JXB Advance Access originally published online on December 12, 2003
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Journal of Experimental Botany, Vol. 55, No. 396, pp. 535-537, February 1, 2004
© 2004 Oxford University Press
GENE NOTE |
Isolation and characterization of a Pti1 homologue from soybean
Received 10 July 2003; Accepted 9 October 2003
1 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
2 National Center for Soybean Improvement, Soybean Research Institute, Nanjing Agricultural University, Nanjing 210095, China
* To whom correspondence should be addressed: Fax: +86 10 6487 3428. E-mail: sychen{at}genetics.ac.cn; jszhang{at}genetics.ac.cn
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Abstract |
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A full-length gene GmPti1 was identified from soybean in an EST sequencing project by its homology to tomato Pti1. It encoded a protein of 366 amino acids. RT-PCR analysis showed that the GmPti1 expression was induced by salicylic acid and wounding. The deduced amino acid sequence had a Ser/Thr/Tyr kinase domain. GmPti1 protein was expressed in E. coli as an MBP fusion, purified by amylose resin and examined for its autophosphorylation ability. The phosphorylation assay in vitro showed that GmPti1 had kinase activity in the presence of Mn2+. These results demonstrated that GmPti1 represented a new Pti1-like gene, unlike the two published genes sPti1a and sPti1b, which encoding proteins had no autophosphorylation ability.
Key words: Autophosphorylation, GmPtil, kinase activity, soybean.
Plants have developed complicated defence mechanisms during evolution to defend themselves from pathogens. In many plant–pathogen interactions, the defence response was activated upon the interaction of a plant resistance (R) gene with the pathogen avirulence gene (Dangl and Jones, 2001; Hammond-Kosack and Jones, 1997; Holub, 2001). To date, most of the cloned R genes, for example, Cf-9 (Jones et al., 1994), RPS2 (Bent et al., 1994), L6 (Lawrence et al., 1995), N (Whitham et al., 1994), and Pto (Martin et al., 1993) genes, confer disease resistance in a ‘gene-for-gene’ manner by recognizing specific avirulent strains of the corresponding pathogen (Flor, 1971; Staskawicz et al., 2001) and encode only five classes of protein (Dangl and Jones, 2001; Holub, 2001). In tomato, Pti1, a functional serine/threonine protein kinase, can physically interact with Pto, which is a serine/threonine kinase and confers resistance to Pseudomonas syringae pv. tomato strains that expresses the avirulence gene avrPto in tomato (Martin et al., 1993; Loh and Martin, 1995; Zhou et al., 1995). Furthermore, overexpression of Pti1 in tobacco enhances the hypersensitive response to Pseudomonas syringae pv. tomato strains. All these facts suggest that Pti1 plays a role in Pto-mediated resistance (Zhou et al., 1995). In soybean, two Pti1-like genes, sPti1a and sPtilb, have been cloned and analysed (Staswick, 2000). However, unlike tomato Pti1, the two encoded proteins cannot autophosphorylate in vitro. In this study, a new member of the Pti1 gene family, designated GmPti1, was cloned from soybean and characterized further.
The GmPti1 gene was identified from soybean (Glycine max L. Merr., Kefeng 1) during an EST sequencing project by its homology to tomato Pti1. Its expression was examined by RT-PCR. For salicylic acid (SA) treatments, Two-week-old seedlings of the soybean cultivar Kefeng 1 were sprayed with 2.0 mM SA until run-off. For wounding treatment, attached leaves were cut. Leaves from the uncut seedlings were used as controls. Samples from all the treatments were harvested at the indicated times and total RNA isolation was performed (Zhang et al., 1995). Total RNA was treated with RNase-free DNAase (Promega) to remove the genomic DNA contamination and the first strand of cDNA was synthesized by using 4 µg total RNA with the cDNA synthesis Kit (Promega) in 20 µl of reaction volume. The specific primers (the sense primer: 5'-CACA TGGGCTACTCCAAGAT-3' and the antisense primer: 5'-CATGT CATCCAATCTCAAAC-3') were designed according to the 3' end of GmPti1. The total volume of the PCR reaction mixture was 25 µl, containing 1 µl of the first strand cDNA, 0.5 µM of each primer, 1x PCR buffer, 0.4 mM dNTP, and 1 U of Taq DNA polymerase. The reaction was denatured at 94 °C for 5 min, and then followed by 35 cycles of 1 min at 94 °C, 1 min at 56 °C and 1 min at 72 °C. PCR was concluded with one step for 10 min at 72 °C. A soybean Tubulin gene, amplified with primers 5'-AACCTCCTCCTCATCGTACT-3' and 5'-GACAGCATCAGCCATGTTCA-3', was used as a control. The PCR products were separated on an agarose gel and quantified using an Imaging DensitoMeter (Bio-Rad). To express GmPti1 protein as maltose-binding protein (MBP) fusion, the GmPti1 coding region was amplified with primers 5'-TCAGAATTCATGCGTC GGTGGCTTTGTTGT-3' and 5'-ATGCTGCAGTCAACTTTCTG GAACAGGAGC-3', digested with EcoRI and PstI, and cloned into the pMAL-c2x expression vector (Biolabs). The recombinant plasmid was transformed into E. coli strain TB1, and the fusion protein was induced and purified by amylose resin (Biolabs). The expression and purification of the fusion protein were confirmed by Western blot using rabbit anti-MBP antibody (Biolabs). One to three µg of the fusion protein was incubated with 30 µCi of [
-32P] ATP (3000 Ci mmol–1) in 25 µl of kinase buffer (50 mM TRIS-HCl [pH 7.0], 1 mM DTT) plus different concentrations of divalent cations Mn2+, Mg2+ or Ca2+ at 22 °C for 1 h. The reaction was terminated by the addition of EDTA to a final concentration of 10 mM. The phosphorylated protein was separated by 10% SDS-PAGE gel, transferred onto PVDF membranes (Amersham) and autoradiographed. Coomassie Blue staining of the same membrane was performed to verify the protein loading (Xie et al., 2003).
