SuperSAGE revealed different classes of early resistance response genes in Capsicum chinense plants harboring L3-resistance gene infected with Pepper mild mottle virus
We used SuperSAGE, an improved version of serial analysis of gene expression, to explore transcriptome changes early in the L3-mediated resistance response of pepper plants against a tobamovirus. Capsicum chinense plants homozygous for the L3 resistance gene were infected with virulent and avirulent strains of Pepper mild mottle virus (PMMoV). Plants were maintained at a temperature nonpermissive for the resistance gene to allow the viruses to spread, then transferred to a permissive temperature for 3 h and subsequently analyzed. In the incompatible reaction, we selected 152 SuperSAGE tags (each 26 nucleotides long) possibly corresponding to upregulated genes, and 84 tags for downregulated genes. Approximately 70% of tags had matching ESTs in the genus Capsicum, other genera within the Solanaceae and/or other families of plants. More than 90% of tags with EST matches could be annotated with either functionally characterized or uncharacterized proteins. We compared genes annotated by SuperSAGE tags and those annotated by partial cDNA that was obtained using the SuperSAGE tag sequences as rapid amplification of the cDNA ends-PCR primers. Of genes annotated by SuperSAGE tags, c. 90% were consistent with those annotated by longer cDNA sequences. We cloned 17 full-length cDNAs from different SuperSAGE tags and confirmed that these genes were upregulated during normal infection in the incompatible interaction. We identified several early resistance response genes including a Ran/TC4 protein and a β-oxidation multifunctional protein, indicating that SuperSAGE is a powerful tool for investigating plant–pathogen interactions.
KeywordsEarly response L3 PMMoV Resistance SuperSAGE Transcriptome
Plants resist pathogen attacks by activating a variety of defense mechanisms such as the hypersensitive response (HR), in which rapid cell death occurs locally at the infection site (Baker et al. 1997). The HR is frequently associated with increased expression of many defense-related genes, i.e., PR-protein genes such as PR1 and PR4b (Bol et al. 1990) and HR-related genes such as HSR203J and HIN1 (Pontier et al. 1999). These proteins are known to be regulated by particular signal molecules involved in defense responses including salicylic acid, jasmonic acid, and ethylene, and therefore have been used as biomarkers to analyze signaling pathways activated in a given plant–pathogen interaction (e.g., Kiba et al. 2006). In addition to the aforementioned defense-related genes, a number of pathogen-induced host genes have been isolated in the plant disease resistance response (e.g., Nakane et al. 2003). However, it is still important to identify more host genes that are specifically upregulated during the resistance response to elucidate the molecular mechanisms of plant disease resistance.
One of the best analyzed experimental systems in plant–virus interaction research is the pathosystem of Tobacco mosaic virus (TMV) and tobacco (Nicotiana tabacum) carrying the TMV-resistance gene, N. Studies have identified several important defense-related genes (e.g., Seo et al. 1995, 2000) in a synchronous defense response system, in which the temperature-sensitive N gene function is activated by shifting to a permissive temperature after TMV has propagated and spread in the inoculated leaves under a nonpermissive temperature (Weststeijn 1981). Although this system is quite powerful to analyze gene expression and biochemical changes during the resistance response, there are few suitable virulent virus isolates that propagate in the host but do not activate the resistance response. Therefore, it is difficult with this system to detect differences between the expression patterns of defense-related genes in compatible and incompatible interactions. A desirable system is one in which we can analyze closely related virulent and avirulent pathogens. A system meeting these requirements is comprised of Pepper mild mottle virus (PMMoV) isolates and Capsicum plants harboring the tobamovirus resistance gene, L3 (Berzal-Herranz et al. 1995; García-Luque et al. 1993; Hamada et al. 2002; Tsuda et al. 1998).
The L3 gene confers resistance against tobamoviruses in Capsicum plants and is accompanied by the induction of necrotic local lesions by an HR (Boukema 1984). L3-gene-mediated resistance is elicited by the coat protein (CP) of tobamoviruses (Berzal-Herranz et al. 1995; Gilardi et al. 2004). Analyses of chimeric and point-mutated viruses have indicated that various single or double amino acid changes in the PMMoV CP can overcome L3-gene-mediated resistance (Berzal-Herranz et al. 1995; Hamada et al. 2002; Tsuda et al. 1998). In our previous study, both the spatial and temporal patterns of defense-related gene induction were critical factors in restricting virus spread by L3-gene-mediated resistance (Hamada et al. 2005), suggesting that the extent of the early response to the viral elicitor is important in determining the overall host response.
