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Takeaki Ishihara, Nozomu Sakurai, Ken-Taro Sekine, Shu Hase, Masato Ikegami, Daisuke Shibata, Hideki Takahashi, Comparative Analysis of Expressed Sequence Tags in Resistant and Susceptible Ecotypes of Arabidopsis thaliana Infected with Cucumber Mosaic Virus, Plant and Cell Physiology, Volume 45, Issue 4, 15 April 2004, Pages 470–480, https://doi.org/10.1093/pcp/pch057
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Abstract
Arabidopsis thaliana ecotype Columbia (Col-0) is susceptible to the yellow strain of cucumber mosaic virus [CMV(Y)], whereas ecotype C24 is resistant to CMV(Y). Comprehensive analyses of ~9,000 expressed sequence tags in ecotypes Col-0 and C24 infected with CMV(Y) suggested that the gene expression patterns in the two ecotypes differed. At 6, 12, 24 and 48 h after CMV(Y) inoculation, the expression of 6, 30, 85 and 788 genes, respectively, had changed in C24, as opposed to 20, 80, 53 and 150 genes in CMV(Y)-infected Col-0. At 12, 24 and 48 h after CMV(Y) inoculation, the abundance of 3, 10 and 55 mRNAs was altered in both ecotypes. However, at 6 h after CMV(Y) inoculation, no genes were co-induced or co-suppressed in both ecotypes. This differential pattern of gene expression between the two ecotypes at an early stage of CMV(Y) infection indicated that the cellular response for resistance may differ from that resulting in susceptibility at the level detectable by the macroarray. According to the expression pattern at various stages of infection, the expression of many genes could be grouped into clusters using cluster analysis. About 100 genes that encode proteins involved in chloroplast function were categorized into clusters 1 and 4, which had a differentially lower expression in CMV(Y)-inoculated C24. The expression of various genes encoding proteins in the endomembrane system belonged to clusters 2 and 4, which were induced in CMV(Y)-inoculated C24 and Col-0 leaves. Characterization of CMV(Y)-altered gene expression in the two ecotypes will contribute to a better understanding of the molecular basis of compatible and incompatible interactions between virus and host plants.
(Received December 25, 2003; Accepted February 19, 2004)
Introduction
Host plants have various responses to virus infection. When a virus overcomes plant defenses, the host plants become systemically infected with the virus and often have systemic symptoms such as chlorosis and stunting. When a cultivar of the host plants is resistant to the virus, the host can avoid systemic infection by localizing the virus at the primary infection site or within the inoculated leaves. This resistance response is often accompanied by the development of necrotic local lesions (NLLs) in virus-inoculated leaves and the expression of a set of defense-related genes (Hooft van Huijsduijnen et al. 1986, Guo et al. 2000). Thirteen defense genes expressed during the induction of the hypersensitive response (HR) in tobacco mosaic virus (TMV)-infected Chenopodium amaranticolor have been characterized with cDNA-ALFP techniques (Cooper 2001). Morphological and physiological changes in the resistance response to viruses suggested that certain pathway(s) that confer a resistance response against the virus may be specifically activated in the resistant cultivar. On the other hand, in susceptible plants systemically infected with a virus, a different set of genes are expressed during the course of systemic symptom expression, even though some pathogenesis- and other stress-related genes are co-regulated during both susceptible and resistant plant responses (Ziemiecki and Wood 1975, Roberts and Wood 1981, Takahashi et al. 1991, Aranda et al. 1996, Escaler et al. 2000, Havelda and Maule 2000). This differential gene expression in the resistant and susceptible interaction between viruses and host plants indicated that the molecular mechanism governing the resistance response may be distinct from the susceptible response. On the other hand, a series of single amino acid substitutions in the coat protein of TMV may induce not only a change in the severity of systemic symptoms but may also induce the HR-like response in tobacco plants, indicating that the coat protein of TMV may have a dual virulent/avirulent function in host plants (Dawson et al. 1988). We have only limited information on the molecular basis for resistant and susceptible responses of plants to viruses.
Cucumber mosaic virus (CMV) has the largest host range of any virus and causes severe agricultural losses in many countries (Palukaitis et al. 1992). CMV is a tripartite, positive-sense, single-strand RNA virus containing four different RNAs (RNA1-4). The virulence determinant encoded on the CMV genome has been well characterized at the amino acid level (Shintaku et al. 1992, Suzuki et al. 1995, Sugiyama et al. 2000, Takahashi et al. 2000). The host responses in CMV-infected tobacco and other host plants also have been extensively studied at the molecular and physiological levels (Ziemiecki and Wood 1975, Roberts and Wood 1981, Takahashi et al. 1991, Takahashi and Ehara 1992, Havelda and Maule 2000).
Arabidopsis thaliana is also susceptible to CMV. For the past 10 years, A. thaliana has been thought to be a useful plant for studying host responses to CMV because of its advantages for molecular genetic approaches (Takahashi et al. 1994, Béclin et al. 1998). In a survey of the response of several ecotypes of A. thaliana to the yellow strain of CMV [CMV(Y)], the C24 ecotype of A. thaliana had a resistance response to CMV(Y), whereas ecotype Columbia (Col-0) was fully susceptible to CMV(Y) (Takahashi et al. 1994). The resistance response to CMV(Y) in C24 was accompanied by the formation of NLLs at the primary site of infection, and systemic spread of the virus was restricted within the inoculated leaves. The coat protein of CMV(Y), which is translated from a subgenomic RNA4 transcribed from a minus strand of RNA3, acts as the avirulence determinant for the induction of the resistance response in CMV(Y)-infected C24 (Takahashi et al. 2001). The CMV(Y)-resistance gene RCY1 in C24 was a single dominant gene, which has been mapped on the long arm of chromosome 5 and encodes the putative 104-kDa CC-NBS-LRR protein (Takahashi et al. 2002). Although A. thaliana ecotype Di-17 is also resistant to turnip crinkle virus (Cooley et al. 2000), to our knowledge, CMV and turnip crinkle virus are the only viruses to induce the HR in Arabidopsis.
