga et al. (1998). Leaf samples were ground with protein extraction buffer {homogenize buffer: 250 mM Tris, 2.5 mM EDTA, 0.1% ascorbic acid, 1mM phenylmethylsulfonyl fluoride (PMSF), pH 7.5} used as 1 g sample/ 5 ml homogenize buffer, then were centrifuged at 12,000 rpm for 5 minutes. The aqueous phases were selected, added with sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 8% 2-mercaptoethanol, 20% glycerol, 0.02% Bromphenol Blue), used as 90 µl supernatant /30 µl sample buffer. Then, the samples were boiled at 100ºC for 5 minutes. The extracted protein samples were separated in 5% stacking gel {4.75% polyacrylamide (acrylamide: bisacrylamide = 73:2), 125 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.033% ammonium peroxodisulfate (APS), 0.1% N,N,N’,N’-tetramethylethylenediamine

(TEMED)} and 10% separating gel (10% polyacrylamide, 375 mM Tris-HCl, pH 8.8, 0.1% SDS, 0.033% APS, 0.1% TEMED) in running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) with 20 mA for 1 hr. After that, samples were transferred to nitrocellulose membrane (NCM) (Bio-Rad, Hercules, CA, USA) with blotting buffer (25 mM Tris, 192 mM glycine, 20% methanol) by ATTO crosspower 500 machine for 30 minutes (NCM 1 cm2/ 20 mA).

(32)

by decolorize buffer overnight. Then gel was washed in DW two times rapidly and washed with DW for 30 minutes. Finally, the gel was dried by vacuum.

The transferred NCM was used in immuno-method. NCM was washed in 0.05% Tween 20 in tris-buffered saline (TTBS) for 10 minutes, then blocked in 3% skim milk in TTBS for 30 minutes. The NCM was washed in TTBS rapidly and then incubated with anti-CMV IgG diluted to 1: 3000 as primary antibody and an alkaline phosphatase-conjugated anti-rabbit immunoglobulin G (IgG) (Chemicon International) diluted to 1: 10000 in TTBS as secondary antibody overnight. After that, it was washed by TTBS for 10 minutes two times and washed by DW rapidly. NCM was incubated in AP 9.5 for 5 minutes and incubated with a color substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Sigma-Aldrich) and nitrotetrazolium blue chloride (Sigma-Aldrich) for 10 minutes. Finally, NCM was hung to dry.

RNA extraction

Tissue samples were kept at –85ºC and then ground in a small mortar and a pestle. The ground tissues were homogenized in TriPure Isolation Reagent (Roche Diagnostics, Mannheim, Germany) (1 ml per a 100 mg ground sample) according to the manufacturer’s instructions. The aqueous phases were transferred to a fresh tube and were incubated for 5 minutes at room temperature. An amount of 0.2 ml of CHCl3/100 mg of sample was added

(33)

temperature. After centrifugation at 13,000 rpm for 15 minutes at 4ºC, the aqueous phase (600 µl) were transferred to a fresh tube, added with 1 volume of 100% isopropanol, and incubated at room temperature for 20 to 30 minutes. The solutions were centrifuged at 13,000 rpm for 15 minutes at 4ºC, the supernatants then were carefully discarded. The pellets were washed with 1 ml of 75% ethanol and centrifuged at 13,000 rpm for 5 minutes at 4ºC. The pellets were briefly dried under vacuum for 5 minutes. The pellets were re-suspended into RNase-free DEPC-treated DW (DDW) in 16 µl. The concentrations of RNA were measured and then stored at –85ºC. The low-molecular-weight RNA were dissolved in 1 volume of 100% formamide and mixed well before use in the Northern blot analysis.

