Cloning and characterization of two rice long-chain base kinase genes and their function in disease resistance and cell death
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- Zhang, H., Li, L., Yu, Y. et al. Mol Biol Rep (2013) 40: 117. doi:10.1007/s11033-012-2040-y
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Sphingolipid metabolites such as long-chain base 1-phosphates (LCBPs) have been shown to play an important role in plants; however, little is known about their function in plant disease resistance and programmed cell death (PCD). In the present study, we cloned and identified two rice long-chain base kinase (LCBK) genes (OsLCBK1 and OsLCBK2), which are involved in biosynthesis of LCBPs, and performed functional analysis in transgenic tobacco. Expression of OsLCBK1 and OsLCBK2 was induced in rice seedlings after treatments with defense signaling molecules and after infection by Magnaporthe grisea, the causal agent of blast disease. Transgenic tobacco plants overexpressing OsLCBK1 were generated and disease resistance assays indicate that the OsLCBK1-overexpressing plants showed enhanced disease resistance against Pseudmonassyringae pv. tabacci, the causal agent of wildfire disease, and tobacco mosaic virus. Expression levels of some defense-related genes were constitutively up-regulated and further induced after pathogen infection in the OsLCBK1-overexpressing plants. Treatment with fungal toxin fumonisin B1, an effective inducer of PCD in plants, resulted in reduced level of cell death in the OsLCBK1-overexpressing plants, as indicated by cell death staining, leakage of electrolyte and expression of hypersensitive response indicator genes. These data suggest that rice LCBKs, probably through regulation of endogenous LCBP level, play important roles in disease resistance response and PCD in plants.
KeywordsRice (Oryza sativa L.) Long-chain base 1-phosphate (LCBP) Long-chain base kinase (LCBK) Transgenic tobacco Disease resistance Programmed cell death
1-Amino cyclopropane-1-carboxylic acid
Long-chain base kinase
Long-chain base 1-phosphate
Programed cell death
Pseudomonas syringae pv. tobacci
Reverse transcriptase-polymerase chain reaction
Tobacco mosaic virus
During long-term co-evolution with pathogens, plants have developed a sophisticated defense network to survive in adverse environmental conditions. Upon infection, plants often activate series of defense response, which are regulated through different signaling pathways, such as salicylic acid (SA)-dependent and jasmonic acid (JA)/ethylene (ET)-dependent signaling pathways [1, 2, 3]. In the past decades, more and more evidence that indicated lipid signaling was part of the complicated network in plant response to adverse stresses emerged [4, 5].
Sphingolipid metabolites such as long-chain base 1-phosphates (LCBPs) including sphingosine-1-phosphate (S1P) have been detected in animals, plants and yeast and have also been demonstrated to function in a plenty of cellular process [6, 7, 8, 9, 10, 11, 12, 13]. It has been shown that LCBPs including S1P play important roles in plant growth/development and stress responses [14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26]. Recent study revealed that LCBPs are sphingolipid intermediates acting as second messengers in modulation of apoptotic-like programmed cell death (PCD) in plants . The biosynthesis of LCBPs is highly conserved among mammalian, yeast and plant cells. Generally, the cellular levels of LCBPs are tightly controlled by LCB kinase (LCBK) activity responsible for their synthesis and the LCBPs phosphatase and lyase activities responsible for their catabolism [28, 29].
Recent studies have revealed the important function for LCBKs in modulation of endogenous cellular LCBP levels and in regulation of stress response in plants. In Arabidopsis, three LCBKs, AtLCBK1 (At5g23450), AtLCBK2/SPHK1 (At4g21540), and At2g46090, have been identified and the biochemical characteristics and biological functions have been studied in detail for AtLCBK2 [30, 31]. AtLCBK2 (AtSphK1) is a functional sphingosine kinase (SphK). SphKs belong to evolutionally conserved lipid kinases, sharing high level of similarity with diglyceride kinase, but contain a special catalytic domain . It was shown that SphK was involved in ABA-induced closure . The Arabidopsis guard-cell lysates exhibited SphK activity and the majority of Arabidopsis leaf SphK activity is associated with membrane fractions [33, 34]. Functional analyses indicate that the stomata of AtLCBK2-knockdown plants are less sensitive, whereas the stomata of AtLCBK2-overexpressed plants are more sensitive to ABA, suggesting that AtLCBK2 is an important component in plant cell ABA signaling . More recently, it was found that phosphatidic acid can bind and stimulate SphKs activity and that SphK and PLDα1A are co-dependent in amplification of response to ABA, mediating stomatal closure [35, 36]. AtLCBK1 can specifically phosphorylate several LCBs present in plants and the AtLCBK1 mRNA is highly expressed in flowers but was slightly increased by low humidity or ABA treatments [30, 37].