Col-0, lacking RCY1, is systemically infected by CMV(Y). At the late stage of virus infection, typical symptoms including chlorosis and stunting developed in CMV(Y)-infected Col-0 (Takahashi et al. 1994). However, whether Col-0 has a specific response to systemic infection of CMV(Y) or whether even systemic infection of CMV(Y) can induce general host defense responses remains unknown. A comparative analysis of host gene expression between resistant C24 and susceptible Col-0 ecotypes to CMV(Y) may improve our understanding of general and specific responses in compatible and incompatible interactions between virus and host plants.
Profiling of transcripts with cDNA arrays has been reported for some aspects of plant–microbe interactions and systemic acquired resistance (Schenk et al. 2000, Maleck et al. 2000, Chen et al. 2002, Colebatch et al. 2002, Itaya et al. 2002, Golem and Culver 2003, Whitham et al. 2003). Comparative analysis of gene expression in A. thaliana after treatment with salicylic acid (SA), jasmonic acid (JA) or ethylene (ET), or infection with the virulent fungal pathogen Alternaria brassicicola indicated the existence of a substantial coordinating network in plant defense responses in various defense signaling pathways (Schenk et al. 2000). Through transcript profiling in A. thaliana after infection with pathogens or treatment with various environmental stresses, a common promoter element in genes of plants responding to a broad set of pathogens was identified (Chen et al. 2002). In tomato plants infected with different pathogenicity strains of potato spindle tuber viroid, the expression of common and unique genes was induced or suppressed (Itaya et al. 2002). Recently, virus-general and virus-specific alterations of gene expression among A. thaliana systemically infected with five distinct RNA viruses were also identified (Whitham et al. 2003). Furthermore, Glazebrook et al. (2003) identified the gene clusters, which co-regulated by SA-dependent or JA/ET-dependent signaling pathways for the HR to Pseudomonas syringae DC3000. A possible model was proposed for cross-talk between the SA-dependent and JA/ET-dependent signaling pathways. However, profiling of transcripts with cDNA array between resistant and susceptible responses to CMV has not been reported.
Here, global gene expression in the resistant and susceptible ecotypes to CMV(Y) at various stages of infection was analyzed using a macroarray membrane composed of 13,000 non-redundant ESTs that corresponded to ~9,000 genes. This report is the first comparative analysis of global gene expression in resistant and susceptible responses to a viral pathogen. A comprehensive analysis of gene expression in CMV(Y)-infected A. thaliana ecotypes should further our knowledge of the molecular basis of the interaction between virus and host plants.
Results
Symptom expression in two ecotypes of A. thaliana infected with CMV(Y)
A. thaliana ecotype C24 had a HR to CMV(Y), although ecotype Col-0 was fully susceptible to CMV(Y) (Takahashi et al. 1994). By 6 and 12 h, no symptoms appeared on inoculated leaves of either ecotype (Fig. 1A), whereas very small macroscopic NLLs appeared on the inoculated leaves of C24 by 24 h after inoculation (Fig. 1B). The NLLs expanded by 48 h after inoculation, although CMV(Y)-infected Col-0 had no symptoms at 48 h (Fig. 1A, B). By 7 d after CMV(Y) inoculation, systemic symptoms developed in CMV(Y)-infected Col-0, but no symptom appeared in CMV(Y)-infected C24 (data not shown).
To study the changes in transcript profiles in C24 and Col-0 infected with CMV(Y), total RNA was isolated from virus-inoculated leaves at five different times: 0, 6, 12, 24 and 48 h after inoculation. CMV RNA was detected in CMV(Y)-inoculated leaves of C24 and Col-0 at 24 and 48 h after inoculation by Northern hybridization (Fig. 1C). The accumulation of CMV RNA was much higher in the inoculated leaves of susceptible Col-0 than resistant C24 at 24 and 48 h after inoculation.
Comparison of global expression profiles between resistant and susceptible responses in CMV(Y)-infected plants
To analyze the gene expression patterns in CMV(Y)-inoculated leaves of resistant C24 and susceptible Col-0, a macroarray membrane composed of 13,000 non-redundant ESTs corresponding to ~9,000 genes was used. The general expression patterns of the genes in CMV(Y)-inoculated C24 and Col-0 in comparison with mock-inoculated C24 as a control were analyzed by three replicate experiments for each treatment at 0, 6, 12, 24 and 48 h after inoculation. The scattered groups display the expression levels of genes in C24 or Col-0 infected with CMV(Y) at the Y axis and those in the mock-inoculated C24 at the X axis (Fig. 2). The distance from each data point to the line log Y = log X (middle broken line) indicates the ratio of induction or suppression. Diagonal black and gray lines represent the 3-fold induction and –3-fold suppression ratios, respectively. The number of genes with more than 3-fold induction or –3-fold suppression increased in resistant C24 more than in susceptible Col-0 during the HR induction (Fig. 2). The higher levels and numbers of altered expression were observed in resistant C24 at 48 h after inoculation (Fig. 2).
To confirm a reproducible alteration of gene expression in CMV(Y)-infected C24 and Col-0 using macroarray analysis, three clones (anthocyanin 5-aromatic acyltransferase, dehydroascorbate reductase 1 and BAG domain-containing protein) were randomly picked up from the clones with altered expression at 6 h after inoculation (Table 1). Their levels of transcription in CMV(Y)-inoculated C24 and Col-0 and mock-inoculated C24 were analyzed at 0, 6, 12, 24 and 48 h after inoculation by Northern hybridization and were compared with their expression values in macroarray analysis (Fig. 3). The altered patterns of gene expression in three selected genes mostly coincided with that in macroarray analysis. Therefore, we judged that the macroarray data were reliable enough for analyzing the change of gene expression in resistant and susceptible ecotypes to CMV(Y).
