2014-06-09T12:52:43Z

Huanghs: /* Evolution */

The rice gene Os06g020880,namely '''LYP6''',is a Lysin Motif-Containing Protain and regarded as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. <ref name="ref1" />

==Annotated Information==
===Function===
*'''Rice LYP6 Is LysM-Containing Protein Localized at the Plasma Membrane'''

The initial goal of this study was to identify the PGN receptors in
rice. As LysM was known as the binding motif for PGN in prokaryotes
and the PGN-related chitin or Nod factor in plants
<ref name="ref2" /><ref name="ref3" />
, we postulated that the potential rice PGN receptor highly likely contains LysM.
Phylogenetic analysis of the LysM-containing proteins (LYPs)
from rice and Arabidopsis indicated that these LYPs could be categorized
into two subgroups '''(Figure 1A)'''. Os-LYP4 (Os09g27890),
Os-LYP5 (Os02g53000), and Os-LYP6 (Os06g10660) belong to
Subgroup I together with At-LYP2 (At1g77630) and At-LYP3
(At1g21880), while CEBiP (Os-LYP1), Os-
LYP2 (Os11g34570), Os-LYP3 (Os09g37600), and At-LYP1
(At2g17120) are members of Subgroup II. Since CEBiP from
subgroup II was previously characterized as the chitin receptor
in rice<ref name="ref4" /><ref name="ref5" />, we reasoned
that the rice PGN receptor may be more likely to exist among
LYP4, LYP5, and LYP6 from another branch (subgroup I) of the
phylogenetic tree '''(Figure 1A)'''. We therefore cloned these three
rice genes, and their protein products consist of 401, 255, and 409 amino acids, respectively. Bioinformatic analysis predicted
that LYP4 and LYP6 both have an N-terminal signal
peptide, two characteristic LysMs, and a putative C-terminal
glycosylphosphatidylinositol (GPI) anchor signal sequence
(v-site) '''(Figure 1A; see Supplemental Figure 1 online)'''. This domain
structure suggests that these proteins are localized at the
plasma membrane through a lipid binding GPI anchor. Although
LYP5 shows a substantial sequence similarity to the N terminus
of LYP6, a long C-terminal portion (;150 amino acids) including
the GPI anchor signal sequence is absent from this protein
(Figure 1A). Therefore, we focused only on LYP4 and LYP6, which
have the GPI anchor for membrane attachment, in the subsequent
study.
All characterized homologs of LYP4 and LYP6 have been
verified as plasma membrane proteins. These LYPs include rice
CEBiP , Arabidopsis LYM1 (At-LYP3), LYM2
(At-LYP1), and LYM3 (At-LYP2) <ref name="ref6" /><ref name="ref7" />, and Medicago truncatula LYM1 and LYM2 <ref name="ref8" />. To confirm the plasma membrane localization of Os-
LYP4 and Os-LYP6, we inserted green fluorescent protein (GFP)
behind the N-terminal signal peptide in both proteins. This
fusion strategy was based on the fact that both the N-terminal signal peptide and the C-terminal GPI anchor signal sequence
eventually would be removed from a mature GPI-anchored protein.
Since plant protoplast systems have been used successfully
to detect the cell surface localization of GPI-anchored proteins
<ref name="ref9" />, we transiently expressed LYP4-GFP or
LYP6-GFP using a monocot-specific constitutive Act1 promoter
in rice green tissue protoplasts. In both cases, colocalization
of GFP signal with the FM4-64–stained plasma membrane was
readily detected under a confocal microscope '''(Figure 1B)'''. To
provide additional evidence for the plasma membrane localization
of these proteins, we isolated the microsomal fraction
from rice cells in which LYP4-GFP or LYP6-GFP was coexpressed
with GFP and CEBiP-HA (HA tag behind the N-terminal signal
peptide of CEBiP). These constructs were all transiently expressed
in rice protoplasts under the control of the constitutive Act1 promoter.
Successful preparation of rice microsomal fractions was
verified in immunoblots showing that CEBiP <ref name="ref4" />was highly enriched in the microsomal fraction while GFP and
endogenous tubulin proteins, as nonmembrane proteins, were
exclusively found in the soluble fraction '''(Figure 1C)'''. As expected,
LYP4 and LYP6 were both visualized in the microsomal
fraction through immunoblotting with anti-GFP antibodies
'''(Figure 1C)'''. Taken together, these data confirmed that LYP4 and
LYP6 localize at the plasma membrane of rice cells.