Northern blot analysis of viral RNA detection

Procedures for blotting onto membrane, hybridization and detection of signal were performed as described previously by Kobori et al. (2002). The total RNAs 1 µg at a concentration in 5 µl were mixed in 16 µl loading dye {1.6 ml formaldehyde, 5.0 ml formamide, 0.5 ml 20X 3-Morpholinopropanesulfonic acid (MOPS), 1.6 ml glycerine}. The total amount of RNA samples (20 µl) were separated in 1.5% agarose gel containing 20X MOPS and 5% formamide. After that, the RNAs were transferred onto Hybond-N+ membrane (GE Healthcare Biosciences, Piscataway, NJ, USA). Next, the membrane was fixed at 120ºC for 30 minutes. After that the membrane was pre-hybridized with

(34)

N-lauroysacorsine, 2% blocking solution (Roche Diagnostics), 50 mM sodium phosphate, pH 7.0} at 68ºC for 1 hr. Then, hybridization was performed using Digoxigenin (DIG)-labeled RNA probes complementary to the conserve 3’-UTR sequence at 68ºC overnight. Afterwards, the membrane was washed with 0.2X SSC (20X SSC containing 20% SDS) once for 10 minutes at room temperature and at 68ºC for 1 hr twice. Then, the membrane was moved to buffer 1 (100 mM maleic acid, 150 mM NaCl, pH 7.5) for 1 minute and blocked with buffer 2 {buffer 1 containing 10% blocking reagent (Roche Diagnostics)} for 40 minutes. Then, DIG-labeled RNA was detected by using anti-Digoxigenin-AP Fab fragment (Roche Diagnostics) specific antibodies in buffer 2 for 30 minutes. After that, the membrane was washed with buffer 1 containing 30% Tween 20 in 12 minutes two times. The membrane was incubated in a solution of alkaline phosphatase pH 9.5 (AP 9.5) 8 minutes and then was incubated in CDP star solution (Biolab, New England, US) 13-17 minutes. Finally, the membrane was performed by X-ray film (RX-U, Fujifilm, Tokyo, Japan) in a dark room for a minute.

1.3 RESULTS

1.3.1 Accumulation of viral CP, viral RNAs and viral distribution in fully expanded tobacco leaves infected with CMV

The cyclic mosaic symptom expression as a result of CMV infection was confirmed for a month. Symptoms appeared on the newly developed upper leaves of the inoculated plants, and the leaves could be divided into three types: mottled, mosaic, and symptomless (Fig.

(35)

1-1a). Mosaic and mottled leaves first appeared on the newly generated leaves after CMV

inoculation, and then symptomless leaves were generated. More mosaic leaves appeared again after the symptomless leaves appeared. Mottled leaves were composed of pale yellow-green tissues, which were uniformly distributed throughout the leaves, while mosaic leaves contained discrete YTs and DGIs (Fig. 1-1a). Symptomless leaves developed no symptoms and looked similar to the healthy controls.

First, the viral CP and distribution in fully expanded leaves (8 to 9 cm in length) was defined by Western blot analysis and immunohistochemical microscopy. In the mosaic leaves, high levels of CP were detected in YTs, which was in contrast with the absence of CP signals in DGIs. Similarly, CP signals were detected in most YT cells, whereas no signals were observed in DGI cells by immunohistochemical microscopy. Note that the boundaries between the infected YTs and uninfected DGIs in the mosaic leaves were distinct (Fig. 1-1c). Mottled leaves contained viral CP, but the CP was less than that in YTs (Fig. 1-1b). The scatter distribution of CMV infected cells in the mottled leaves was observed (Fig. 1-1c). The accumulation of CP was not detected in the symptomless leaves by Western blot analysis, and very few infected cells were detectable by immunohistochemical microscopy (Fig. 1c). These results confirmed that the different viral content among YTs, DGIs, mottled, and symptomless leaves was due to the differences in CMV distribution, indicating that the different symptom severity among the mottled, symptomless, and mosaic leaves corresponded with the differences in CMV distribution in the tissues.