Unique genes induced and suppressed in CMV(Y)-infected C24 and Col-0
The number of genes with altered expression level in resistant C24 and susceptible Col-0 at differential stages after CMV(Y) inoculation, are shown in a Venn diagram in Fig. 4. At 6 h after inoculation, six genes had more than a 3-fold induction or a –3-fold suppression in resistant C24 in comparison with their expression in mock-inoculated plants, whereas 20 genes were altered in their expression in susceptible Col-0 at 6 h after inoculation (Fig. 4). However, none of the genes with altered expression level were the same in both ecotypes at 6 h after inoculation. This distinct differential alteration of gene expression in resistant C24 and susceptible Col-0 suggests that the cellular response at an early stage of CMV(Y) infection might be different in the resistance response in C24 and the susceptible response in Col-0 at the level detectable by the macroarray.
The induced and suppressed genes in C24 and Col-0 at 6 h after inoculation are listed in Table 1 with expression levels, AGI numbers and functional classification determined by TAIR A. thaliana database. Two genes that were specifically induced in CMV(Y)-infected C24, encoded a homologue of the monosaccharide transporter AtSTP3 and an unknown protein; whereas four genes, specifically suppressed in CMV(Y)-infected C24, encoded a putative dehydroascorbate reductase, a germin-like protein and two unknown proteins. The expression of six genes was temporally altered in CMV(Y)-inoculated C24 at 6 h after inoculation and recovered to the level of the mock-inoculated at 12, 24 and 48 h after inoculation (data not shown). At 6 h after inoculation in CMV(Y)-infected Col-0, the expression of 20 genes was altered (Table 1). Of these 20 genes, the expression of 16 genes was induced, whereas four genes were suppressed. Although, in Table 1, At3g48360, which was one of the 16 induced genes in CMV(Y)-infected Col-0, showed 3.3-fold induction in CMV(Y)-infected C24, comparative analysis of At3g48360 among CMV(Y)-infected C24 and Col-0 and mock-inoculated plant by the Fisher’s least significant difference procedure in three replicate experiments indicated that the expression of At3g48360 did not significantly increase in CMV(Y)-infected C24 but CMV(Y)-infected Col-0. Encoded by 14 induced genes and two suppressed genes are an F-box protein, a transporter, a putative protein related to oxidative stress and cell death (germin-like protein and BAG domain-containing protein), an R gene-like protein and proteins containing other diverse function. The functions of two induced and two suppressed gene products are unknown. The altered expression patterns of these 20 genes in CMV(Y)-infected Col-0 differed at a late stage (24–48 h after inoculation) (see Table 2 in Supplementary Material located at www.pcp.oupjournals.org).
At 12 h after CMV(Y) inoculation, the level of expression of 30 genes changed in resistant C24, whereas the level of expression of 80 genes changed in susceptible Col-0 (Fig. 4). A total of 107 genes had an altered expression level, and the expression of three genes was co-induced in both ecotypes. At 24 h after inoculation, the expression of 85 genes was altered in resistant C24, whereas 53 were altered in susceptible Col-0 (Fig. 4). Among these genes, eight genes were co-induced in both ecotypes, while two were co-suppressed in them.
At 48 h after CMV(Y) inoculation, the expression of 150 genes was altered in susceptible Col-0, although the expression of 788 genes changed in resistant C24 (Fig. 4). Of these, 29 genes were induced in both ecotypes at 48 h after inoculation, whereas 26 genes were suppressed in both (Fig. 4). These co-alterations of gene expression in CMV(Y)-infected C24 and Col-0 at the late stage of CMV(Y) infection indicated that, in addition to the alteration of unique gene expression, the expression of a set of common genes might also be altered in both resistant and susceptible interactions.
Cluster analysis of co-regulated genes
The altered expression patterns of the genes in Fig. 4 were categorized into four groups using cluster analysis (Fig. 5 and Table 2 in Supplementary Material). Although some temporal induced or suppressed genes were not sortable into any of the four clusters, 813 genes were categorized. Gene expression in cluster 1 was suppressed only in CMV(Y)-inoculated C24. Although the expression of genes in cluster 4 was suppressed in both CMV(Y)-inoculated C24 and Col-0, the suppression of many genes in C24 in cluster 4 was greater than for those in Col-0 (Fig. 5). The expression of several genes in cluster 2 was induced only in CMV(Y)-infected C24, whereas cluster 3 contained genes that were gradually induced in CMV(Y)-infected Col-0 (Fig. 5). Cluster 2 contained many genes for defense-related proteins such as chitinase, peroxidase, hevein-like protein, superoxidase dismutase, glutathione S-transferase and cysteine protease inhibitor (see Table 2 in Supplementary Material). However, because some defense-related genes, ex. PR-1, PR-5 and PDF1.2, which are generally induced in incompatible interaction, were not spotted in this macroarry membrane, we could not detect their up-regulation in this experiment. The large number of genes in clusters 1, 2 and 4 were co-regulated in CMV(Y)-infected C24 (Table 2). Therefore, the HR seemed to be associated with a comprehensive induction and suppression of a set of unique genes.
When the cellular structure or localization of the products encoded on 813 genes in these four clusters was further analyzed by TAIR gene ontology annotations (http://arabidopsis.org/tools/bulk/go/index.jsp), the 813 genes were divided into 19 groups (data not shown). The genes included in two of 19 groups: chloroplast proteins and endomembrane system (endoplasmic reticulum, Golgi bodies, vesicles, cell membrane and nuclear envelope), are shown in Tables 3 and 4, respectively. In combined analysis of two categorizations based on gene expression pattern and cellular localization, 102 of 143 genes encoding chloroplast proteins were categorized in clusters 1 and 4, and 81 of 121 genes that encoded proteins in the endomembrane system were categorized in clusters 2 and 3 (see Tables 3 and 4 in Supplementary Material). These results suggested that the expression of many genes encoding protein involved in chloroplast function were more suppressed in CMV(Y)-inoculated C24 at the differential level than CMV(Y)-inoculated Col, but the genes encoding the endomembrane system were induced in CMV(Y)-inoculated C24 and/or Col. Other groups of cellular components contained similar number of genes categorized in clusters 1 to 4 (data not shown).