*'''LYP6 Can Physically Bind PGN and Chitin'''
As we speculated that LYP4 and LYP6 are rice PGN receptors,
we next addressed the question whether these proteins could
physically bind PGN. Insoluble PGN purified from three different
bacterial pathogens, X. oryzae pv oryzae (PGNXoo), X. oryzae pv
oryzicola (PGNXoc), and Pseudomonas syringae pv tomato (Pto)
DC3000 (PGNPto), was individually used to pull down the purified
6His-tagged recombinant LYP4 and LYP6 proteins in solution.
Indeed, both LYP4 and LYP6 could coprecipitate with these
PGNs '''(Figure 2A)'''. We further found that the HPLC-purified
soluble PGNXoo muropeptides, which are lysostaphin-digested
products of PGNXoo, could compete with the insoluble PGNXoo
for binding to these proteins, as the increase of muropeptides
in solution was coupled with a decrease of PGNXoo-precipitated
LYP4 and LYP6 '''(Figure 2B)'''. Moreover, the analysis of PGN
binding kinetics suggested that the association of PGNXoo to
these proteins occurred as early as within 1 min and reached
saturation in ;30 min (see Supplemental Figure 4 online). In
parallel, we conducted a PGN pull-down assay for the purified
6His-tagged recombinant Os-CERK1 (Os-LysM-RLK9) extracellular
domain. In contrast with LYP4 and LYP6, CERK1 extracellular
domain could be barely precipitated by any of these
PGNs '''(Figure 2A)'''. An effort to test CEBiP for PGN binding was
hindered by the difficulty in producing 6His-tagged recombinant
CEBiP proteins in Escherichia coli (data not shown).
These data revealed a specific physical interaction between
bacterial PGN and the two rice LYPs.
As PGN is structurally related to chitin<ref name="ref3" />, we
tested whether LYP4 and LYP6 could also physically bind to chitin.
Chitin beads (NEB) and insoluble crab shell chitin (Sigma-Aldrich)
were used to pull down the purified LYP4 and LYP6 proteins in
solution. Surprisingly, these proteins were readily precipitated
by either commercial chitin products '''(Figure 2A)'''. Furthermore,
when highly purified N-acetylchitohexaose (Isosep), a soluble
hexamer of chitin oligosaccharide, was included in the pull-down
assay to compete with the chitin beads, we observed a clear
negative correlation between the amount of N-acetylchitohexaose
added and the amounts of LYP4 and LYP6 precipitated
by chitin beads '''(Figure 2B)'''. Moreover, the assay of chitin binding
kinetics suggested that the binding of chitin to these proteins
occurred within 5 min and became saturated in ;20 min
'''(see Supplemental Figure 4 online)'''. In parallel, we also performed
the same chitin pull-down assay for the purified CERK1 extracellular
domain. In agreement with the previous suggestion<ref name="ref5" />, we did not detect any coprecipitation of CERK1 with
chitin beads or the crab shell chitin '''(Figure 2A)'''. Our data suggested
that LYP4 and LYP6 physically bind chitin in addition to PGN.
We next introduced cross-competition into the pull-down
experiments and found that addition of excess soluble PGNXoo
muropeptides disrupted the precipitation of these proteins by
chitin beads '''(Figure 2C)'''. Likewise, the presence of excess soluble
N-acetylchitohexaose blocked the coprecipitation of these
proteins with PGNXoo '''(Figure 2C)'''. By contrast, addition of excess
soluble LPS (Sigma-Aldrich) failed to affect the amounts of LYP4
and LYP6 precipitated by PGNXoo or chitin beads '''(Figure 2D)'''. These data reinforced that LYP4 and LYP6 selectively bind
PGN and chitin but not LPS. Furthermore, LYP4 and LYP6 expressed in rice protoplasts
also could be precipitated by different bacterial PGNs and
commercial chitin products '''(Figure 2E)''', confirming the physical
association of PGN and chitin to LYPs in rice. By contrast, the
CEBiP proteins successfully expressed in rice protoplasts could
be precipitated only by chitin but not PGN, while the full-length
rice CERK1 proteins expressed in rice protoplasts could be pulled
down by neither MAMP '''(Figure 2E)'''.

*'''Silencing of LYP6 Compromises Diverse PGN- and Chitin-Induced Defense Responses in Rice'''

The findings regarding the plasma membrane localization of LYP4
and LYP6 and their physical interactions with both PGN and chitin
pointed to a more exciting possibility that these proteins may be
not only the PGN receptors but also previously unknown chitin
receptors in rice. This motivated us to evaluate both PGN- and
chitin-induced defense responses in LYP4 or LYP6 RNAi or
overexpressing (OX) transgenic rice. Two representative lines of RNAi or OX transgenic rice were used for each gene in the
subsequent analysis. The empty vector transgenic rice and
the wild-type rice were used as controls. Although LYP4 and
LYP6 genes share ;80% identity, the LYP4 and LYP6 transcripts
were specifically reduced by ;80% by their cognate
RNAi construct in the silencing lines '''(see Supplemental Figure 5A
online)'''. Notably, the expression of CEBiP and CERK1, the two
known genes involved in rice chitin perception, was affected by
neither RNAi construct '''(see Supplemental Figure 5B online)'''. On
the other hand, the expression of LYP4 in its OX lines was increased
by 5.2- to 6.8-fold and that of LYP6 in its OX lines was
increased by 14- to 15-fold '''(see Supplemental Figure 5C online)'''.
Using LYP RNAi or OX transgenic rice, we investigated three
different cell responses occurring at different defense time points
after PGN or chitin exposure. These defense responses included
ROS generation (a very early response), defense gene activation
(an early response), and callose deposition (a late response). LPS,
as another glycoconjugate elicitor, was used as control MAMP
during the examination of the two earlier defense responses. Indeed, the amounts of ROS triggered by the purified PGNXoo or
its HPLC-purified muropeptides declined by ;40% in either LYP4
or LYP6 RNAi transgenic rice when compared with that produced
in the control rice '''(Figure 3A)'''. Similarly, the amounts of ROS induced
by chitin or its soluble fragment N-acetylchitohexaose also
decreased by 37 to 42% in LYP RNAi transgenic rice '''(Figure 3A)'''.
By contrast, LYP4 and LYP6 OX transgenic rice showed comparable
ROS production after PGN or chitin treatment '''(Figure 3A)'''.
Notably, all transgenic rice lines and the wild-type rice treated with
LPS exhibited no significant difference in ROS production
'''(Figure 3A)'''. Since ROS generation is one of the earliest defense
responses<ref name="ref10" />, these results suggested that
LYP4 and LYP6 function quite upstream within the PGN- and
chitin-induced defense signaling pathways and strongly corroborated
the notion that these proteins are potential receptors
for the two MAMPs.
Moreover, the activation of four representative defense marker
genes, namely, Beta-Glu, MLO, WRKY13, and PAL, by 30 min
PGN/muropeptides or chitin/N-acetylchitohexaose treatment
in wild-type rice was substantially suppressed in LYP4 or LYP6
RNAi transgenic rice '''(Figure 3B)'''. However, these defense
marker genes responded to 30-min LPS treatment equally well
in both wild-type and LYP RNAi transgenic rice '''(Figure 3B)'''.
Furthermore, the callose staining spots on rice young leaves
after PGNXoo muropeptides or N-acetylchitohexaose treatment
were dramatically reduced in LYP4 or LYP6 RNAi transgenic
rice when compared with those in the control rice '''(Figure 3C)'''. By contrast, PGNXoo muropeptides or N-acetylchitohexaose
treatment resulted in more callose deposition in LYP4 or LYP6
OX transgenic rice than in the control rice '''(Figure 3C)'''. These
data further strengthened the notion that LYP4 and LYP6 play
crucial roles in PGN- and chitin-induced defense signaling in rice.

