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Cloning of a putative hypersensitive induced reaction gene from wheat infected by stripe rust fungus
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Gene 407 (2008) 193 – 198 www.elsevier.com/locate/gene
Cloning of a putative hypersensitive induced reaction gene from wheat infected by stripe rust fungus Xiu-Mei Yu1 , Xiu-Dao Yu, Zhi-Peng Qu, Xin-Jie Huang, Jun Guo, Qing-Mei Han, Jie Zhao, Li-Li Huang, Zhen-Sheng Kang⁎ College of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A and F University, Yangling, Shaanxi, 712100, PR China Received 17 June 2007; received in revised form 12 September 2007; accepted 5 October 2007 Available online 13 October 2007
Abstract The hypersensitive response (HR) is one of the most efficient forms of plant defense against biotrophic pathogens and results in localized cell death and the formation of necrotic lesions. In this study, a novel putative hypersensitive induced reaction (HIR) gene from wheat leaves infected by incompatible stripe rust pathogen CY23, designated as Ta-hir1, was identified by using rapid amplification of cDNA ends (RACE). Ta-hir1 encodes 284 amino acids, with a predicted molecular mass of 31.31 KDa. A phylogenetic analysis showed that Ta-hir1 was highly homologous to Hv-hir1 from barley at both cDNA and deduced amino-acid levels. Amino-acid sequence analysis of the wheat HIR protein indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain typical for stomatins which served as a negative regulator of univalent cation permeability, especially for potassium. The expression profile of the Ta-hir1 transcript detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real time-PCR), respectively, showed that the highest expression occurred 48 h post inoculation (hpi), which is consistent with our previous histopathology observations during the stripe rust fungus–wheat incompatible reaction. © 2007 Elsevier B.V. All rights reserved. Keywords: RACE; Sequence characterization; Suppression subtractive hybridization; Transcripts expression profiles
1. Introduction Hypersensitive response (HR), one of the most efficient forms of plant defense against biotrophic pathogens, is usually defined as ‘the rapid death of plant cells in association with the restriction of pathogen growth’ (Goodman and Novacky, 1994). It occurs in resistant plant cultivars in response to incompatible viruses, fungi, bacteria (Lam et al., 2001; Vleeshouwers et al., 2000; Baker et al., 1993), and causes a series of biochemical processes that result in Abbreviations: cDNA, DNA complementary to RNA; HR, hypersensitive response; HIR, hypersensitive induced reaction; RACE, rapid amplification of cDNA ends; SPFH, Stomatins, Prohibitins, Flotillins and HflK/C; RT-PCR, reverse transcriptase-polymerase chain reaction; real time-PCR, real timepolymerase chain reaction; bp, base pair(s); hpi, hours post inoculation; SSH, suppression subtractive hybridization; BLAST, basic local alignment search tool. ⁎ Corresponding author. Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China. Tel.: +86 02987091312; fax: +86 02987091312. E-mail address: [email protected] (Z.-S. Kang). 1 Present address: College of Life Science, Agriculture University of Hebei, Baoding, Hebei, 071001, PR China. 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.10.010
cell death in the adjacent living cells thus causing an adverse environment for the pathogens. Elicitors of HR cause intracellular ion influx, membrane dysfunction, and increase in the generation of reactive oxygen species and salicylic acid (Heath, 1998, 2000). A cascade of events triggered within the signaling pathway results from the interaction of ligands originating from pathogens with intra or extra-cellular plant receptors in an incompatible reaction which will further lead to modifications of plant cell walls, accumulation of pathogenesis-related (PR) proteins or phytoalexins, and accumulation of oxidized phenolic compounds that can cause browning of the dead cells. In addition, the HR typically induces systemic changes throughout the plant, including induced resistance to a variety of previously compatible pathogens (Kombrink and Sossich, 1995). Karrer et al. (1998) used a functional screening method to isolate several genes whose products elicited the HR in tobacco challenged by the tobacco mosaic virus. One of the isolated cDNAs, NG1, was able to induce the formation of lesions and expression of PR-2 protein, an acid β-glucanase. Three maize hypersensitive induced reaction (HIR) genes, Zm-hir1, Zm-hir2
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and Zm-hir3, have been isolated based on their amino-acid homology to the tobacco NG1 sequence (Nadimpalli et al., 2000). Studies on the regulation of the Zm-hir3 transcript indicated higher level of expression of this gene in the maize disease lesion mimic mutant Les9 when compared to the wild type, and reduced expression in maize line which suppressed the Les9 lesion mimic phenotype. Many other data also suggested participation of the maize HIR genes in HR. In addition to three Zm-hir genes, Nadimpalli et al. (2000) also isolated and identified four genes closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4) and one to stomatins (Zm-stm1). They further used PHI-BLAST searches combined with multiple sequence alignments and neighbor-joining tree construction to show that maize HIR proteins, along with prohibitins and stomatins, formed the superfamily PID (proliferation, ion, and death). Prohibitins are involved in proliferation and cell cycle control, stomatins in ion channel regulation, and HIR genes are involved in cell death. Although members of this superfamily are involved in diverse functions, their structural similarity suggested a conserved molecular mechanism, which Nadimpalli et al. (2000) postulated to be involved in ion channel regulation. Recently, four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hvhir3, Hv-hir4 were identified (Rostoks et al., 2003). Sequence analyses of the barley HIR proteins indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain (Tavernarakis et al., 1999). Barley HIR genes were expressed in all organs and development stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression thus implicating HIR genes in the induction of HR (Rostoks et al., 2003). In a previous study, we isolated from an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis, a cDNA fragment, WSRP1878, homologous to the barley Hv-hir1 gene (Yu et al., 2007). In the present study, we cloned a full length wheat HIR gene using rapid amplification of cDNA ends (RACE). Based on the sequence of the WSRP1878 fragment and designated it as Ta-hir1. The characteristics of Ta-hir1 cDNA and deduced amino-acid sequences were identified by a series of bioinformatic softwares. In addition, the expression profile of Ta-hir1 at different post-inoculation time points was investigated by reverse transcriptase-polymerase chain reaction (RTPCR) and real time-polymerase chain reaction (real time-PCR). 2. Materials and methods 2.1. Plant materials and inoculation Wheat (Triticum aestivum L.) cultivar Suwon11 and stripe rust race CY23 were the biological materials used for the full length cloning of the wheat HIR1 gene and its expression analysis. Suwon11 was presumed to contain a stripe rust resistant gene YrSu (Cao et al., 2003; Li et al., 2006). Plants were grown and maintained as described in Kang and Li (1984). Freshly collected urediospores were applied with a paintbrush to the surface of the primary leaf of seven-day old wheat seedlings.