Discussion
The cDNA array technology allowed the simultaneous analysis of altered gene expression of ~9,000 Arabidopsis genes in response to CMV infection. However, variations in results are inherent with this method as a result of external interference and technical and biological sources. To counter this variation, three independent experiments were conducted for the analysis. When the value of gene expression was ≤0.3 once in any three independent experiments, the gene was eliminated from the analysis because low density data are less reproducible. Furthermore, when the ratio of gene expression between C24 and Col-0 was already altered at 0 h, the gene was also omitted from the analysis because the level of that gene expression seemed to be essentially different between two ecotypes. Results consistent with the expression pattern in the Northern hybridizations for three selected genes in Fig. 3 were obtained. In addition, the induction of many defense-related genes such as chitinase and peroxidase which were generally recognized as infection-inducible genes, were observed in cluster 2 containing up-regulated genes in only CMV(Y)-infected C24. Therefore, the macroarray data presented in this paper is reliable and adequate enough to select candidate genes for further study of the interaction between CMV and A. thaliana.
A complex of genes expressed in resistant plants in response to viruses differs both generally and specifically from those expressed in susceptible plants. The analysis of global gene expression using cDNA array gives us a better understanding about the molecular basis of these host responses to virus infection. At 48 h after CMV inoculation, the expression of 150 genes was altered in susceptible Col-0, whereas the expression of 788 genes changed in resistant C24. Thus, systemic infection of CMV may not dramatically affect gene expression in A. thaliana, in comparison to the resistance response to CMV(Y) expressed as the HR. A similar low level of gene expression was obtained in array studies of a susceptible response in A. thaliana and tomato infected with TMV, related tobamoviruses and four other distinct RNA viruses (Golem and Culver 2003, Whitham et al. 2003).
In our study, we could identify specifically two induced and four suppressed genes in CMV(Y)-infected resistant C24 at 6 h after inoculation using cDNA macroarray. Because NLLs never developed in CMV(Y)-inoculated C24 leaves at 6 h after inoculation, the altered expression of these six genes is clearly not caused by the NLL formation. The expression of these genes was not altered in Col-0 systemically infected with CMV(Y) at 6 h after inoculation. The expression of 20 other genes had changed in susceptible Col-0 by 6 h after inoculation (Table 1). This distinct altered gene expression between two ecotypes at an early stage of CMV(Y) infection suggested that the cellular response for resistance to CMV(Y) may be different from that resulting in susceptibility to CMV(Y) in Arabidopsis at the level detectable by the macroarray. On the other hand, recent analyses of the avirulent pathogens suggest that the avirulence product has not only an avirulence function but can also act as a virulence determinant in host plants (Luderer and Joosten 2001, Van der Hoorn et al. 2002). The coat protein of CMV(Y) acts as not only an avirulence determinant for inducing the HR in C24 but also a virulence determinant for systemic symptoms in tobacco (Suzuki et al. 1995, Sugiyama et al. 2000, Takahashi et al. 2000, Takahashi et al. 2002). This dual function of the coat protein of CMV to host plants may be based on a single biochemical reaction. Therefore, a single biochemical mechanism may occur at the primary interaction between the coat protein of CMV (avirulence/virulence determinant) and host plants. A distinct cellular response for either resistance or susceptibility to CMV then seems to progress in the host plants.
Two genes induced in resistant C24 by CMV(Y) at 6 h after inoculation encoded monosaccharide transporter AtSTP3 and an unknown protein (Table 1). A. thaliana has at least 14 homologous genes coding for putative monosaccharide-H+ symporters (STPs: sugar transporter proteins). The transcript of AtSTP1 is most abundant in leaves and is also found in stems, flowers and roots (Sauer et al. 1990), whereas AtSTP2 was specifically expressed in developing pollen (Truernit et al. 1999). Enhanced expression of AtSTP4 was observed in plants treated with wounding and elicitors and infected with pathogens (Truernit et al. 1996). AtSTP is a low-affinity transporter that accumulates in leaves, and the level of AtSTP3 mRNA also increased after wounding, although the response of AtSTP3 to other treatments including pathogen challenge has yet to be investigated (Buttner et al. 2000). Here, AtSTP3 was up-regulated before NLL formation of the HR in CMV(Y)-infected C24. The function of AtSTP3 in CMV(Y)-infected C24 may be to recover sugars from the apoplast after sugars were consumed as an energy source for the resistance response to CMV(Y). Although direct evidence for monosaccharide import into the HR tissue for the resistance to CMV(Y) has not been found, starch accumulation is correlated to chlorotic lesion formation at the primary infection site of CMV-inoculated cotyledons of Cucubita pepo (Técsi et al. 1994).
Four CMV(Y)-suppressed genes in C24 encoded putative dehydroascorbate reductase, germine-like protein (GLP2a) and two unknown proteins (Table 1). Dehydroascorbate reductase and GLP2a are functionally involved in degradation of active oxygen species. The decrease of these two proteins in CMV(Y)-infected C24 may contribute to the accumulation of active oxygen species as the signal molecule for inducing the resistance to CMV(Y).
On the other hand, although the expression of 20 genes changed in susceptible Col-0 at 6 h after inoculation, the function of their gene products varied (Table 1). However, some of them may have a role in systemic infection by CMV(Y). For example, the expression of BAG domain-containing protein was induced in CMV(Y)-infected Col-0 (Table 1, Fig. 3). A BAG protein is thought to interact with BCL2, a member of the Bcl-2 family, which regulates the signaling cascade for apoptosis in mitochondria of human cells (Hanada et al. 1995). Apoptosis is suppressed by the binding of BAG to domain 4 of BCL2 (Takayama et al. 1995). Therefore, the temporary induction of BAG domain-containing protein in CMV(Y)-infected Col-0 only at 6 h may be associated with the suppression of the HR induction, thereby accelerating systemic spread of CMV(Y).