(36)

Figure 1-1. Cyclic mosaic symptom expression in Cucumber mosaic virus (CMV) Pepo strain-infected tobacco (Nicotiana tabacum cv. Samsun NN). Photographs were obtained at 30 days post inoculation. The uninoculated upper leaves of a CMV infected tobacco plant alternately showed mottled, mosaic, and symptomless leaves. b: Accumulation of CMV coat protein (CP) in each symptomatic leaf type as assessed by Western blot analysis with an anti-CMV IgG antibody. Note that the accumulation of CMV is detectable in the samples from the mottled leaf and the yellow-green tissues (YTs) of the mosaic leaf but not in the symptomless leaves, dark green islands (DGIs) of the mosaic leaf, and healthy leaf. CBB: coomassie brilliant blue. c: CMV distribution in each symptomatic leaf type. Transverse sections were obtained from fully expanded mottled, mosaic, and symptomless leaves at 30 days postinoculation and treated with anti-CMV IgG antibody. Infected cells are darkly stained. Dotted lines indicate the obvious boundary between YTs and DGIs in a mosaic leaf. Arrowheads indicate infected cells in a symptomless leaf. M: mesophyll cells, UE: Upper epidermal cells, LE: Lower epidermal cells. Bar = 200 µm.

(37)

1.3.2 Viral CP and RNAs in young developing leaves shifted along leaf positions in CMV-infected tobacco

Because the expression of mosaic symptoms has been implicated in viral distribution in YDLs, I focused on the viral CP and RNAs in YDLs (1 to 2 cm) at all leaf positions in the CMV-infected tobacco to analyze the cyclic mosaic symptom expression. It was impossible to detect the viral CP and RNAs sequentially in all of the YDLs using one individual plant. Therefore, the largest leaves (L0) of about thirty tobacco plants of similar size were inoculated with CMV, 1 to 2 cm sized developing leaves (L6-14) above inoculated leaf (L0) were periodically collected from 3 individual plants between 5 and 25 days postinoculation (dpi), and the amounts of CP and viral RNAs were examined by Western and Northern blot analyses. Three plants inoculated at the same time were also used for symptom observation for a month; these plants exhibited leaves that were mosaic at leaf position 6 to 8, mottled at leaf position 9, symptomless at leaf position 10 to 11, and then mosaic again at leaf position 12 to 14.

The amount of CP in the YDLs was dependent on the leaf positions (Fig. 1-2a). The CP was detected at high concentrations in L6-8, reduced in L9-11, and increased again in L12-14. Similar fluctuation of the amount of viral RNA was obtained by Northern blot analysis (Fig. 1-2b). These results showed that viral concentration in YDLs shifted along the leaf positions. The L6-8 and L12-14 YDLs that contained high CMV levels usually developed into mosaic leaves, and the L9 YDLs that contained reduced CMV usually developed into mottled leaves. In contrast, the L10-11 that became symptomless leaves contained very low CMV concentrations. Although the L10 of the three observed plants

(38)

1-2). In addition, the fluctuation of the viral load according to the leaf positions was

different between CP and RNAs (Fig. 1-2a vs. 1-2b). This result might be due to difference among test plants because the viral CP and RNAs was assessed with three individual YDLs; the timing of the viral CP and RNAs shift in the YDLs might be variable among test plants. However, all of the experiments were repeated three times and showed similar results. Therefore, it can be concluded that the shift in viral concentration in the YDLs along the different leaf positions was related to symptom severity in the fully expanded leaves.

Figure 1-2. Accumulation of CMV CP (a) and viral RNAs (b) in YDLs 1 to 2 cm in length

in CMV-Pepo infected tobacco plants. The numbers indicates leaf positions above the inoculated leaf (L0). The amounts of viral RNAs and CP were analyzed by Northern and Western blot analyses, respectively. CMV CP was detected with an anti-CMV IgG antibody. The viral RNA was detected with a DIG-labelled riboprobe complementary to the conserved 3’-UTR sequence. Total proteins and ribosomal RNA were stained with coomassie brilliant blue (CBB) and methylene blue, respectively, to ensure equal loading. H: healthy plant, Mo: mottled, M: mosaic, and S: symptomless.

