Os08g0398400

Gene Os08g0398400，namely OsHIR1，triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1.

Annotated Information

Function

OsHIR1 triggers hypersensitive cell death and its localization to the plasma membrane is enhanced by OsLRR1.HIR1 (OsHIR1) as an interacting partner of the OsLRR1 (rice Leucine-Rich Repeat protein 1). OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptakes.

Expression

"Figure1.Schematic representation of the conserved structural domains in OsHIR1 and its homologues.(from reference "

"Figure2.Phylogenetic analysis of OsHIR1 and its published plant homologues.(from reference "

"Figure3.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.(from reference "

"Figure4.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).(from reference "

"Figure5.Semi-quantitative analysis of OsHIR1 and OsLRR1 electron microscopy signals in the untransformed control and the OsLRR1 overexpressing rice line. The immunogold-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.(from reference "

"Figure6.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.(from reference "

"Figure10.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).(from reference "

"Figure13.Arabidopsis seeds of OsHIR1-overexpressing lines and control (empty vector) were germinated and grown on ½ MS agar plates with 1.5 % sucrose in the absence or presence of 150 μM As(V) or 50 μM Cd in a controlled environment with a 16 h light/8 h dark photoperiod at 22/18 °C. Root length was analyzed using the Image J software at 12 days after planting (DAP). The data are presented as the mean ± SD of three independent experiments (total n = 30 seedlings per treatment).Asterisks represent significant differences for each mean value of OsHIR1-overexpressing plants compared to the control (*P < 0.05 and **P < 0.01, t test).(from reference "

OsHIR1 was identified as a putative interacting partner ofOsLRR1. The OsHIR1 protein exhibits high similarity (from 84% to 96% identity) to homologues from dicots and monocots (Figure 11), including maize, barley, wheat, pepper, and A. thaliana. For all the close homologues of OsHIR1, computational analysis reveals a putative N-myristoylation site at the N-terminus, followed by a transmembrane domain that is embedded within a Band 7-domain, which covers most of the OsHIR1 protein (Figure 1). In an unrooted phylogenetic tree (Figure 2), HIR proteins can be further divided into two branches: dicots and monocots. Among HIR homologues from monocots, the OsHIR1 shares the highest similarity with the maize ZmHIR1 (96% identity).

To show that OsHIR1 is related to the plant defense response, we investigated whether its gene expression is responsive to pathogen challenge. Northern and western blot analyses showed that both the mRNA and protein levels of OsHIR1 increased after the rice plant was inoculated with the pathogen Xoo LN44 (Figure 3). On the other hand, no such change was observed after mock treatment (Figure 3).

A transgenic line that exhibited a high level of OsLRR1 gene expression was chosen for subsequent electron microscopy analysis (Figure 4). Interestingly, in addition to the elevated level of OsLRR1 mRNA, the expression of the OsHIR1 gene in the OsLRR1 transgenic line was also enhanced (Figure 4).

Immuno-gold electron microscopy studies showed that not only the signal density of the OsLRR1 proteins, but also that of the OsHIR1 proteins, in the plasma membrane, was increased in the OsLRR1 overexpressing line by at least two folds, when compared to the untransformed control (Figure 5). On the other hand, there was no significant difference (Student’s t-test, p < 0.05) between the number of OsHIR1 signals in the tonoplast of the OsLRR1 overexpressing line and that in the untransformed control. These results indicated that OsLRR1 enhanced the plasma membrane localization of OsHIR1.

Proximal occurrences of large and small gold particles were detected in the plasma membrane (Figure 6), supporting the notion that OsLRR1 and OsHIR1 co-localized and interacted in the plasma membrane. Ectopic expression of the OsHIR1 can cause spontaneous hypersensitive response lesions in the leaves of transgenic A. thaliana. To perform a rapid gain-of-function test of QsHIR1,transgenic A. thaliana plants ectopically expressing OsHIR1 were generated. Three weeks after germination, the leaves of about 20% of the OsHIR1 transgenic plants (Col-0/OsHIR1) exhibited white spontaneous HR lesions located randomly at the margins and tips (Figure 3a, red arrows). As negative controls, the untransformed wild type (Col-0) and transgenic plants with the empty vector (Col-0/V7) exhibited normal growth. Transgenic plants expressing OsLRR1 (Col-0/OsLRR1) did not exhibit visible differences in the size, shape, or color of the leaves, when compared to the negative controls (Figure 7).To further observe the effect of OsHIR1 on cell death, lactophenol-trypan blue staining was performed using the leaves of the transgenic A. thaliana. The expression of OsHIR1 caused extensive spontaneous cell death (Figure 8,black arrows). On the other hand, the expression of OsLRR1 only resulted in very mild spontaneous cell death (Figure 8). This explains the lack of visible lesions found in OsLRR1 transgenic plants (Figure 7). No spontaneous cell death was observed in the untransformed control and transgenic plants containing the empty vector (Figure 8).