* '''LYP6 Affects Rice Susceptibility to Both Bacterial and Fungal Pathogens'''

To validate the importance of LYP4 and LYP6 in rice innate
immunity, we conducted pathogen growth assay in LYP RNAi or
OX transgenic rice using the bacterial blight pathogen X. oryzae,
the bacterial streak pathogen Xanthomonas oryzicola, and the
fungal blast pathogen Magnaporthe oryzae. As expected,
compared with the lesion area caused by X. oryzae infection in
wild-type rice, the lesion areas in LYP4- or LYP6-silencing rice
were significantly enlarged '''(Figures 4A and 4B)'''. Accordingly,
bacterial growth was increased by 25- to 50-fold in LYP silencing
rice (Figure 4C). Similarly, the lesion area due to X.
oryzicola infection also expanded significantly in LYP4- or LYP6-
silencing rice relative to that in wild-type rice '''(Figures 4D and 4E)'''.
Consistent with this, more X. oryzicola growth was detected in
LYP silencing rice '''(Figure 4F)'''. In addition, the LYP silencing rice
appeared to be more susceptible to fungal M. oryzae infection as
the lesion size per leaf was considerably larger in LYP silencing
rice than in wild-type rice '''(Figures 4G and 4H)'''. Accordingly, the
lesion number per leaf was increased by 0.9- to 1.4-fold in LYP
silencing rice compared with that in wild-type rice '''(Figure 4I)'''.
These data suggested that knockdown of LYP4 and LYP6
expression in rice results in an increased susceptibility to both
bacterial and fungal pathogens. Conversely, the lesion area caused by X. oryzae infection
decreased to 41 to 58% in LYP4 or LYP6 OX rice relative to that
in wild-type rice '''(Figure 4B)''' and the X. oryzae growth in these
LYP OX lines was reduced by more than 80% '''(Figure 4C)'''. More
significantly, the lesion size caused by X. oryzicola infection
decreased to 15 to 38% in LYP OX rice relative to that in wildtype
rice '''(Figure 4E)''', and the bacterial growth in these LYP OX
rice dropped by more than 90% '''(Figure 4F)'''. Moreover, the lesion
area due to M. oryzae infection also shrank to 19 to 40% in LYP
OX rice '''(Figure 4H)''', and the lesion number per leaf was reduced
to 50 to 63% in comparison with that in wild-type rice '''(Figure 4I)'''.
These results indicated that upregulation of LYP4 and LYP6
expression in rice leads to an enhanced resistance against both
bacterial and fungal infection.

[[File:69F1.jpg|frame|Figure 1. LysM-Containing LYP4 and LYP6 Are Rice Plasma Membrane Proteins.
(A) Phylogenetic tree and domain structure diagram of LYPs in rice and Arabidopsis. The phylogenetic tree was generated using MEGA4. Full-length
amino acids sequences of plant LYPs were selected for generating a bootstrap neighbor-joining phylogenetic tree. Creye2 from Chlamydomonas
reinhardtii was used as an outgroup. Bootstrap probabilities were obtained from 1000 replicates. A scale bar is indicated. A text file of the alignment
used to generate this tree is available as Supplemental Data Set 1 online. The N-terminal signal peptide, LysM, and the C-terminal GPI anchor signal
(v-site) of each LYP are colored in orange, green, and yellow, respectively.
(B) LYP4 and LYP6 localize at rice plasma membrane. Os-LYP4 and Os-LYP6 with GFP inserted behind the N-terminal signal peptide were individually
expressed in rice protoplasts and visualized by confocal microscopy. FM4-64 dye was used to stain the plasma membrane.
(C) LYP4 and LYP6 localize in the microsomal fraction. LYP4-GFP and LYP6-GFP were individually coexpressed with CEBiP-HA and GFP in rice
protoplasts. Microsomal and soluble fractions of protoplast lysates were separated through Suc gradient centrifugation. Distribution of individual
proteins and the endogenous tubulin was analyzed through immunoblotting with anti-GFP, anti-HA, and antitubulin antibodies.
(D) Relative expression levels of LYP4 and LYP6 in different rice tissues and developmental stages. The expression levels of these genes were
determined by qPCR, and the expression level of each gene in rice calli is set as 100%.
(E) Upregulation of LYP4 and LYP6 transcripts in rice seedlings by X. oryzae. Five-day-old rice seedlings were incubated with X. oryzae suspension (105
cells/mL) or mock treated (sterile water) for the indicated period. The expression levels of these genes were examined by qPCR, and the induction fold
of each gene was calculated by the gene expression level in X. oryzae–treated seedlings relative to that in mock-treated seedlings at the same time
point. hpi, h postinoculation.
(F) Induction of LYP4 and LYP6 expression in mature rice leaf and root by bacterial pathogen X. oryzae. The mature leaf (the fourth leaf) and the primary
root from the indicated Promoter:GUS transgenic rice at the five-leaf stage were immersed into X. oryzae suspension (105 cells/mL) for 2 h before GUS
staining. Bars = 1 mm.
(G) Induction of LYP4 and LYP6 expression by diverse MAMPs. Five-day-old rice seedlings were treated with 100 mg/mL of insoluble PGNXoo, soluble
PGNXoo muropeptides, insoluble crab shell chitin, soluble chitin fragment N-acetylchitohexaose, soluble LPS, or 100 nM flg22 for 1 h, and the induction
of each gene was examined by qPCR.
The experiments in (B) to (G) were repeated three times with similar results.
The data in (D), (E), and (G) represent the mean 6 SD of nine samples from three independent tests.]]