CP CBB RNA1, 2 RNA3 RNA4 Leaf positions 6 7 8 9 10 H 11 12 13 14 M M M Mo S S M M M

b

a

rRNA Representative symptoms

(39)

1.3.3 Kinetics of viral distributions during leaf development in CMV-infected tobacco

To clarify when CMV distribution in mosaic, mottled, or symptomless leaves were formed during leaf development, the CMV distribution in the LP and YDLs was investigated. The CMV distribution of representative leaf positions at L7, 9, 11 and 13 were observed with immunohistochemical microscopy. Each LP or YDLs was sampled from three different plants corresponding to the three different symptomatic states in fully expanded leaves. Three plants inoculated at the same time were used for symptom observation, confirming that L7, 9, 11 and 13 were mosaic, mottled, symptomless, and mosaic, respectively. Five transverse sections from the top to the base of each leaf obtained from three LP and three YDLs for each leaf position were examined, and representative images are shown in Fig.

1-3.

Signals from CP in L7 (mosaic) LP were detected in vascular tissues and leaf blades, and they were distributed across the whole tissues of L7 (mosaic) YDLs. Dense CP signals in the L9 (mottle) LP were detected in vascular tissues and leaf blades at the leaf tip, and weak CP signals were detected in the leaf base, resulting in CP signals in all parts of the L9 (mottle) YDLs. These signals at L11 (symptomless) were rarely detected in the LP, and few signals appeared in the vascular tissues and in the mesophyll cells of the YDLs. The CP signals at L13 (mosaic) were detected in vascular tissues in the LP, and uninfected cells were also found in the leaf blades of the YDLs. Thus, in contrast with L7 (mosaic) and L9 (mottle), the boundaries between infected and uninfected cells in the L13 (mosaic) LP were maintained in the YDLs. These results showed that the shift of the viral CP and RNAs in the YDLs, as shown in Fig. 1-2, was due to the change in the viral distribution in the

(40)

In mosaic leaves, separate CMV distribution was observed in the L13 (mosaic) LP/YDLs, while CMV was present in most cells of the L7 (mosaic) YDLs. This result indicated that the separate CMV distribution in L13 (mosaic) has already been determined at the LP stage but not in L7 (mosaic).

Figure 1-3. CMV distribution in the LP and YDLs of each symptomatic type of the

CMV-Pepo infected tobacco plants. Transverse sections obtained from LP or YDLs at leaf positions 7, 9, 11 and 13 were treated with anti-CMV IgG antibody. Infected cells are darkly stained. Dotted lines indicate the boundaries between virus-infected tissues (YTs) and virus-free tissues (DGIs). Blank arrowheads show CMV infected cells, while filled arrowheads point to uninfected cells. Bars indicate 1 mm (LP) and 1 cm (YDLs).

L7: Mosaic L9: Mottle L 1 1 : Symptomless L13: Mosaic

Fig.

3

Healthy LP YDLs LP YDLs

(41)

1.4 DISCUSSION

Here, I revealed that the CMV CP and RNAs in the YDLs shifted with leaf positions and was related to symptom severity on the expanded leaves. I also observed the kinetics of CMV distribution in the LP/YDLs stages and showed that the shift of the viral concentration in the YDLs resulted from the viral distribution in the LP/YDLs. Furthermore, I revealed that the distinct CMV distribution in the fully expanded symptomless leaves and mosaic leaves at the late infection period was already determined at the leaf primordia stage of leaf development. In contrast, the CMV distribution in the early mosaic leaves was not yet determined at the YDLs stage. The mechanisms that induced mosaic, mottled, and symptomless leaves are discussed as follows.

I confirmed that the viral CP and RNAs decreased in the LP/YDLs stage of symptomless leaves (Fig. 1-2). Immunohistochemical study further revealed that the decrease in viral concentration in the LP/YDLs of symptomless leaves was due to the reduced viral distribution in the LP/YDLs (Figs. 1-2 and 1-3 L11). For the L11 that developed as symptomless leaves, very few viral signals in the LP/YDLs were observed (Fig. 1-2, L11 and Fig. 1-3, L11), indicating that a virtually virus-free state in the L11 (symptomless) LP was maintained during leaf development and that L11 finally developed as symptomless leaves. Thus, present results verified Tomaru’s suggestion; the virus-free state of symptomless leaves was determined at the LP stage.

