ISSN : 2287-5174(Online)
Cloning and Characterization of Pathogenesis-related Gene 10a (OgPR10a) Derived from Wild Rice (Oryza grandiglumis)
As sessile organisms, plants are constantly exposed to diverse biotic and abiotic stresses. Thus, plants need to develop unique defense mechanisms to continue their life under unfavorable conditions. Plant cells possess both preformed structural characteristics that act as physical barriers for inhibiting the pathogen from entrance and spread and an inducible biochemical reaction that takes place in the infected cells and naive distal cells. The recognition of external stimuli activates subsequent cellular responses including expression of pathogenesis-related (PR) proteins, production of antimicrobial compounds, and so on. (van Loon, 1985). Since Van Loon (1970) identified PR-proteins from tobacco plants showing hypersensitive local cell death to infection with tobacco mosaic virus (TMV), many PR proteins have been described in a wide variety of plant species (Bol et al., 1990). So far PR proteins have been classified at least into 14 different groups, based on their sequence identities at amino acid level (Van Loon and Van Strien, 1999). A certain kind of PR proteins possessed direct anti-microbial activities against plant pathogenic microorganisms (Niderman et al., 1995; Lee and Hwang, 2006). Additionally, genetically modified plants carrying PR genes were resistant against pathogen infection in various plant species (Alexander et al., 1993; Datta et al., 1999). However, how PR genes act as crucial defenders during defense response remained unclear.
PR10 protein was firstly identified as a major pollen allergen (Bet v1) in white birch plants (Breiteneder et al., 1989). PR10 genes encoding small acidic protein were differentially transcribed in a number of plant species after pathogen infection (Somssich et al., 1988; Matton and Brisson, 1989; Barratt and Clark, 1991; Linthorst, 1991; Crowell et al., 1992; Warner et al., 1993; Lo et al., 1999; Midoh and Iwata, 1996; McGee et al., 2001). Enzymatically, PR10 proteins had ribonuclease activity and/or cytokinin- binding ability in plants (Bantignies et al., 2000; Fujimoto et al., 1998). The pathogen-induced expression patterns suggested that PR10 could be involved in either disease development or plant defense response. A stable expression of pea PR10 conferred salinity tolerance on canola plants under high salt condition, whereas this pea PR10 expression did not protect transgenic canola plants against blackleg (Leptosphaeria maculans) disease (Srivastava et al., 2004; Wang et al., 1999). On the other hand, knockdown of PR10 expression induced accumulation of other PR proteins in root tissues of Medicago truncatula infected with Aphanomyces euteiches and the PR10-silenced plants showed disease resistance in response to A. euteiches infection (Colditz et al., 2007). However, the experimental clues were still insufficient for proving molecular function of PR10 gene family during plant defense response, compared with those for other PR genes.
A rice PR10 gene family was classified into three different clades; PR10a (PROBENAZILE-INDUCBLE PROTEIN, PBZ1), PR10b, and PR10c, that is a non-functional pseudogene (Midoh and Iwata 1996; McGee et al., 2001). Transcription of rice PR10 gene was up-regulated by diverse stimuli, including diverse biotic and abiotic stresses, as other PR genes did (Midoh and Iwata, 1996; Rakwal et al., 1999; McGee et al., 2001; Agrawal et al., 2002; Tanaka et al., 2003). Additionally, exogenous treatment of some plant hormones, such as jasmonic acid (JA), salicylic acid (SA), abscisic acid (ABA), and kinetin could regulate OsPR10 and JIOsPR10 gene expression (Rakwal et al., 2001, Jwa et al., 2001).