CMV seems to multiply extensively in the inoculated leaves at 12 h after inoculation. At 12 h after CMV(Y) inoculation, the level of expression of 30 genes changed in resistant C24, whereas the level of expression of 80 genes changed in susceptible Col-0. These genes encoded proteins containing diverse function (Table 2). As some genes encoding proteins for signal transduction, such as F-box protein, Ran-binding protein and serine threonine-protein kinase, were included in them, a distinct biochemical response for either resistance or susceptibility to CMV may progress at this stage.
Gene expression in resistant C24 had changed dramatically by 48 h after inoculation. Because the NLLs had expanded in CMV(Y)-inoculated C24 leaves by 48 h after inoculation, numerous genes may have been altered in their expression during the plant response leading to HR cell death and senescence.
To further characterize the genes identified by macroarray analysis, the pattern of altered expression of each gene was categorized by hierarchical clustering analysis. The expression of 813 genes had totally changed by 6, 12, 24 or 48 h in either CMV(Y)-inoculated resistant C24 or susceptible Col-0. The differential patterns of 813 genes were grouped mostly into four clusters (Fig. 5). Gene expression in cluster 1 was suppressed only in CMV(Y)-inoculated C24, whereas in cluster 4, gene expression in CMV(Y)-inoculated C24 was suppressed more than in CMV(Y)-inoculated Col-0. About 100 genes encoding proteins in the chloroplast were categorized in clusters 1 and 4. Because the expression of the genes in clusters 1 and 4 was suppressed during the progress of the HR in CMV(Y)-inoculated C24 leaves, a functional change in the chloroplast may be required for the induction of the HR. In TMV-infected tobacco carrying the N gene, a decreased level of chloroplast function is also associated with the induction of the HR (Seo et al. 2000).
The genes encoding the proteins located in the endomembrane system were categorized in clusters 2 or 3 and were induced in CMV(Y)-inoculated C24 or Col-0. In general, the RNA virus replicates on certain areas of the endomembrane system (Hagiwara et al. 2003). Therefore, some of the genes induced in CMV(Y)-inoculated C24 or Col-0 may be associated with virus replication. In general, a set of defense-related genes are expressed by pathogen attack, oxidative stress, HR cell death or senescence. The level of defense-related gene expression was also elevated in CMV(Y)-inoculated C24. In addition to specific induction of defense-related gene expression, oxidative stress, HR cell death and senescence may also cause coordinated changes in gene expression similar to those during the HR in C24.
The profile of transcripts presented in this study demonstrated that the cellular responses involved in resistance may be essentially different from those for susceptibility at an early stage of CMV(Y) infection at the level detectable by the macroarray, whereas common and specific responses that may be caused either directly or indirectly by the interaction between virus and plant are induced in resistant and susceptible ecotypes at a late stage of CMV(Y) infection. However, even with information about the proteins encoded by the up- and down-regulated genes in CMV(Y)-infected C24, the molecular mechanisms determining the resistant or susceptible response to CMV remain unknown. To further understand the molecular basis of virus–host plant interactions, the function of each gene identified by cDNA macroarray must be analyzed. C24 plants, rendered deficient in gene function by T-DNA tagging, are helping us to better understand the role of these up- and down-regulated genes in resistant and susceptible responses to CMV(Y).
Materials and Methods
Plant and virus
A yellow strain of CMV [CMV(Y)] (Tomaru and Hidaka 1960) was propagated in tobacco (Nicotiana tabacum cv. Xanthi nc), then purified as described previously (Takahashi and Ehara 1993). A. thaliana ecotypes C24 and Col-0 were grown at 24°C under the continuous fluorescent light (8,000 lux) condition in the mixture of vermiculite and perlite (1 : 1 mixture). Fully expanded leaves of 3-week-old A. thaliana plants were inoculated with 100 µg ml–1 of CMV(Y). At 0, 6, 12, 24 and 48 h after virus inoculation, the inoculated leaves were harvested to extract total RNA.
cDNA array preparation
The 13,000 non-redundant ESTs corresponding to ~9,000 genes (Asamizu et al. 2000, Hirai et al. 2003) were composed of three nylon membranes (8×12 cm each) by a BIOMEK 2000 robotic workstation (Beckman Instruments, Inc., Fullerton, CA, U.S.A.) according to the manufacturer’s instruction manual. Some sets of macroarray membranes were supplied by the Japanese Consortium for A. thaliana DNA Array (JCAA).
Preparation of probes and hybridization
Total RNA was isolated from A. thaliana leaves using an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA, U.S.A.). Total RNA was reverse transcribed to synthesize [α33P]dCTP-labeled cDNA probes. Total RNA (20 µg) was then suspended in 9 µl of deionized distilled water and mixed with 1 µl of 50 µM oligo(dT)20. After heat-denaturing at 65°C for 5 min, 5 µl of 5× cDNA synthesis buffer (Invitrogen, CA, U.S.A.), 2 µl of 0.1 M DTT, 1 µl of dNTP mixture (20 mM dGTP, 20 mM dATP, 20 mM dTTP and 0.125 mM dCTP each), 5 µl of [α33P]dCTP (Amersham Pharmacia Biotech, U.S.A.), 1 µl of 40 U µl–1 RNaseOUT (Invitrogen, CA, U.S.A.) and 1 µl of 15 U µl–1 Thermoscript RT (Invitrogen, CA, U.S.A.) were added and incubated at 50°C for 60 min. After termination by heating at 85°C for 5 min, 2 U of RNase H was added and the solution then incubated at 37°C for 20 min. The synthesized cDNA was purified by gel-filteration through a ProbeQuant G-50 Micro column (Amersham Pharmacia Biotech, U.S.A.) according to the manufacturer’s instructions. The ratio of [α33P]dCTP incorporation to cDNA was measured according to Sasaki et al. (2001).
