OsBIRH1, a DEAD-box RNA helicase with functions in modulating defence responses against pathogen infection and oxidative stress

Abstract

DEAD-box proteins comprise a large protein family with members from all kingdoms and play important roles in all types of processes in RNA metabolism. In this study, a rice gene OsBIRH1, which encodes a DEAD-box RNA helicase protein, was cloned and characterized. The predicted OsBIRH1 protein contains a DEAD domain and all conserved motifs that are common characteristics of DEAD-box RNA helicases. Recombinant OsBIRH1 protein purified from Escherichia coli was shown to have both RNA-dependent ATPase and ATP-dependent RNA helicase activities in vitro. Expression of OsBIRH1 was activated in rice seedling leaves after treatment with defence-related signal chemicals, for example benzothiadiazole, salicylic acid, l-aminocyclopropane-1-carboxylic acid, and jasmonic acid, and was also up-regulated in an incompatible interaction between a resistant rice genotype and the blast fungus, Magnaporthe grisea. Transgenic Arabidopsis plants that overexpress the OsBIRH1 gene were generated. Disease resistance phenotype assays revealed that the OsBIRH1-overexpressing transgenic plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv. tomato DC3000. Meanwhile, defence-related genes, for example PR-1, PR-2, PR-5, and PDF1.2, showed an up-regulated expression in the transgenic plants. Moreover, the OsBIRH1 transgenic Arabidopsis plants also showed increased tolerance to oxidative stress and elevated expression levels of oxidative defence genes, AtApx1, AtApx2, and AtFSD1. The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in defence responses against biotic and abiotic stresses.

Fig. 1.

Structure of OsBIRH1 protein. (A) Alignment of OsBIRH1 with Arabidopsis AtRH50 (At3g06980) and human BAD18438. Conserved motifs are indicated by short thick lines and the conserved phenylalanine residue is indicated by an inverted filled triangle. (B) Domain organization of the OsBIRH1 protein. The amino acid positions are indicated. (C) Comparison of the conserved motifs in OsBIRH1 with consensus from other DEAD-box RNA helicase proteins.

Fig. 2.

Phylogenetic tree analysis of OsBIRH1 with DEAD-box RNA helicase protein from other organisms. Phylogenetic trees were constructed using the Neighbor–Joining method and genetic distances were calculated using the Kimura two-parameter model. Bootstrap values from 1000 replicates were used to assess the robustness of the trees. The DEAD-box RNA helicase proteins used were: Arabidopsis thaliana At3g06980 (AAO00880, AY113064), At4g09730 (BAD43116, AK175435), AtLOS4 (At3g53110, BT002444), AtSTRS1 (At1g31970, AY080680), AtSTRS2 (At5g08620, AY035114), AtISE2 (At1g70070, AF387007), AtUPF1 (At5g47010, AF484122), AtDCL1 (At1g01040, AF292940), AtPMH1 (At3g22310, AY091091), AtPMH2 (At3g22330, AY062502), AtHEN2 (At2g06990, AY080791), and AtDRH1 (At3g01540, AY062591); Oryza sativa Os01g0184500 (BAD67795); Homo sapiens Hs-BAD18438 (BAD18438); Zea mays ZmDRH1 (AAR29370, AY466159); and Nicotinana tabacum NtVDL (AAG34873, AF261032). The OsBIRH1 protein is underlined.

Fig. 3.

RNA-dependent ATPase and ATP-dependent RNA helicase activities of OsBIRH1. (A) ATPase activity was measured by a spectrophotometric assay in the presence or absence of total rice RNA. Oxidation of NADH, which is directly proportional to the rate of ATP hydrolysis, was continuously monitored by measuring the absorbance at 338 nm. Diamonds, OsBIRH1 (150 nM) without RNA; squares, OsBIRH1 (150 nM) with RNA (50 mg ml⁻¹). (B) Scheme of the partial RNA duplexes used for unwinding activity assays. (C) RNA unwinding activity of OsBIRH1. Activity was measured by the ability of the protein to dissociate the partial RNA duplex. Fifty femtomol of labelled substrate was incubated with increasing concentrations of OsBIRH1 in the presence or absence of 2 mM ATP. The products of the reaction were separated by a 10% native PAGE and visualized by autoradiography. ss probe, Single strand probe as positive control; Boiled ds probe, the duplex was boiled before loading on the gel.