In the fully expanded mosaic leaves, the boundaries between the CMV-infected YTs and the uninfected DGIs were distinct (Fig. 1-1). The viral distribution in these YDLs

(42)

was distributed in most cells of the L7 (mosaic) YDLs, while CMV distribution in the LP was restricted around vascular tissues and leaf blades (Fig. 1-3, L7). This result indicated that the boundaries between infected and uninfected tissues for L7 (mosaic) were not established at the YDLs stage. Similar to L7 (mosaic), most cells in the L9 (mottle) YDLs were infected with CMV (Fig. 1-3, L9). Because L7 and L9 developed into mosaic and mottled leaves, their distinct viral distributions were determined after the YDLs stage. In contrast to L7 (mosaic) and L9 (mottle), the separate CMV distribution in L13 (mosaic) that developed as late mosaic leaves was observed both in the LP and YDLs (Fig. 1-3, L13), indicating that the boundaries between infected and uninfected cells in the L13 (mosaic) LP were maintained during leaf development. In addition, Fig. 1-2 showed that the viral CP and RNAs in the L12-14 YDLs did not reach the same levels as in the L6-8 YDLs, although these YDLs became mosaic leaves. These results supported the immunohistochemical observations that a distinct CMV distribution was maintained at the L13 (mosaic) YDLs stage for the late mosaic leaves, while CMV was distributed to most cells at the L7 (mosaic) YDLs stage for early mosaic leaves (Fig. 1-3, L7 vs. L13). In the fully expanded mosaic leaves, the boundary between YTs and DGIs corresponded with the boundary between infected and uninfected cells (Fig. 1-1). Therefore, it appears that the boundaries between the infected and uninfected cells in the late mosaic leaves became established at the LP stage (Fig. 1-3, L13), and then CMV-infected and uninfected tissues in the LP develop into YTs and DGIs in the fully expanded mosaic leaves, respectively (Fig. 1-1). Taken together, I consider two mechanisms of mosaic leaf generation: (i) viral-free DGI cells are generated from infected tissues after the YDLs stage in the early mosaic leaves and (ii) YTs and DGIs are already formed at the LP stage in the late mosaic leaves.

(43)

When and how uninfected cells were generated in L7 (mosaic) and L9 (mottle) has not been clarified in this study. The Western and Northern blot analyses showed viral CP and RNAs decreased in the L9 (mottle) YDLs (Fig. 1-2), but the immunohistochemical analysis showed that most cells of the L9 (mottle) YDLs were infected with CMV (Fig. 1-3, L9). For this reason, there is a possibility that the CMV concentration in some cells was decreased in the L9 (mottle) YDLs. This reduction of the viral concentration in some cells might have resulted in a dispersion of the CMV distribution in the fully expanded mottled leaves. To elucidate the generation of early mosaic and mottled leaves, detailed observation of the kinetics of viral distribution after the YDLs stage is needed.

Previously, it has been demonstrated that the CMV infection in tobacco meristem decreased with time (Mochizuki & Ohki 2004). The virus-free state of the L11 (symptomless) leaf primordia in symptomless leaves might differentiate from virus-free meristem. In contrast with the continuity of the virus-free stage of meristem (Mochizuki & Ohki 2004), the leaf primordia in the late infection period were infected with CMV again and then developed into the late mosaic leaves (Figs. 1-2 & 1-3). The reason why the viral kinetics was different at leaf positions was not clarified in this study.