To explore the molecular basis of L3-mediated resistance with special emphasis on the early responses, we attempted to identify host genes induced only by avirulent PMMoV isolates at early stages after the onset of the resistance response. We used a temperature shift procedure similar to that of the TMV-N gene system mentioned to obtain plant material that has synchronous resistance responses.
A number of methods have been used to isolate differentially expressed plant genes. We selected SuperSAGE, an improved version of serial analysis of gene expression (Matsumura et al. 2003, 2005), to profile mRNAs from infected Capsicum chinense plants. SuperSAGE is a powerful tag-based method that provides a quantitative profile of genes expressed in the biological material of interest. SuperSAGE gives 26-base tag sequences of expressed genes, which are almost twice as long as those obtained using the original SAGE technique. Because the longer tag sequences enable tag-to-gene annotation with higher specificity, it is especially useful for expression profiling in organisms in which little genome information is available (Coemans et al. 2005; Nasir et al. 2005). In this report, we describe the genes identified by SuperSAGE that are up- or downregulated during the early resistance response to PMMoV in C. chinense plants. These plant genes belong to different classes of genes; the involvement of some genes identified is not well known in the plant disease response. The importance of the genes identified here in the resistance response is discussed.
Materials and methods
Plants and viruses
C. chinense PI159236, a pepper that is homozygous for L3 (L3-Cc), was grown in pots containing commercial soil (Sakata Seed, Yokohama, Japan) in a plant growth room at 24°C with 16 h illumination per day. An infectious cDNA clone of PMMoV P1,2,3 pathotype, pPIW138N, was constructed by introducing an amino acid substitution, M138N, into the infectious cDNA clone of PMMoV-Iw (pathotype P1,2), as described previously (Hamada et al. 2007). The oligonucleotide primers used were as follows: 138NF (5′-CGTGGCACGGGAAATTACAATCAAGCTCTG-3′) and 138NR (5′-ATTGTAATTTCCCGTGCCACGAACTAACTC-3′). Purified PMMoV virions, namely, PIW and PIW-138N, were prepared from N. benthamiana plants inoculated with in vitro RNA transcripts from the cDNA clones pPIW and pPIW-138N, respectively, as previously described (Hamada et al. 2007).
Induction of synchronous resistance response
L3-Cc plants mechanically inoculated with purified PMMoV (0.2 μg/ml in 10 mM phosphate buffer, pH 7.2) were maintained in a growth chamber at 32°C for 7 days, then transferred to a growth chamber at 22°C. The inoculated leaves were harvested or observed at indicated times after transferring the plants to 22°C. Spread of the viruses was examined by hammer blot analysis as described previously (Kobayashi et al. 2002).
Generation of SuperSAGE libraries and data analysis
Total RNA was extracted from inoculated leaves 3 h after the temperature shift described. We isolated mRNA from total RNA using the PolyATract mRNA isolation system (Promega, Madison, WI, USA), according to the manufacturer’s protocol. SuperSAGE was carried out as described (Matsumura et al. 2003) using 5 μg mRNA starting material. Briefly, double-stranded cDNA synthesized from the mRNA with biotinylated oligo(dT) primer was digested with the restriction enzyme NlaIII. The biotin-labeled, digested cDNA fragments were collected using streptavidin-magnetic beads. Two linkers, each carrying an EcoP15I recognition site, were ligated to the end of cDNA fragments on the beads. Linker-ligated cDNA on the beads was digested with EcoP15I, and the released fragments (linker and 26–27 bp cDNA) were ligated after blunt-ending to form randomly paired ditags. These ditags were PCR amplified, and linker fragments were removed by NlaIII digestion and gel purification. Purified ditags (without linkers) were concatenated by ligation, and longer (>500 bp) concatemers were cloned into plasmids and sequenced. Extraction and counting of SuperSAGE tags and statistical analysis by a Monte–Carlo simulation method were performed using SAGE2000 software kindly provided by Dr. K. W. Kinzler, Johns Hopkins University.
Cloning of cDNA corresponding to SuperSAGE tags
To isolate longer cDNAs fragments adjacent to SuperSAGE tags, we performed 5′- and 3′-rapid amplification of the cDNA ends (RACE) with a GeneRacer kit (Invitrogen, Carlsbad, CA, USA) using total RNA from C. chinense leaves infected with PIW for 3 days. RACE PCR was performed using either GeneRacer 5′ or 3′ primers, together with the 22-nucleotide primers consisting of SuperSAGE tag sequences without common four-bases NlaIII sites. Full-length cDNA was amplified with gene-specific PCR primers that were designed based on the sequence data from 5′- and 3′-RACE analyses. The PCR products were cloned into a pSTBlue vector (Novagen, Madison, WI, USA) and sequenced.
