Phenotypical and molecular characterization of the Tomato mottle Taino

virus–Nicotiana megalosiphon interaction

Cyrelys Collazo a, Pedro Luis Ramos a, Osmany Chacon b, Carlos Javier Borroto a, Yunior Lopez a,

Merardo Pujol a, Bart P.H.J. Thomma c, Ingo Hein d, Orlando Borras-Hidalgo a,*

a Center for Genetic Engineering and Biotechnology, Plant Functional Genomic, P.O. Box 6162, Havana, Calle 31, 10600, Cubab Tobacco Research Institute, Carretera de Tumbadero Km. 8, P.O. Box 6063, San Antonio de los Banos, Havana, Cuba

c Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlandsd Scottish Crop Research Institute, Invergowrie, Dundee, Scotland DD25DA, UK

Accepted 22 February 2006

Abstract

Tomato mottle Taino virus (ToMoTV) infection causes significant yield losses in plants of various Solanaceous species. In this study, the

interaction between Nicotiana megalosiphon and ToMoTV was characterized on a phenotypical and molecular basis. In order to isolate genes that

are differentially expressed during the interaction of N. megalosiphon with ToMoTV, a PCR-based suppression subtractive hybridization (SSH)

was utilized. RNA dot-blot analysis confirmed induction of representative genes upon ToMoTV inoculation at different time points. Interestingly,

most of the genes identified are reported here for the first time to be involved in the response of N. megalosiphon to begomovirus infection.

q 2006 Elsevier Ltd. All rights reserved.

Keywords: Plant defense; ToMoTV; Nicotiana megalosiphon; Suppression subtractive hybridization; SSH; Begomovirus

1. Introduction

Geminiviruses are an emerging group of plant viruses that

affect horticultural crops in tropical and subtropical areas

around the world. The family Geminiviridae is divided into

four genera (Curtovirus, Mastrevirus, Topocuvirus and

Begomovirus) based on the genome organization, host range

and insect vectors [34]. The Begomovirus genus comprises

viruses that are characterized by monopartite or bipartite DNA

genomes that infect dicotyledonous plants and are transmitted

by whiteflies (Bemisia tabaci). Since 1989, begomoviruses

have caused epidemics that have been directly proportional to

the increase in whitefly populations in several crops in Cuba

[6].

Tomato mottle Taino virus (ToMoTV) is a begomovirus

with a single stranded DNA (ssDNA) bipartite genome

comprising two components: DNA-A and DNA-B. Component

A includes the genes associated with the replication and

encapsidation of the virus, whereas component B harbors genes

0885-5765/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.pmpp.2006.02.003

* Corresponding author. Tel.: C53 7 271 6022; fax: C53 7 273 1779.

E-mail address: orlando.borras@cigb.edu.cu (O. Borras-Hidalgo).

related to the viral movement through the plant [24]. This virus

is a pathogen of various Solanaceous species such as tomato,

potato, and tobacco. Infected plants show characteristic

symptoms that include yellow mosaics, plant stunting,

dwarfing, chlorotic mottle and curling of the leaves [6].

Understanding how virus-induced symptoms arise in plants

remains a longstanding challenge as it is still largely unclear

how virus infection impacts plant cells and tissues biochemi-

cally as well as physiologically [21]. Systemic infection of

plants by viruses requires the modification of host cells in order

to facilitate compatibility. These modifications allow viral

replication, propagation and movement, as well as suppression

of host defense responses and are associated with changes in

host gene expression. Indeed, it has been shown that induction

of these mechanisms involves a complex network of signal

perception, amplification and transduction, in which several

molecules and defense related genes participate [37]. It is

conceivable that a broader understanding of the transcriptional

changes associated with viral infection will reveal important

details on how plants respond to viruses and how viruses infect

plants.

Several studies have investigated genes expressed during

Begomovirus–host interactions. In Arabidopsis, numerous

defense-associated genes have been identified and shown to

be coordinately regulated in response to infection with various

Physiological and Molecular Plant Pathology 67 (2006) 231–236

www.elsevier.com/locate/pmpp

C. Collazo et al. / Physiological and Molecular Plant Pathology 67 (2006) 231–236232

viruses [36]. In Nicotiana benthamiana, Tomato golden mosaic

virus (TGMV) induces the accumulation of proliferating

cell nuclear antigen mRNA, which is the processivity factor

of a host DNA polymerase, in mature plant cells [10].