After inoculation, the plants were kept at high humidity in the dark for 24 h and followed by a regular day-night cycle in a growth chamber. A control inoculation was made with sterile water. Wheat leaves were excised at 0, 24, 36, 48, 60, 72 and 96 hour post-inoculation (hpi), and quickly frozen in liquid nitrogen and stored at − 80 °C prior to extraction of total RNA. 2.2. Total RNA isolation and SSH Isolation of total RNA was carried out according to the instructions of the RNeasy Plant Mini kit (Qiagen, Germany). Total RNA for SSH and RACE analyses was extracted from wheat leaves at 24, 48 and 72 hpi, while RNA for gene expression analysis was obtained from all sampling points. DNase I treatment was applied to remove contaminating genomic DNA. SSH was done using the PCR-Select™ cDNA Subtraction kit (Clontech, USA) according to the manufacturer's instructions. RNA from wheat leaves infected by P. striiformis was used as the tester, while RNA from mock-inoculated leaves was used as the driver. 2.3. Analysis of SSH library After two rounds of subtractive hybridization and two rounds of suppressive PCR, PCR products were ligated into the pGEM-T Easy Vector (Promega, USA) to construct an incompatible SSH cDNA library (Yu et al., 2007). To identify wheat differentially expressed genes after infection by the stripe rust fungus, all positive clones from the library were subjected to sequencing, clustering, BLAST alignment, functional annotation and classification into different categories (unpublished data). 2.4. Rapid amplification of cDNA ends (RACE) Through BLAST searches, a 479 bp cDNA, WSRP1878, was identified and showed the highest similarity to the HIR protein 1 from Hordeum vulgare. To obtain a full-length cDNA of the wheat HIR1 gene, a pair of gene specific primers (forward primer: 5′-CAAGAGGGCTGAAGGTGAGGCAGAATCG-3′, reverse primer: 5′-TTTCCAACACTCC TCAAACCATCCTGCC-3′) was designed based on WSRP1878 sequence and synthesized (Shanghai GeneCore BioTechnologies Co., Ltd.). 3′and 5′ RACE were performed using a SMART™ RACE cDNA Amplification Kit (Clontech, USA). PCRs were done in a PTC-200 thermocycler (MJ Research, USA) with the following parameters: 5 cycles of 95 °C for 30 s, 72 °C for 3 min, 5 cycles of 95 °C for 30 s, 70 °C for 30 s, 72 °C for 3 min, 25 cycles of 95 °C for 30 s, 68 °C for 30 s, 72 °C for 3 min, then final extension at 72 °C for 10 min. PCR products were gel purified and cloned into pGEM-T Easy Vector (Sambrook et al., 1989). An ABI PRISM 3130XL Genetic analyzer (Applied Biosystems, USA) was used to determine the nucleotide sequence of the positive clones. 2.5. Sequence analysis and phylogenetic analysis DNA sequence data were analyzed using BLAST (http:// www.ncbi.nlm.nih.gov/blast/), CAP3 Sequence Assembly Program (http://pbil.univ-lyon1.fr/cap3.php), ORF Finder (http://
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www.ncbi.nlm.nih.gov/gorf/gorf.html), InterProScan (http:// www.ebi.ac.uk/InterProScan/) and PROSITE (http://au.expasy. org/prosite/) network services. The alignment of the deduced protein sequences and phylogenetic tree were computed using DNAMAN and ClustalX version1.81 program employing standard parameters, respectively. 2.6. Reverse Transcriptase-PCR (RT-PCR) analysis Wheat 18S rRNA (GenBank accession no. AY049040) and tubulin (GenBank accession no. U76558) genes were used as reference genes. Primer sequences used in RT-PCR analysis were as follows: wheat 18S rRNA gene forward primer: 5′CTTCTTAGAGGGACTATGGC-3′, reverse primer: 5′-TACGGAAACCTTGTTACGAC-3′; tubulin gene forward primer: 5′CTCCTTCCCCATTTCGC-3′, reverse primer: 5′-CCAGAGCCAGTTCCACCT-3′; wheat Ta-hir1 gene forward primer: 5′CTGGCCTTCTCTGAGAATGT-3′, reverse primer: 5′GGAGCTGACCATCTCGTATC-3′. PCR was performed with a PCR Master Mix Kit (Takara, Japan) using 2 μl (10 × dilution) of first strand cDNA as template and 0.2 μmol/L of each primer (final concentration) in a final volume of 25 μl. PCR was performed on a PTC-200 thermocycler (MJ, USA) with an initial denaturation step at 95 °C 10 s, followed by 30 or 35 cycles of denaruration at 95 °C 5 s, annealing and extension at 60 °C 10 s. The PCR products were detected on 1% agarose gel.
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CYCLER∣∣quantitative PCR detection system (Cepheid, USA). Wheat 18S rRNA gene was used as a standard control in the real time-PCR. Primer and TaqMan probe sequences for wheat Tahir1 gene and the reference gene are as follows: wheat 18S rRNA gene forward primer: 5′-CGTCCCTGCCCTTTGTACAC-3′, reverse primer: 5′-AACACTTCACCGGACCATTCA3′, probe sequence: 5′-FAM-CCGCCCGTCGCCCTACCGTAMRA-3′; wheat Ta-hir1 gene forward primer: 5′-GCCATG A AT G A G AT C A AT G C A - 3 ′ , r e v e r s e p r i m e r : 5 ′ TTCGGCCTCGGCTTTCT-3′, probe sequence: 5′-FAMAGTGGCTGCCAGCCTCATCCTAGC-TAMRA-3′. With the exception of the addition of 0.04 μmol/L TaqMan probe to the reaction system, PCR amplification and the thermal cycling conditions were the same as those for RT-PCR. Quantification of the target gene was assessed using relative standard curves. In our study, the plasmid with the Ta-hir1 gene was used to prepare standard curves and target gene quantity being determined from the standard curve. Real time-PCR experiments were done in triplicate, and the Ta-hir1 gene transcript level was normalized to the average amount of 18S rRNA transcript by expressing it as a percentage. Normalized values of three replicated experiments were used to calculate the average amount of a wheat Ta-hir1 gene transcript. 3. Results 3.1. Characterization of Ta-hir1 cDNA
2.7. Real time-PCR analysis Expression of the Ta-hir1 gene in wheat leaves was further analyzed by real time-PCR using TaqMan probes in a SMART
Following 3′ and 5′ RACE analyses, two DNA fragments around 600 and 1000 bp, respectively, were detected on 1% agarose gel. Following plasmid construction, transformation
Fig. 1. Comparison of the predicted amino acid sequences of HIR1 gene of barley, wheat and maize. Amino acid sequence of wheat was deduced from the Ta-hir1 gene. Amino acid substitutions were marked in grey. The consensus amino acid sequences are highlighted in black. Hv, Hordeum vulgare (GenBank accession no. AY137511), Ta, Triticum aestivum (GenBank accessession no. EF514209), Zm, Zea mays (GenBank accessession no. AF236373).
