Os01g0771200 - Revision history

Zhennan: /* Extension */

2016-06-24T09:09:15Z

‎Extension

Zhennan: /* Extend */

2016-06-24T09:00:40Z

‎Extend

Zhennan at 08:15, 24 June 2016

2016-06-24T08:15:28Z

Zhennan at 08:12, 24 June 2016

2016-06-24T08:12:13Z

Zhennan at 08:10, 24 June 2016

2016-06-24T08:10:37Z

Zhennan at 08:09, 24 June 2016

2016-06-24T08:09:14Z

Zhennan at 08:05, 24 June 2016

2016-06-24T08:05:57Z

Zhennan at 08:05, 24 June 2016

2016-06-24T08:05:44Z

Zhennan at 08:04, 24 June 2016

2016-06-24T08:04:34Z

Zhennan: /* Evolution */

2016-06-24T08:02:36Z

‎Evolution

@@ Line 41: / Line 41: @@
 * 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.
-== Extension ==
+== Knowledge extension ==
 * The association between XB24 and XA21 is compromised upon inoculation of the Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP <ref name="ref7"/>. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice plants silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice plants overexpressing XB24 are compromised for immunity. XA21 is degraded in the presence of Ax21 when XB24 is overexpressed. These findings reveal that XB24 negatively regulates XA21 PRR function.