This indicates that such viruses alter developmental controls

to activate the transcription of host genes whose products are

required for viral DNA replication. Also spatial analysis of

gene expression in cucurbit plants infected with Cucumber

mosaic virus (CMV) revealed both local and systemic

effects [16].

Nicotiana megalosiphon is a wild tobacco species that is

generally used as a parent in genetic tobacco breeding

programs because of its high resistance towards several

important diseases [12]. For example, N. megalosiphon has

been shown to be highly resistant to Peronospora hyoscyami

f.sp. tabacina and Phytophthora parasitica [4,11]. On the other

hand, this species has been used in studies with viral pathogens

such as potato virus A, and shown to be highly susceptible [32].

However, nothing is known about its susceptibility towards

ToMoTV infection.

Although begomoviruses cause important economic losses

in crops, little is known about transcriptional changes in the

host plant following infection. This study is aimed at gaining a

broader insight into the responses elicited by ToMoTV

infection in N. megalosiphon, on both a phenotypical and a

molecular basis. PCR-based suppression subtractive hybridi-

zation (SSH) was used to generate a cDNA library enriched for

virus-induced genes.

2. Materials and methods

2.1. Plant material and virus inoculation

N. megalosiphon plants (seed provided by the Tobacco

Research Institute, Havana, Cuba), were grown from seed in

6-in pots containing black turf and rice husk (4:1) [23]. Six-

week-old N. megalosiphon plants were inoculated with a

ToMoTV strain provided by the Plant Virology Laboratory

from the Centre for Genetic Engineering and Biotechnology

(Havana, Cuba) [6]. The cloned DNA components of the virus

were obtained as a product from the digestion of pZErOe-2.1

vector (Invitrogen, San Diego, CA) with the appropriate

restriction endonucleases: ToMoTV-A, XbaI; ToMoTV-B,

PstI–HindIII. Viral infection was established using a particle

delivery system (PDS 1000, Bio-Rad, US). The inoculation

mix was prepared as described by Sandford [27], using 10 mg

DNA from each genomic viral component (DNAs A and B),

deposited on the surface of 3 mg of gold microparticles, under

vacuum conditions (28 mm Hg) at 550 kPa of pressure for each

bombardment [13]. The particle delivery was carried out over

the apical meristem zone of the plants. Fifteen plants were

bombarded with the virus-containing mix, and another 15

plants were mock-inoculated. After bombardment, plants were

transferred to a greenhouse at 25–28 8C until symptom

development. The phenotype was evaluated at 5, 15, 25 and

40 days post-inoculation (dpi).

2.2. Assessment of infection progress

All leaves (inoculated as well as systemic) from ToMoTV-

infected and mock-inoculated plants were collected after 0, 5,

15 and 25 dpi. Five plants were used per treatment and per time

point. Total DNA was extracted according to the procedure

described by Dellaporta [8]. In order to detect viral DNA in the

inoculated plants, DNA blotting was performed. Plant DNA

(100 ng) collected after virus inoculation was spotted on

Hybond NC nylon membrane (Amersham-Pharmacia, UK),

hybridized with a radioactive labeled fragment of 2.5 kb from

ToMoTV component A and washed according to the protocol

accompanying the Rapid-Hyb buffer (Amersham-Pharmacia,

UK). Radioactive signals were detected by exposure to X-ray

film (Eastman-Kodak, Rochester, NY). Prior to re-use,

membranes were stripped by washing twice with 0.13 SSC/

0.5% (w/v) SDS at 95 8C for 30 min.