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Fig. 2. Phylogenetic tree of higher plant SPFHs. Branches of the neighbor-joining phylogenetic tree were labeled with species name (Hv — Hordeum vulgare, Os — Oryza sativa, Zm — Zea mays, Ta — Triticum aestivum, Pc — Pennisetum ciliare, Ca — Capsicum annuum, Cs — Cucumis sativus and Lj — Lotus japonicus) followed by the gene name and GenBank accession number.
and cDNA clones sequencing, 501 bp for 3′RACE and 944 bp for 5′RACE fragments were obtained by removing vector and adapter sequences. Furthermore, CAP3 software was used to combine the two fragments into an 1132 bp consensus sequence including a poly-A signal region in the 3′-UTR. BLAST analysis showed that the 1132 bp sequence shared high similarity (93%) to Hv-hir1 (GenBank accession no. AY137511) (data not shown). The region from 77 to 931 nucleotides was the open reading frame (ORF) encoding a polypeptide of 284 amino acids. Moreover, the sequence surrounding the start codon, ACCATGG, agreed with that of the Kozak consensus initiator ANNATGG, which was identical to the sequence reported for Hv-hir1. The deduced molecular mass of the peptide was 31.31 KDa with a predicted pI of 5.29. Based on the strong identity to barley Hv-hir1, we designated the wheat 1132 bp sequence as Ta-hir1 (GenBank accession no. EF514209). A comparison of the deduced amino acid sequences of HIR1 genes from barley, maize and wheat was performed using DNAMAN. The result showed strong homology among these HIR1 sequences, especially for Hv-hir1 and Ta-hir1 (only two amino acid residues different) (Fig. 1). All available plant HIR sequences plus Zm-phb and Zm-stm were used to construct a neighbor-joining tree (Fig. 2). These results demonstrated a high degree of similarity of the HIR1, HIR2 and HIR3 groups across the species analyzed, while Zm-phb and Zm-stm also showed high degree of similarity with HIR3 and HIR4. Ta-hir1 grouped together with the proteins encoded by Hv-hir1, Zm-hir1 and Oshir1, whereas, Hv-hir2, Pc-hir and Zm-hir2 were clustered together, and Hv-hir3, Ca-hir and Zm-hir3 were in another group. The results suggested a high level of conservation among HIR1, HIR2, HIR3 and Zm-phb from different species. The protein conserved domain search at the EBI web page indicated that Ta-hir1 gene contained the signature of stomatins, but no apparent transmembrane region or signal peptide sequences (InterPro IPR001107 family). Additionally, results predicted from PROSITE showed that Ta-hir1 protein contained
an N-myristoylation site, protein kinase C, tyrosine kinase and casein kinase II phosphorylation sites, which were absolutely consistent with the Hv-hir1 protein. 3.2. RT-PCR analysis To determine the expression profile of wheat Ta-hir1gene at each time point after inoculation, RT-PCR was performed in which the amount of total RNA for each time point was standardized to contain equivalent amounts of wheat 18S rRNA and tubulin genes. Fig. 3 showed the detection results of RTPCR for Ta-hir1, 18S rRNA and tubulin genes in wheat leaves infected by stripe rust fungus at different time points. Wheat Tahir1 gene showed elevated amount of transcripts from 24 until 48 hpi at which time the maximal expression level was reached. From 48 to 72 hpi, the expression of wheat Ta-hir1 began to decrease steadily with an apparent increase at 96 hpi. The expression of 18S rRNA and tubulin of wheat leaves sampled in our study showed almost the same level of transcripts during the time-course experiment. 3.3. Real time-PCR analysis To confirm the reliability and reproducibility of RT-PCR analysis results and to accurately quantify the transcripts of Tahir1 in the different samples, real time-PCR was performed. The
Fig. 3. RT-PCR analysis of Ta-hir1 in wheat leaves infected by stripe rust fungus at different time points. Numbers in parentheses indicate PCR cycles.
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Fig. 4. Real time-PCR analysis of expression profiles of Ta-hir1 gene in wheat leaves infected by stripe rust fungus at different time points. The amount of transcript was normalized to 18S rRNA genes and was expressed as the percentage of the reference gene transcripts. Error bars represent the variation among three independent replications.
expression profile of Ta-hir1 in wheat leaves after inoculation is shown in Fig. 4. The y-axis indicates the amount of wheat Tahir1 transcript that was normalized to 18S rRNA gene and was expressed as the percentage of the reference gene, and the x-axis indicates the different sampling points. Real time-PCR analysis showed that Ta-hir1 of wheat was strongly up-regulated as early as 24 h after infection with P. striiformis race CY23. The maximum induction occurred at 48 hpi, whose transcripts were 18.77 fold over that of the control (0 h). From 48 to 72 hpi, the accumulation of transcripts decreased steadily and was followed by a slightly increase at 96 hpi. However, the amount of transcripts at every time point was much higher than that of control. The RT-PCR and real time-PCR results showed a consistent expression profile and that a maximal accumulation of wheat Ta-hir1 gene transcripts occurred at 48 hpi. 4. Discussion In the present study, we initially identified a gene fragment isolated from an incompatible SSH library of wheat leaves infected by the stripe rust fungus which showed a high similarity to the barley Hv-hir1 gene. In order to understand whether it would be involved in HR of wheat, a full-length cDNA sequence of 1132 bp with a complete ORF was isolated from infected wheat leaves using the RACE technique. We designated it as Ta-hir1 because many characteristics of this gene predicted by different bioinformatic softwares were highly similar to the deduced results of the barley Hv-hir1 gene. Studies on HIR proteins have been performed on various plant species, such as barley, maize, rice and cucumber (Rostoks et al., 2003, Nadimpalli et al., 2000, De los Reyes et al., 2003, Kim et al., 2004). The length of HIR proteins either from monocotyledons or dicotyledons ranges from 283 to 288 aa. Moreover, the neighbor-joining trees of all available HIR sequences (before August 15th, 2006) showed a high degree of relationship among the HIR1, HIR2, and HIR3 groups across the species analyzed. Amino-acid sequence analysis of the HIR proteins indicated the presence of the characteristic SPFH protein domain (Nadimpalli et al., 2000, Rostoks et al., 2003). In the present study, wheat Ta-hir1 gene possessed the stomatins domain which was thought to act as a negative regulator of