@@ Line 41: / Line 41: @@
 * 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.
-== Extend ==
+== Extension ==
-The association between XB24 and XA21 is compromised upon inoculation of the Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP <ref name="ref7"/>. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice plants silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice plants overexpressing XB24 are compromised for immunity. XA21 is degraded in the presence of Ax21 when XB24 is overexpressed. These findings reveal that XB24 negatively regulates XA21 PRR function.
+* The association between XB24 and XA21 is compromised upon inoculation of the Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP <ref name="ref7"/>. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice plants silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice plants overexpressing XB24 are compromised for immunity. XA21 is degraded in the presence of Ax21 when XB24 is overexpressed. These findings reveal that XB24 negatively regulates XA21 PRR function.
-'''XB24 Physically Associates with XA21 in Vivo.''' The author isolated XB24 as an XA21 interacting protein through yeast two-hybrid screening. The XB24 cDNAis expressed froma unique rice gene,Os01g56470, 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. Although 38 human proteins, 43 Arabidopsis proteins, and 67 additional rice proteins are annotated to contain a conserved ATPase motif, 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, they 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. They established a homozygous line, A114, with a single transgene insertion and demonstrated that it confers full resistance to Xoo strain PXO99. 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)'''.
+* The XB24 cDNAis expressed froma unique rice gene,Os01g56470, 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. Although 38 human proteins, 43 Arabidopsis proteins, and 67 additional rice proteins are annotated to contain a conserved ATPase motif, 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, they 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 ('''Figure 1A''').
 [[File:1HF6.jpg|400px|thumb|right|'''Figure. 6.''' ''Effects of excess XB24 on XA21 protein stability.'']]
-'''XB24 Dissociates from XA21 in Response to PXO99 Inoculation.'''To determine whether XB24 is degraded in response to Xoo strain PXO99  inoculation, they performed a Western blot analysis to detect the XB24 protein before and after inoculation. They found that a similar amount of XB24 protein was detected in Xa21 and Kitaake plants before inoculation and 1 day or 2 days after inoculation. This result shows that XB24 is not degraded in response to Ax21. They next investigated whether Ax21 recognition affects the interaction of XA21 and XB24. We performed coimmunoprecipitation experiments withPAP(targeting ProA-XA21)using rice leaf tissues fromtheXa21line inoculated with Xoo strain PXO99 or Xoo strain PXO99ΔraxST, which lacks Ax21 activity due to a knockout of the raxST gene. They then carried out immunoblotting to detect XB24. A similar coimmunoprecipitation was performed using Kitaake rice leaves as a control. As shown in '''Fig. 1D''', they observed a sharp decrease in the amount of XB24 associated with ProA-XA21 post-PXO99 inoculation, whereas, no decrease in the amount of XB24 associated with ProA-XA21 was observed after PXO99ΔraxST inoculation. These results clearly indicate that the physical interaction between XB24 and XA21 disassociates specifically in response to Xoo strains expressing Ax21 activity.
+* To determine whether XB24 is degraded in response to Xoo strain PXO99  inoculation, they performed a Western blot analysis to detect the XB24 protein before and after inoculation. They found that a similar amount of XB24 protein was detected in Xa21 and Kitaake plants before inoculation and 1 day or 2 days after inoculation. This result shows that XB24 is not degraded in response to Ax21. They next investigated whether Ax21 recognition affects the interaction of XA21 and XB24. We performed coimmunoprecipitation experiments withPAP(targeting ProA-XA21)using rice leaf tissues fromtheXa21line inoculated with Xoo strain PXO99 or Xoo strain PXO99ΔraxST, which lacks Ax21 activity due to a knockout of the raxST gene.
 [[File:1HF7.jpg|400px|thumb|right|'''Figure. 7.''' ''A model for XB24-mediated regulation of XA21 function.'']]
-'''ATPase Activity Is Essential for XB24-Mediated Regulation of XA21 Function.''' The author tested whether XB24 ATPase activity was required for XB24 to regulate XA21 function. They developed Xa21/Xb24ox and Xa21/Xb24S154Aox plants using NtapXb24ox and NtapXb24S154Aox plants, respectively, to cross with 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, these results demonstrate that XB24 requires S154 to repress XA21 function. Thus, we conclude that the ATPase activity of XB24 is essential for XB24 to regulate XA21-mediated defense response.
+* ATPases are abundant in most species. ATPases have been classified into four superfamilies, F-, V-, A-, and P-ATPases, based on their structures (33–36). There are some other proteins that cannot be classified into these subfamilies but have ATPase activity, such as heat shock proteins(HSPs), includingHSP60 (33),HSP70 (34), and HSP72 (35). XB24 does not belong to any of these previously described superfamilies of ATPases or HSPs. The only conserved structure in XB24 is the region composed of 10 amino acids PSINERES154SS that is predicted as the ATPase motif, (P-[SAP]-[LIV]-[DNH]-{LKGN}-{F}-{S}-S-{DCPH}-S).
+* The ATPasemotif in the F1,V1, and A1complexes of F-, V-, and AATPases is also essential for ATPase activities, whereas the PATPases and theHSPs do not contain thismotif.However,whether this motif is enough for the ATPase activity of proteins is unclear. Here, They show that XB24, a protein with an ATPase motif but no other motifs or domains, functions as anATPase. Proteins with this conserved motif that cannot be classified into the previously identified ATPases exist in many species, including bacteria, fungi,human, Arabidopsis, and rice.
-'''XB24 Represents a Previously Undescribed Class of ATPases'''. ATPases are abundant in most species. ATPases have been classified into four superfamilies, F-, V-, A-, and P-ATPases, based on their structures (33–36). There are some other proteins that cannot be classified into these subfamilies but have ATPase activity, such as heat shock proteins(HSPs), includingHSP60 (33),HSP70 (34), and HSP72 (35). XB24 does not belong to any of these previously
+* Here, they show that XA21 function is enhanced when XB24 expression is reduced and that XA21 function is compromised when XB24 is overexpressed.XB24 regulation on XA21 is tightly associated with its ATPase activity. Thus, they conclude that XB24 regulates XA21 function via its ATPase activity.In '''Figure. 7''', they present a model to summarize these results. They hypothesize that the XA21 protein is present on the plasma membrane [after transit from theER(30)], where it recognizes the Ax21PAMP.
-described superfamilies of ATPases or HSPs. The only conserved structure in XB24 is the region composed of 10 amino acids PSINERES154SS
+* XB24 physically associates with XA21 and uses ATP to promote phosphorylation of certain Ser/Thr sites onXA21, keeping theXA21 protein in an inactive state.Upon recognition ofAx21, the XA21 kinase becomes activated, triggering downstream defense responses. The mechanism(s) for XA21 activation following perception of Ax21 likely requires dissociation of XA21 from XB24 and/or removal of theXB24-promoted autophosphorylation. In this model, XA21 autophosphorylation occurs on multiple residues,some of which stimulate XA21 function and others of which inhibit XA21 function. For example, autophosphorylation of the JM residues,Ser-686, Thr-688, and Ser-689, is required forXA21-mediated resistance.
-that is predicted as the ATPase motif, (P-[SAP]-[LIV]-[DNH]-{LKGN}-{F}-{S}-S-{DCPH}-S).The ATPasemotif in the F1,V1, and A1complexes of F-, V-, and AATPases is also essential for ATPase activities, whereas the PATPases and theHSPs do not contain thismotif.However,whether this motif is enough for the ATPase activity of proteins is unclear. Here, They show that XB24, a protein with an ATPase motif but no other motifs or domains, functions as anATPase. Proteins with this conserved motif that cannot be classified into the previously identified ATPases exist in many species, including bacteria, fungi,human, Arabidopsis, and rice. However, none of these have previously been functionally characterized. Thus, our results demonstrating that XB24 is an ATPase with an important function in XA21-mediated immunity will facilitate functional studies of XB24-type ATPases in other species.
+* Autophosphorylation of Thr-705 is also needed for XA21 function. Multisite phosphorylation has been previously demonstrated for the function of insulin receptor substrate 1 (IRS1) in human. In this case, the activation of protein kinase B in response to insulin propagates insulin signaling and promotes the phosphorylation of IRS1 on serine residues, generating a positivefeedback loop for insulin action. Insulin also activates other kinases that induce the phosphorylation of IRS1 on specific sites and inhibit its functions. There is thus a delicate balance existing between positive IRS1 tyrosine/serine phosphorylation and negative IRS1 serine phosphorylation, which can regulate the IRS1.When XB24 is overexpressed (a nonphysiological state), the XA21 protein may not dissociate from XB24 readily or the XB24-promoted phosphorylation may not be easily removed.
+* In this case,binding of Ax21 to XA21 may lead to a conformational change in XA21, exposing the XA21 protein to degradation by endogenous proteases. Alternatively, a protease activity could be induced by Ax21/XA21 binding. In either case, overexpressed XB24 would result in degradation of XA21 when challenged by Ax21. They have previously reported that XB15, a PP2C phosphatase,dephosphorylates autophosphorylated XA21 and negatively regulates the XA21-mediated innate immune responses. Our findings that XB24 promotes XA21 autophosphorylation and inhibits XA21-mediated immune response to the Ax21 PAMP further demonstrate that the phosphorylation state of XA21 is critical for XA21-mediated signaling.
-'''A Model for XB24-Mediated Regulation of XA21.''' Here, they show that XA21 function is enhanced when XB24 expression is reduced and that XA21 function is compromised when XB24 is overexpressed.XB24 regulation on XA21 is tightly associated with its ATPase activity. Thus, they conclude that XB24 regulates XA21
+* Phosphorylation of certain residues on XA21 negatively regulates XA21 function, whereas phosphorylation on other residues may be required for activation of XA21 function. These latter residues are likely dephosphorylated by XB15 to down-regulate XA21 activity. Together with our results that the association between XB24 and XA21 is compromised but the association between XB15 and XA21 is enhanced upon PXO99 inoculation, our model suggests that the regulation by XB24 occurs before Ax21 recognition but that regulation by XB15 occurs after Ax21 recognition.
 ==Labs working on this gene==