Little is known about the involvement of LCBPs and LCBKs in plant disease resistance response and PCD. In the present study, we cloned two rice LCBK genes, OsLCBK1 and OsLCBK2 and performed functional analysis of OsLCBK1 in transgenic tobacco. Expression of both OsLCBK1 and OsLCBK2 was induced by defense-related signal molecules and in an incompatible interaction between rice and Magnaporthe grisea. Overexpression of OsLCBK1 in transgenic tobacco plants resulted in enhanced disease resistance against Pseudmonassyringae pv. tabacci and tobacco mosaic virus, and reduced PCD after treatment with fumonisin B1 (FB1), an inducer of PCD in plants. Our results suggest that LCBKs play important roles in regulating plant disease resistance response and PCD.
Materials and methods
Plant growth and treatments
Rice (Oryza sativa L.) cv. Yuanfengzao and a pair of isogenic lines (H8R and H8S) were used in this study. Seedlings were grown in a growth room for 3 weeks, in which the photoperiod is 10 h day/14 h night with temperature of 22–27 °C. Seedlings of cv. Yuanfengzao were treated by spraying with solutions of 0.3 mm benzothidiazole (BTH, Novartis Crop Protection Inc., Research Triangle Park, NC, USA), 100 μM JA (Sigma-Aldrich, St. Louis, MO, USA) or 100 μM 1-amino cyclopropane-1-carboxylic acid (ACC) (Sigma-Aldrich, St. Louis, MO, USA), respectively. JA and ACC were dissolved in 0.1 % ethanol. The controls were treated in the same way as spraying with 0.1 % ethanol or distilled sterilized water. Seedlings of H8R and H8S inoculated with Magnaporthe grisea (strain 85-14B1, race ZB1) was done as described previously . Leaf samples were collected at different time points as indicated and stored at −80 °C until use.
Cloning of the OsLCBK1 and OsLCBK2
The full-length cDNAs of putative rice OsLCBK1 and OsLCBK2 were amplified with gene-specific primers OsLCBK1-orf-1F (ATG GAA ATT CAG AAG TCT GAT C)/OsLCBK1-orf-1R (CTA CTG TAC AGG ATT TTT GGC TG) and OsLCBK2-orf-1F (ATG GAA GTA GCT GAT CTA GA)/OsLCBK2-orf-1R (TAA GTA GAT TGT CTG CTT TGC) using phage DNA prepared from a rice cDNA library as template. Amplified PCR products were purified using DNA Gel Purification Kit (Sangon, Shanghai, China) and cloned into pUCm-T-vector (Sangon, Shanghai, China) by T/A cloning, yielding plasmids pUCm-OsLCBK1-1 and pUCm-OsLCBK2-1.
DNA sequencing was performed by the Invitrogen sequencing company (Shanghai, China). Similarity searches on nucleotide and amino acid sequences were carried out using BLAST at the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). Sequences of other LCBK proteins from other organisms were retrieved from GenBank database. Sequence alignments were carried out using ClustalW program in DNAstar software (LaserGene, Madison, WI, USA).
Construction of binary vector and transformation of tobacco
The coding sequence of the OsLCBK1 gene was amplified with plasmids pUCm-OsLCBK1 as templates, using primers OsLCBK1-ORF-2F (GTA GAA TTC ATG GAA ATT CAG AAG TCT GAT) (a EcoRI site underlined)/OsLCBK1-ORF-2R (ATA GTC GAC TAC TGT ACA GGA TTT TTG GCT) (a SalI site underlined). The PCR products were digested with EcoRI/SalI, and ligated into plant binary vector CHF3 pp2p212 digested with same restriction enzymes. The correct recombinant plasmid was transformed into Agrobacterium tumefaciens strain EHA105 by electroporation. Transformation of tobacco was performed using Agrobacterium-mediated leaf disc transformation method as described previously . Seeds of the transgenic lines were germinated on 1/2 MS medium containing 200 μg/mL kanamycin and the survivors were transferred into natural soil and grown to set seeds. The transgenic lines were allowed to grow for 3 generations and homozygous and single-copy lines were screened and confirmed by segregation of antibiotic resistance marker.