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univalent cation permeability, especially for potassium. Stomatins has a single membrane-spanning region near its N terminus, while wheat Ta-hir1 and barley Hv-hir1, Hv-hir2, Hv-hir3 genes did not have apparent transmembrane regions. However, the PROSITE search indicated that the N-termini of these proteins had an N-myristoylation motif and thus, these HIR proteins could be anchored to membranes (Rostoks et al., 2003). Taken together, the molecular function and mode of action for the wheat Ta-hir1 need to be further evaluated in the incompatible interaction between wheat and the obligate stripe rust pathogen. Two different methods were utilized to characterize the expression profile of the Ta-hir1 gene during the infection timecourse. RT-PCR was a method of endpoint detection, whilst real time-PCR monitor fluorescence emitted during the reaction as an indicator of the amount of amplicon produced after each PCR cycle (Heid et al., 1996). For the RT-PCR, we selected two control genes, the tubulin and 18S rRNA genes, which have been reported to be consistently expressed in Arabidopsis (Czechowski et al., 2005), to eliminate RNA differences among the different samples and thus avoiding unreliable results. To ensure the reliability of control genes, we selected 18S rRNA gene in wheat which had been used in a few previous studies to value the expression of targeted gene in real time-PCR (Scofield et al., 2005). The results from the two detection methods were consistent, and the maximal accumulation of wheat Ta-hir1 transcripts occurred at 48 h post inoculation. In addition, real time-PCR enabled a quantification of the transcripts. Besides Ta-hir1, two additional cDNAs sequences with complete ORF were obtained via RACE analysis. All three genes encoded 284 amino acids, and were highly similar to the barley Hv-hir1 gene. Their deduced amino acids were absolutely consistent, but there were important differences in sequences length and 5′nucleotide sequences. The longest sequence (1212 bp) had 80 nucleotides more than the shortest one (1132 bp) and 49 bases more than the intermediate size fragment (1163 bp). At present, only the cDNA sequence with 1132 bp was submitted to the GenBank due to its highest similarity to the barley Hv-hir1 gene. Differences among the three nucleotide sequences may result from alternative splicing or different transcription initiation sites. Additionally, short-cut phenomenon which was caused by poor mRNA or degradation of mRNA by RNase H, particular secondary structures of mRNA and inappropriate conditions for amplification (Schaefer, 1995; Zhang and Frohman, 1997), often make it difficult to obtain complete 5′RACE fragments. Another DNA fragment of 750 bp was also obtained during the 5′RACE amplification. This fragment showed high homology to Hv-hir1, but was 250 bp shorter than the real and complete amplification product. This suggests strongly that the 750 bp fragment results from a kind of short-cut phenomenon. These results reinforce the needs for high quality mRNA and optimal conditions for amplification of full length sequence when using RACE. Our previous histopathological observations (unpublished data) showed that in the incompatible interactions between wheat cultivar Suwon11 and stripe rust race CY23, the host cells in the infection site in the inoculated leaves started to become necrotic 24 hpi, and the numbers of necrotic host cells at each
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infection site increased until 60 hpi. Meanwhile proliferation of stripe rust fungus was markedly inhibited in the host leaves. These results seem to coincide with the expression pattern of Tahir1 transcripts in the incompatible interaction between wheat cultivar Suwon11 and stripe rust race CY23. Previous studies showed that some HIR genes were over-expressed in most of the disease lesion mimic mutation lines, which implied the vital role of HIR gene in HR (Nadimpalli et al., 2000; Rostoks et al., 2003). The numbers of wheat Ta-hir1 transcripts at each time point of post-inoculation were much higher than that of noninoculated control. We can postulate that wheat Ta-hir1 plays an important role in wheat protection against incompatible stripe rust pathogens. Functional characterization of the candidate wheat HIR gene Ta-hir1 is in progress. Acknowledgements This study was supported financially by the National Basic Research Program of China (No. 2006CB101901), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (NO.2005295), the Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education of China (No.200558), the Nature Science Foundation of China (No.30671350), and the 111 Project from Ministry of Education of China (B07049). References Baker, C.J., Orlandi, E.W., Mock, N.M., 1993. Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cells. Plant Physiol. 102, 1341–1344. Cao, Z.J., Jing, J.X., Wang, M.N., et al., 2003. Relation analysis of stripe rust resistance gene in wheat important cultivar suwon 11, suwon 92 and hybrid 46. Acta Bot. Boreal-Occident. Sin. 23, 64–68 (In Chinese). Czechowski, T., Stitt, M., Altmann, T., et al., 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17. Goodman, R.N., Novacky, A.J., 1994. The hypersensitive reaction in plants to pathogens. A resistant phenomenon. American Phytopathological Society, St. Paul, Minn., p. 244. De los Reyes, B.G., Morsy, M., Gibbons, J., et al., 2003. A snapshot of the low temperature stress transcriptome of developing rice seedlings (Oryza sativa L.) via ESTs from subtracted cDNA library. Theor. Appl. Genet. 107, 1071–1082. Heath, M.C., 1998. Apoptosis, programmed cell death and the hypersensitive response. Eur. J. Plant Pathol. 104, 117–124.
Heath, M.C., 2000. Hypersensitive response-related death. Plant Mol. Biol. 44, 321–334. Heid, C.A., Stevens, J., Livak, K.J., et al., 1996. Real time quantitative PCR. Genome Res. 6, 986–994. Kang, Z.S., Li, Z.Q., 1984. Discovery of a normal T. type new pathogenic strain to Lovrin10. Acta Cllegii Septentrionali Occidentali Agriculturae 4, 18–28 (in Chinese). Karrer, E.E., Beachy, R.N., Holt, C.A., 1998. Cloning of tobacco genes that elicit the hypersensitive response. Plant Mol. Biol. 36, 681–690. Kim, M.S., Kim, Y.C., Cho, B.H., 2004. Gene expression analysis in cucumber leaves primed by root colonization with pseudomonas chlororaphis O6 upon challenge-inoculation with Corynespora cassiicola. Plant biol. (Stuttg.) 6, 105–108. Kombrink, E., Somssich, I.E., 1995. Defense responses of plants to pathogens. Adv. Bot. Res. 21, 2–33. Lam, E., Kato, N., Lawton, M., 2001. Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848–853. Li, Z.F., Xia, X.C., Zhou, X.C., et al., 2006. Seedling and slow rusting resistance to stripe rust in Chinese common wheat. Plant Dis. 90, 1302–1312. Nadimpalli, R., Yalpani, N., Johal, G.S., et al., 2000. Prohibitins, stomatins, and plant-disease response genes compose a protein superfamily that controls cell proliferation, ion channel regulation, and death. J. Biol. Chem. 275, 29579–29586. Rostoks, N., Schmierer, D., Kudrna, D., et al., 2003. Barley putative hypersensitive induced reaction genes: genetic mapping, sequence analyses and differential expression in disease lesion mimic mutants. Theor. Appl. Genet. 107, 1094–1101. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. Schaefer, B.C., 1995. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends. Anal. Biochem. 227, 255–273. Scofield, S.R., Huang, L., Brandt, A.S., et al., 2005. Development of a virusinduced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol. 138, 2165–2173. Tavernarakis, N., Driscoll, M., Kyrpides, N.C., 1999. The SPFH domain: implicated in regulating targeted protein turnover in stomatins and other membrane-associated proteins. Trends Biochem. Sci. 24, 425–427. Vleeshouwers, V.G.A.A., Dooijeweert, W.V., Govers, F., et al., 2000. The hypersensitive response is associated with host and non host resistance to Phytophthora infestans. Planta 210, 853–864. Yu, X.M., Yu, X.D., Qu, Z.P., et al., 2007. Construction of wheat SSH cDNA library induced by Puccinia striiformis and analysis of expressed sequence tags. Acta Phytopathol. Sinica 37, 50–55 (In Chinese). Zhang, Y., Frohman, M.A., 1997. Using rapid amplification of cDNA ends (RACE) to obtain full-length cDNAs. Methods Mol. Biol. 69, 61–87.
