Os01g0771200

Gene RAP-DB of Os01g0771200,namely XB24, is a XA21 binding protein gene and XB24 is a unique ATPase from a previously unclassified subclass. It does not belong to any of these previously described superfamilies of ATPases or HSPs.

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Annotated Information

Function

• XB24 ATPase Enhances Autophosphorylation of XA21K668. We tested whether XB24 is a substrate ofXA21 or affectsXA21 kinase autophosphorylation. Purified His-XB24 and GST-XA21K668 were co-incubated in the presence of [32P]ATP for kinase analysis.For a control, the purified His-XB24 was co-incubated with GSTXA21K668K736E,a catalytically inactive mutant . As Fig. 2B shows, the GST-XA21K668 autophosphorylates as expected, whereas His-XB24 does not autophosphorylate or become transphosphorylated by GST-XA21K668. The phosphorylation of GSTXA21K668 is highly enhanced in the presence ofHis-XB24 protein.No phosphorylation of GST-XA21K668K736E can be detected in reactions carried out in the presence of absence of His-XB24. These results demonstrate that XB24 promotes XA21K668 autophosphorylation.To test whether XB24 promotes autophosphorylation of intact, native XA21 protein, the immunoprecipitated ProAXA21 protein from rice tissue described above (0, 1, or 2 days post-PXO99 inoculation) was co-incubated with the purified His-XB24 for kinase autophosphorylation analyses.These results demonstrate that XB24 promotes autophosphorylation of the native XA21 protein. Furthermore, XB24 is not transphosphorylated by the XA21 protein with or without PXO99 inoculation (Fig. S5). To test whether the ATPase activity of XB24 is required for promoting XA21K668 autophosphorylation, the purified Ntap-XB24 and NtapXB24S154A were incubatedwithGST-taggedXA21K668 in the presence of [32P]ATP for kinase analyses. Autophosphorylation of GST-XA21K668 is enhanced in the presence of rice-expressed Ntap-XB24 but not Ntap-XB24S154A (Fig. 2C). Autophosphorylation of theGST-XA21K668 fusion protein is also enhanced in the presence of the His-XB24 protein but not His-XB24S154A (Fig. S6). These results demonstrate that XB24 enhances XA21 autophosphorylation and that itsATPase activity is required for this function.

• Silencing of Xb24 Enhances Xa21-Mediated Resistance. To investigate the biological function of XB24, we used the RNA interference(RNAi) approach to silence the Xb24 gene and monitored its effects on disease resistance. We developed two independent lines, Xb24RNAi-3 and Xb24RNAi-9, each containing a single-locus insertion, using the rice cultivar Kitaake as the transgene recipient.RT-PCR analysis revealed that Xb24 transcript levels were significantly reduced in these two lines (Fig. S7A). Both lines show similar disease lesion lengths compared to the control line Kitaake after challenge with PXO99 (Fig. S7B), indicating that silencing of Xb24 does not affect the susceptibility of Kitaake to Xoo. To explore the role of XB24 in XA21-mediated signaling, we crossed Xb24RNAi-3 and Xb24RNAi-9 with Xa21 lines and obtained one progeny form the Xa21/Xb24RNAi-3 cross and three from the Xa21/Xb24RNAi-9 cross. Our initial results indicated that silencing of Xb24 enhanced resistance (Fig. S7B). To confirm these results,we developed anF4 line (A176) from one of the F1 plants.TheA176 line carries homozygous Xa21 and homozygous Xb24RNAi-9. We then inoculated 3-week-old A176 plants.As shown in Fig. 3A, these plants developed much shorter lesion lengths (3 ± 0.9 cm) than the wildtype Xa21 plants (6.8±1.2 cm), which show only partial resistance at the 3-weeks-old (tilling) stage (28). A t test gave a P value of 8.62 ×10−13, showing a highly significant difference.Rice line Xb24RNAi-9 showed similar disease lesion lengths (16 ± 2.5 cm) as Kitaake (P =0.56). Bacterial growth curve analysis revealed that Xa21/Xb24RNAi-9 lines harbor 3.2-fold less Xoo bacteria (1.48 × 107 ±1.2 × 106) in their leaves than the Xa21 lines (4.8 × 107±4.4 × 106) at 12 days postinoculation (Fig. 3B), consistent with the leaf lesion length measurements described above. This experiment was repeated three times, and similar results were obtained each time. These results demonstrate that silencing of Xb24 expression enhances XA21-mediated disease resistance.