2.3. Suppression subtractive library construction and

initial screening

A subtracted and normalized cDNA library was constructed

based on subtractive suppression hybridization (SSH) accord-

ing to the PCR-Select Subtractive Hybridization Kit (Clontech,

Palo Alto, CA). All leaves (inoculated as well as systemic)

from plants inoculated with ToMoTV and collected at 5, 15

and 25 dpi were pooled and total RNA was extracted using the

SV Total RNA Isolation Kit (Promega, Madison, USA). Five

plants were used per treatment per time point. The same

procedure was followed for mock-inoculated plants. Poly

(A)CRNA was isolated using the Dynabeadsw mRNA

Purification Kit (Dynal A.S., Oslo, Norway), according to the

manufacturer’s instructions. First strand cDNA was syn-

thesized using the Reverse Transcription Kit (Promega,

Madison, USA), followed by incubating first strand products

with RNAse H and DNApol I at 16 8C for 2 h to generate double

stranded cDNA. The cDNA prepared from N. megalosiphon

inoculated with ToMoTV was used as the ‘tester’ and that from

the mock-inoculated sample as the ‘driver’ for the forward

subtraction. Following the subtraction and PCR amplification,

cDNA fragments putatively induced by ToMoTV infection

were isolated, cloned into the pGEM-T Easy vector and

transformed into Escherichia coli XL1-blue cells (Promega,

Madison, USA). Positive bacterial clones were picked and

grown in Petri plates containing LB medium with 100 mg/L

ampicillin. DNA from 96 clones was amplified using M13

forward and reverse primers to check for the presence and size

of individual inserts. PCR products were spotted onto nylon

membranes and subjected to hybridization with the same tester

and driver cDNA samples as described above.

2.4. DNA sequencing and sequence data analysis

DNA sequencing was performed using an automated ABI

Model 377 DNA sequencer (Applied Biosystems, Warrington,

UK) according to manufacturer’s instructions. The M13

forward and reverse primers were used to generate sequences

Fig. 2. Detection of viral DNA in infected Nicotiana megalosiphon plants.

DNA dot-blot of N. megalosiphon plants inoculated with ToMoTV (A) or mock

inoculated (B) and harvested at 5, 15 and 15 dpi, respectively. Ten micrograms

of DNA was dot-blotted to Hybond NC membrane and the membrane was

hybridized with a 2.5 kb fragment of ToMoTV component A.

Fig. 1. Nicotiana megalosiphon is a host for Tomato mottle Taino virus. The

phenotype of 6-week-old Nicotiana megalosiphon plants mock-inoculated

(left) or inoculated with ToMoTV (right) at 40 dpi.

C. Collazo et al. / Physiological and Molecular Plant Pathology 67 (2006) 231–236 233

for all cDNAs isolated (Perkin Elmer ABI PRISM Dye

Terminator Cycle sequencing kit). Analyses of cDNA

sequence similarity to database sequences was conducted by

comparison with non-redundant protein and nucleotide

databases using BLASTX and BLASTN searches provided

through the NCBI database [2]. The degree of sequence

similarity between the cDNA clone and known sequences was

represented by the expect (E) value. E value scores below 10K5

were considered as significant.

2.5. Analysis of transcript levels by RNA dot-blot analysis

Differential expression of clones selected following the

initial screening and sequence analysis was confirmed by RNA

dot-blot utilizing RNA extracted from plant material collected

in an independent experiment. Total RNA (10 mg) from

N. megalosiphon plants inoculated with ToMoTV and mock-

inoculated and harvested at 0, 5, 15 and 25 dpi was spotted on

Hybond NC nylon membrane (Amersham-Pharmacia, UK)

and probed with 32P-labeled (ICN Biomedicals, Irvine, CA)

PCR products from selected clones that showed significant

homology to genes with known function using the ReadyPrime

random primed DNA labeling kit (Amersham-Pharmacia).

Membranes were hybridized, washed and prepared for re-use

as described above.

3. Results

3.1. Phenotypical characterization of the N. megalosiphon–

ToMoTV interaction

In order to determine whether or not N. megalosiphon is a

host for ToMoTV, 6-week-old plants were infected and

subsequently analyzed. No symptoms of viral infection were

visible on any of the inoculated leaves or on systemic leaves at

5 and 15 dpi, respectively, and the first symptoms of disease

were observed by 25 dpi. Furthermore, a striking dwarfing and

remarkable reduction of nodes, internodes and foliar area were

observed by 40 dpi (Fig. 1). The inoculated plants as well as the

control plants exhibited curling of the lower leaves (Fig. 1),

which should be attributed to the biolistic treatment with virus-

coated particles. DNA dot-blot analysis in order to detect

presence of ToMoTV in infected plants confirmed that

ToMoTV was detectable at 15 dpi (Fig. 2).