Gene 407 (2008) 193 – 198 www.elsevier.com/locate/gene
Cloning of a putative hypersensitive induced reaction gene from wheat infected by stripe rust fungus Xiu-Mei Yu1 , Xiu-Dao Yu, Zhi-Peng Qu, Xin-Jie Huang, Jun Guo, Qing-Mei Han, Jie Zhao, Li-Li Huang, Zhen-Sheng Kang⁎ College of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A and F University, Yangling, Shaanxi, 712100, PR China Received 17 June 2007; received in revised form 12 September 2007; accepted 5 October 2007 Available online 13 October 2007
Abstract The hypersensitive response (HR) is one of the most efficient forms of plant defense against biotrophic pathogens and results in localized cell death and the formation of necrotic lesions. In this study, a novel putative hypersensitive induced reaction (HIR) gene from wheat leaves infected by incompatible stripe rust pathogen CY23, designated as Ta-hir1, was identified by using rapid amplification of cDNA ends (RACE). Ta-hir1 encodes 284 amino acids, with a predicted molecular mass of 31.31 KDa. A phylogenetic analysis showed that Ta-hir1 was highly homologous to Hv-hir1 from barley at both cDNA and deduced amino-acid levels. Amino-acid sequence analysis of the wheat HIR protein indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain typical for stomatins which served as a negative regulator of univalent cation permeability, especially for potassium. The expression profile of the Ta-hir1 transcript detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time polymerase chain reaction (real time-PCR), respectively, showed that the highest expression occurred 48 h post inoculation (hpi), which is consistent with our previous histopathology observations during the stripe rust fungus–wheat incompatible reaction. © 2007 Elsevier B.V. All rights reserved. Keywords: RACE; Sequence characterization; Suppression subtractive hybridization; Transcripts expression profiles
1. Introduction Hypersensitive response (HR), one of the most efficient forms of plant defense against biotrophic pathogens, is usually defined as ‘the rapid death of plant cells in association with the restriction of pathogen growth’ (Goodman and Novacky, 1994). It occurs in resistant plant cultivars in response to incompatible viruses, fungi, bacteria (Lam et al., 2001; Vleeshouwers et al., 2000; Baker et al., 1993), and causes a series of biochemical processes that result in Abbreviations: cDNA, DNA complementary to RNA; HR, hypersensitive response; HIR, hypersensitive induced reaction; RACE, rapid amplification of cDNA ends; SPFH, Stomatins, Prohibitins, Flotillins and HflK/C; RT-PCR, reverse transcriptase-polymerase chain reaction; real time-PCR, real timepolymerase chain reaction; bp, base pair(s); hpi, hours post inoculation; SSH, suppression subtractive hybridization; BLAST, basic local alignment search tool. ⁎ Corresponding author. Northwest A&F University, Yangling, Shaanxi, 712100, P.R. China. Tel.: +86 02987091312; fax: +86 02987091312. E-mail address: [email protected] (Z.-S. Kang). 1 Present address: College of Life Science, Agriculture University of Hebei, Baoding, Hebei, 071001, PR China. 0378-1119/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2007.10.010
cell death in the adjacent living cells thus causing an adverse environment for the pathogens. Elicitors of HR cause intracellular ion influx, membrane dysfunction, and increase in the generation of reactive oxygen species and salicylic acid (Heath, 1998, 2000). A cascade of events triggered within the signaling pathway results from the interaction of ligands originating from pathogens with intra or extra-cellular plant receptors in an incompatible reaction which will further lead to modifications of plant cell walls, accumulation of pathogenesis-related (PR) proteins or phytoalexins, and accumulation of oxidized phenolic compounds that can cause browning of the dead cells. In addition, the HR typically induces systemic changes throughout the plant, including induced resistance to a variety of previously compatible pathogens (Kombrink and Sossich, 1995). Karrer et al. (1998) used a functional screening method to isolate several genes whose products elicited the HR in tobacco challenged by the tobacco mosaic virus. One of the isolated cDNAs, NG1, was able to induce the formation of lesions and expression of PR-2 protein, an acid β-glucanase. Three maize hypersensitive induced reaction (HIR) genes, Zm-hir1, Zm-hir2
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and Zm-hir3, have been isolated based on their amino-acid homology to the tobacco NG1 sequence (Nadimpalli et al., 2000). Studies on the regulation of the Zm-hir3 transcript indicated higher level of expression of this gene in the maize disease lesion mimic mutant Les9 when compared to the wild type, and reduced expression in maize line which suppressed the Les9 lesion mimic phenotype. Many other data also suggested participation of the maize HIR genes in HR. In addition to three Zm-hir genes, Nadimpalli et al. (2000) also isolated and identified four genes closely related to prohibitins (Zm-phb1, Zm-phb2, Zm-phb3, and Zm-phb4) and one to stomatins (Zm-stm1). They further used PHI-BLAST searches combined with multiple sequence alignments and neighbor-joining tree construction to show that maize HIR proteins, along with prohibitins and stomatins, formed the superfamily PID (proliferation, ion, and death). Prohibitins are involved in proliferation and cell cycle control, stomatins in ion channel regulation, and HIR genes are involved in cell death. Although members of this superfamily are involved in diverse functions, their structural similarity suggested a conserved molecular mechanism, which Nadimpalli et al. (2000) postulated to be involved in ion channel regulation. Recently, four distinct barley HIR genes, Hv-hir1, Hv-hir2, Hvhir3, Hv-hir4 were identified (Rostoks et al., 2003). Sequence analyses of the barley HIR proteins indicated the presence of the SPFH (Stomatins, Prohibitins, Flotillins and HflK/C) protein domain (Tavernarakis et al., 1999). Barley HIR genes were expressed in all organs and development stages analyzed, indicating a vital and non-redundant function. Barley fast-neutron mutants exhibiting spontaneous HR (disease lesion mimic mutants) showed up to a 35-fold increase in Hv-hir3 expression thus implicating HIR genes in the induction of HR (Rostoks et al., 2003). In a previous study, we isolated from an incompatible suppression subtractive hybridization (SSH) cDNA library of wheat leaves infected by Puccinia striiformis, a cDNA fragment, WSRP1878, homologous to the barley Hv-hir1 gene (Yu et al., 2007). In the present study, we cloned a full length wheat HIR gene using rapid amplification of cDNA ends (RACE). Based on the sequence of the WSRP1878 fragment and designated it as Ta-hir1. The characteristics of Ta-hir1 cDNA and deduced amino-acid sequences were identified by a series of bioinformatic softwares. In addition, the expression profile of Ta-hir1 at different post-inoculation time points was investigated by reverse transcriptase-polymerase chain reaction (RTPCR) and real time-polymerase chain reaction (real time-PCR). 2. Materials and methods 2.1. Plant materials and inoculation Wheat (Triticum aestivum L.) cultivar Suwon11 and stripe rust race CY23 were the biological materials used for the full length cloning of the wheat HIR1 gene and its expression analysis. Suwon11 was presumed to contain a stripe rust resistant gene YrSu (Cao et al., 2003; Li et al., 2006). Plants were grown and maintained as described in Kang and Li (1984). Freshly collected urediospores were applied with a paintbrush to the surface of the primary leaf of seven-day old wheat seedlings.