(44)

The 2b protein of CMV is necessary for cyclic mosaic symptom expression in tobacco

CHAPTER II

The 2b protein of CMV is necessary for cyclic

mosaic symptom expression in tobacco

(45)

CHAPTER II

The 2b protein of CMV is necessary for cyclic mosaic symptom expression in tobacco

2.1 INTRODUCTION

The 2b protein of CMV has been shown to be one of the suppressor protein (Brigneti et al. 1998). Guo and Ding (2002) demonstrated that 2b restricted the long-distance spread of RNA silencing, and Goto et al. (2007) suggested that 2b protein interfered with the RNA silencing pathway by binding to small interfering RNA (siRNA). Furthermore, Zhang et al. (2006) showed that 2b interacted directly with Argonaute 1, a component of the antiviral RNA-induced silencing complex. Earlier studies suggested that 2b controlled systemic viral movement, and a lack of 2b protein was reported to be associated with reduced pathogenicity (Ding et al. 1995a, b).

A 2b protein-defective mutant of the CMV Pepo strain (∆2b) induces only mild symptom and no cyclic symptoms in systemically infected tobacco plants (Ryang et al. 2004). Because the expression of mosaic symptoms has been implicated in virus distribution in leaf primordia (LP) of infected plants (Hosokawa et al. 1990), 2b mediation of virus distribution in developing tissues including the shoot apical meristem (SAM) and LP was hypothesized. To elucidate the role of 2b in CMV distribution in developing tissues and cyclic symptom development, the sequential distribution of Pepo and ∆2b in the SAM and LP as well as in inoculated leaves was compared.

(46)

2.2 MATERIALS AND METHODS

Plant materials, CMV and inoculation

The Pepo strain of CMV (Pepo) was originally obtained from Cucurbita pepo in Japan (Osaki et al. 1973). ∆2b is a modified Pepo strain that lacks translation of an intact 2b protein (Ryang et al. 2004). Five to seven-leaf-stage tobacco plants (Nicotiana tabacum cv.Xanthi-nc) were inoculated with the viruses. The largest leaf was mechanically inoculated with 50 µl (50 µg/ml) purified Pepo or ∆2b per plant, and the inoculated plants were grown in a greenhouse at 24º-30ºC.

Immunohistochemical microscopy

Procedures for the preparation of SAM and LP tissues from inoculated tobacco plants, immunohistochemistry were conducted using method described in Chapter I.

Protein analysis

Plant tissues collected from inoculated SAM and LP were sequentially collected at 7, 10, 14, 18 and 21 dpi. The procedures for protein extraction, SDS-PAGE, blotting onto nitrocellulose membranes and detection of CMV coat protein (CP) were performed as described in Chapter I.

(47)

RNA analysis

Total RNA was extracted from SAM and LP tissues using TRI Reagent (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer’s instructions. For detection of CMV genomic RNAs, 1 µg total RNA was loaded onto a 1.5% denaturing agarose gel. The procedures for blotting onto Hybond-N+ membrane (GE Healthcare Biosciences, Piscataway, NJ, USA), hybridization with DIG-labelled RNA probe complementary to the conserved 3’-UTR sequence, and detection of signals were performed as described in Chapter I.

Northern blot analysis for short RNA detection

The procedures for siRNA were performed as described previously (Goto et al. 2003). The total RNAs 20 µg at a concentration in 7.5 µl were dissolved in 1 volume of 100% formamide (7.5 µl) and mixed well. The RNA samples then were boiled in a water bath at 95ºC for 5 minutes and moved into ice rapidly. The RNA samples were mixed with 5 µl loading buffer {2X tris-buffered saline (TBS), 40% sucrose, 0.1% SDS, 0.15 (w/v) Bromphenol Blue}. The total amounts of RNA samples (20 µl) were separated on 40% acylamide gel containing 7 M urea. After that, the low-molecular weigh RNAs were transferred by electro-blotting onto a Hybond-NX membrane (Amersham Biosciences, Uppsala, Sweden). Next, the membrane was moved into 2X SSC 15 minutes and hung it to dry for 10 minutes, then was fixed at 120ºC for 30 minutes. After that the membrane was pre-hybridized at 40ºC for 1 hr. Then, hybridization was performed using an antisense

(48)

RNAs transcripts complementary to the CMV minus-strands RNAs obtained from the pepo-CMV clones (Saitoh et al. 1999) at 40ºC overnight. The procedures for detection of the signals were performed as described in Chapter I. An oligo DNA (5’-GGATCCTTTCAGAAAGCACCT TCC-3’) labeled with DIG using a DIG oligonucleotide Kit (Roche Diagnostics, Mannheim, Germany) was used as a low molecular weight (25 bp-long) DNA marker.