One set of macroarray membranes (three membranes) was prehybridized with 12 ml of 0.5 M Church phosphate buffer (Church and Gilbert 1984) containing 1 mM EDTA, 7% SDS and 12 µg of oligo(dA)18 in a hybridization bag at 55°C for 6 h. Heat-denatured 33P-labeled cDNA was mixed with 1 ml of the Church phosphate buffer, and then added to the hybridization bag. After incubation at 55°C for 20 h, membranes were washed twice with 2× SSC containing 0.1% SDS at 25°C for 5 min, once with 1× SSC containing 0.1% SDS at 65°C for 15 min, and twice with 0.1× SSC containing 0.1% SDS at 65°C for 15 min. The membranes were wrapped with plastic film and exposed to an IP image plate (Fuji Photo Film, Tokyo, Japan) for 3 d.
Data analysis
Signals on the IP image plates were scanned with FX (Bio-Rad, U.S.A.) and quantified using Array Vision 5.1 software (IMAGING Research Inc., Ontario, Canada). Signal intensity was calculated by subtracting raw signal intensity (vol) with local background (bg) quantified in the corners around individual spots. If the subtracted signal intensity was minus, the clone was flagged as undetectable and discarded from the analysis. The median value (MED) of subtracted signal intensity in each membrane was calculated. Normalization of signal intensity in each membrane was calculated using the following formula: normalized value = (vol – bg)/MED. Three replicate experiments for one set of membranes were conducted. The average of the normalized value of the signal intensity for each gene in three replicate experiments was adopted as the expression value of the gene. The genes with altered expression at 0 h after CMV(Y) inoculation were omitted from the analysis because these genes already seemed to be differentially expressed in the two ecotype backgrounds. To select differentially expressed genes among three treatments: CMV(Y)-infected C24 and Col-0 and mock-inoculated plant, stringent criteria were applied as described next. When the value of gene expression was ≤0.3 in one of three treatments, the gene was eliminated for further analysis, because such low intensity data are less reproducible. One-way analysis of variance of the expression values was performed to identify the genes having similar altered expression patterns in three replicate experiments. Gene which expression level was significantly different among three treatments, was selected using the Fisher’s least significant difference procedure. The expression value for CMV(Y)-inoculated C24 or Col-0 divided by that of mock-inoculated C24 indicated the ratio of induction or suppression. Ratios of <1 were transformed to –1/ratio. Then, when the value of gene expression increased more than 3-fold or decreased less than –3-fold in the CMV(Y)-inoculated plant, we identified the gene expression as altered reproducibly between two treatments. The data was analyzed using Microsoft Excel.
The genes with altered expression among three treatments were categorized by hierarchical clustering analysis using TIGR Multiple Expression Viewer (MEV, http://www.tigr.org/). Clone names of JCAA and their corresponding AGI number were assigned using the Database for Arabidopsis Research and Tools (DART; http://biochem.agr.nagoya-u.ac.jp/atgenome/). Functional classification and cellular structure or localization of each clone were determined by using TAIR database (http://arabidopsis.org/) and TAIR gene ontology annotations (http://arabidopsis.org/tools/bulk/go/index.jsp), respectively. The results obtained for each gene categorized by cluster analysis and gene ontology annotations are shown in Table 2–4 in Supplementary Material.
Northern hybridization
Total RNA was extracted from virus-inoculated leaves by the acid guanidium-phenol-chloroform method (Chomczynski and Sacchi 1987). Northern hybridization was performed by the standard procedure (Sambrook and Russell 2001). To detect transcripts from three ESTs encoding anthocyanin 5-aromatic acyltransferase-like protein (AGI number At5g39050), dehydroascorbate reductase 1-like protein (At1g19570) and BAG domain-containing protein (At5g52060) by northern hybridization, respectively, their probe DNA fragments were amplified by PCR with primers, 5′-TTGCATTGAAGATTCCAGAGATT-3′ and 5′-GAAGCAAATCAACAAGAACATCC-3′ for anthocyanin 5-aromatic acyltransferase-like protein, 5′-ATTCCGACGTCATCGTTGGTATA-3′ and 5′-GACCTTGAACAACATTACCACAC-3′ for dehydroascorbate reductase 1-like protein, 5′-GTGAAGAGAGTGCAGAATTATGT-3′ and 5′-AAGAATTCCCAATTAAACCTGGG-3′ for BAG domain-containing protein. To detect CMV RNA1-4, cDNA to CMV RNA was used. The probe cDNAs were labeled with [32P]dCTP using the Megaprime™ DNA labeling system (Amersham-Pharmacia, U.S.A.).
Supplementary Material
Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oupjournals.org.
Acknowledgments
We thank The Japanese Consortium for Arabidopsis thaliana DNA Array (JCAA) for providing macroarray membranes. We also thank Dr. Beth E. Hazen for correcting the English in this paper. We are gratefully acknowledge to Dr. Ichiro Uyeda, Hokkaido University, for useful suggestions. This work was supported in part by a grant-in-aid for scientific research on priority areas (Molecular Mechanisms of Plant-Pathogenic Microbe Interaction–Toward Production of Disease Resistant Plants) by the Ministry of Education, Culture, Sports and Arts, Japan, and by the Sasakawa Scientific Research Grant from The Japan Science Society.
Corresponding author: E-mail, takahash@bios.tohoku.ac.jp; Fax, +81-22-717-8659.