Ectopic expression of OsHIR1 in transgenic A. thaliana enhances resistance to P. syringae pv. tomato DC3000 (Pst DC3000) When the untransformed wild type (Col-0) or A. thaliana transformed with the empty vector cassette (Col-0/V7) was inoculated with the pathogen Pst DC3000, disease symptoms (yellowing and necrosis) gradually appeared and the infected areas spread out from the original inoculation sites (Figure 9). Such symptoms were alleviated in the transgenic line expressing OsLRR1, consistent with the results of our previous study . The spread of pathogen infection was also suppressed by the ectopic expression of OsHIR1 (Figure 9). Consistent with these visible symptoms, transgenic plants expressing either OsLRR1 or OsHIR1 exhibited a lower titer of pathogens when compared to Col-0 and the empty vector control (Figure 10).However, the OsHIR1 transgenic lines showed a stronger effect on lowering the pathogen titer when compared to the OsLRR1 transgenic line (Figure 10).

The expression levels of PR1 and PR2, two defense marker genes in the salicyclic acid pathway related to the defense against biotrophic pathogens such as Pst DC3000, were monitored in both mock- (Figure 11)and pathogen-inoculated (Figure 12) plants. In both mock-treated and pathogen-inoculated plants, the expression levels of PR1 and PR2 were elevated in both OsHIR1 and OsLRR1 transgenic plants when compared to Col-0 and transgenic plants containing the empty vector cassette. However, the OsHIR1 transgenic plants exhibited significantly higher levels of PR1 and PR2 gene induction than the OsLRR1 transgenic plants (p < 0.05).

OsHIR1‑overexpressing Arabidopsis enhances As and Cd tolerance The finding that the OsHIR1 gene was upregulated in response to As and Cd treatments raises the question of the function of OsHIR1 associated with both elemental stress responses. Three independent transgenic lines were selected depending on the expression level of the OsHIR1 by RT-PCR and then compared to the control plants under 150 μM As and 50 μM Cd (Figure 13). The OsHIR1-overexpressing lines exhibited no apparent phenotypic differences from the vector control lines under normal conditions. However, OsHIR1-overexpressing plants showed significantly longer root lengths than those of the control plants under As and Cd conditions (Fig. 4a). To investigate the possible mechanism by which OsHIR1 ubiquitin E3 ligase affects As or Cd uptake in the transgenic line, we compared As and Cd accumulation between the OsHIR1-overexpressing lines and the control plants. The concentrations of As in the shoots and roots of the OsHIR1-overexpressing plants were 14.1 and 46.4 %, respectively, which were lower than those of the control plants (Figure 14). Cd uptake by the OsHIR1-overexpressing plants was also 3.8 and 12.3 % lower than the control plants in the shoots and roots, respectively(Figure 15). These results suggest that the heterogeneous overexpression of the OsHIR1 gene in Arabidopsis may reduce both As and Cd uptakes by regulating its substrate protein.

Evolution

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.

OsHIR1 E3 ligase positively regulates OsTIP4;1 related to As and Cd uptakes.

Labs working on this gene

• State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China

• Plant Genomics Lab, Department of Applied Plant Sciences, Kangwon National University

