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. 2010 Dec 30;10:290.
doi: 10.1186/1471-2229-10-290.

Rice Hypersensitive Induced Reaction Protein 1 (OsHIR1) Associates With Plasma Membrane and Triggers Hypersensitive Cell Death

Free PMC article

Rice Hypersensitive Induced Reaction Protein 1 (OsHIR1) Associates With Plasma Membrane and Triggers Hypersensitive Cell Death

Liang Zhou et al. BMC Plant Biol. .
Free PMC article


Background: In plants, HIR (Hypersensitive Induced Reaction) proteins, members of the PID (Proliferation, Ion and Death) superfamily, have been shown to play a part in the development of spontaneous hypersensitive response lesions in leaves, in reaction to pathogen attacks. The levels of HIR proteins were shown to correlate with localized host cell deaths and defense responses in maize and barley. However, not much was known about the HIR proteins in rice. Since rice is an important cereal crop consumed by more than 50% of the populations in Asia and Africa, it is crucial to understand the mechanisms of disease responses in this plant. We previously identified the rice HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). Here we show that OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1.

Result: Through electron microscopy studies using wild type rice plants, OsHIR1 was found to mainly localize to the plasma membrane, with a minor portion localized to the tonoplast. Moreover, the plasma membrane localization of OsHIR1 was enhanced in transgenic rice plants overexpressing its interacting protein partner, OsLRR1. Co-localization of OsHIR1 and OsLRR1 to the plasma membrane was confirmed by double-labeling electron microscopy. Pathogen inoculation studies using transgenic Arabidopsis thaliana expressing either OsHIR1 or OsLRR1 showed that both transgenic lines exhibited increased resistance toward the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. However, OsHIR1 transgenic plants produced more extensive spontaneous hypersensitive response lesions and contained lower titers of the invading pathogen, when compared to OsLRR1 transgenic plants.

Conclusion: The OsHIR1 protein is mainly localized to the plasma membrane, and its subcellular localization in that compartment is enhanced by OsLRR1. The expression of OsHIR1 may sensitize the plant so that it is more prone to HR and hence can react more promptly to limit the invading pathogens' spread from the infection sites.


Figure 1
Figure 1
Structural domains and phylogenetic relationships of OsHIR1 homologues and expression of OsHIR1 under pathogen inoculation. (a) Alignment of OsHIR1 homologues in plants. "*" represents conserved amino acid residues, ":" conserved substitutions, and "." semi-conserved amino acid substitutions. (b) Schematic representation of the conserved structural domains in OsHIR1 and its homologues. (c) Phylogenetic analysis of OsHIR1 and its published plant homologues. (d) The mRNA and protein levels of OsHIR1 0, 2, 4 and 6 days after inoculation of Xanthomonas oryzae pv. oryzae (Xoo) race LN44 or mock treatment by a leaf-clipping method. Ten μg total RNA and 10 μg total protein were loaded onto each lane.
Figure 2
Figure 2
Regulation of the subcellular localization of OsHIR1 by OsLRR1. (a) Immuno-gold electron microscopy studies. Anti-OsLRR1 and anti-HIR1 antibodies were used to detect the subcellular localization of OsLRR1 and OsHIR1, respectively, in rice leaves. PM: Plasma membrane; TN: Tonoplast. (b) Expression of OsLRR1 and OsHIR1 in an OsLRR1 overexpressing rice line. Real-time RT-PCR analysis was performed to compare the relative gene expression (expression in untransformed control was set to 1). Error bars show the standard errors (N = 3). (c) Semi-quantitative analysis of OsHIR1 and OsLRR1 electron microscopy signals in the untransformed control and the OsLRR1 overexpressing rice line. The immuno-gold-labeled signal counting was described in Methods. Error bars show the standard errors (N = 10). * in (b) and (c) indicates that the difference is significant (p < 0.05, Student's t-test) between the transformants and the untransformed wild type. (d) Double labeling of OsHIR1 and OsLRR1. Two independent photos were shown to illustrate the co-localization of OsHIR1 (15 nm gold particles) and OsLRR1 (6 nm gold particles) to the plasma membrane. PM: Plasma membrane; CW: Cell wall.
Figure 3
Figure 3
Hypersensitive response lesions and spontaneous cell death due to the overexpression of OsHIR1. (a) Hypersensitive response lesions in some OsHIR1 transgenic plants. Three weeks after germination, white necrotic lesions located randomly at the margins and tips of leaves (red arrows) were observed in about 20% of the OsHIR1 transgenic plants. Such a phenomenon was not found in untransformed wild type (Col-0), empty vector transgenic control (Col-0/V7), or OsLRR1 transgenic plants (Col-0/OsLRR1). (b) Lactophenol-trypan blue staining showing spontaneous cell death. Leaves of 3-week-old plants were stained with lactophenol-trypan blue to detect dead cells. Spontaneous cell death found on the leaves of OsHIR1 and OsLRR1 transgenic plants were indicated by black arrows. Bars = 100 μm
Figure 4
Figure 4
Pathogen inoculation test of transgenic A. thaliana expressing OsHIR1. (a) Disease symptoms after pathogen inoculation. Six-week-old seedlings of the untransformed wild type (Col-0), the empty vector-transformed control (Col-0/V7), and the OsLRR1 (Col-0/OsLRR1) and OsHIR1 transgenic lines (Col-0/OsHIR1) were challenged with Pst DC3000. The symptoms were recorded 5 days after inoculation. (b) Pathogen titers 5 days after pathogen inoculation. Rosette leaves were collected from inoculated plants for pathogen titer determination. Statistical analysis using ANOVA followed by Fisher's LSD Test (p < 0.05) reveals 3 groups: 1, the untransformed wild type and the vector-only control; 2, OsLRR1 transgenic plants; and 3, OsHIR1 transgenic plants. The error bars indicate standard errors (N = 3). (c) and (d) Expression of defense marker genes without (mock) or with Pst DC3000 inoculation. Real-time RT-PCR was performed using reverse-transcribed RNA samples. Relative expression levels of PR1 and PR2 in all plants were compared to the mock-inoculated untransformed wild type parent (Col-0; expression level set to 1). Both the expressions of PR1 and PR2 can be categorized into different groups using ANOVA followed by Fisher's LSD Test (p < 0.05). In (d), the gene expression in mock-treated Col-0 was used just to set the reference for gene expression and was not included in the statistical analysis. The error bars indicate standard errors (N = 3). Three independent OsHIR1 transgenic lines (Col-0/HIR1-1, Col-0/HIR1-2, and Col-0/HIR1-3) were used for quantitative studies in (b), (c), and (d).

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