3.2. Construction of the subtracted cDNA library

In order to isolate host genes that are induced in the interaction

between N. megalosiphon and ToMoTV, a ‘forward’ subtracted

cDNA library was generated in which cDNA that was isolated

from infectedN.megalosiphonwas used as tester and cDNA from

mock-inoculated plants as driver. Leaves from N. megalosiphon

were collected at different intervals after the inoculation and

pooled before RNA extraction. Since for many viruses inoculated

leaves do not show full symptoms and can be considered as being

qualitatively and quantitatively different from systemically

infected leaves, we have chosen to collect all leaves (inoculated

as well as systemic leaves) from the test plants. Samples were

collected at 5, 15 and 25 dpi. PCR amplification of the subtracted

cDNA resulted in fragments ranging in size from 250 to 1300 bp,

with an average size of 350 bp. PCR products from 96 clones

were transferred to nylon membrane and subjected to hybridiz-

ation with the tester and driver cDNA samples. This screen helped

identifying 84 genes that were indeed differentially expressed and

which were therefore selected for sequence analysis.

Table 1

Summary of identified SSH clones and their BLAST search results

Clone Accession number Sequence homology/match Functional cat-

egories

E value

GV02 DW587328 Glutamate decarboxylase [Nicotiana tabacum] U54774 Metabolism 1!10K83

GV10 DW587336 Drought-induced protein [Arabidopsis thaliana] CAB78633 Defense response 9!10K10

GV12 DW587338 Receptor-like protein kinase [Arabidopsis thaliana] AAA32857 Defense response 4!10K44

GV32 DW587358 Oxygen-evolving enhancer protein [Bruguiera gymnorrhiza] AB043962 Metabolism 2!10K75

GV34 DW587360 Ubiquitin-conjugating enzyme [Nicotiana tabacum] AB026056 Protein synthesis 3!10K47

GV42 DW587368 Glycine rich protein [Nicotiana tabacum] AB041513 Defense response 7!10K58

GV66 DW587392 Auxin-binding protein [Nicotiana tabacum] X70902 Metabolism 5!10K90

GV69 DW587395 Cytokinin-specific binding protein [Vigna radiata] AB012218 Metabolism 3!10K22

GV73 DW587399 Osmotic protein ODE1 [Capsicum annuum] AF169203 Defense response 6!10K17

GV74 DW587400 Cysteine protease inhibitor [Oryza sativa] J03469 Defense response 3!10K12

GV78 DW587404 Environmental stress-induced protein [Medicago sativa] M74191 Defense response 4!10K76

GV80 DW587406 Superoxide dismutase [Nicotiana plumbaginifolia] X55974 Defense response 7!10K16

GV82 DW587408 Dehydration-induced protein [Arabidopsis thaliana] BAD94687 Defense response 3!10K82

GV83 DW587409 Cellulose synthase [Arabidopsis thaliana] NP_171773 Metabolism 1!10K41

C. Collazo et al. / Physiological and Molecular Plant Pathology 67 (2006) 231–236234

3.3. Sequence analysis of differentially expressed cDNAs

Upon sequencing of the 84 selected cDNA clones (GenBank

accession numbers DW587327 to DW587410), 20% of the

sequences were found to be redundant and 67 diffentially induced

transcripts were retained. These cDNA fragments were categor-

ized according to their homology assigned by database homology

searches (http://mips.gsf.de/proj/thal/db/). About 25% of the

clones were found to encode proteins that displayed insufficient

similarity to known proteins, and were therefore classified as

unknown. The majority of cDNA clones from the subtracted

library for which a putative function could be assigned exhibited

homology to genes associated with defense, signal transduction,

transport, metabolism, protein synthesis and energy.

Fig. 3. RNA dot-blot analysis of cDNA clones at different time points. Six-

week-old plants of N. megalosiphon were inoculated with ToMoTV (A), mock-

inoculated (B) and untreated (C). The RNA (10 mg) was isolated at indicated

times post-inoculation. The RNA was spotted on a membrane and hybridized

with the cDNA clones indicated at the left.