Wild species of crop plants might have many useful alleles for crop improvement, which we have lost during plant domestication processes. Wild rice (Oryza grandiglumis, CCDD genome) originated from tropical America (Akimoto 1998) is highly resistant against the infection of Magnaporthe grisea and Xanthomonas oryzae (Vaughan 1994). In spite of its potential, only several defense-related genes were molecularly cloned in the wild rice (Kim et al., 2005; Jeon et al., 2008). A truncated fragment of PR10 gene (OgPR10) was isolated from O. grandiglumis by the combination of suppression subtractive hybridization (SSH) and cDNA macroarray (Kim et al., 2005). We obtained a full length OgPR10 cDNA clone from leaves of wild rice plants by using rapid amplified cDNA ends (RACE). OgPR10 mRNA was distinctively expressed in leaves treated with either yeast extract containing fungal elicitor or probenazole as a chemical elicitor for plant defense response. Interestingly, heterologous expression of OgPR10 gene not only inhibited growth of Escherichia coli (E. coli), but also made seedling lethal in Arabidopsis plants. Furthermore we showed that OgPR10-expression conferred disease ressitance on rice plants (cv. Dongjin) against M. grisea infection.
MATERIALS AND METHODS
Plants and chemicals
Wild rice (O. grandiglumis) grew in the National Crop Experiment Station (NCES) within the Rural Development Administration in Korea. The fully expanded leaves of 14-day-old plants were used in this study. Approximately 2 cm-long leaf segments were cut with clean scissors and floated on solutions supplemented with any given chemical. The leaf segments were incubated under 16 h of light and 8 h of darkness at 24℃, harvested at the times indicated, and used immediately or stored at -80℃ until extraction of total RNA. Cantharidin (CN), endothall (En), JA, and SA were purchased from Sigma-Aldrich (St. Louis, MO). All the chemicals (100 uM of working concentration) were prepared by dissolving appropriate solvent or water (Rakwal et al., 2001).
For northern blot analysis, total RNA was isolated from leaves of wild rice plants using the plant RNA Reagent (Invitrogen, USA). Total RNA (20 ㎍) was separated on a 1.2% formaldehyde-denaturing agarose gel, blotted onto a nylon membrane (Hybond-N+, Amersham), and hybridized with a 32P-labeled partial OgPR10 probe using a LaddermanTM kit (Takara, Japan). Hybridization was carried out for 18 h at 4℃, washed with 2 X SSC and 0.1% SDS at 65℃ for 1 h, and exposed to an X-ray film (Kodak) using two intensifying screens for 3 days at -80℃.
Recombinant OgPR10 protein
For producing OgPR10 protein in E. coli, full length OgPR10 gene was inserted into the BamHI site of the pET32 vector (Novagen, Germany). The resulting in-frame fusion plasmid was transformed into E. coli strain BL21 (DE3). Overexpression of OgPR10 tagged with six histidine residues at the N-terminus was induced by 0.4 mM isopropyl-ß-D-thiogalactoside (IPTG) at 30℃ for 3 h. The total soluble protein concentration was quantified by the Bradford assay, separated on a 12% SDS-polyacrylamide gel to visualize recombinant OgPR10 proteins expressed in the system. To monitor bacterial growth after adding IPTG into cultured cell (OD600=0.5), we measured optical density 2.5 hours after induction using spectrophotometer (Amersham).
Subcelluar localization of OgPR10 in plant
The termination codon of the OgPR10 gene was removed using gene-specific oligonucleotide primers, 5’-GGGTCTAGAATGGCTCCGGCCTGCGTCTCC-3’ and 5’-GGGGGATCCGGGCGTACTCGGTAGGGTGAG-3’. The amplified product was fused in frame to the coding region of soluble- modified green fluorescent protein (smGFP) (Davis and Viestra, 1998). The smGFP fusion constructs (pOgPR10- smGFP) was introduced into the epidermal cell of onion using microprojectile bombardment system, PDS 1000 (Bio-Rad, USA). Fluorescence photographs of cells were taken using a fluorescence microscope using UV-blue light excitation (Zeiss, Germany).