@@ Line 18: / Line 18: @@
 [[File:Fig. 4.png|400px|thumb|right|'''Figure 4.''' ''Effects of overexpression of Xb24 on Xa21-mediated resistance.'']]
 * To explore the role of XB24 in XA21-mediated signaling, they 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. To confirm these results, they developed an F4 line (A176) from one of the F1 plants.TheA176 line carries homozygous Xa21 and homozygous Xb24RNAi-9. They then inoculated 3-week-old A176 plants.As shown in （'''Figure. 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<ref name="ref5"/>.
 * At 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 ('''Figure. 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<ref name="ref2"/>.
@@ Line 29: / Line 30: @@
 * 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<ref name="ref2"/>.
 ==Expression==
@@ Line 73: / Line 46: @@
 '''XB24 Physically Associates with XA21 in Vivo.''' The author isolated XB24 as an XA21 interacting protein through yeast two-hybrid screening. The XB24 cDNAis expressed froma unique rice gene,Os01g56470, 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. Although 38 human proteins, 43 Arabidopsis proteins, and 67 additional rice proteins are annotated to contain a conserved ATPase motif, 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, they 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. They established a homozygous line, A114, with a single transgene insertion and demonstrated that it confers full resistance to Xoo strain PXO99. 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)'''.
+[[File:1HF6.jpg|400px|thumb|right|'''Figure. 6.''' ''Effects of excess XB24 on XA21 protein stability.'']]
 '''XB24 Dissociates from XA21 in Response to PXO99 Inoculation.'''To determine whether XB24 is degraded in response to Xoo strain PXO99  inoculation, they performed a Western blot analysis to detect the XB24 protein before and after inoculation. They found that a similar amount of XB24 protein was detected in Xa21 and Kitaake plants before inoculation and 1 day or 2 days after inoculation. This result shows that XB24 is not degraded in response to Ax21. They next investigated whether Ax21 recognition affects the interaction of XA21 and XB24. We performed coimmunoprecipitation experiments withPAP(targeting ProA-XA21)using rice leaf tissues fromtheXa21line inoculated with Xoo strain PXO99 or Xoo strain PXO99ΔraxST, which lacks Ax21 activity due to a knockout of the raxST gene. They then carried out immunoblotting to detect XB24. A similar coimmunoprecipitation was performed using Kitaake rice leaves as a control. As shown in '''Fig. 1D''', they observed a sharp decrease in the amount of XB24 associated with ProA-XA21 post-PXO99 inoculation, whereas, no decrease in the amount of XB24 associated with ProA-XA21 was observed after PXO99ΔraxST inoculation. These results clearly indicate that the physical interaction between XB24 and XA21 disassociates specifically in response to Xoo strains expressing Ax21 activity.
+[[File:1HF7.jpg|400px|thumb|right|'''Figure. 7.''' ''A model for XB24-mediated regulation of XA21 function.'']]
 '''ATPase Activity Is Essential for XB24-Mediated Regulation of XA21 Function.''' The author tested whether XB24 ATPase activity was required for XB24 to regulate XA21 function. They developed Xa21/Xb24ox and Xa21/Xb24S154Aox plants using NtapXb24ox and NtapXb24S154Aox plants, respectively, to cross with 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, these results demonstrate that XB24 requires S154 to repress XA21 function. Thus, we conclude that the ATPase activity of XB24 is essential for XB24 to regulate XA21-mediated defense response.