3.4. Analysis of transcript levels by RNA dot-blot analysis

RNA dot-blot analysis was performed on a selection of the

differentially expressed cDNAs. Clones were selected that

displayed high homology to genes with known function (for

clones that appeared to have database homologues with a

significant E-value scores below 10K5). Fourteen cDNA clones

were used as probes on RNA dot-blots prepared with total RNA

extracted fromN.megalosiphon plants inoculated with ToMoTV

or mock inoculated at 0, 5, 15 and 25 dpi, and collected (Table 1).

As predicted, expression levels of the selected transcripts were

significantly elevated in N. megalosiphon plants inoculated with

ToMoTV compared to basal expression levels in mock-

inoculated plants (Fig. 3). Several clones (GV02, GV10, GV12,

GV42, GV66, GV73, GV74, GV78 and GV82) exhibited strong

induction already 5 dpi in ToMoTV infected plants when

compared to the mock-inoculated plants. It is interesting to note

that the accumulation of different transcripts occurs with different

kinetics. Several genes are induced after 5 days, and while the

expression of some of then declined by 15 days (GV02, GV10,

GV66 and GV73), for others the expression levels stay elevated

(GV12, GV74, GV80 and GV82). GV32, with homology to an

oxygen-evolving enhancer protein, and GV69, with homology to

cytokinin-specific binding protein, were induced only after

15 dpi. Finally, GV34, with homology to an ubiquitin-conjugat-

ing enzyme was only significantly induced after 25 days.

4. Discussion

N. megalosiphon is used in Cuban tobacco breeding

programs for the introduction of disease resistance against

several fungal pathogens into new tobacco cultivars. One

aspect of this study was to determine if N. megalosiphon is

C. Collazo et al. / Physiological and Molecular Plant Pathology 67 (2006) 231–236 235

susceptible to ToMoTV. N. megalosiphon has been used in

several studies with viral pathogens such as Potato Virus A

(PVA) [32], Tobacco Mild Green Mosaic Virus (TMGMV) and

Tobacco Mosaic Virus (TMV) [30]. These studies have shown

that N. megalosiphon is a highly susceptible host for these

RNA viruses. However, little is known about the resistance of

N. megalosiphon to DNA viruses. We now show that severe

viral symptoms caused by ToMoTV were observed at 40 dpi.

Infected plants exhibited chlorotic mottle and a striking

dwarfing as well as a remarkable reduction of nodes, internodes

and foliar areas (Fig. 1). These symptoms are similar to those

observed in other susceptible hosts such as potato, bean, pepper

and Nicotiana tabacum [6]. Furthermore, the presence of viral

DNA in systemic tissues that was demonstrated as early as

15 dpi (so before symptom appearance) by DNA dot-blot

analysis, indicated a successful viral infection, replication and

movement under our experimental conditions (Fig. 2).

A second aspect of this study focused on the listing of

transcriptional changes occurring in the host plant in

response to ToMoTV infection. It has been shown that

viruses use a variety of strategies to promote their infections

in susceptible hosts. In plants, these strategies involve well-

documented modifications that enhance infections, such as

the formation of replication complexes [17], suppression of

post-transcriptional gene silencing [35], alteration of cell-to-

cell trafficking [20], and interference with regulation of the

plant cell cycle [15]. Plants can resist viral attack if they are

capable of activating appropriate defense mechanisms, such

as systemic acquired resistance [3]. These responses are

typically accompanied by dramatic changes in host gene

expression that include up-regulation of pathogenesis related

proteins.