Structured Information

Gene Name	Os08g0398400
Description	Similar to Hypersensitive-induced response protein
Version	NM_001068279.1 GI:115476295 GeneID:4345499
Length	4031 bp
Definition	Oryza sativa Japonica Group Os08g0398400, complete gene.
Source	Oryza sativa Japonica Group ORGANISM Oryza sativa Japonica Group Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta; Spermatophyta; Magnoliophyta; Liliopsida; Poales; Poaceae; BEP clade; Ehrhartoideae; Oryzeae; Oryza.
Chromosome	Chromosome 8
Location	Chromosome 8:19099911..19103941
Sequence Coding Region	19101742..19101930,19102201..19102321,19102445..19102536,19102609..19102705,19103258..19103613
Expression	GEO Profiles:Os08g0398400
Genome Context	<gbrowseImage1> name=NC_008401:19099911..19103941 source=RiceChromosome08 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008401:19099911..19103941 source=RiceChromosome08 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgggtcaagcactcggtttggtacaagtagatcaatcgacagtagccatcaaggagtcctttgggaagtttgatgaggttcttgagcctggatgccactttttaccctggtgcatagggaagcagatcgctggatatctttccttgcgtgtgcagcagctggatgtccgttgtgaaacaaagactaaggacaatgtttttgtcaatgttgtggcatctgtgcaataccgtgctcttgctgagaaggcatcagacgccttttacaggcttagcaacaccagggagcaaatccagtcgtatgtttttgatgtgatcagggctagtgttccaaagatgaacttggatgatgcatttgagcagaagaatgagatcgcaaaagctgtggaagatgagcttgaaaaggcaatgtcaatgtatggttatgagattgtgcaaactcttattgttgatattgaaccagatgagcatgttaagagagcaatgaatgaaatcaacgcagctgctaggctgagggtggcagccaatgagaaagctgaagcagagaagattcttcagatcaagagagctgaaggagatgcagaatccaagtacttggctggtctgggtatcgcaaggcagcgtcaggctatagtggatgggctgagagacagtgttcttgccttctccgagaacgtgcctgggacctctgccaaggatgtcatggatatggttctggttacgcaatacttcgacaccatgaaggagataggagcctcatccaagtcctcttcagtgttcatccctcacgggccaggtgccgtcaaagatatcgctgcgcagataagagatggtcagctccaggccaaattgatctaa</cdnaseq>
Protein Sequence	<aaseq>MGQALGLVQVDQSTVAIKESFGKFDEVLEPGCHFLPWCIGKQIA GYLSLRVQQLDVRCETKTKDNVFVNVVASVQYRALAEKASDAFYRLSNTREQIQSYVF DVIRASVPKMNLDDAFEQKNEIAKAVEDELEKAMSMYGYEIVQTLIVDIEPDEHVKRA MNEINAAARLRVAANEKAEAEKILQIKRAEGDAESKYLAGLGIARQRQAIVDGLRDSV LAFSENVPGTSAKDVMDMVLVTQYFDTMKEIGASSKSSSVFIPHGPGAVKDIAAQIRD GQLQAKLI</aaseq>
Gene Sequence	<dnaseqindica>1832..2020#2291..2411#2535..2626#2699..2795#3348..3703#agaatttgccctcctcacttctatacttcaatccccttcctttatttttttttttgggaggggccgaggcaaccgtcgccactccgacgatcccgtcccatccgccgccgccgccggagatttcctcgaggtaagccctaccccgctcctctccagcctctctccccttcggcgggcgcccagcccgagccccgacctcgatttcttggaatttcttggcgagcgtttgtttcttggggtttcctctagattttttcttttttttattattttggggacggatttagctctcgtggaggagattgggcggataatttcgtcggaggggggagggggagggggatgttggaatcgggtggtgggtaatttcgtcgattctccggtccttttccttgagtttagcctgggaattgcggttggttcttggacgcggtttgagttcttggttttttccactttctgtgcggacagatttagggcttgcgctttgcgagtgggggtaaaattcgatggtgtgctgttccagctttgtccacatactgcttggaaatttcaggaggttttttcagtcaagtcaagcacatcttttttgctagactaggccatgagatggaggttaacgtcgaactgtacccacctcagctaaaaataataataattctcaacaacagtatccaagtggctgtattgtttattcttgtttagttatgacgcagatgaatactactcgctaaagattgtgactttcttagttgtcacctcgtcgtgtggagccctgtgggttcttgctcttgacttttatcaaacaatgattgcttggcaagtgtcttgtttgattcgtctgccctttggcttgcatcgccagagtttgctggcttgcgctggcatttaatcaaaagtataatcattcccccaagtataatcaggattagcataatctttagcttcaactatttatatgatatatttggcttgtctgtagtctgtagtggccaagaacatcctgaagcagatttatcctatcaggctatcagcactaagttgatcggcacgtataggttttcttttgcgtcattctttttcattactgcatttctatggtgaattgttttcttttcttttgtatggctacatctattaccctgttgacttctgtgagcctgtgatgtactccctccgggctgataatacttgtcattttggacaagcgcacggtcttcaaaaaacaactttgaccattatttcctattataatatgtaaaaaagtgttaacaaatatatgatcatattaaagtactttttaagaccaatctatatatgtagtcaccatatttgaaagacaaatattttaaaaatgatttatataatcaaatattctaaagtttgacctcacccttgtccaaaacgacaagtattatcagcccggagggagtagtaatcttaggcatttttcagggaatgattacacagtataagtttgagtaatgtgttatcatgatatgcttttcaacaagatatacatattttttgcaacactgtttggtctaagttatctgaaattgcaattgcttgacttttcatttgggatgcgttgggcaatctattgtcctgacatacattcatcttcatttgaagccatggctttatcttttttaattcatatcttttgtttctgtttgtatgtactaaaaacttgaaatatgaatttccattggagaagttaacaccaacggagtataagcaagattatggcaaaacattcagttgttagttgtttcgttagcatatggcaccatttgtaattctgcatttcttcctttctagataagccatgggtcaagcactcggtttggtacaagtagatcaatcgacagtagccatcaaggagtcctttgggaagtttgatgaggttcttgagcctggatgccactttttaccctggtgcatagggaagcagatcgctggatatctttccttgcgtgtgcagcagctggatgtccgttgtgaaacaaagactaaggttccccagtttattttcttaactctggtttcttttatatttcatggcaagtgtctggagcaattgtcaggcatgataaaaaatggttctcaaaattcttgcattttgttccattcgtttactcctttatgataacatactgatgatggttatgtgcacatatctctgttttccttattcttcacttacactaatgcatgatgcatcaaagaataaacccaccttttctgagctctctgacgatgtataccattttgtggatatctgcaggacaatgtttttgtcaatgttgtggcatctgtgcaataccgtgctcttgctgagaaggcatcagacgccttttacaggcttagcaacaccagggagcaaatccagtcgtatgtttttgatggtaaggttcctgtatgctgtgaacattcatgcatccgatgcatttgcagttcatactcttctgattaccttctctatgttttgcctctattgttcaatgctaacagaactttcttcctcgcagtgatcagggctagtgttccaaagatgaacttggatgatgcatttgagcagaagaatgagatcgcaaaagctgtggaagatgagcttgaaaaggtttgtgttcaataattggtggtcatctagagcatagtggagttttatactgacctagtatatactctgtaggcaatgtcaatgtatggttatgagattgtgcaaactcttattgttgatattgaaccagatgagcatgttaagagagcaatgaatgaaatcaacgcaggtaagtattaattaaaacatcaattcattccatttcttcagagacattatttggcttggtgtttatgcttgacatttgagcttgcattggaaacaattttctcctgtttagacatgaaaatgttaaattctctttccgagaaacaaaaccatgtttatattgtctagttttattcaagttttatactgaaacactgaaaattgttgccacaaatatttttaggctttcaacacagtaacactgtaagttatatatagatggaacactgaaaagttatatatagatgtacataacatattatgaatattaacttcgtgaatacttgcaccataacagtcataagtaacacaggcacatgtaactgcaacctattgattacattgtgtgcaacaaaaaatgtgttggtcaactagtcaagtaaatatttcgtgctttttaagggcctgcaacatatatgtttggttggtctggtgtttttctaaagatgagctgtatggctgtaaatgtgctggacggcaacatagttttaactgctgttatttgatgatacagctgctaggctgagggtggcagccaatgagaaagctgaagcagagaagattcttcagatcaagagagctgaaggagatgcagaatccaagtacttggctggtctgggtatcgcaaggcagcgtcaggctatagtggatgggctgagagacagtgttcttgccttctccgagaacgtgcctgggacctctgccaaggatgtcatggatatggttctggttacgcaatacttcgacaccatgaaggagataggagcctcatccaagtcctcttcagtgttcatccctcacgggccaggtgccgtcaaagatatcgctgcgcagataagagatggtcagctccaggccaaattgatctaagaggatggcagggaatggatgttctggatttcttgggcattacgaaagtctgcagtaataccttatctacgtattcgatattttgtgagataaaaacttaggcaaaagaagttctattgtaagtatcacatgcacgcgtgcttcagtaccgtttgttcatagtaattcctgaggttatttccctagacgagtgtcaatatacttcttgaattgcatgtttctgtaaataattcctaagtgttaccgggctgtttggaattatttgcatgttgatgttagtgaccttaagtaatgtcccatcttattatgtgatgtcatgatgatttcc</dnaseqindica>
External Link(s)	NCBI Gene:Os08g0398400, RefSeq:Os08g0398400