Transgenic plants and fungal infection
Development of OgPR10-overexpressing Arabidopsis with pCAMBIA 2301 was done as described previously (Jeon et al., 2008). To create inducible gene expression system the full length OgPR10 was inserted into pTA7002 and both constructs were used for Arabidopsis (Col-0) transformation by vacuum infiltration method (Clough and Bent 1998). Dexamethasone (DEX, Sigma-Aldrich) was dissolved in ethanol and appropriately diluted to 1 to 50 µM. Seeds from the transformed plants were sown in Petri dishes containing 1/2 strength MS liquid media and transferred to multi-well cell culture plate containing DEX solution. Transgenic rice lines (Oryza sativa L. cv. Dongjin) were also generated by Agrobacterium-mediated transformation with a expression vector pCAMBIA 1301-OgPR10. To induce the formation of conidia, the rice blast fungus was cultured on rice polish agar medium at 25℃ for 7 days and scraped with a sterilized rubber spoon. The cultures were irradiated with BLB light for 2 days. The rice seedlings were inoculated with conidia (5×105 spores/ml) of M. grisea KI-1113a isolate. Following the incubation of the seedlings in the dark for 1 day at 25±2℃ and 100% RH, they were transferred to a growth chamber maintained at 25±2℃ and 70-80% RH with 12 hours of daylight per day. Disease severity to rice blast was scored by the number of lesions on the sprayed leaves 7 days after inoculation.
RESULTS AND DISCUSSION
Through SSH and cDNA macroarray experiments for identifying defense-related genes from wild rice plants, we identified a gene consisting of 480 bp nucleotide. The gene encodes 160 amino acids with a predicted molecular mass of 16.944 kDa and an isoelectric point (pI) of 4.91 (Fig. 1). In silico analysis showed that a putative small acidic protein might localize at cytoplasm in plant cells (WoLF PSORT, Horton et al., 2007). Significant sequence homology was found between the predicted OgPR10 protein and PR10 proteins from rice (Fig. 2). The OgPR10 gene showed the highest homology to the rice root specific pathogenesis-related 10 gene (RSOsPR10) with 90% identity and nearly 70% identities to the rice probenazole-inducible gene (OsPBZ1) and rice PR10a gene (OsPR10a) at amino acid level. OgPR10 and OsPR10 proteins contained a GXGGXXG motif that is found in many nucleotide- binding proteins (Gajhede et al. 1996). Thus we named the gene OgPR10. The genomic organization of OgPR10 gene confirmed by Southern blot analysis is likely to be single copy gene for each genome (data not shown).
Fig. 1.Nucleotide and deduced amino acid sequence of the Oryza grandigumis PATHOGENESIS-RELATED 10 (OgPR10) protein. The deduced amino acid is designated at the bottom of the sequence. An asterisk represents the stop codon. The sequence was deposited in GeneBank accession No. CK429147.
Fig. 2.A comparative alignment of the deduced amino acid sequence of Oryza grandigumis PR10 (OgPR10) with homologous sequences. Identical amino acid residues are highlighted in black. Arrowheads indicate the strictly conserved residues and the conserved motif GXGGXG is underlined. Residue numbers are indicated to the right. Origin of sequences: RSOsPR10 (BAD03969), PR10 (AAF85972), PBZ1 (BAA24277), respectively.
Probenazole is a well-known elicitor, not a fungicide, for triggering plant defense response against rice blast disease. Exogenous application of probenazole induced OsPR10/PBZ1 gene expression in rice plants (Midoh and Iwata, 1996). OgPR10 mRNA was weakly expressed in mock-treated plants at least over the experimental period. However, the expression of OgPR10 mRNA was induced within 3 days after treatment with probenazole, and reached the highest level at 6 days (Fig. 3A). Several signaling molecules, such as ABA, JA and SA, could activate their own signaling cascade in order to resist the stress under environmental changes (Reymond and Farmer 1998). To determine whether these molecules have an effect on OgPR10 expression, OgPR10 mRNA levels were determined in wild rice seedlings treated with JA, SA, CN, EN, and yeast extract as a general fungal elicitor (Fig. 3B). Both JA and SA had no positive effect on OgPR10 induction. On the contrary, protein phosphatase inhibitors, CN and EN, induced the OgPR10 gene expression as they activated antifungal defense responses in soybean and rice (MacKintosh et al., 1994). Since wounding did not have any effect on induction of OgPR10 transcript accumulation (data not shown), the induction of gene expression solely attributed to direct effect from CN and EN application. This also suggests an effect of CN and EN, probably through hyperphosphorylation of certain signal transducing proteins, in causing an increased expression of the OgPR10 transcript. Additionally, 20% yeast extract solution was the strongest exogenous signal for triggering OgPR10 transcription among all the treatment tested in this study. Yeast extract is often used as a general elicitor for triggering defense- signaling pathway in plants. The fact that yeast extract, not SA and JA, activated OgPR10 expression suggested that recognition of microbe-associated molecular patterns (MAMPs) mainly regulate OgPR10 expression in wild rice plants.