• Overexpression of XB24 Compromises XA21-Mediated Resistance. To further investigate the involvement ofXB24 in theXA21-mediated signaling, we created construct Ubi-Xb24 to overexpress XB24 using the maize Ubi-1 promoter. We introduced the Ubi-Xb24 construct directly into an Xa21 (in the TP309 genetic background)line by Agrobacterium-mediated transformation using mannose Selection and generated five independent T0 plants. After PCR-based genotyping and RT-PCR-based transcripts expression analyses to confirm that Xb24 is overexpressed, we challenged 6-week-old Xa21 lines withPXO99.Wefound that all of the five lines have longer disease lesion lengths compared with the wild-type Xa21 plants (Fig. S8).Two homozygous lines (Xa21/Xb24ox-1 and -2) fromtwo of these five independent lines were then developed. Overexpression of XB24 (XB24ox) in the progeny from these homozygous lines was confirmed by protein gel blotting analysis(Fig. 4A). Six-week-old plants were challenged with PXO99. Disease lesion lengths on both the Xa21/Xb24ox-1 and -2 lines (7.3 ± 0.5 cmfor line 1 and 6.0 ± 0.5 cmfor line 2) were longer than those observed on Xa21 lines (1.3 ±0.4 cm) (Fig. 4 A and B). The low P values (5.02 × 10−21 for Xa21/Xb24ox-1 and 2.06 × 10−23 for Xa21/Xb24ox-2) indicate that these differences are statistically significant. At 12 days postinoculation,the accumulation of bacterial populations, as measured by bacterial growth curve analysis, in the two Xa21/Xb24ox lines (1.23 × 108 ±1.88 × 107 for Xa21/Xb24ox-1 and 1.08 × 108 ± 1.97 × 107 for Xa21/Xb24ox-2)was clearly higher (>2-fold) than in theXa21 lines (5.20×107 ± 8.9 × 105) (Fig. 4C). Again, the low P values (8.27 × 10−4 forXa21/Xb24ox-1 and 2.72 × 10−3 for Xa21/Xb24ox-2) of bacterial accumulation at 12 days postinoculation indicate that these differences are statistically significant. Rice lines overexpressing Xb24 display similar levels of susceptibility as control lines lacking overexpressed Xb24 in three independent biological replicates (Fig. S8).These results demonstrate that overexpression of XB24 compromises XA21-mediated resistance.

• Overexpression of Xb24 Causes XA21 Instability Following Ax21 Recognition. To gain insight into the mechanism of XB24-mediated regulation of XA21 function, we tested whether XB24 affects the amount of the XA21 protein after Xoo inoculation. As shown inFig. 6 A and B, without Xoo inoculation (Mock treatment). Overexpression ofXB24(Xa21/Ntap-Xb24) causednosignificant decrease in the ProA-XA21 protein level compared to overexpression of Ntap (Xa21/Ntap) alone. In contrast, after inoculation with PXO99,the Xa21/Xb24ox line showed a sharp decrease in the ProA-XA21 protein level. The Xa21/Ntap control line showed amarked increase.When inoculated with the Xoo strain PXO99ΔraxST, the Xa21/Xb24ox sample showed an increase in the ProA-XA21 level similar to that of the Xa21/Ntap control. Similar results were obtained from three biological repeats of this experiment.These results indicate that the sharp decrease in the XA21 protein level is Ax21-specific.