After inoculation, the plants were kept at high humidity in the dark for 24 h and followed by a regular day-night cycle in a growth chamber. A control inoculation was made with sterile water. Wheat leaves were excised at 0, 24, 36, 48, 60, 72 and 96 hour post-inoculation (hpi), and quickly frozen in liquid nitrogen and stored at − 80 °C prior to extraction of total RNA. 2.2. Total RNA isolation and SSH Isolation of total RNA was carried out according to the instructions of the RNeasy Plant Mini kit (Qiagen, Germany). Total RNA for SSH and RACE analyses was extracted from wheat leaves at 24, 48 and 72 hpi, while RNA for gene expression analysis was obtained from all sampling points. DNase I treatment was applied to remove contaminating genomic DNA. SSH was done using the PCR-Select™ cDNA Subtraction kit (Clontech, USA) according to the manufacturer's instructions. RNA from wheat leaves infected by P. striiformis was used as the tester, while RNA from mock-inoculated leaves was used as the driver. 2.3. Analysis of SSH library After two rounds of subtractive hybridization and two rounds of suppressive PCR, PCR products were ligated into the pGEM-T Easy Vector (Promega, USA) to construct an incompatible SSH cDNA library (Yu et al., 2007). To identify wheat differentially expressed genes after infection by the stripe rust fungus, all positive clones from the library were subjected to sequencing, clustering, BLAST alignment, functional annotation and classification into different categories (unpublished data). 2.4. Rapid amplification of cDNA ends (RACE) Through BLAST searches, a 479 bp cDNA, WSRP1878, was identified and showed the highest similarity to the HIR protein 1 from Hordeum vulgare. To obtain a full-length cDNA of the wheat HIR1 gene, a pair of gene specific primers (forward primer: 5′-CAAGAGGGCTGAAGGTGAGGCAGAATCG-3′, reverse primer: 5′-TTTCCAACACTCC TCAAACCATCCTGCC-3′) was designed based on WSRP1878 sequence and synthesized (Shanghai GeneCore BioTechnologies Co., Ltd.). 3′and 5′ RACE were performed using a SMART™ RACE cDNA Amplification Kit (Clontech, USA). PCRs were done in a PTC-200 thermocycler (MJ Research, USA) with the following parameters: 5 cycles of 95 °C for 30 s, 72 °C for 3 min, 5 cycles of 95 °C for 30 s, 70 °C for 30 s, 72 °C for 3 min, 25 cycles of 95 °C for 30 s, 68 °C for 30 s, 72 °C for 3 min, then final extension at 72 °C for 10 min. PCR products were gel purified and cloned into pGEM-T Easy Vector (Sambrook et al., 1989). An ABI PRISM 3130XL Genetic analyzer (Applied Biosystems, USA) was used to determine the nucleotide sequence of the positive clones. 2.5. Sequence analysis and phylogenetic analysis DNA sequence data were analyzed using BLAST (http:// www.ncbi.nlm.nih.gov/blast/), CAP3 Sequence Assembly Program (http://pbil.univ-lyon1.fr/cap3.php), ORF Finder (http://
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www.ncbi.nlm.nih.gov/gorf/gorf.html), InterProScan (http:// www.ebi.ac.uk/InterProScan/) and PROSITE (http://au.expasy. org/prosite/) network services. The alignment of the deduced protein sequences and phylogenetic tree were computed using DNAMAN and ClustalX version1.81 program employing standard parameters, respectively. 2.6. Reverse Transcriptase-PCR (RT-PCR) analysis Wheat 18S rRNA (GenBank accession no. AY049040) and tubulin (GenBank accession no. U76558) genes were used as reference genes. Primer sequences used in RT-PCR analysis were as follows: wheat 18S rRNA gene forward primer: 5′CTTCTTAGAGGGACTATGGC-3′, reverse primer: 5′-TACGGAAACCTTGTTACGAC-3′; tubulin gene forward primer: 5′CTCCTTCCCCATTTCGC-3′, reverse primer: 5′-CCAGAGCCAGTTCCACCT-3′; wheat Ta-hir1 gene forward primer: 5′CTGGCCTTCTCTGAGAATGT-3′, reverse primer: 5′GGAGCTGACCATCTCGTATC-3′. PCR was performed with a PCR Master Mix Kit (Takara, Japan) using 2 μl (10 × dilution) of first strand cDNA as template and 0.2 μmol/L of each primer (final concentration) in a final volume of 25 μl. PCR was performed on a PTC-200 thermocycler (MJ, USA) with an initial denaturation step at 95 °C 10 s, followed by 30 or 35 cycles of denaruration at 95 °C 5 s, annealing and extension at 60 °C 10 s. The PCR products were detected on 1% agarose gel.