@@ Line 16: / Line 16: @@
 ===Enhances Xa21-Mediated Resistance===
 * To investigate the biological function of XB24, they used the RNA interference(RNAi) approach<ref name="ref3"/><ref name="ref4"/> to silence the Xb24 gene and monitored its effects on disease resistance. They 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. Both lines show similar disease lesion lengths compared to the control line Kitaake after challenge with PXO99, indicating that silencing of Xb24 does not affect the susceptibility of Kitaake to Xoo.
 * To explore the role of XB24 in XA21-mediated signaling, they 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. To confirm these results, they developed an F4 line (A176) from one of the F1 plants.TheA176 line carries homozygous Xa21 and homozygous Xb24RNAi-9. They then inoculated 3-week-old A176 plants.As shown in （'''Figure. 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<ref name="ref5"/>.
 * At 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 ('''Figure. 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<ref name="ref2"/>.
@@ Line 30: / Line 31: @@
-[[File:Fig. 4.png|frame|Figure 4.Effects of overexpression of Xb24 on Xa21-mediated resistance. (A)
 [[File:1HF5.jpg|frame|Fig. 5. Requirement of XB24 ATPase activity for regulation of XA21-mediated
@@ Line 74: / Line 68: @@
 * 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.
-== Other associations with XA21 ==
+== Extend ==
 The association between XB24 and XA21 is compromised upon inoculation of the Xanthomonas oryzae pv. oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP <ref name="ref7"/>. XB24 promotes autophosphorylation of XA21 through its ATPase activity. Rice plants silenced for Xb24 display enhanced XA21-mediated immunity, whereas rice plants overexpressing XB24 are compromised for immunity. XA21 is degraded in the presence of Ax21 when XB24 is overexpressed. These findings reveal that XB24 negatively regulates XA21 PRR function.