Other associations with XA21

• XB24 Physically Associates with XA21 in Vivo. WeisolatedXB24 as an XA21 interacting protein through yeast two-hybrid screening.The XB24 cDNAis expressed froma unique rice gene,Os01g56470(Fig. S1A), and encodes a 198-aa protein. The predicted secondary structure has no significant motifs except for a C-terminal ATP synthase α- and β-subunits signature (ATPase) motif with the sequence PSINERESSS (Fig. S1B). Although 38 human proteins,43 Arabidopsis proteins, and 67 additional rice proteins are annotated to contain a conserved ATPase motif (Fig. S2), none share similarity beyond the ATPase motif with XB24 and most are not functionally characterized. Thus, XB24 belongs to a previously uncharacterized class of ATPases.To confirm the specificity of the XB24-XA21interaction, we performed yeast two-hybrid analysis and foundthatXB24 associates with XA21K668 (containing the entire juxtamembrane and the kinase domains of XA21) but not with XA21K668K736E , a catalytically inactive mutant of XA21K668 (Fig. 1A Left). These results indicate that the association between XB24 and XA21 requires XA21 kinase activity. The ATPase motif of XB24 is not required for the XB24-XA21 interaction in yeast because XB24(1-146), lacking the ATPase motif, retains the ability to interact with XA21, whereas XB24(146-198), containing the ATPase motif, is incapable of interacting with XA21 (Fig. 1A Right).To determine whether XB24 physically associates with XA21 in vivo, we created transgenic plants that express a protein A domaintagged XA21 (ProA-XA21) under control of the native Xa21 promoter in the rice cultivar Kitaake. We established a homozygous line, A114, with a single transgene insertion and demonstrated that it confers full resistance to Xoo strain PXO99 (Fig. S3). A complex associated with ProA-XA21 was immunoprecipitated from total extracts from A114 leaves. Ntap (N-terminal tandem affinity purification, which contains the same protein A domain) transgenic plants, under control of the maizeUbi-1 promoter, were used as the control. The immunoprecipitateswere separated on anSDS/PAGEgel and analyzed by Western blotting using the PAP antibody to probe ProA-XA21 and Ntap, and anti-XB24 antibody for XB24, separately. The PAP probe detected full-length ProA-XA21 and a cleaved XA21 product (marked by an asterisk in Fig. 1B) in the ProA-XA21 immunoprecipitate.Aclear band of endogenousXB24 was detected from the immunoprecipitate of ProA-XA21 but not from the precipitates of Ntap (Fig. 1B).

• ATPase Activity Is Essential for XB24-Mediated Regulation of XA21 Function. We next tested whether XB24 ATPase activity was required for XB24 to regulate XA21 function.We developed Xa21/Xb24ox and Xa21/Xb24S154Aox plants using NtapXb24ox andNtapXb24S154Aox plants, respectively, to crosswith ProAXa21 plants,and inoculated these plantswith PXO99.As shown in Fig. 5A, all Xa21/Xb24ox plants display compromised resistance, whereas Xa21/Xb24S154Aox plants show similar disease lesion lengths compared to Xa21 plants. The lesion length difference between Xa21 and Xa21/Xb24ox is highly significant (P = 1.40 × 10−10),whereas the difference between Xa21 and Xa21/Xb24S154Aox is not (P = 0.12). Bacterial growth curve analysis revealed that the amount of Xoo bacteria accumulation in Xa21/Xb24ox plants(2.65 × 108 ± 5.74 × 107) is higher (∼2.45-fold) than that of Xa21 plants (1.08 × 108 ± 6.55 × 106) at 12 days postinoculation(Fig. 5B). The amount of Xoo bacterial accumulation in Xa21/Xb24S154Aox plants (0.91 × 108 ± 1.65 × 107) is similar to that measured in Xa21 plants (Fig. 5B). The low P values of bacteria accumulation at 12 days postinoculation in Xb24ox plants (0.033against Xa21 and 0.028 against Xa21/Xb24S154Aox, respectively)indicate that these differences are statistically significant. This experiment was repeated two times and similar results were obtained each time. Because ProA-XA21 was expressed to similar levels in Xa21/Xb24ox, Xa21/Xb24S154Aox, and Xa21 plants (Fig. S9), these results demonstrate that XB24 requires S154 to repress XA21 function. Therefore, we conclude that the ATPase activity of XB24 is essential for XB24 to regulate XA21-mediated defense response.

Expression

The XB24 cDNAis expressed from a unique rice gene,Os01g56470 (Fig. S1A), and encodes a 198-aa protein. The predicted secondarystructure has no significant motifs except for a C-terminal ATP synthase α-and β-subunits signature (ATPase) motif with the sequence PSINERESSS (Fig. S1B).Although 38 human proteins, 43 Arabidopsis proteins, and 67 additional rice proteins are annotated to contain a conserved ATPase motif (Fig. S2), none share similarity beyond the ATPase motif with XB24 and most are not functionally characterized. Thus, XB24 belongs to a previously uncharacterized class of ATPases.The only conserved structure in XB24 is the region composed of 10 amino acids PSINERES154SS (Fig. S1B) that is predicted as the ATPase motif, (P-[SAP]-[LIV]-[DNH]-{LKGN}-{F}-{S}-S-{DCPH}-S) (Fig. S1C).

Silencing of Xb24 Enhances Xa21-Mediated Resistance.

Overexpression of XB24 Compromises XA21-Mediated Resistance.

Evolution

• XB24 ATPase enzyme activity is required for XB24 function. XA21 is degraded in the presence of the pathogen-associated molecular pattern Ax21 when XB24 is overexpressed. These results demonstrate a function for this large class of broadly conserved ATPases in PRR-mediated immunity.XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice lines silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice lines overexpressing XB24 are compromised for immunity.