Genomic DNA extraction and PCR detection
After frozen leaf samples collected from transgenic tobacco plants were ground to a fine powder with liquid nitrogen, the powder samples were transferred into 200 μL TPS buffer (100 mmol/L Tris–Cl, pH 8.0; 10 mmol/L EDTA, pH8.0; 1 M KCl). After incubation for 20 min at 75 °C, the samples were extracted by centrifugation for 15 min at 15,000 rpm. Transferring the supernatant to a new Eppendorf tube and adding 1 volume of cooled isoamyl alcohol. The DNA in supernatant was precipitated at −20 °C for 30 min and centrifuged, followed by washing with 70 % ethanol. The genomic DNA was dissolved in 20–30 μL 1/10 TE buffer (10 mmol/L Tris–Cl, pH 8.0; 1 mmol/L EDTA, pH 8.0). About 100 ng of genomic DNA was used as template to amplify transgenes by PCR using primers of OsLCBK1-ORF-1F/1R.
Eight-week-old wild-type and transgenic tobacco plants were used for disease assays. Pseudomonassyringae pv. tabaci was cultured in King’s B broth at 28 °C for 2 days, and a bacterial suspension was prepared in 10 mM MgCl2 (5 × 104 colony forming units mL−1). Inoculation was performed by infiltration of the bacterial suspension using a 1-mL needleless syringe into leaves of tobacco plants. To determine bacterial titers, 10 infected leaves were collected from different plants after inoculation. Leaf discs made by a hole puncher were scattered in 10 mM MgCl2, then serial-diluted by 1:10, and plated onto King’s B medium. After placed at 28 °C for 2 days, bacterial colonies appeared were counted. For tobacco mosaic virus (TMV) disease assay, purified TMV was diluted in a potassium phosphate buffer (50 mM, pH 7.0) to a final concentration of 10 ng/μL. Fully expanded leaves were dusted with dry carborundum and inoculated by gently rubbing the upper leaf surface with 100 μL of the viral suspension inoculum, followed by rinsing with distill sterilized water immediately. The inoculated plants were maintained at 23–26 °C under continuous illumination provided by fluorescent lamps and lesion numbers on inoculated leaves were counted 5 days after inoculation. Leaf samples were collected to detect viral accumulation by analyzing levels of the coat protein genes using PCR with gene-specific primers. All experiments were repeated three times and at least six plants were used in each treatment.
Cell death assays
Fully expanded leaves of eight-week-old wild-type and transgenic tobacco plants grown in soil were infiltrated with approximately 20 μL of 10 μM fumonisin B1 (Sigma-Aldrich, St. Louis, MO, USA) in 10 mM MgSO4 solution or mock treated with only 10 mM MgSO4 solution using a 1-mL needleless syringe. To detect cell death, Evans blue staining was done as described previously  with minor modifications. Briefly, detached leaves were vacuum infiltrated with 0.1 % w/v Evans blue solution for three 5 min cycles, remained them incubation under vacuum for 20 min rinsed the leaves with water and cleared by boiling in alcoholic lactophenol (95 % ethanol:lactophenol, 2:1) for 2 min, rinsed with 50 % ethanol, then distilled water. For measurement of electrolyte leakage, four leaf discs (10 mm diameter) were putted into 4 mL of distilled water for 3 h at room temperature and primary conductivity values were measured and recorded using a DDS-IIAT type conductivity meter. The leaf discs were then boiled for 5 min and water were added to make the same volume as the starting volumes, the total conductivity was recorded. The percentage of electrolyte leakage was calculated as 100 % × (initial conductivity of the samples)/(total conductivity after boiling). TUNEL assay was performed using in situ Apoptosis Detection Kit (TaKaRa, Dalian, China) according to the manufacturer’s instruction.