[[File:69F2.jpg|frame|Figure 2. LYP4 and LYP6 Selectively Bind PGN and Chitin but Not LPS.
(A) LYP4 and LYP6 coprecipitate with insoluble PGN or chitin. Sixty micrograms of PGN purified from bacterial pathogens X. oryzae (Xoo), X. oryzicola
(Xoc), or P. syringae pv tomato DC3000 (Pto), commercial chitin beads (NEB), and crab shell chitin (Sigma-Aldrich) were individually used to pull down 1
mg purified 6His-tagged recombinant Os-LYP4, Os-LYP6, or Os-CERK1 in solution.
(B) Soluble PGN muropeptides and N-acetylchitohexaose compete with insoluble PGN and chitin, respectively, for LYP4 and LYP6 binding. Fifty
micrograms of PGNXoo (left panel) or chitin beads (right panel) were used to pull down 1 mg of purified recombinant Os-LYP4 or Os-LYP6 in the presence
of increasing amounts (0, 40, and 80 mg) of PGNXoo muropeptides (left panel) or N-acetylchitohexaose (right panel).
(C) Soluble PGN muropeptides and N-acetylchitohexaose cross-compete with insoluble chitin and PGN, respectively, for LYP4 and LYP6 binding. Fifty
micrograms of PGNXoo (left panel) or chitin beads (right panel) were used to pull down 1 mg of purified recombinant Os-LYP4 or Os-LYP6 in the presence
of increasing amounts (0, 40, and 80 mg) of N-acetylchitohexaose (left panel) or PGNXoo muropeptides (right panel).
(D) Soluble LPS shows no binding to LYP4 and LYP6. Fifty micrograms of PGNXoo (left panel) or chitin beads (right panel) was used to pull down 1 mg of
purified recombinant Os-LYP4 or Os-LYP6 in the presence of increasing amounts (0, 40, and 80 mg) of LPS.
(E) LYP4 and LYP6 produced in planta coprecipitate with insoluble PGN or chitin. One hundred micrograms of PGN or chitin was individually used to
pull down MYC-tagged LYP4 and LYP6 or HA-tagged CEBiP and CERK1, which was individually expressed in 2 mL of rice protoplasts. Triton X-100
(0.5%) was used to lyse protoplasts and solubilize membrane proteins.
One of the three biological repeats with similar results is shown, and results were obtained by immunoblotting using anti-His, anti-MYC, or anti-HA
antibodies.]]

[[File:69F3.jpg|frame|Figure 3. RNAi Silencing or Overexpressing of LYP4 and LYP6 Specifically Modulate PGN- and Chitin- but Not LPS-Induced Defense Responses in Rice.
(A) ROS generation induced by PGN or chitin is significantly suppressed in LYP4 or LYP6 RNAi transgenic rice lines. Roots from 4-d-old wild-type or
transgenic rice seedlings were incubated with 100 mg/mL PGNXoo, PGNXoo muropeptides, crab shell chitin, N-acetylchitohexaose, LPS, or sterile water (mock)
for 30 min before ROS measurement. The data represent the mean 6 SD of nine samples from three independent tests. CK, control (empty vector transgenic
rice); RLU, relative light units; WT, the wild type.
(B) Defense gene activation induced by PGN or chitin is compromised in LYP4 or LYP6 RNAi transgenic rice lines. Callus cells from wild-type or RNAi
transgenic rice lines were incubated with 100 mg/mL PGNXoo, PGNXoo muropeptides, crab shell chitin, N-acetylchitohexaose, LPS, or sterile water
(mock) for 30 min, and the induction of four representative defense marker genes Beta-Glu, MLO, WRKY13, and PAL was determined by qPCR. The
data represent the mean 6 SD of nine samples from three independent tests.
(C) Callose deposition induced by PGN or chitin is substantially impaired in LYP4 and LYP6 RNAi transgenic rice but enhanced in LYP OX rice. The first
leaf of 5-d-old wild-type, LYP4-RNAi-1, LYP6-RNAi-2, LYP4-OX-2, or LYP6-OX-3 rice seedling was immersed into solution containing 10 mg/mL
PGNXoo muropeptides or N-acetylchitohexaose and vacuumed for 30 min. Callose staining was conducted 18 h later.
At least three biological repeats were conducted for individual experiments.]]

===Expression===
* LYP6 Expression Can Be Induced Quickly by Bacterial Pathogen Infection or Diverse MAMPs
To understand better the function of LYP4 and LYP6, we
checked the expression profiles of these genes in different
rice tissues and developmental stages by quantitative realtime
PCR (qPCR). LYP4 and LYP6 were most abundantly
expressed in rice callus cells, and both transcripts progressively
decreased during maturation '''(Figure 1D)'''. Furthermore, analysis of
LYP4 and LYP6 expression patterns in Promoter:GUS (for
b-glucuronidase) transgenic rice demonstrated strong GUS
staining in young seedlings, particularly in the root meristem region
and the lateral root primordium '''(see Supplemental Figure 2
online)''', resembling the expression patterns of their ortholog LYM1
in M. truncatula <ref name="ref8" />. Interestingly, expression
of LYP4 and LYP6 in rice seedlings, mature leaves, and roots
could be quickly induced upon exposure to the rice bacterial
pathogen Xanthomonas oryzae pv oryzae. In 5-d-old rice seedlings,
1 h X. oryzae treatment induced a 24- and 26-fold increase
of endogenous LYP4 and LYP6 transcripts, respectively '''(Figure
1E)'''. Moreover, incubation with X. oryzae suspension for 2 h
rendered a strong GUS activity in mature leaves and roots of
the Promoter:GUS transgenic rice, while incubation with sterile
water had no effect on the GUS activity '''(Figure 1F)'''. The possibility
of false positive GUS staining due to pathogen contamination
could be excluded as no GUS activity could be detected in the
empty vector (pCAMBIA-1391Z containing an intact GUS gene.