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CYCLER∣∣quantitative PCR detection system (Cepheid, USA). Wheat 18S rRNA gene was used as a standard control in the real time-PCR. Primer and TaqMan probe sequences for wheat Tahir1 gene and the reference gene are as follows: wheat 18S rRNA gene forward primer: 5′-CGTCCCTGCCCTTTGTACAC-3′, reverse primer: 5′-AACACTTCACCGGACCATTCA3′, probe sequence: 5′-FAM-CCGCCCGTCGCCCTACCGTAMRA-3′; wheat Ta-hir1 gene forward primer: 5′-GCCATG A AT G A G AT C A AT G C A - 3 ′ , r e v e r s e p r i m e r : 5 ′ TTCGGCCTCGGCTTTCT-3′, probe sequence: 5′-FAMAGTGGCTGCCAGCCTCATCCTAGC-TAMRA-3′. With the exception of the addition of 0.04 μmol/L TaqMan probe to the reaction system, PCR amplification and the thermal cycling conditions were the same as those for RT-PCR. Quantification of the target gene was assessed using relative standard curves. In our study, the plasmid with the Ta-hir1 gene was used to prepare standard curves and target gene quantity being determined from the standard curve. Real time-PCR experiments were done in triplicate, and the Ta-hir1 gene transcript level was normalized to the average amount of 18S rRNA transcript by expressing it as a percentage. Normalized values of three replicated experiments were used to calculate the average amount of a wheat Ta-hir1 gene transcript. 3. Results 3.1. Characterization of Ta-hir1 cDNA
2.7. Real time-PCR analysis Expression of the Ta-hir1 gene in wheat leaves was further analyzed by real time-PCR using TaqMan probes in a SMART
Following 3′ and 5′ RACE analyses, two DNA fragments around 600 and 1000 bp, respectively, were detected on 1% agarose gel. Following plasmid construction, transformation
Fig. 1. Comparison of the predicted amino acid sequences of HIR1 gene of barley, wheat and maize. Amino acid sequence of wheat was deduced from the Ta-hir1 gene. Amino acid substitutions were marked in grey. The consensus amino acid sequences are highlighted in black. Hv, Hordeum vulgare (GenBank accession no. AY137511), Ta, Triticum aestivum (GenBank accessession no. EF514209), Zm, Zea mays (GenBank accessession no. AF236373).
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Fig. 2. Phylogenetic tree of higher plant SPFHs. Branches of the neighbor-joining phylogenetic tree were labeled with species name (Hv — Hordeum vulgare, Os — Oryza sativa, Zm — Zea mays, Ta — Triticum aestivum, Pc — Pennisetum ciliare, Ca — Capsicum annuum, Cs — Cucumis sativus and Lj — Lotus japonicus) followed by the gene name and GenBank accession number.
and cDNA clones sequencing, 501 bp for 3′RACE and 944 bp for 5′RACE fragments were obtained by removing vector and adapter sequences. Furthermore, CAP3 software was used to combine the two fragments into an 1132 bp consensus sequence including a poly-A signal region in the 3′-UTR. BLAST analysis showed that the 1132 bp sequence shared high similarity (93%) to Hv-hir1 (GenBank accession no. AY137511) (data not shown). The region from 77 to 931 nucleotides was the open reading frame (ORF) encoding a polypeptide of 284 amino acids. Moreover, the sequence surrounding the start codon, ACCATGG, agreed with that of the Kozak consensus initiator ANNATGG, which was identical to the sequence reported for Hv-hir1. The deduced molecular mass of the peptide was 31.31 KDa with a predicted pI of 5.29. Based on the strong identity to barley Hv-hir1, we designated the wheat 1132 bp sequence as Ta-hir1 (GenBank accession no. EF514209). A comparison of the deduced amino acid sequences of HIR1 genes from barley, maize and wheat was performed using DNAMAN. The result showed strong homology among these HIR1 sequences, especially for Hv-hir1 and Ta-hir1 (only two amino acid residues different) (Fig. 1). All available plant HIR sequences plus Zm-phb and Zm-stm were used to construct a neighbor-joining tree (Fig. 2). These results demonstrated a high degree of similarity of the HIR1, HIR2 and HIR3 groups across the species analyzed, while Zm-phb and Zm-stm also showed high degree of similarity with HIR3 and HIR4. Ta-hir1 grouped together with the proteins encoded by Hv-hir1, Zm-hir1 and Oshir1, whereas, Hv-hir2, Pc-hir and Zm-hir2 were clustered together, and Hv-hir3, Ca-hir and Zm-hir3 were in another group. The results suggested a high level of conservation among HIR1, HIR2, HIR3 and Zm-phb from different species. The protein conserved domain search at the EBI web page indicated that Ta-hir1 gene contained the signature of stomatins, but no apparent transmembrane region or signal peptide sequences (InterPro IPR001107 family). Additionally, results predicted from PROSITE showed that Ta-hir1 protein contained
an N-myristoylation site, protein kinase C, tyrosine kinase and casein kinase II phosphorylation sites, which were absolutely consistent with the Hv-hir1 protein. 3.2. RT-PCR analysis To determine the expression profile of wheat Ta-hir1gene at each time point after inoculation, RT-PCR was performed in which the amount of total RNA for each time point was standardized to contain equivalent amounts of wheat 18S rRNA and tubulin genes. Fig. 3 showed the detection results of RTPCR for Ta-hir1, 18S rRNA and tubulin genes in wheat leaves infected by stripe rust fungus at different time points. Wheat Tahir1 gene showed elevated amount of transcripts from 24 until 48 hpi at which time the maximal expression level was reached. From 48 to 72 hpi, the expression of wheat Ta-hir1 began to decrease steadily with an apparent increase at 96 hpi. The expression of 18S rRNA and tubulin of wheat leaves sampled in our study showed almost the same level of transcripts during the time-course experiment. 3.3. Real time-PCR analysis To confirm the reliability and reproducibility of RT-PCR analysis results and to accurately quantify the transcripts of Tahir1 in the different samples, real time-PCR was performed. The
Fig. 3. RT-PCR analysis of Ta-hir1 in wheat leaves infected by stripe rust fungus at different time points. Numbers in parentheses indicate PCR cycles.