Gene expression analysis by RT-PCR
Total RNA was extracted using TRIZOL reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Total RNA (500 ng) was reverse-transcribed using the SuperScript III Kit (Invitrogen, Shanghai, China). One microliter of the RT reaction and 10 pmol of each primer were used for semi-quantitative RT-PCR in a total volume of 25 μL. PCR conditions were set as 94 °C 30 s, 54–62 °C 30 s and 72 °C 30 s for 25–35 cycles based on the abundance of transcript for each gene, followed by 5 min of final extension at 72 °C. PCR products were electrophoresed on a 1.2 % agarose gel. Gene-specific primers used were as follow: OsLCBK1-rt-1F, GGA ACC TAT ATT CAA GCT TGC A; OsLCBK1-rt-1R, CTG AGA GTT CAA GAA CAT CAC T; OsLCBK2-rt-1F, GGA TGT CAT CTC TGT ATC TTA C; OsLCBK2-rt-1R, CAG AAA GCT CCA GGA CAT CAC T; OsActin-1F, ACT GCT CCC ATC TAT GAA GGA; OsActin-1R, CTG CTG GAA TGT GCT GAG AGA; NtPR1-1F, GAT GCC CAT AAC ACA GCT CGT GC; NtPR1-1R, GCC TCT ATA ATT ACC TGG AGG ATC; NtPR2-1F, GCA ACA TAT TCA GGG ATC; NtPR2-1R, ATT GAA ATT GAG TTG ATA; NtPR3-1F, CCA GAG TGA CAG ATA TTA; NtPR3-1R, GCC CTG GCC GAA GTT CCT; NtPR5-1F, GTC AAC CAA TGC ACC TAC; NtPR5-1R, GGT GGA TCA TCC TGT GGA; Nthsr203J-1F, TAG CCA CGC AGA TGC AAA CC; Nthsr203J-1R, GTG ACA ATC AAG ACG GTA C; NtHIN1-1F, GAG CCA TGC CGG AAT CCA AT; NtHIN1-1R, CTA CCA ATC AAG ATG GCA TCT GG; NtActin-1F, CTA TTC TCC GCT TTG GAC TTG GCA; NtActin-1R, ACC TGC TGG AAG GTG CTG AGG GAA.
Characterization of rice OsLCBK1 and OsLCBK2
By database searching using LCBKs including SphKs from human, mouse and Arabidopsis as queries, two genes (Os10g37280 and Os04g45800) were found to encode putative LCBKs and designated as OsLCBK1 and OsLCBK2, respectively. We cloned by PCR and sequenced the full-length cDNAs for the OsLCBK1 and OsLCBK2 genes. The obtained sequences, together with the full-length cDNAs retrieved from GenBank database (AK070811 and AK121309 for OsLCBK1, and AK121106 for OsLCBK2), confirmed the annotations of predicted coding sequences of these two genes.
Expression of OsLCBK1 and OsLCBK2 in response to signaling molecules and pathogen infection
Generation of OsLCBK1-overexpressing transgenic lines
To study further the biological function of OsLCBK1 in defense response, a functional analysis in transgenic tobacco plants was performed. The coding sequence of OsLCBK1 was cloned into a plant binary vector CHF3 under the control of CaMV 35S promoter and was introduced into tobacco using the Agrobacterium-mediated leaf disc transformation method. Transgenic lines with single copy of the OsLCBK1 gene were screened based on segregation of antibiotic resistance marker in progenies. These transgenic lines with single copy were allowed to grow for 3 generations, and homozygous lines were used for downstream studies. RT-PCR analysis indicated that OsLCBK1 was expressed in these transgenic tobacco lines (Fig. 2c). During our experiments, we did not observe any morphological and developmental changes in the OsLCBK1-overexpressing tobacco plants (data not shown).