===Evolution===
The promiscuity of Os-LYP4 and Os-LYP6 in binding carbohydrate
MAMPs PGN and chitin is reminiscent of At-FLS2
binding three different ligands, including flagellin and Ax21 as
well as the endogenous CLV3 peptide <ref name="ref11" />
<ref name="ref12" /> The promiscuity of PRRs in sensing multiple
MAMPs provides a distinct physiological advantage to the
host so that a limited number of PRRs would be able to perceive
a maximum number of MAMPs. Considering plants in nature
are often exposed concurrently to several groups of microbial
pathogens, the advantage brought by LYP4 and LYP6 in rice is
particularly spectacular in that these PRRs could detect PGN
and chitin derived individually from the two major microbial
groups, bacteria and fungi. As the expression of LYP4 and
LYP6 genes could be rapidly upregulated upon recognition of
either MAMP'''(Figure 1G)''', it seems that either type of microbial
infection would quickly sensitize rice for further infection by
both groups of microbes. Interestingly, although the transgenic
rice overexpressing LYP4 or LYP6 indeed demonstrated
an enhanced pathogen resistance '''(Figure 4)''', the PGN- or chitininduced
ROS production in these rice plants did not show significant
difference compared that in wild-type rice '''(Figure 3A)'''.
Pathogen resistance is a complicated consequence of innate
immunity, whereas ROS production is just one of the very early
defense responses in plant innate immunity. The biological significance
of ROS production in plant defense is not fully understood.
It is very likely that the intensity of ROS generation is not linearly correlated with the magnitude of pathogen resistance, as
the latter is shaped by complex interplay between multiple layers
of defense responses that are induced by a cocktail of MAMPs
from pathogens and occur with distinct dynamics.
The functions of other three evolutionarily related Os-LYPs
(LYP2, LYP3, and LYP5) still remain enigmatic. LYP2 and LYP5
are presumably located in the apoplastic space rather than the
plasma membrane due to lack of the GPI anchor '''(Figure 1A)''',
whereas LYP3 likely resides at the plasma membrane like LYP4
and LYP6. LYP2, LYP3, and LYP5 all contain two intact LysMs
'''(Figure 1A; see Supplemental Figure 1 online)'''; thus, their binding
capacity to PGN or chitin cannot be excluded at this moment.
By inference, they may serve certain regulatory functions in PGN
or chitin signaling in rice, similar to the case of Eix1 and Eix2
proteins in tomato. Both Sl-Eix1 and Sl-Eix2 were found to bind
the fungal elicitor xylanase, where only the Eix2 receptor mediated
defense signaling while Eix1 acted as a decoy receptor to
attenuate the xylanase-induced defense signaling <ref name="ref13" /><ref name="ref14" />.Both Os-LYP4 and Os-LYP6 are likely to be N-glycosylated
when expressed in rice cells as the GFP hybrids of both proteins
showed an actual molecular mass around 100 kD instead of the
predicted molecular mass of 65 kD '''(Figure 1C)''', reminiscent of
other LYP proteins expressed in planta <ref name="ref4" />
<ref name="ref8" /><ref name="ref7" /> Intriguingly, the
N-glycosylation of these receptors appeared to be dispensable for
ligand binding since the recombinant receptors expressed in E. coli were competent in PGN and chitin binding (Figure 2A). Similar
findings were also obtained for their orthologs in Arabidopsis
<ref name="ref7" />. Recently, it has been revealed that the Nglycosylation
of NFP, the LysM-RLK for Nod factor perception,
was not essential for its biological activity including the ligand
binding <ref name="ref15" />. Our work and others thus suggest
a role of N-glycosylation in regulating the protein trafficking of
these receptors.
Although Os-LYP4 or Os-LYP6 are each able to bind PGN
and chitin, our data suggest that they are not functionally
redundant. This was because knockdown of single LYP gene
expression in rice was sufficient to impair both PGN- and
chitin-induced defense responses significantly '''(Figure 3)''' and
to cause severe bacterial or fungal infection phenotypes '''(Figure 4)'''.
Similar observations recently have been made for Arabidopsis
PGN receptors LYM1 and LYM3, where lym1 lym3 double mutant
did not show increased susceptibility to bacterial pathogens when
compared with lym single mutants <ref name="ref7" />. Both
studies favor a cooperative relationship between the pair of PRRs,
suggesting that they may work in the same receptor complex.
The identification of LYP4 and LYP6 as additional chitin
receptors in rice raises the question regarding their relationship with the previously identified rice chitin receptor CEBiP<ref name="ref4" />. Kaku and coworkers found that 70% of the genes
activated by chitin in wild-type rice cells lost responsiveness in
CEBiP-RNAi rice cells, suggesting that CEBiP might play a major
role in rice chitin perception. In line with this speculation,
RNAi silencing of CEBiP diminished the chitin-induced ROS
generation by 85% , while silencing of LYP4/6
only reduced the chitin-induced ROS generation by ;40% '''(Figure
3A)'''. However, as 30% of the upregulated genes and 20% of the
downregulated genes could respond to chitin equally well in wildtype
and in CEBiP-RNAi rice cells, CEBiP and LYP4/6 proteins are
very likely to work in different chitin receptor complexes. In support
of this speculation, a major portion of CEBiP proteins were
visualized as homodimers in blue native PAGE analysis <ref name="ref5" />. Nevertheless, a small fraction of CEBiP proteins
did exist as larger-size oligomers<ref name="ref5" />, which
makes it ambiguous whether some CEBiP proteins can be in the
same complexes with LYP4/6. Further investigation of the composition
and stoichiometry of rice chitin receptor complexes as
well as chitin responses in CEBiP and LYP4/6 triple knockdown
rice will be necessary to dissect fully their contributions in rice
chitin perception and signaling. In sum, two LysM-containing proteins,
LYP4 and LYP6, are dual function receptors for bacterial PGN
and fungal chitin in rice. We provided three key lines of evidence
pertaining to the function of these proteins. First, they are localized
at plant cell surface. Second, they can specifically bind
PGN and chitin. Third, knockdown of their expression perturbs
the PGN- and chitin-induced defense responses in rice. LYP4
and LYP6 are unique among known PRRs in that they can
recognize MAMPs across microbial groups. Future investigation
of the LysMs in these PRRs will be meaningful not only for understanding
the biochemical basis of LysM-PGN and LysM-chitin
interactions, but also for guiding the engineering of promiscuous
PRRs into other crop species to improve disease resistance.
Moreover, comparison of the similarities and differences in PGN
and chitin perception machineries between rice and Arabidopsis
will provide valuable evolutionary insights for understanding critical
mechanisms underlying innate immunity signaling in plants.