Fig. 1. Association of XB24 with XA21 in yeast and in rice plants. (A) Interaction of XB24 with XA21K668 in yeast. K668, truncated XA21 (XA21K668) containing the entire JM and kinase domains (23); K668K736E, kinase catalytically inactive mutant XA21K668K736E; XB24(1-146), truncated XB24 containing amino acids 1–146; XB24(146-198), truncated XB24 containing amino acids 146–198 including the ATPase motif. Blue, positive interaction. Expression proteins were detected using antibodies as indicated in Western blotting. (B) Detection of XA21 and XB24 in immunoprecipitates of ProA-XA21 from rice tissues using the Peroxidase Anti-Peroxidase (PAP) probe and anti-XB24, respectively. *, Cleaved form of ProA-XA21 (23, 24). (C) Analysis of XB24 protein levels in plants before and after PXO99 inoculation using anti-XB24 in Western blot analysis. A duplicate protein gel was stained with Coomassie brilliant blue (CBB) as loading control. (D) Dissociation of XB24 from XA21 in response to PXO99 inoculation. Detection of ProA-XA21 and XB24 in the immunoprecipitates of ProA-XA21 from rice leaf tissues not treated or treated (1 or 2 days) with Xoo strains as indicated. IP, immunoprecipitate.

Fig. 2. An ATPase activity is associated with XB24 and effects XA21 autophosphorylation. (A) ATPase activity assay on purified Ntap-XB24 and Ntap-XB24S154A protein from transgenic plants. The same amount of proteins was used. (Left) Representative autoradiogram. (Right) Quantitative results of three independent experiments. ATP hydrolysis was quantified based on radioactivity of the reaction product Pi. Error bars indicate SDs. (B) Effects of XB24 on XA21 autophosphorylation. In vitro autophosphorylation assays were performed on GST-XA21K668 and GST-XA21K668K736E, respectively, in the presence of the purified His-XB24 protein. (C) Effects of XB24 ATPase on XA21 autophosphorylation. An in vitro autophosphorylation assay was performed on GST-XA21K668 in the presence of the same amount of rice expressed Ntap-XB24 or Ntap-XB24S154A. (Upper) Representative autoradiogram. (Lower) Quantitative results (mean + SD) from three independent experiments. CK, control provided using autophosphorylation assay on GST-XA21K668 in the absence of XB24. The autophosphorylation level from CK was arbitrarily set as “1.”

Fig. 3. Effects of reduced expression of Xb24 on Xa21-mediated resistance. (A) Quantitative lesion length measurements of rice leaves at 14 days after PXO99 inoculation. The means ± SD of each sample was calculated from 24 infected leaves of 8 plants. (B) Bacterial growth curves after PXO99 inoculation. Error bars indicate SDs.

Fig. 4. Effects of overexpression of Xb24 on Xa21-mediated resistance. (A)Photograph of rice leaves 14 days after inoculation with PXO99 (Top). The disease lesions are indicated from the top of the leaf cuts to the arrows. The XB24 protein was detected by anti-XB24 (Middle). A duplicate protein gel was stained with CBB as control (Bottom). (B) Quantitative lesion length measurements of rice leaves at 14 days after Xoo inoculation. The average of each sample was calculated from 40 infected leaves of 10 plants. (C) Growth curves of Xoo postinoculation. Error bars in B and C indicate SDs.

Fig. 5. Requirement of XB24 ATPase activity for regulation of XA21-mediated immunity. (A) Lesion lengths were measured for Xa21, Kitaake, Xa21/Xb24ox, and Xa21/Xb24S154Aox at 14 days after PXO99 inoculation. The mean and SD of each sample were determined using 32 infected leaves from 8 plants. (B) Bacterial growth curve analysis after PXO99 inoculation. Error bars indicate SDs.

Fig. 6. Effects of excess XB24 on XA21 protein stability. (A) Protein immunodetection after SDS/PAGE separation. Protein samples were prepared from ProA-Xa21 transgenic rice plants overexpressing Ntap-Xb24 (labeled Xa21/Ntap-Xb24) or Ntap (labeled Xa21/Ntap) before or after inoculation (1 day) with PXO99 or PXO99ΔRaxST (lacking Ax21 activity). Protein accumulation detected by the PAP probe is shown in (Top) (for ProA-XA21) and (Middle) (for Ntap-XB24 or Ntap), respectively. (Bottom) CBB-stained gel as a loading control. (B) Quantification of XA21 protein levels. The average and SD are calculated from three biological replicate experiments of (A).