RNA extraction, RT-PCR and real-time PCR analysis
RNA was extracted using the RNAqueous kit (Ambion, Austin, TX, USA) for RT-PCR and real-time PCR. RT-PCR was carried out using the PrimeScript 1st strand cDNA synthesis kit and ExTaq DNA polymerase (Takara, Otsu, Japan) with primer pairs shown in Table S1. Real-time PCR analysis was carried out using QuantiTect SYBR Green RT-PCR (Qiagen, ABI Prism 7000 sequence detection systems) and 100 ng of total RNA as template. The primers were designed based on sequence data from 3′- or 5′-RACE analysis of SuperSAGE-tagged genes (Table S1). For internal reference, 18S ribosomal RNA levels were measured in parallel.
Results and discussion
Synchronous HR-inducing system by temperature shift in pepper plants
Generation of SuperSAGE libraries and annotation of SuperSAGE tags
Summary of SuperSAGE libraries
Total no. of different tagsb
Total tag countsc
Specific tags to each libraryd
Tags common to both librariese
R = Sf
R or S ≥ 4g
3 > R/S > 1/3h
R/S ≥ 3i
S/R ≥ 3j
Summary for EST-guided annotation of SuperSAGE tags
Classification of SuperSAGE tags
R/S ≥ 3
S/R ≥ 3
With annotation/with EST (%)c
EST of other Solanaceaee
EST of other familiesf
Tags with no EST matches (% of total)g
Long cDNA clonedh
Matched annotation (%)i
Non-Capsicum Solanaceae ESTk
Functional annotation of SuperSAGE-tagged genes
R/S ≥ 3
S/R ≥ 3
Stress and aging
Functional categories of SuperSAGE tag annotations
Table 3 categorizes up- or downregulated genes in the resistance response corresponding to SuperSAGE tags as mentioned. It also includes an additional ten tags, which did not have homologous ESTs in the database but were annotated instead by partial cDNA cloning. Striking changes in the transcriptome after the onset of the resistance response presented here include (1) upregulation of genes involved in primary and secondary metabolism, (2) upregulation of signaling genes, stress- and aging-related genes and transporter genes, (3) up- and down-regulation of different photosynthetic genes, and (4) up- and downregulation of different ribosomal protein genes. Elicitation of primary and secondary metabolism was demonstrated in potatoes treated with a fungal elicitor and is a common feature of the resistance response (Nakane et al. 2003). Upregulation of signaling, stress/aging and transporter genes has been documented in several studies (for review, see Farmer 2000). Photosynthetic genes have been reported to be generally downregulated (Matsumura et al. 2003). However, we observed upregulation of a small set of photosynthetic genes, suggesting that functional and structural changes in photosynthetic machinery occur during the defense response. Similarly, translational machinery could also change its structure and function during the resistance response. Further analyses of each photosynthetic and ribosomal protein would determine the functional and structural alteration in these supermolecular machineries that are essential for plant cells.
Full-length cDNA cloning, expression analysis and annotation of SuperSAGE-tagged genes
Partial list of SuperSAGE-tagged genes that were upregulated under normal avirulent Pepper mild mottle virus infection conditions
Cu/Zn superoxide dismutase
NADP-dependent malic enzyme
Multidrug resistance-associated protein 2
CONSTANS-interacting protein 2b
MATE family transporter
C2 domain-containing protein
4-Methyl-5(B-hydroxyethyl)-thiazol monophosphate biosynthesis enzyme
Glyoxysomal fatty acid beta-oxidation multifunctional protein
26S Proteasome subunit, RPN2a
Erwinia carotovora subsp. atroseptica response gene 1, phosphate-induced protein 1 (phi-1)
Zinc finger (C3HC4-type RING finger) family protein
For validating cDNA amplification by RACE using tag primers, we took a cautious approach for full-length cDNA cloning: (1) determination of both end sequences by 3′- and 5′-RACE, (2) amplification of almost the entire mRNA sequence using gene-specific primers designed from both end sequences, and (3) confirmation of the presence of SuperSAGE tag sequence within the amplified sequence. Thus, we isolated and confirmed the annotation of 17 SuperSAGE-tagged genes (Table 4). Supplementary table S2 lists the accession numbers of these genes and of their homologues referred to in their annotation.