Fig. 3.OgPR10 mRNA expression in wild rice plants. (A) Accumulation with time of OgPR10 mRNA after treatment with probenazole. Total RNA was isolated from leaves of wild rice after treatment for the indicated times with a solvent only (control: 0.05% Tween 20, 1% acetone) or a solvent containing 100 mg/L probenazole by submerged application. (B) Expression OgPR10 gene transcript in wild rice leaves treated with mock (C), JA, SA, cantharidin (CN), endothall (EN), and Yeast extract (F). Northern blot analysis of RNA from leaf treated with 100 μM JA, 100 μM SA, 100 μM CN, 100 μM EN, and 20% Yeast extract solution. Twenty ㎍ of total RNA were applied to each lane in a formaldehyde agarose gel. After electrophoresis, RNA was transferred onto a nylon membrane and allowed to hybridized with OgPR10 cDNA as probe.
In order to test RNase activity or cytokinine binding activity, we were trying to generate recombinant OgPR10 protein in E. coli. Unexpectedly, we could not get enough amounts of recombinant OgPR10 protein to do this experiment. The failure seemed to be due to the retarded growth of bacteria. To check the possibility, we tested the growth of E. coli carrying the expression cassette under inducible condition. After addition of IPTG to the bacterial cultures, clear growth retardation was observed in recombinant E. coli carrying OgPR10 gene (Table 1). This strongly suggested that the amount of recombinant OgPR10 protein may not be large enough to be detected by staining but enough to inhibit bacterial growth with its predicted function, RNAse activity.
Table. 1.E. Coli (BL21) growth with OgPR10 expression.
The study of subcellular localization of OgPR10 revealed that OgPR10 might be nearly inside cell including nucleus (Fig. 4). The result is coincided with in silico analysis. However, we can not exclude the possibility that the OgPR10-GFP fusion protein is not active, because the localization pattern of OgPR10-GFP protein was very similar to smGFP alone.
Fig. 4.Subcellular localization of OgPR10 in onion epidermal cells. GFP was fused to the C-terminus of the full ORF of OgPR10. (A) The bright-field images of onion epidermal cells. (B) The image observed by fluorescence microscopy under UV-blue light excitation (Zeiss).
To understand in vivo OgPR10 function, we developed transgenic Arabidopsis plants constitutively expressing wild rice OgPR10 under the control of the CaMV 35S promoter. Seeds (T1 plants) from the T0 plants were harvested and germinated on selection media containing kanamycin (50 ug/ml). During the seed germination, all the seedlings became dying within 14 days (Fig. 5. upper panel). At the beginning it was attributed to low transformation rate of Arabidopsis. However, after getting no survivals from many rounds of trials, it was doubted that the dying seedling might be resulted from OgPR10 expression in Arabidopsis. Following northern blot analysis revealed that dead plants exhibited high level of OgPR10 transcripts (Fig. 5. low panel). Recently, it was reported that a PR10 proteins from hot pepper (Capsicum annuum) exhibits ribonuclease activity (Park et al., 2004). Based on previous studies about PR10’s RNase activity, we could predict that that OgPR10 might also have RNase activity and cause the cell death of germinating Arabidopsis plants. Even though all the data presented seems very convincing, we still could not provide direct evidence whether the gene had RNase activity and gene expression was real cause of plant death. To solve the seedling lethality in OgPR10-expressing Arabidopsis controlled by 35S promoter, we decided to use glucocorticoid-inducible promoter for OgPR10 expression in Arabidopsis (Fig. 6A) (Aoyama and Chua, 1997). The application of dexamethasone (DEX), commercial glucocorticoid hormone, to transgenic Arabidopsis plants also caused cell death even in 1 μM concentration as stable expression of OgPR10 did (Fig. 6B). Two independent experiments showed exactly same result and untransformed control plants does not showed any of cell death lesions. With all the results presented here it is strongly suggested that over-expression of OgPR10 with RNase activity resulted in cell death in transgenic Arabidopsis plants.