[[File:69F4.jpg|frame|Figure 4. RNAi Silencing or Overexpressing of LYP4 and LYP6 Affects Rice Innate Immunity against Bacterial and Fungal Pathogens.]]

[[File:69F5.jpg|frame|Figure 5. PGN and Chitin Engage Overlapping Receptor Components in
Rice.
(A) Rice cells pretreated with excess PGN have a dramatically attenuated
alkalinization response to subsequent chitin treatment. Rice callus cells
were first treated with 600 mg/mL insoluble PGNXoo for 40 min (endpoint
marked by the arrow) and then treated with 100 mg/mL PGNXoo muropeptides
(green curve), 100 mg/mL N-acetylchitohexaose (blue curve),
or 10 nM flg22 (purple curve).
(B) Rice cells pretreated with excess chitin have a dramatically attenuated
alkalinization response to subsequent PGN treatment. Rice callus
cells were first treated with 600 mg/mL crab shell chitin for 40 min
(endpoint marked by the arrow) and then treated with 100 mg/mL PGNXoo
muropeptides (green curve), 100 mg/mL N-acetylchitohexaose (blue
curve), or 10 nM flg22 (purple curve).(C) PGNXoo muropeptides and N-acetylchitohexaose used in (A) and (B)
are active. Rice callus cells were first treated with 1 mM flg22 for 40 min
(endpoint marked by the arrow) and then treated with 100 mg/mL PGNXoo
muropeptides (green curve), 100 mg/mL N-acetylchitohexaose (blue
curve), or 10 nM flg22 (purple curve).
Three biological replicates were conducted for (A) to (C), and similar
results were obtained.]]

==Others about this gene==
PGN and Chitin Engage Overlapping Perception
Components in Rice
The aforementioned data suggested that LYP4 and LYP6 may
be shared by both PGN and chitin perception systems in rice. If
this were the case, the rice cells saturated with one of the two
elicitors would become temporarily less responsive to another
because the receptors, once occupied by the first elicitors,
would not become immediately available for binding the second.
To test this speculation, we set up three sets of tandem
MAMP treatments for rice callus cells and closely followed the
elevation of the medium pH that indicates the magnitude of
alkalinization response of rice cells. In the first set of experiments,
the rice cells were initially saturated with 600 mg/mL
insoluble PGNXoo and then treated with 100 mg/mL PGNXoo
muropeptides, 100 mg/mL N-acetylchitohexaose, or 10 nM
flg22 (GenScript). It was obvious that the flg22 treatment after
the saturating PGN treatment induced a second spike of medium
alkalinization, while the N-acetylchitohexaose treatment
led to only a slight increase of medium pH '''(Figure 5A)'''. The
alkalinization response to flg22 excluded the possibility that
disappearance of the alkalinization response to chitin was due
to depletion of certain components necessary for medium alkalinization
by the saturating PGN treatment. Hence, these
results suggested that chitin and PGN are perceived through
overlapping receptor systems. In the second set of experiments,
the rice cells were first saturated with 600 mg/mL crab
shell chitin before the subsequent treatment with 100 mg/mL
PGNXoo muropeptides, 100 mg/mL N-acetylchitohexaose, or
10 nM flg22. Likewise, although the flg22 treatment following the
saturating chitin treatment could induce another dramatic elevation of medium pH, the PGNXoo muropeptides gave rise to
only a negligible alkalinization response '''(Figure 5B)'''. These
results repeatedly suggested that chitin and PGN are sharing
overlapping perception systems. In the third set of experiments,
the rice cells were presaturated with 1 mM flg22 and then treated
individually with 100 mg/mL PGNXoo muropeptides, 100 mg/mL
N-acetylchitohexaose, and 10 nM flg22. In contrast with flg22,
both PGNXoo muropeptides and N-acetylchitohexaose could
provoke a further medium alkalinization '''(Figure 5C)''', verifying
that PGNXoo muropeptides and N-acetylchitohexaose used in
these experiments '''(Figures 5A to 5C)''' were active elicitors.
Taken together, these data indirectly support the notion that LYP4 and LYP6 are dual function receptors for PGN and chitin
in rice.

==Labs working on this gene==
*State Key Laboratory of Biocontrol, Key Laboratory of Gene Engineering of the Ministry of Education and Guangdong Key Laboratory
of Plant Resources, School of Life Sciences, Sun Yat-sen University, 510275 Guangzhou, People’s Republic of China

