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Phenotypical and molecular characterization of the Tomato mottle Taino virus–Nicotiana megalosiphon interaction
Physiological and Molecular Plant Pathology 67 (2006) 231–236 www.elsevier.com/locate/pmpp
Phenotypical and molecular characterization of the Tomato mottle Taino virus–Nicotiana megalosiphon interaction Cyrelys Collazo a, Pedro Luis Ramos a, Osmany Chaco´n b, Carlos Javier Borroto a, Yunior Lo´pez a, Merardo Pujol a, Bart P.H.J. Thomma c, Ingo Hein d, Orlando Borra´s-Hidalgo a,* a
Center for Genetic Engineering and Biotechnology, Plant Functional Genomic, P.O. Box 6162, Havana, Calle 31, 10600, Cuba b Tobacco Research Institute, Carretera de Tumbadero Km. 8, P.O. Box 6063, San Antonio de los Ban˜os, Havana, Cuba c Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands d 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 * Corresponding author. Tel.: C53 7 271 6022; fax: C53 7 273 1779. E-mail address: [email protected] (O. Borra´s-Hidalgo).
0885-5765/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2006.02.003
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 biochemically 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
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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 hybridization (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]. Sixweek-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 ToMoTVinfected 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) according 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 synthesized 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
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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 mockinoculated 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
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.
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 hybridization 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.
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 viruscoated 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 infected N. megalosiphon was 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
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.
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Table 1 Summary of identified SSH clones and their BLAST search results Clone
Accession number
Sequence homology/match
Functional categories
E value
GV02 GV10 GV12 GV32 GV34 GV42 GV66 GV69 GV73 GV74 GV78 GV80 GV82 GV83
DW587328 DW587336 DW587338 DW587358 DW587360 DW587368 DW587392 DW587395 DW587399 DW587400 DW587404 DW587406 DW587408 DW587409
Glutamate decarboxylase [Nicotiana tabacum] U54774 Drought-induced protein [Arabidopsis thaliana] CAB78633 Receptor-like protein kinase [Arabidopsis thaliana] AAA32857 Oxygen-evolving enhancer protein [Bruguiera gymnorrhiza] AB043962 Ubiquitin-conjugating enzyme [Nicotiana tabacum] AB026056 Glycine rich protein [Nicotiana tabacum] AB041513 Auxin-binding protein [Nicotiana tabacum] X70902 Cytokinin-specific binding protein [Vigna radiata] AB012218 Osmotic protein ODE1 [Capsicum annuum] AF169203 Cysteine protease inhibitor [Oryza sativa] J03469 Environmental stress-induced protein [Medicago sativa] M74191 Superoxide dismutase [Nicotiana plumbaginifolia] X55974 Dehydration-induced protein [Arabidopsis thaliana] BAD94687 Cellulose synthase [Arabidopsis thaliana] NP_171773
Metabolism Defense response Defense response Metabolism Protein synthesis Defense response Metabolism Metabolism Defense response Defense response Defense response Defense response Defense response Metabolism
1!10K83 9!10K10 4!10K44 2!10K75 3!10K47 7!10K58 5!10K90 3!10K22 6!10K17 3!10K12 4!10K76 7!10K16 3!10K82 1!10K41
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 categorized 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.
15 dpi. Finally, GV34, with homology to an ubiquitin-conjugating 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
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 from N. 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 mockinoculated 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
Fig. 3. RNA dot-blot analysis of cDNA clones at different time points. Sixweek-old plants of N. megalosiphon were inoculated with ToMoTV (A), mockinoculated (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.
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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 welldocumented modifications that enhance infections, such as the formation of replication complexes [17], suppression of post-transcriptional gene silencing [35], alteration of cell-tocell 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
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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 Trichoderma 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 cytokininspecific 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
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Phenotypical and molecular characterization of the Tomato mottle Taino virus–Nicotiana megalosiphon interaction Cyrelys Collazo a, Pedro Luis Ramos a, Osmany Chaco´n b, Carlos Javier Borroto a, Yunior Lo´pez a, Merardo Pujol a, Bart P.H.J. Thomma c, Ingo Hein d, Orlando Borra´s-Hidalgo a,* a
Center for Genetic Engineering and Biotechnology, Plant Functional Genomic, P.O. Box 6162, Havana, Calle 31, 10600, Cuba b Tobacco Research Institute, Carretera de Tumbadero Km. 8, P.O. Box 6063, San Antonio de los Ban˜os, Havana, Cuba c Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands d 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 * Corresponding author. Tel.: C53 7 271 6022; fax: C53 7 273 1779. E-mail address: [email protected] (O. Borra´s-Hidalgo).
0885-5765/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.pmpp.2006.02.003
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 biochemically 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
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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 hybridization (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]. Sixweek-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 ToMoTVinfected 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) according 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 synthesized 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
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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 mockinoculated 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
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.
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 hybridization 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.
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 viruscoated 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 infected N. megalosiphon was 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
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.
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Table 1 Summary of identified SSH clones and their BLAST search results Clone
Accession number
Sequence homology/match
Functional categories
E value
GV02 GV10 GV12 GV32 GV34 GV42 GV66 GV69 GV73 GV74 GV78 GV80 GV82 GV83
DW587328 DW587336 DW587338 DW587358 DW587360 DW587368 DW587392 DW587395 DW587399 DW587400 DW587404 DW587406 DW587408 DW587409
Glutamate decarboxylase [Nicotiana tabacum] U54774 Drought-induced protein [Arabidopsis thaliana] CAB78633 Receptor-like protein kinase [Arabidopsis thaliana] AAA32857 Oxygen-evolving enhancer protein [Bruguiera gymnorrhiza] AB043962 Ubiquitin-conjugating enzyme [Nicotiana tabacum] AB026056 Glycine rich protein [Nicotiana tabacum] AB041513 Auxin-binding protein [Nicotiana tabacum] X70902 Cytokinin-specific binding protein [Vigna radiata] AB012218 Osmotic protein ODE1 [Capsicum annuum] AF169203 Cysteine protease inhibitor [Oryza sativa] J03469 Environmental stress-induced protein [Medicago sativa] M74191 Superoxide dismutase [Nicotiana plumbaginifolia] X55974 Dehydration-induced protein [Arabidopsis thaliana] BAD94687 Cellulose synthase [Arabidopsis thaliana] NP_171773
Metabolism Defense response Defense response Metabolism Protein synthesis Defense response Metabolism Metabolism Defense response Defense response Defense response Defense response Defense response Metabolism
1!10K83 9!10K10 4!10K44 2!10K75 3!10K47 7!10K58 5!10K90 3!10K22 6!10K17 3!10K12 4!10K76 7!10K16 3!10K82 1!10K41
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 categorized 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.
15 dpi. Finally, GV34, with homology to an ubiquitin-conjugating 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
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 from N. 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 mockinoculated 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
Fig. 3. RNA dot-blot analysis of cDNA clones at different time points. Sixweek-old plants of N. megalosiphon were inoculated with ToMoTV (A), mockinoculated (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.
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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 welldocumented modifications that enhance infections, such as the formation of replication complexes [17], suppression of post-transcriptional gene silencing [35], alteration of cell-tocell 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
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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 Trichoderma 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 cytokininspecific 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
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