Fig. 7. A model for XB24-mediated regulation of XA21 function. Before Ax21 recognition (left), XB24 physically associates with XA21 and uses ATP to promote phosphorylation of certain Ser/Thr sites on XA21, keeping the XA21 protein in an inactive state. Upon recognition of Ax21 (center), Xb24 dissociates from Xa21 leading to activation of the XA21 kinase, resulting in resistance. Once the signal has been relayed, XA21 binds the XB15 phosphatase (right), which attenuates the immune response, likely by dephosphorylation of amino acids required for XA21 function.

Labs working on this gene

• College of Life Science, Zhejiang Sci-Tech University, Hangzhou 310018, China.
• Department of Plant Pathology, University of California, Davis, CA 95616,USA.
• Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA.

Structured Information

Gene Name	Os01g0771200
Description	Similar to Mal d 1-associated protein
Version	NM_001050918.1 GI:115440206 GeneID:4324614
Length	1888 bp
Definition	Oryza sativa Japonica Group Os01g0771200, 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 1
Location	Chromosome 1:34299150..34301037
Sequence Coding Region	34299283..34299494,34300420..34300804
Expression	GEO Profiles:Os01g0771200
Genome Context	<gbrowseImage1> name=NC_008394:34299150..34301037 source=RiceChromosome01 preset=GeneLocation </gbrowseImage1>
Gene Structure	<gbrowseImage2> name=NC_008394:34299150..34301037 source=RiceChromosome01 preset=GeneLocation </gbrowseImage2>
Coding Sequence	<cdnaseq>atgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaa</cdnaseq>
Protein Sequence	<aaseq>MGWRWHDDGDDGGRGLGDIPDLAGGGGGGDGERCATRRVVQSRC HTEEVEPGRFVRKCEKTEQLLRDCVGRPSELVESKTENTEEDVTDEMKSGSLSLGFPT NEPFAFPGLRSDIEALEKGLFGSIGSFLDDAERMTNDFLKSFGVPSINERESSSFDGQ PTGRHIGGQPAGRHIEEGTAKDTKQNDYAEFSSKITDV</aaseq>
Gene Sequence	<dnaseqindica>134..345#1271..1655#agaaacgagccggccggattgctctccaagccaaacacggcccagagaggcgagagcccccacaccgccaaacccgacccggaaatcaactgcacacgctccgatcccctctctccagatcgattcggaccccatgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggtatatacatatactccatggcctcgcgcttttccctgatctgttccttcttttcctgcgaggatcccctctttgcatattactgttcgttgttttagtgatgaattggtgataacatgtcatgcgcagttgtgttactattagtcccgtgtatgattgtgagctgtgtatagccgcctctctgtggacgattttcgtgagtctgtcctcgctgatttgatacaaacacgaattcattaagaatggggaatgtgccgtgatgatcgagacgtttgagttctgtggtgaatagtatgatcaaatgtgtgtaataacttgggcttttgaattttggtagctaattagatatggaatttctatccagttttaggtgcccgtgccatcgacatgggttaaaagttattgtgggctattttgcgctcaacttgttaatcaacaaattttggacggaggcagtactttgtaggcatggggtaaatttgtatgatacacttgttgcttgaagatgttcgtgaactcatgggatttctagacacctacaaccaattacattacagtgttattatattctaacgatatgtaaagagcagctgtgttcgtgttgtctacactaaagattcaacaactagtggtcatcatcaaaccgatagcatatcctcattttgcaggattttgaaaactcacatctcgttagcatccttccttttatttcttcacatgcctcatgttaaacttttaagtgcatgaaacccaaatttctgtatttcgtagctccttgtaaccaatgtacaacactatgattagtattctggatagattttgtacttcccaaagtattttggttgttgacaagctattacaataatctgatggataacctaccatacattatttctcctaaccgatcacaataatcttcttctaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaaggatctacagttagctgacgcacctttgggagcagcttgccaattttgtattttgaacatctccatggttgtaattggaaggggaaggatcagtttgactgttttataagcagagtcgtctgaagtctgaagtgttgcgcttataagaacaattgtgtatattactgttttagataagcctgtttgtgttcctcaacaatgagatcaattatggatggtttttttctctgctt</dnaseqindica>
External Link(s)	NCBI Gene:Os01g0771200, RefSeq:Os01g0771200