==References==
<references>
<ref name="ref1">Bing Liu, Jian-Feng Li, Ying Ao,Jinwang Qu, Zhangqun Li, Jianbin Su, Yang Zhang, Jun Liu, Dongru Feng,Kangbiao Qi, Yanming He, Jinfa Wang, and Hong-Bin Wang.(2012) .Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual
Roles in Peptidoglycan and Chitin Perception in Rice
Innate Immunity. The Plant Cell 24: 3406–3419. </ref>
<ref name="ref2">Bateman, A., and Bycroft, M. (2000). The structure of a LysM domain
from E. coli membrane-bound lytic murein transglycosylase D (MltD).
J. Mol. Biol. 299: 1113–1119.</ref>
<ref name="ref3">Silipo, A., Erbs, G., Shinya, T., Dow, J.M., Parrilli, M., Lanzetta, R.,
Shibuya, N., Newman, M.A., and Molinaro, A. (2010). Glycoconjugates
as elicitors or suppressors of plant innate immunity.
Glycobiology 20: 406–419.</ref>
<ref name="ref4">Kaku, H., Nishizawa, Y., Ishii-Minami, N., Akimoto-Tomiyama, C.,
Dohmae, N., Takio, K., Minami, E., and Shibuya, N. (2006). Plant cells
recognize chitin fragments for defense signaling through a plasma
membrane receptor. Proc. Natl. Acad. Sci. USA 103: 11086–11091.</ref>
<ref name="ref5">Shimizu, T., Nakano, T., Takamizawa, D., Desaki, Y., Ishii-Minami,
N., Nishizawa, Y., Minami, E., Okada, K., Yamane, H., Kaku, H.,
and Shibuya, N. (2010). Two LysM receptor molecules, CEBiP and
OsCERK1, cooperatively regulate chitin elicitor signaling in rice.
Plant J. 64: 204–214.</ref>
<ref name="ref6">Borner, G.H., Lilley, K.S., Stevens, T.J., and Dupree, P. (2003).
Identification of glycosylphosphatidylinositol-anchored proteins in
Arabidopsis. A proteomic and genomic analysis. Plant Physiol. 132:
568–577.</ref>
<ref name="ref7">Willmann, R., et al. (2011). Arabidopsis lysin-motif proteins LYM1
LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity
to bacterial infection. Proc. Natl. Acad. Sci. USA 108: 19824–
19829.</ref>
<ref name="ref8">Fliegmann, J., Uhlenbroich, S., Shinya, T., Martinez, Y., Lefebvre,
B., Shibuya, N., and Bono, J.J. (2011). Biochemical and phylogenetic
analysis of CEBiP-like LysM domain-containing extracellular
proteins in higher plants. Plant Physiol. Biochem. 49: 709–720.</ref>
<ref name="ref9">Takos, A.M., Dry, I.B., and Soole, K.L. (1997). Detection of glycosylphosphatidylinositol-
anchored proteins on the surface of Nicotiana
tabacum protoplasts. FEBS Lett. 405: 1–4.</ref>
<ref name="ref10">Boller, T., and Felix, G. (2009). A renaissance of elicitors: Perception
of microbe-associated molecular patterns and danger signals by
pattern-recognition receptors. Annu. Rev. Plant Biol. 60: 379–406.</ref>
<ref name="ref11">Danna, C.H., Millet, Y.A., Koller, T., Han, S.W., Bent, A.F., Ronald, P.C.,
and Ausubel, F.M. (2011). The Arabidopsis flagellin receptor FLS2
mediates the perception of Xanthomonas Ax21 secreted peptides. Proc.
Natl. Acad. Sci. USA 108: 9286–9291</ref>
<ref name="ref12">Lee, H., Chah, O.K., and Sheen, J. (2011). Stem-cell-triggered immunity
through CLV3p-FLS2 signalling. Nature 473: 376–379.</ref>
<ref name="ref13">Bar, M., Sharfman, M., and Avni, A. (2011). LeEix1 functions as
a decoy receptor to attenuate LeEix2 signaling. Plant Signal. Behav.
6: 455–457.</ref>
<ref name="ref14">Bar, M., Sharfman, M., Ron, M., and Avni, A. (2010). BAK1 is required
for the attenuation of ethylene-inducing xylanase (Eix)-induced defense
responses by the decoy receptor LeEix1. Plant J. 63: 791–800.</ref>
<ref name="ref15">Lefebvre, B., Klaus-Heisen, D., Pietraszewska-Bogiel, A., Hervé, C.,
Camut, S., Auriac, M.C., Gasciolli, V., Nurisso, A., Gadella, T.W.J.,
and Cullimore, J. (2012). Role of N-glycosylation sites and CXC motifs
in trafficking of Medicago truncatula Nod factor perception protein to
plasma membrane. J. Biol. Chem. 287: 10812–10823</ref>
</references>

==Structured Information==
{{JaponicaGene|
GeneName = Os06g0208800|
Description = Hypothetical protein|
Version = NM_001063640.1 GI:115467011 GeneID:4340448|
Length = 1483 bp|
Definition = Oryza sativa Japonica Group Os06g0208800, 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 = [[:category:Japonica Chromosome 6|Chromosome 6]]|
AP = Chromosome 6:5560449..5561931|
CDS = 5560451..5560506,5560594..5560755,5560834..5560865,5561590..5561831|
GCID = <gbrowseImage1>
name=NC_008399:5560449..5561931
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008399:5560449..5561931
source=RiceChromosome06
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>tatgcactaactgctggaaactgtgtgcagtgcagctgtggaccaggagatctcaaattgtattgcacaccagcttcattaacagcatcatgttcaagtatgcagtgccccaatagcaacctcatgcttgggaatgtgactgcccaatccaccagtggtggatgcaatgtctcatcttgcagctatgctggacttgttaatggaaccattgccacgtcgctctcctctggtcttcaacccacatgcccaggaccacatcagttccccccactcagggcgacaccaattgcagtaaaccaaggatcataccttgctccatcacctgcaccaggagctggggaggctggcggcgatattccgggcttccctgggagctccaacgtttctcctgcaaacggcccttctgggagtgtctcccaagcggcttcggtgaatcggccacaccaaatcgtcgccctcattctttctgtagccttgtacttccagatgtga</cdnaseq>|
AA = <aaseq>YALTAGNCVQCSCGPGDLKLYCTPASLTASCSSMQCPNSNLMLG NVTAQSTSGGCNVSSCSYAGLVNGTIATSLSSGLQPTCPGPHQFPPLRATPIAVNQGS YLAPSPAPGAGEAGGDIPGFPGSSNVSPANGPSGSVSQAASVNRPHQIVALILSVALY FQM</aaseq>|
DNA = <dnaseqindica>3..58#146..307#386..417#1142..1383#cctatgcactaactgctggaaactgtgtgcagtgcagctgtggaccaggagatctcaagtaatgctgccttttccttgttcaatgcaattctattgcttctacatgttttggaatatagtcctcattttctctcttcttctgcagattgtattgcacaccagcttcattaacagcatcatgttcaagtatgcagtgccccaatagcaacctcatgcttgggaatgtgactgcccaatccaccagtggtggatgcaatgtctcatcttgcagctatgctggacttgttaatggaaccattgccacgtcgtaagtaaaaaataagtctaataaacctttcttttttatattcttacaatagatgattcttacctaacttatgcgcaggctctcctctggtcttcaacccacatgcccaggtaattacagtgttatcttcatgatatctcttcctatgtgctgcatacggaatatgcaacactatcacttatttgctctatccatctagacccgtcttcttgaaaagatgcacttggtattgtctgatcaaaaagtgaacacataatgatactaaagaagaaaagaagctcaaattaaattaaaaatgttcaattatgcatcactggcattctttaaataatagtcatttgatcaaggatcatatgtaaaatccaagtaattaagtgcgttatgttcatttaaagttaaatgtgaagagtgaaagtgctgtagtagctattaaaagcatgtttctggaatactagtcgcaaacaatgaaacaaagccacatcaaggtttccgttcatgatgtatcattgaatgaggaacttatgattttatccgagtcactgaatgcattatattcatttcaagaaaaaaggtgtaggacagtaggagtggcggcaatttttctgtttcttattgaaaacatgtttctggcatatttatcggcattgcaattttaagccaaaccacatcagtctttctggagtctgatcaaatcacaccaaaaccagaataataaaggtttaaaccaacttggtattctaaaattccttcacttgcctgttggccatgcatcatcaattcctatatttatgatctgatctatatattgcttctcactcaaacctctcaacacaggaccacatcagttccccccactcagggcgacaccaattgcagtaaaccaaggatcataccttgctccatcacctgcaccaggagctggggaggctggcggcgatattccgggcttccctgggagctccaacgtttctcctgcaaacggcccttctgggagtgtctcccaagcggcttcggtgaatcggccacaccaaatcgtcgccctcattctttctgtagccttgtacttccagatgtgaattcgtgtgcagagtattgttgttgttgctggctgtgtgtattgaacagggggccctcatttgggcctggtggaaccctaatgtcaaggtcctttctatc</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001063640.1 RefSeq:Os06g0208800]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 6]]
[[Category:Chromosome 6]]