Tissue printing analysis

A leaf tissue was brushed to eliminate epidermis by celites. The brushed leaf was then pressed directly onto nitrocellulose membrane (NCM) (Bio-Rad, Hercules, CA, USA) that had been treated with 0.2 M CaCl2 prior to blotting. It was then blocked NCM by 3% skim

milk in 0.05% Tween 20 in TBS (TTBS) for 30 minutes. The CMV CP was detected in tissue printing by using specific antibodies diluted 1:3000, as the primary antibody, and an alkaline phosphatase-conjugated goat anti-rabbit IgG (Chemical International, Temicula, CA, USA) diluted 1:10000, as the secondary antibody in TTBS at 4ºC overnight. After that, it was washed by TTBS for 10 minutes three times and incubated into a solution of an alkaline phosphatase (AP) pH 9.5. Afterward an alkaline phosphatase was detected by using a color substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Sigma-Aldrich) and nitrotetrazolium blue chloride (Sigma-Aldrich) for 10 minutes. Finally, NCM was washed by DW for 10 minutes.

(49)

2.3 RESULTS

2.3.1 Involvement of 2b protein in efficient CMV distribution in SAM and LP

To assess the role of 2b protein in CMV infection of the SAM and LP, the sequential distribution of Pepo and ∆2b in these tissues was compared. Five shoot meristem tissue samples and three LP of 2-3 mm in length from different plants were sampled at 4, 7, 10, 14, 18 or 21 dpi. The distribution of Pepo and ∆2b was observed by immunohistochemical microscopy.

Figure 2-1. Distribution of Pepo and ∆2b in shoot meristem at 4, 7, 10, 14 and 18 dpi, as

indicated. The distribution of CMV CP was observed by immunohistochemical Healthy

(50)

Fig. 2-1 Shows the distribution of Pepo and ∆2b in the shoot meristem. Pepo

signals were detected in most SAM tissues at 4-10 dpi. The Pepo signals gradually decreased at 14 dpi and no signal were detected at 18 dpi. However, some signals were still detectable in the cells below the SAM and within primordia. Conversely, ∆2b CP signals were first detected in the shoot meristem at 7 dpi, but signals were not detected in the SAM. Furthermore, ∆2b signals disappeared in the shoot meristem at 10 dpi and were rarely detectable in the shoot meristem at 18 dpi. CMV CP signals were not observed in the shoot meristem from uninfected tobacco samples.

Fig. 2-2 Shows typical images of Pepo and ∆2b distribution in LP from 4 to 21

dpi, Pepo CP signals were detected in vascular tissues at 4 dpi and in the surrounding tissues at 7 dpi. By 10-21 dpi, the Pepo signals had spread throughout the LP, with the strongest signals at 14 dpi. The Pepo signals gradually decreased from 18 to 21 dpi. In the ∆2b-inoculated LP, a few ∆2b CP signals were detected at 14 and 21 dpi only. CMV CP signals were not detectable in the LP from uninfected tobacco samples.

(51)

2.3.2 Accumulation of vRNA and siRNA in the SAM and LP

Because CMV 2b protein acts as a silencing suppressor, the RNA silencing activity directed

Figure 2-2. Distribution of Pepo and ∆2b in LP at 4, 7, 10, 14, 18 and 21 dpi, as indicated.

The distribution of CMV CP was observed by immunohistochemical microscopy. Infected cell are darkly stained. CP signals were not observed in the LP from uninfected tobacco samples. Arrows indicate CMV CP signals in LP. Bars = 500 µm.