AGI number | JCAA clone number | Putative function | Induction fold | |
R | S | |||
At5g61520 | FB090e02F | hexose transporter, putative | 3.0 a | –1.3 |
At5g42900 | SQ050c09 | expressed protein | 3.2 a | –1.5 |
At1g19570 | RZ69e11 | dehydroascorbate reductase, putative | –3.2 b | –1.4 |
At5g39190 | SQ019h11 | germin-like protein (GLP2a) | –3.6 b | 1.0 |
– | SQ049d12 | unknown | –3.8 b | –1.1 |
– | RZ165h01 | unknown | –3.3 b | –1.3 |
At1g25170 | FB011h10 | anthranilate synthase beta subunit and unknown protein (chimera) | –1.5 | 3.5 a |
At5g24150 | APD48h01 | squalene monooxygenase gene homolog | –1.5 | 4.5 a |
At5g39050 | APZ17a04 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.1 a |
At5g39050 | APD07c12 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.8 a |
At5g49730 | APD12g08 | similar to ferric-chelate reductase (FRO1) (Pisum sativum) | 1.0 | 3.1 a |
At5g47560 | APD40a08 | sodium/dicarboxylate cotransporter | 1.2 | 3.1 a |
At5g45490 | RZ193d07 | disease resistance protein -related | –1.5 | 3.2 a |
At1g58602 | FB058e07 | disease resistance protein (CC-NBS-LRR class), putative | –1.1 | 5.7 a |
At1g25141 | APD52f01 | F-box protein-related | 1.4 | 4.9 a |
At5g52060 | SQ046h08 | BAG domain containing protein | 1.2 | 3.2 a |
At3g48360 | RZ176d04 | expressed protein, MEL-26, Caenorhabditis elegans, U67737 | 3.3 | 5.7 a |
At1g61670 | FB095g08F | expressed protein, similar to membrane protein PTM1 precursor isolog | 1.2 | 3.4 a |
At5g52810 | RZ54b02 | expressed protein, contains similarity to ornithine cyclodeaminase | 1.3 | 4.0 a |
At4g09980 | RZ200e01 | expressed protein, m6A methyltransferase – Homo sapiens, | 1.3 | 5.0 a |
At5g03350 | APZ44c11 | expressed protein | 1.3 | 11.1 a |
– | APD41c08 | unknown | 1.2 | 3.3 a |
At5g61970 | RZL17g10 | signal recognition particle – related | 1.2 | –3.2 b |
At4g29190 | FB010d03 | expressed protein, zinc finger transcription factor | –1.4 | –3.2 b |
At5g53420 | SQ089h05 | expressed protein | –1.5 | –3.0 b |
At3g10480 | SQ162d12 | expressed protein | –1.6 | –3.6 b |
AGI number | JCAA clone number | Putative function | Induction fold | |
R | S | |||
At5g61520 | FB090e02F | hexose transporter, putative | 3.0 a | –1.3 |
At5g42900 | SQ050c09 | expressed protein | 3.2 a | –1.5 |
At1g19570 | RZ69e11 | dehydroascorbate reductase, putative | –3.2 b | –1.4 |
At5g39190 | SQ019h11 | germin-like protein (GLP2a) | –3.6 b | 1.0 |
– | SQ049d12 | unknown | –3.8 b | –1.1 |
– | RZ165h01 | unknown | –3.3 b | –1.3 |
At1g25170 | FB011h10 | anthranilate synthase beta subunit and unknown protein (chimera) | –1.5 | 3.5 a |
At5g24150 | APD48h01 | squalene monooxygenase gene homolog | –1.5 | 4.5 a |
At5g39050 | APZ17a04 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.1 a |
At5g39050 | APD07c12 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.8 a |
At5g49730 | APD12g08 | similar to ferric-chelate reductase (FRO1) (Pisum sativum) | 1.0 | 3.1 a |
At5g47560 | APD40a08 | sodium/dicarboxylate cotransporter | 1.2 | 3.1 a |
At5g45490 | RZ193d07 | disease resistance protein -related | –1.5 | 3.2 a |
At1g58602 | FB058e07 | disease resistance protein (CC-NBS-LRR class), putative | –1.1 | 5.7 a |
At1g25141 | APD52f01 | F-box protein-related | 1.4 | 4.9 a |
At5g52060 | SQ046h08 | BAG domain containing protein | 1.2 | 3.2 a |
At3g48360 | RZ176d04 | expressed protein, MEL-26, Caenorhabditis elegans, U67737 | 3.3 | 5.7 a |
At1g61670 | FB095g08F | expressed protein, similar to membrane protein PTM1 precursor isolog | 1.2 | 3.4 a |
At5g52810 | RZ54b02 | expressed protein, contains similarity to ornithine cyclodeaminase | 1.3 | 4.0 a |
At4g09980 | RZ200e01 | expressed protein, m6A methyltransferase – Homo sapiens, | 1.3 | 5.0 a |
At5g03350 | APZ44c11 | expressed protein | 1.3 | 11.1 a |
– | APD41c08 | unknown | 1.2 | 3.3 a |
At5g61970 | RZL17g10 | signal recognition particle – related | 1.2 | –3.2 b |
At4g29190 | FB010d03 | expressed protein, zinc finger transcription factor | –1.4 | –3.2 b |
At5g53420 | SQ089h05 | expressed protein | –1.5 | –3.0 b |
At3g10480 | SQ162d12 | expressed protein | –1.6 | –3.6 b |
Arabidopsis Genome Initiative (AGI) numbers were assigned using Web site at http://www-biology.ucsd/labs/secchroeder/downloads/probe_agi_descr.txt and JCAA clone numbers at http://biochem.agr.nagoya-u.ac.jp/atgenome/. Putative function of each gene product was determined using the TAIR database (http://arabidopsis.org/). Induction fold of each gene in CMV(Y)-inoculated C24 (R) and Col-0 (S) at 6 h after inoculation is shown. Ratios of <1 were transformed to –1/ratio. Genes with induced or suppressed expression in R or S are divided by dashed line.
a Values represent statistically significant induction.
b Values represent statistically significant suppression.