Proteases, such as those encoded by SS12 and SS21, have roles in resistance signaling and HR (Gilroy et al. 2007; Hatsugai et al. 2004; Kruger et al. 2002; Xia et al. 2004). The NADP-dependent malic enzyme (SS14) was highly upregulated in both temperature-shift experiments and in a normal incompatible interaction. This gene and NADPH oxidoreductase (SS48) are reportedly upregulated by treatment of potato tubers with an oomycete elicitor (Nakane et al. 2003), indicating that they are commonly upregulated during the resistance response, at least in solanaceous plants. A recent study has shown that aconitase (SS84) has a role in the regulation of oxidative stress resistance via regulation of superoxide dismutase (SS13) gene expression (Moeder et al. 2007). Hsp90 (SS18) is a well-studied molecular chaperone and reportedly has roles in resistance signaling (Takahashi et al. 2003). Recent studies have shown that transporters have roles in resistance (for review, see Rea 2007). The EST-guided annotation of SuperSAGE tags suggested that five transporters including SS29 and SS45 were upregulated, supporting a crucial role for some transporters in plant defense. Studies have shown that various C2 domain-containing proteins (SS52) are induced by biotic and abiotic stresses (Kim et al. 2003; Ouelhadj et al. 2006). One of them, Arabidopsis BAP, was shown to negatively regulate defense responses (Yang et al. 2006, 2007), but others still need to be functionally characterized. Vitamin B was reported to activate plant defense mechanisms (Ahn et al. 2005, 2007), and the induction of an enzyme involved in its biosynthesis (SS62) during the resistance response is presented here for the first time. A subunit of the 26S proteasome (SS85) and a ring-finger protein (SS124) that are involved in the ubiquitin–proteasome pathway are known to be important for defense responses (Goritschnig et al. 2007). SS122 encodes a protein similar to one induced by bacterial infection in potato (Dellagi et al. 2000) and also to one induced by phosphate in tobacco (Sano et al. 1999).
Unlike those mentioned, the fatty acid β-oxidation multifunctional protein (SS77) and Ran/TC4 (SS86) have not been described in the context of disease resistance. Fatty acid β-oxidation is a process of energy production from storage lipids and is known to be activated during seed germination (Eastmond and Graham 2000; Rylott et al. 2006). The significance of the upregulation of a key enzyme in this process during the resistance response remains unclear. However, a possible role of this enzyme in the resistance response is the biosynthesis of lipid-derived signaling molecules, which reportedly have important roles in plant disease resistance (Shah 2005). Ran is a member of the Ras GTPase superfamily and is well conserved throughout the eukaryotes. Ran is known to have roles in nucleocytoplasmic macromolecular transport and spindle assembly during cell division (Joseph 2006). Overexpression of Ran in transgenic plants resulted in altered cell division and development (Wang et al. 2006), but it has not been analyzed in the context of disease resistance. Recent studies have identified a GTPase-activating protein of Ran, RanGAP, as an essential interaction partner of the virus resistance gene Rx (Sacco et al. 2007; Tameling and Baulcombe 2007). The Ran system, including Ran itself, RanGAP and a Ran guanine nucleotide exchange factor, might have a crucial role in resistance signaling or pathogen perception in plants.
We have identified in this study a number of SuperSAGE tags with reliable tag-to-gene annotations, which were up- or downregulated during the early resistance response to PMMoV in pepper plants harboring the L3 resistance gene. Because C. chinense, which we used in this study, is not a widely cultivated crop, genomic information for this species is not available. Nevertheless, SuperSAGE enabled us to identify a number of early resistance response genes with efficient EST-guided annotation procedures. Moreover, cloning of full-length cDNA can be carried out following SuperSAGE because the tag sequence is long enough to use as a PCR primer for 5′- and 3′-RACE cloning. We successfully isolated several full-length cDNA clones using SuperSAGE tag-derived primers and found some new resistance-associated genes. The functions of these genes in the resistance response are under investigation. Our results emphasize that SuperSAGE is a powerful tool to analyze plant–pathogen interactions in wide variety of crop plants, including those lacking genomic information.
We thank Dr. K. W. Kinzler for SAGE2000 software, Harumi Takahashi and Kazue Obara for technical assistance and all members of IBRC for fruitful discussion. This study was supported in part by the Iwate Prefecture Government and in part by a grant-in-aid for Scientific Research (C) (18580047) from the Japan Society for the Promotion of Science.
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