Increased resistance of OsLCBK1-overexpressing plants to Pst and TMV
Up-regulated expression of PR genes in OsLCBK1-overexpressing plants
Reduced level of FB1-induced cell death in OsLCBK1-overexpressing plants
Sphingolipids are critical components of eukaryotic cells and sphingolipid metabolites such as LCBPs, S1P and ceramide (Cer) are important regulators in animal cells [42, 43, 44]. Identification of some Arabidopsis mutants with altered disease resistance response and/or PCD phenotypes have revealed that enzymes involved in the sphingolipid metabolism, e.g. serine palmitoyltransferase and ceramide kinase, play important roles in plant defense responses and PCD [45, 46, 47, 48, 49]. However, direct evidence supporting a role for LCBKs in plant disease resistance and PCD is lacking. In the present study, we studied in detail the involvement of a rice LCBK gene OsLCBK1 in disease resistance and PCD using ectopic overexpression in transgenic tobacco lines. Our results showed that overexpression of OsLCBK1 in transgenic tobacco plants resulted in enhanced disease resistance against different type of pathogens and reduced cell death, suggesting that LCBKs-mediated synthesis of LCBPs plays an important role in regulation of defense response and PCD in plants. These new findings not only extend our understanding of the biological function of LCBPs and LCBKs, but also provide new insights into the physiological and molecular mechanisms of plant disease resistance response and regulation of PCD.
Expression of OsLCBK1 was not only induced by some well-known defense-related signal molecules, but also activated by infection with M. grisea. Notably, induced expression of OsLCBK1 was only observed in an incompatible interaction between rice and M. grisea. These observations indicate that the LCBPs metabolism in rice plants is responsive to infection from incompatible pathogen, supporting a role for LCBKs involved in LCBPs biosynthesis in disease resistance response. This conclusion is further supported by our observations that the OsLCBK1-overexpressing tobacco plants displayed enhanced resistance to Pst and TMV (Figs. 3, 4). Generally, up-regulated expression of defense genes is involved in regulation of defense responses against different types of pathogens . A constitutively up-regulated expression and a further increase in expression of defense genes tested were observed in the OsLCBK1-overexpressing plants before and after pathogen infection (Fig. 5), indicating that elevated levels of endogenous LCBPs, probably synthesized by OsLCBK1, activate or potentiate endogenous signaling pathway(s) that modulate defense responses.
It was previously found that disruption of sphingolipid metabolism is lethal to the plants because of the over-accumulation of cytotoxic sphingolipid metabolites [46, 50]. Mutation or overexpression of genes encoding enzymes involved in sphingolipid synthetic enzymes can affect PCD in plants [45, 46, 48, 51, 52, 53, 54]. In Arabidopsis accelerated cell death 5 (acd5) mutant plants, spontaneous PCD-like lesions are caused by the disruption of Cer kinase . In the present study, overexpression of OsLCBK1 in transgenic plants, which may contain elevated level of endogenous LCBPs, attenuated FB1-induced PCD as verified by Evans blue staining and TUNEL staining of dead cells, measurement of electrolyte leakage and analysis of HR indicator gene expression (Fig. 6). In animals, ceramide and sphingosine activate PCD, whereas sphingosine-1-phosphate (S1P) suppresses PCD [7, 55, 56, 57]. Similarly, it was also found that ceramide and sphingosine induced plant PCD while S1P blocked PCD in Arabidopsis leaves or reduced PCD in Arabidopsis cell culture [27, 45, 46]. However, our results are contrary to previous observation that the Arabidopsis mutants lacking LCBPs lyase are FB1-sensitive . Degradation of LCBPs is catalyzed by LCBPs lyase or phosphatase [28, 29]. Theoretically, lack of LCBPs lyase should increase the endogenous level of LCBPs, implying that elevated level of endogenous LCBPs increases PCD; however, the exact level of endogenous LCBPs might be not changed due to the existence of LCBPs phosphatase in the mutants. Taken together, our results suggest an important role for LCBKs, probably through modulation of endogenous LCBP level, in regulation of plant PCD.
In summary, evidence presented in this study suggests that OsLCBK1 plays important roles in regulation of defense response against pathogen infection and PCD. Overexpression of OsLCBK1 results in constitutive expression of defense-related genes in transgenic tobacco plants and thus OsLCBK1 appears to be a positive regulator of defense response. Because of the involvement of OsLCBK1 in LCBPs synthesis, we thus speculate that endogenous level of LCBPs may play important roles in regulation of defense signaling pathways in plants. However, biochemical analysis of endogenous LCBPs in plant defense responses needs to be further explored.
This study was supported by the National Key Project for Research on Transgenic Plant (2011ZX08001-002), the National Natural Science Foundation of China (No. 30971880 and No. 31101397), and the National High-Tech R & D Program (No. 2012AA101505).