AGI number | JCAA clone number | Putative function | Induction fold | |
R | S | |||
At5g61520 | FB090e02F | hexose transporter, putative | 3.0 a | –1.3 |
At5g42900 | SQ050c09 | expressed protein | 3.2 a | –1.5 |
At1g19570 | RZ69e11 | dehydroascorbate reductase, putative | –3.2 b | –1.4 |
At5g39190 | SQ019h11 | germin-like protein (GLP2a) | –3.6 b | 1.0 |
– | SQ049d12 | unknown | –3.8 b | –1.1 |
– | RZ165h01 | unknown | –3.3 b | –1.3 |
At1g25170 | FB011h10 | anthranilate synthase beta subunit and unknown protein (chimera) | –1.5 | 3.5 a |
At5g24150 | APD48h01 | squalene monooxygenase gene homolog | –1.5 | 4.5 a |
At5g39050 | APZ17a04 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.1 a |
At5g39050 | APD07c12 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.8 a |
At5g49730 | APD12g08 | similar to ferric-chelate reductase (FRO1) (Pisum sativum) | 1.0 | 3.1 a |
At5g47560 | APD40a08 | sodium/dicarboxylate cotransporter | 1.2 | 3.1 a |
At5g45490 | RZ193d07 | disease resistance protein -related | –1.5 | 3.2 a |
At1g58602 | FB058e07 | disease resistance protein (CC-NBS-LRR class), putative | –1.1 | 5.7 a |
At1g25141 | APD52f01 | F-box protein-related | 1.4 | 4.9 a |
At5g52060 | SQ046h08 | BAG domain containing protein | 1.2 | 3.2 a |
At3g48360 | RZ176d04 | expressed protein, MEL-26, Caenorhabditis elegans, U67737 | 3.3 | 5.7 a |
At1g61670 | FB095g08F | expressed protein, similar to membrane protein PTM1 precursor isolog | 1.2 | 3.4 a |
At5g52810 | RZ54b02 | expressed protein, contains similarity to ornithine cyclodeaminase | 1.3 | 4.0 a |
At4g09980 | RZ200e01 | expressed protein, m6A methyltransferase – Homo sapiens, | 1.3 | 5.0 a |
At5g03350 | APZ44c11 | expressed protein | 1.3 | 11.1 a |
– | APD41c08 | unknown | 1.2 | 3.3 a |
At5g61970 | RZL17g10 | signal recognition particle – related | 1.2 | –3.2 b |
At4g29190 | FB010d03 | expressed protein, zinc finger transcription factor | –1.4 | –3.2 b |
At5g53420 | SQ089h05 | expressed protein | –1.5 | –3.0 b |
At3g10480 | SQ162d12 | expressed protein | –1.6 | –3.6 b |
AGI number | JCAA clone number | Putative function | Induction fold | |
R | S | |||
At5g61520 | FB090e02F | hexose transporter, putative | 3.0 a | –1.3 |
At5g42900 | SQ050c09 | expressed protein | 3.2 a | –1.5 |
At1g19570 | RZ69e11 | dehydroascorbate reductase, putative | –3.2 b | –1.4 |
At5g39190 | SQ019h11 | germin-like protein (GLP2a) | –3.6 b | 1.0 |
– | SQ049d12 | unknown | –3.8 b | –1.1 |
– | RZ165h01 | unknown | –3.3 b | –1.3 |
At1g25170 | FB011h10 | anthranilate synthase beta subunit and unknown protein (chimera) | –1.5 | 3.5 a |
At5g24150 | APD48h01 | squalene monooxygenase gene homolog | –1.5 | 4.5 a |
At5g39050 | APZ17a04 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.1 a |
At5g39050 | APD07c12 | similar to anthocyanin 5-aromatic acyltransferase | –1.0 | 3.8 a |
At5g49730 | APD12g08 | similar to ferric-chelate reductase (FRO1) (Pisum sativum) | 1.0 | 3.1 a |
At5g47560 | APD40a08 | sodium/dicarboxylate cotransporter | 1.2 | 3.1 a |
At5g45490 | RZ193d07 | disease resistance protein -related | –1.5 | 3.2 a |
At1g58602 | FB058e07 | disease resistance protein (CC-NBS-LRR class), putative | –1.1 | 5.7 a |
At1g25141 | APD52f01 | F-box protein-related | 1.4 | 4.9 a |
At5g52060 | SQ046h08 | BAG domain containing protein | 1.2 | 3.2 a |
At3g48360 | RZ176d04 | expressed protein, MEL-26, Caenorhabditis elegans, U67737 | 3.3 | 5.7 a |
At1g61670 | FB095g08F | expressed protein, similar to membrane protein PTM1 precursor isolog | 1.2 | 3.4 a |
At5g52810 | RZ54b02 | expressed protein, contains similarity to ornithine cyclodeaminase | 1.3 | 4.0 a |
At4g09980 | RZ200e01 | expressed protein, m6A methyltransferase – Homo sapiens, | 1.3 | 5.0 a |
At5g03350 | APZ44c11 | expressed protein | 1.3 | 11.1 a |
– | APD41c08 | unknown | 1.2 | 3.3 a |
At5g61970 | RZL17g10 | signal recognition particle – related | 1.2 | –3.2 b |
At4g29190 | FB010d03 | expressed protein, zinc finger transcription factor | –1.4 | –3.2 b |
At5g53420 | SQ089h05 | expressed protein | –1.5 | –3.0 b |
At3g10480 | SQ162d12 | expressed protein | –1.6 | –3.6 b |
Arabidopsis Genome Initiative (AGI) numbers were assigned using Web site at http://www-biology.ucsd/labs/secchroeder/downloads/probe_agi_descr.txt and JCAA clone numbers at http://biochem.agr.nagoya-u.ac.jp/atgenome/. Putative function of each gene product was determined using the TAIR database (http://arabidopsis.org/). Induction fold of each gene in CMV(Y)-inoculated C24 (R) and Col-0 (S) at 6 h after inoculation is shown. Ratios of <1 were transformed to –1/ratio. Genes with induced or suppressed expression in R or S are divided by dashed line.
a Values represent statistically significant induction.
b Values represent statistically significant suppression.
Abbreviations
- CMV
cucumber mosaic virus
- ET
ethylene
- HR
hypersensitive response
- JA
jasmonic acid
- NLL
necrotic local lesion
- SA
salicylic acid
- TMV
tobacco mosaic virus.
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