Fig. 5.Overexpression of OgPR10 in Arabidopsis controlled by CaMV 35S promoter caused seedlings lethal in Arabidopsis. To verify OgPR10 mRNA expression in transgenic plants, total RNA was isolated and analyzed by Northern hybridization using OgPR10 specific probe. C: wild-type Col-o plants, 1: dying OgPR10-expressing plants showing green leaves, 2: dead OgPR10-expressing plants.
Fig. 6.Glucocorticoid-induced death of OgPR10-expressing Arabidopsis seedlings. (A) Schematic representation of the glucocorticoid-inducible system. 35S, the CaMV 35S promoter; GVG, the chimeric GVG transcription factor; E9, pea rbcS-E9 poly(A) addition sequence; 6ⅩGAL4, six copies of the DNA binding sites for GAL4; OgPR10, OgPR10 coding sequence; 3A, pea rbcS-3A poly(A) addition sequence. (B) Untransformed wild-type (right panel) and transgenic OgPR10 (left panel) seeding grown for 7 days, on MS media containing the concentrations of dexamethasone indicated.
We also generated transgenic rice plants expressing OgPR10 gene. The transformed rice plants showed various phenotypes from normal growth to retarded. Among them, morphologically normal and hygromycin-resistant T0 plants were initially screened for expression of the OgPR10 gene by GUS analysis. The expression level of GUS activity in the OgPR10-overexpression lines varied markedly among the transformants (data not shown). To test if OgPR10 expression rendered rice plants resistant against pathogen infection, we inoculated transgenic rice plants with a virulent isolate KI-1113a of M. grisea. Inoculated untransformed and OgPR10-expressing rice plants incubated under humid conditions for 1 day for successful colonization and transferred to a growth chamber. We monitored disease severity 7 days after inoculation. Differences in the degree of disease symptoms between OgPR10 transgenic and untransformed plants were distinctly identified. Symptoms on control leaves appeared around 3 days after inoculation whereas in leaves of OgPR10-expressing plants disease symptoms were consistently absent 3 days after inoculation. Severe symptoms appeared on leaves of untransformed plants, while OgPR10- expressing rice plants showed overall enhanced disease resistance against the infection of rice blast fungus (Fig. 7).
Fig. 7.Enhanced disease resistance in OgPR10-expressing rice plants. Conidial suspensions were spray-inoculated onto rice seedlings between the 5th and 6th leaf stages. (A) Phenotype of representative OgPR10 transgenic lines (M30, M35, M38, M43, M47). (B) Disease severity as inferred from the mean lesion size leaves in wild type and transgenic plants after inoculating the fungal spores. Lesion sizes were measured 7 days after inoculation.
In summary, we identified OgPR10 gene from wild rice plants. The OgPR10 gene was transcribed in leaves treated with probenazole, yeast extract, and protein phosphatase inhibitors, but not in leaves with SA and JA, which are well-known plant defense signals. This strongly suggested that the OgPR10 gene might be regulated by MAMPs- triggered defense response. We failed to get both recombinant OgPR10 protein and transgenic Arabidopsis plants constitutively expressed OgPR10 gene, which indicating that either its unknown activity or RNase activity resulted in inhibiting the growth of bacteria and the seedling development in plants. Unlike Arabidopsis, stable expression of OgPR10 could be selected among rice transformants and no alteration of visible phenotype were observed in transgenic rice plants, exhibiting disease resistance in response to the infection of rice blast fungus. Thus we propose that further process using the OgPR10-expressing rice plants to breed fungal blast-resistant rice plants will be beneficial to real breeding program against the most severe disease in Asia.
This work was supported by Basic Science Research Program (Scientists in Local Universities) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology to H.W.J (2011- 0006134), by Next-generation BioGreen21 program funded by Rural Development Administration (RDA) to S.H.S. and C-S. K. (PJ00800602).
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