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Fig. 4. Real time-PCR analysis of expression profiles of Ta-hir1 gene in wheat leaves infected by stripe rust fungus at different time points. The amount of transcript was normalized to 18S rRNA genes and was expressed as the percentage of the reference gene transcripts. Error bars represent the variation among three independent replications.
expression profile of Ta-hir1 in wheat leaves after inoculation is shown in Fig. 4. The y-axis indicates the amount of wheat Tahir1 transcript that was normalized to 18S rRNA gene and was expressed as the percentage of the reference gene, and the x-axis indicates the different sampling points. Real time-PCR analysis showed that Ta-hir1 of wheat was strongly up-regulated as early as 24 h after infection with P. striiformis race CY23. The maximum induction occurred at 48 hpi, whose transcripts were 18.77 fold over that of the control (0 h). From 48 to 72 hpi, the accumulation of transcripts decreased steadily and was followed by a slightly increase at 96 hpi. However, the amount of transcripts at every time point was much higher than that of control. The RT-PCR and real time-PCR results showed a consistent expression profile and that a maximal accumulation of wheat Ta-hir1 gene transcripts occurred at 48 hpi. 4. Discussion In the present study, we initially identified a gene fragment isolated from an incompatible SSH library of wheat leaves infected by the stripe rust fungus which showed a high similarity to the barley Hv-hir1 gene. In order to understand whether it would be involved in HR of wheat, a full-length cDNA sequence of 1132 bp with a complete ORF was isolated from infected wheat leaves using the RACE technique. We designated it as Ta-hir1 because many characteristics of this gene predicted by different bioinformatic softwares were highly similar to the deduced results of the barley Hv-hir1 gene. Studies on HIR proteins have been performed on various plant species, such as barley, maize, rice and cucumber (Rostoks et al., 2003, Nadimpalli et al., 2000, De los Reyes et al., 2003, Kim et al., 2004). The length of HIR proteins either from monocotyledons or dicotyledons ranges from 283 to 288 aa. Moreover, the neighbor-joining trees of all available HIR sequences (before August 15th, 2006) showed a high degree of relationship among the HIR1, HIR2, and HIR3 groups across the species analyzed. Amino-acid sequence analysis of the HIR proteins indicated the presence of the characteristic SPFH protein domain (Nadimpalli et al., 2000, Rostoks et al., 2003). In the present study, wheat Ta-hir1 gene possessed the stomatins domain which was thought to act as a negative regulator of
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univalent cation permeability, especially for potassium. Stomatins has a single membrane-spanning region near its N terminus, while wheat Ta-hir1 and barley Hv-hir1, Hv-hir2, Hv-hir3 genes did not have apparent transmembrane regions. However, the PROSITE search indicated that the N-termini of these proteins had an N-myristoylation motif and thus, these HIR proteins could be anchored to membranes (Rostoks et al., 2003). Taken together, the molecular function and mode of action for the wheat Ta-hir1 need to be further evaluated in the incompatible interaction between wheat and the obligate stripe rust pathogen. Two different methods were utilized to characterize the expression profile of the Ta-hir1 gene during the infection timecourse. RT-PCR was a method of endpoint detection, whilst real time-PCR monitor fluorescence emitted during the reaction as an indicator of the amount of amplicon produced after each PCR cycle (Heid et al., 1996). For the RT-PCR, we selected two control genes, the tubulin and 18S rRNA genes, which have been reported to be consistently expressed in Arabidopsis (Czechowski et al., 2005), to eliminate RNA differences among the different samples and thus avoiding unreliable results. To ensure the reliability of control genes, we selected 18S rRNA gene in wheat which had been used in a few previous studies to value the expression of targeted gene in real time-PCR (Scofield et al., 2005). The results from the two detection methods were consistent, and the maximal accumulation of wheat Ta-hir1 transcripts occurred at 48 h post inoculation. In addition, real time-PCR enabled a quantification of the transcripts. Besides Ta-hir1, two additional cDNAs sequences with complete ORF were obtained via RACE analysis. All three genes encoded 284 amino acids, and were highly similar to the barley Hv-hir1 gene. Their deduced amino acids were absolutely consistent, but there were important differences in sequences length and 5′nucleotide sequences. The longest sequence (1212 bp) had 80 nucleotides more than the shortest one (1132 bp) and 49 bases more than the intermediate size fragment (1163 bp). At present, only the cDNA sequence with 1132 bp was submitted to the GenBank due to its highest similarity to the barley Hv-hir1 gene. Differences among the three nucleotide sequences may result from alternative splicing or different transcription initiation sites. Additionally, short-cut phenomenon which was caused by poor mRNA or degradation of mRNA by RNase H, particular secondary structures of mRNA and inappropriate conditions for amplification (Schaefer, 1995; Zhang and Frohman, 1997), often make it difficult to obtain complete 5′RACE fragments. Another DNA fragment of 750 bp was also obtained during the 5′RACE amplification. This fragment showed high homology to Hv-hir1, but was 250 bp shorter than the real and complete amplification product. This suggests strongly that the 750 bp fragment results from a kind of short-cut phenomenon. These results reinforce the needs for high quality mRNA and optimal conditions for amplification of full length sequence when using RACE. Our previous histopathological observations (unpublished data) showed that in the incompatible interactions between wheat cultivar Suwon11 and stripe rust race CY23, the host cells in the infection site in the inoculated leaves started to become necrotic 24 hpi, and the numbers of necrotic host cells at each
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infection site increased until 60 hpi. Meanwhile proliferation of stripe rust fungus was markedly inhibited in the host leaves. These results seem to coincide with the expression pattern of Tahir1 transcripts in the incompatible interaction between wheat cultivar Suwon11 and stripe rust race CY23. Previous studies showed that some HIR genes were over-expressed in most of the disease lesion mimic mutation lines, which implied the vital role of HIR gene in HR (Nadimpalli et al., 2000; Rostoks et al., 2003). The numbers of wheat Ta-hir1 transcripts at each time point of post-inoculation were much higher than that of noninoculated control. We can postulate that wheat Ta-hir1 plays an important role in wheat protection against incompatible stripe rust pathogens. Functional characterization of the candidate wheat HIR gene Ta-hir1 is in progress. Acknowledgements This study was supported financially by the National Basic Research Program of China (No. 2006CB101901), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (NO.2005295), the Program for Changjiang Scholars and Innovative Research Team in University, Ministry of Education of China (No.200558), the Nature Science Foundation of China (No.30671350), and the 111 Project from Ministry of Education of China (B07049). References Baker, C.J., Orlandi, E.W., Mock, N.M., 1993. Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cells. Plant Physiol. 102, 1341–1344. Cao, Z.J., Jing, J.X., Wang, M.N., et al., 2003. Relation analysis of stripe rust resistance gene in wheat important cultivar suwon 11, suwon 92 and hybrid 46. Acta Bot. Boreal-Occident. Sin. 23, 64–68 (In Chinese). Czechowski, T., Stitt, M., Altmann, T., et al., 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 139, 5–17. Goodman, R.N., Novacky, A.J., 1994. The hypersensitive reaction in plants to pathogens. A resistant phenomenon. American Phytopathological Society, St. Paul, Minn., p. 244. De los Reyes, B.G., Morsy, M., Gibbons, J., et al., 2003. A snapshot of the low temperature stress transcriptome of developing rice seedlings (Oryza sativa L.) via ESTs from subtracted cDNA library. Theor. Appl. Genet. 107, 1071–1082. Heath, M.C., 1998. Apoptosis, programmed cell death and the hypersensitive response. Eur. J. Plant Pathol. 104, 117–124.
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