@@ Line 12: / Line 12: @@
 * 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. To test whether the ATPase activity of XB24 is required for promoting XA21K668 autophosphorylation, the purified Ntap-XB24 and NtapXB24S154A were incubated with GST-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 ('''Figure. 2C''').
 * Autophosphorylation of the GST-XA21K668 fusion protein is also enhanced in the presence of the His-XB24 protein but not His-XB24S154A. These results demonstrate that XB24 enhances XA21 autophosphorylation and that itsATPase activity is required for this function<ref name="ref2"/>.
 ===Enhances Xa21-Mediated Resistance===
@@ Line 28: / Line 29: @@
 [[File:Fig. 4.png|frame|Figure 4.Effects of overexpression of Xb24 on Xa21-mediated resistance. (A)

@@ Line 9: / Line 9: @@
 ===Enhances Autophosphorylation of XA21K668===
 * The author tested whether XB24 is a substrate of XA21 or affects XA21 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 GST-XA21K668K736E, a catalytically inactive mutant<ref name="ref1"/>. As ( '''Figure. 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. To test whether the ATPase activity of XB24 is required for promoting XA21K668 autophosphorylation, the purified Ntap-XB24 and NtapXB24S154A were incubated with GST-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 ('''Figure. 2C''').
 * Autophosphorylation of the GST-XA21K668 fusion protein is also enhanced in the presence of the His-XB24 protein but not His-XB24S154A. These results demonstrate that XB24 enhances XA21 autophosphorylation and that itsATPase activity is required for this function<ref name="ref2"/>.
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-[[File:Fig. 2B.png|frame|Figure 2.An ATPase activity is associated with XB24 and effects XA21 auto-
 [[File:Fig. 3.png|frame|Figure 3.Effects of reduced expression of Xb24 on Xa21-mediated resistance.

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 *Cell-surface pattern recognition receptors (PRRs) are key components of the innate immune response in animals and plants. These receptors typically carry or associate with non-RD kinases to control early events of innate immunity signaling. Despite their importance, the mode of regulation of PRRs is largely unknown. Here we show that the rice PRR, XA21(Activator of XA21-mediated immunity), interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase. To date, three XA21 binding (XB) proteins—XB3 (an E3 ubiquitinligase), XB10 (OsWRKY62), and XB15 (a PP2C phosphatase)—have been shown to regulate XA21-mediated immunity. XB24 associates with XA21 in vivo and modulates XA21 function. XB24 belongs to a large class of broadly conserved ATPases of unknown function. The association between XB24 and XA21 is compromised upon inoculation of the Xanthomonas oryzae pv.
-[[File:1HF1.jpg|300px|thumb|right|'''Figure. 1'''. ''Association of XB24 with XA21 in yeast and in rice plants.'']]
+[[File:1HF1.jpg|400px|thumb|right|'''Figure. 1'''. ''Association of XB24 with XA21 in yeast and in rice plants.'']]
 ==Annotated Information==