In order to identify differentially expressed genes in plants

upon viral infection, cDNA subtractive hybridisation was

employed. Database searches revealed that many of the 67

different N. megalosiphon cDNAs displayed significant

homology to genes with known or predicted function and

comprised genes encoding proteins related to defense, signal

transduction, transport, metabolism, protein synthesis and

energy. Subsequent RNA dot-blot analysis confirmed that

most of the cDNA clones indeed showed differential

expression patterns at various time points in infected versus

mock-inoculated plants. Several of those genes are thought to

play a role in stress responses or pathogen defense. Glutamate

decarboxylase protein (GV02) is a cytosolic enzyme that has

been shown to be a calmodulin-binding protein that is

Ca2C/calmodulin activated [38]. Glutamate decarboxylase

catalyzes the conversion of glutamate to gamma-aminobutyric

acid (GABA). GABA accumulation is induced in response to a

sudden decrease in temperature, in response to heat shock,

mechanical manipulation, and water stress. Rapid GABA

accumulation in response to wounding may play a role in plant

defense against insects [25].

Also, a gene showing homology to cysteine protease

inhibitor genes (GV74) was identified. Cysteine protease

inhibitors are presumed to have a role in plant defense due to

their role in the regulation of cysteine proteases which are key

enzymes in apoptosis processes [29]. They are induced in

various stress conditions such as plant–pathogen interaction in

rice upon Magnaporthe grisea infection [19] and in bean by

wounding and methyl jasmonate treatment [5]. Besides,

cysteine protease inhibitors have been isolated from pearl

millet that exhibit potent antifungal activity against Tricho-

derma reesei and other important phytopathogenic fungi,

namely, Claviceps, Helminthosporium, Curvularia, Alternaria

and Fusarium species [18].

We found a clone (GV80) with homology to mRNA

superoxide dismutase which has an important role during the

hypersensitive response (HR). Compatible interactions, which

lead to chlorotic phenotypes, are typically associated with a

loss of photosynthetic capacity, an increase in respiration, a

change in carbohydrate partitioning and altered starch

accumulation [28]. The systemic induction of superoxide

dismutase adds to a list of genes that are similarly induced in a

range of compatible host–virus interactions. These include

peroxidase, catalase [26], pathogenesis-related (PR) genes [7],

and glutathione-S-transferase [14]. Some of these genes also

contribute to signatures for the HR and for systemic acquired

resistance, and may indicate the involvement of a general stress

response or the invocation of the senescence pathway in

response to infection [1].

A putative receptor-like protein kinase (GV12) was

induced at all the time points evaluated after infection

with an apparent peak of expression at about 15 dpi. Protein

kinases play a central role in signaling during pathogen

recognition and the subsequent activation of plant defense

mechanisms [22]. However, also in other signaling processes

receptor-like protein kinases play crucial roles. Interesting,

an auxin-binding protein (GV66) was up-regulated early in

the interaction but returned to a lower expression by 15 and

25 dpi. Also other genes that are linked to plant growth and

development were found to be upregulated, such as a

cellulose synthase catalytic chain (GV83) and a cytokinin-

specific binding protein (GV69). Though auxin is mainly

known as a key hormone that controls plant growth and

development, evidence is accumulating that this plant

hormone might play a role in pathogen defense. In several

pathosystems auxin-related genes are found induced upon

pathogen challenge [9]. In addition, in Arabidopsis the

auxin-resistance locus AXR1 has been shown to be required

for pathogen resistance [33].

Obviously, it needs to be recognized that, despite defense

genes being induced, N. megalosiphon is a susceptible host to

ToMoTV, and induction of the defense genes does not lead to

incompatibility. In general it can be stated that the speed at

which a defense response is activated, together with the

efficacy of that defense response to a particular pathogen,

determines the outcome of the interaction [31]. To a large

extent, the difference between compatible and incompatible

interactions should be found not so much in the induction of

different defense responses, but rather in the speed at which

those responses are activated in those interactions. Therefore,

the inventory of defense genes induced in a compatible

interaction is still valuable and might lead to defense genes

C. Collazo et al. / Physiological and Molecular Plant Pathology 67 (2006) 231–236236

that can be used to engineer disease resistance. Furthermore,

characterization and functional analysis of the genes that have

been identified in this study can lead to a more comprehensive

understanding of Nicotiana megalosiphon–Begomovirus

interactions.

Acknowledgements

The authors would like to thank Dr Philip Smith for

comments and critical reading of the manuscript. B.P.H.J.T. is

supported by a VENI grant of the Research Council for Earth

and Life sciences (ALW) of the Netherlands Organization for

Scientific Research (NWO).

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