Fig. 4.

Expression patterns of OsBIRH1 in rice seedlings after treatment with different resistance signal chemicals and in interactions between rice and Magnaporthe grisea. (A) Expression of OsBIRH1 by disease resistance-related signal molecules. Rice seedlings were treated by spraying with solutions of 0.3 mM BTH, 1 mM SA, 100 μM JA, 100 μM ACC, or water. (B) Induced expression of OsBIRH1 in rice and the blast fungus interactions. Three-week-old rice seedlings of H8R and H8S were inoculated with M. grisea. Rice leaf samples were collected at each time point (hours) as indicated after treatment or inoculation. Twenty micrograms of total RNA were fractionated on a 1.2% agarose formaldehyde gel and hybridized with the α-³²P-labelled 856 bp fragment of OsBIRH1 as a probe. The corresponding ethidium bromide gel images show equal loading of total samples.

Fig. 5.

Southern blot and RT-PCR analysis of the OsBIRH1 transgenic plants. (A) Southern blot analysis of the transgenic lines. M, Non-transgenic plant as a negative control; 1–10, individual OsBIRH1 transgenic plants. (B) RT-PCR analysis of OsBIRH1 expression in homozygous transgenic lines. WT, Non-transgenic plant as a negative control (CK-); V, pCAMBIA 99-1 transgenic plants; #6, #7, #9, and #2, individual OsBIRH1 transgenic plants carrying a single copy of the OsBIRH1 gene.

Fig. 6.

Enhanced disease resistance of OsBIRH1-overexpressing Arabidopsis plants against Pseudomonas syringae pv. tomato and Alternaria brassicicola. (A) Representative leaves showing disease symptoms (left panel) and lesion size (right panel) after inoculation with Alternaria brassicicola. Leaves from wild-type (Wt) and OsBIRH1 transgenic plants were inoculated with a 4 μl droplet of spores (10⁶ spores ml⁻¹) and photographs were taken at 5 d after inoculation. Lesion size was measured 3 d after inoculation. Data presented are means ±SD from a minimum of 40 lesions. (B) Representative leaves showing disease symptoms (left panel) and bacterial growth (right panel) after infiltration with Pseudomonas syringae pv. tomato DC3000. Bacterial growth [colony-forming units (CFU) cm⁻² leaf area] in leaves at 0, 2, and 4 d after inoculation was measured. Photographs were taken 3 d after inoculation. Data presented are means ±SD from three independent experiments. Different letters above the columns indicate significant differences at P=0.05. WT, Wild type; #2 and #9, OsBIRH1-overexpressing plants.

Fig. 7.

Expression of defence-related genes in the OsBIRH1-overexpressing plants. Two-week-old seedlings grown on MS medium were collected for analysis of gene expression by RT-PCR. WT, Non-transgenic plant as a negative control (CK-); V, pCAMBIA 99-1 transgenic plants; #2, #6, #7 and #9, individual transgenic plants carrying single copy of OsBIRH1 gene.

Fig. 8.

Enhanced tolerance to oxidative stress of OsBIRH1-overexpressing plants. (A) Percentage of seed germination of the wild-type (WT) and transgenic Arabidopsis lines on 1/2 MS agar medium containing 100 μM MV. Germination was scored when the radical tip had fully emerged from the seed coat. (B) Enhanced tolerance to MV of the transgenic plants at the seedling stage. Representative seedlings 14 d after MV treatment are shown. (C) Relative fresh weight of the WT or transgenic lines grown in the absence or presence of MV. Seedlings grown without MV are given a value of 100%. (D) Representative leaves from WT and transgenic plants 5 d after MV treatment are shown. (E) Chlorophyll concentration of the leaf tissues of the WT or transgenic lines was measured in the presence or absence of different MV concentrations. Data presented are means ±SD from three independent experiments. Different letters above the columns indicate significant differences at P=0.05.

Fig. 9.

Expression of oxidative stress defence genes in the OsBIRH1-overexpressing plants. Two-week-old seedlings grown on MS medium were collected for analysis of gene expression by RT-PCR. WT, Non-transgenic plant as a negative control (CK-); #2, #6, #7, and #9, individual transgenic plants carrying single copy of the OsBIRH1 gene.