The full-length GmPti1 gene (Accession No. AY263347 [GenBank] ) was 1430 bp, containing an open reading frame of 1101 bp plus 135 bp of 5'-untranslated region and 194 bp of 3'-untranslated region. The GmPti1 gene encoded a protein of 366 amino acids with a calculated molecular weight of 40.38 kDa. The deduced GmPti1 amino acid sequence was compared with other homologous proteins (supplementary data can be found at JXB online). It was found that GmPti1 shared 68% identity with tomato Pti1 (Zhou et al., 1995), 65% identity with the two sPtils from soybean (Staswick, 2000), and 30% identity with a protein kinase APK1 from Arabidopsis (Hirayama and Oka, 1992). Using the SMART program (Schultz et al., 1998), a Ser/Thr/Tyr kinase domain was identified in GmPti1 (data not shown). This feature was similar to those of tomato Pti1 and soybean sPti1a and sPti1b. Southern analysis was also performed and two polymorphic loci of the GmPti1 gene were detected. The two loci were mapped onto the linkage group I and B1, respectively, using a RIL population NJRIKY (Wu et al., 2001).
Salicylic acid (SA) has been considered to be involved in various signal transduction pathways, especially in leading to systemic acquired resistance against pathogen attack (Malamy and Klessig, 1992) as well as PR protein expression (Dempsey et al., 1999). The GmPti1 expression in response to SA treatment by RT-PCR was investigated (Fig. 1A). Upon SA treatment, GmPti1 expression was gradually increased and reached a maximum level at 60 h after the initiation of the SA treatment, and then declined (Fig. 1A). GmPti1 expression, as indicated in Fig. 1B, was also induced by wounding in the first 2–6 h. The induction by wounding occurred at an earlier stage than that by SA. The expression pattern of the GmPti1 gene resembles that of a TIR-NBS-LRR type resistance gene KR1 from soybean in response to the same treatments (He et al., 2003), suggesting that these two genes may have some relevance in co-ordinating plant defence responses.
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To address the biochemical property of GmPti1, the MBP–GmPti1 fusion protein was expressed, purified and confirmed on Western blots using the rabbit anti-MBP antibody (data not shown). The purified MBP fusion protein was assayed for phosphorylation activity. The result showed that GmPti1 had protein kinase activity in the presence of Mn2+ and the autophosphorylation decreased slightly when the concentration of Mn2+ increased from 10 mM to 20 mM (Fig. 2). No detectable phosphorylation was observed when Mg2+ or Ca2+ was used as a source of divalent cations (Fig. 2). In tomato, Pti1 can autophosphorylate and can be phosphorylated by Pto, but cannot phosphorylate Pto (Zhou et al., 1995). In soybean, two sPti1s can be phosphorylated by tomato Pto, but lack autophosphorylation activity (Staswick, 2000). In the present study, GmPti1 showed autophosphorylation activity in the presence of Mn2+. Such a feature made GmPti1 different from the other two sPti1s. Therefore, two classes of Pti1-like genes were present in soybean. However, it is not known whether GmPti1 can be phosphorylated by soybean Pto and the function of GmPti1 in the defence response remains to be investigated. The different properties might indicate different roles of the Pti1 genes in soybean defence responses, and further study should reveal its function by overexpression of the GmPti1 gene or silencing of the GmPti1 gene.
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Supplementary data |
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Comparison of the deduced amino acid sequence of the GmPti1 (AY263347) with other proteins can be found at Journal of Experimental Botany online. The compared proteins were tomato Pti1 (U28007 [GenBank] ), soybean sPti1a (AF249317), soybean sPti1b (AF249318), and Arabidopsis APK1 (D12522 [GenBank] ). The alignment was generated using the DNASTAR program. Gaps were introduced to maximize the alignment.
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Acknowledgement |
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This work was supported by National Key Basic Research Special Funds, PR China (2002CB111301), (G1998010209), and High Tech Program (2002AA211051).
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