Os06g0208800

2014-06-09T12:51:00Z

2014-06-09T04:05:47Z

Huanghs: /* Other associations with XA21 */

Gene Os01g0771200,namely '''XB24''', is a XA21 binding protein gene .'''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.

==Annotated Information==
===Character===
(Don't Delete)
*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, interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase.

*The rice PRR, XA21, recognizes the PAMP, Ax21 (Activator of XA21-mediated immunity), which is highly conserved in all sequenced genomes of Xanthomonas and in Xylella . Previous studies have shown that the intracellular non-RD cytoplasmic kinase domain of XA21 contains intrinsic kinase activity. Phosphorylation of amino acids Ser-686, Thr-688, and Ser-689 of XA21 is required to stabilize the XA21 protein . 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. Oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP . 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.

===Function===

*'''XB24 ATPase 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 ( '''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 the GST-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<ref name="ref2"/>.

*'''XB24 Possesses ATPase Activity.''' Because XB24 contains aC-terminal ATPasemotif and the residue serine (Ser) 154 is a predicted key site for this motif, we tested whether it indeed possesses intrinsic ATPase activity.Wepurified Ntap-XB24 andNtap-XB24S154A(containing a single amino acid change of Serine 154 by Alanine) from Ntap-Xb24 and Ntap-XB24S154A transgenic plants, respectively, and performed the ATP hydrolysis assay. As shown in Fig. 2A, Ntap-XB24 displayed significant ATP hydrolysis activity, whereas Ntap-XB24S154A had only negligible ATPase activity.We also found that E. coli-produced recombinant protein His-XB24 possesses ATPase activity and that the S154A mutant completely abolished the ATPase activity of XB24 '''(Fig. S4)'''. Taken together, these results show that the XB24 protein possesses an ATPase activity and that amino acid S154 is essential for its ATPase activity.

*'''Silencing of Xb24 Enhances Xa21-Mediated Resistance.''' To investigate the biological function of XB24, we 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 （'''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<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 ('''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<ref name="ref2"/>.

*'''Overexpression of XB24 Compromises XA21-Mediated Resistance.''' To investigate the involvement of XB24 in the XA21-mediated signaling, the author created construct Ubi-Xb24 to overexpress XB24 using the maize Ubi-1 promoter. They introduced the Ubi-Xb24 construct directly into an Xa21 (in the TP309 genetic background)line by Agrobacterium-mediated transformation using mannose selection<ref name="ref6"/> 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 with PXO99.They found that all of the five lines have longer disease lesion lengths compared with the wild-type Xa21 plants. 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.These results demonstrate that overexpression of XB24 compromises XA21-mediated resistance<ref name="ref2"/>.

*'''Overexpression of Xb24 Causes XA21 Instability Following Ax21 Recognition.''' To gain insight into the mechanism of XB24-mediated regulation of XA21 function, they tested whether XB24 affects the amount of the XA21 protein after Xoo inoculation. As shown in '''Fig. 6 A and B''', without Xoo inoculation (Mock treatment). Overexpression of XB24(Xa21/Ntap-Xb24) caused no significant 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<ref name="ref2"/>.

[[File:1HF1.jpg|frame|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.]]

[[File:Fig. 2B.png|frame|Figure 2.An ATPase activity is associated with XB24 and effects XA21 auto-
phosphorylation. (A) ATPase activity assay on purified Ntap-XB24 and Ntap-
XB24S154Aprotein 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 auto-
radiogram. (Lower) Quantitative results (mean + SD) from three independ-
ent 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.”|left]]

[[File:Fig. 3.png|frame|Figure 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.|right]]

[[File:Fig. 4.png|frame|Figure 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.]]

[[File:1HF5.jpg|frame|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.]]

[[File:1HF6.jpg|frame|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).]]

===Expression===
*The XB24 cDNAis expressed from a unique rice gene, Os01g56470, 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.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.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).

*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.

== Other associations with XA21 ==

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'''(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)'''.

'''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. Wenext 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''', we 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.

'''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.

(Don't Delete)
'''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
described superfamilies of ATPases or HSPs. 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)'''.TheATPasemotif in the F1,V1, andA1complexes 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, we 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 ofXB24-type ATPases in other species.

==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.

==References==
<references>

<ref name="ref1">Liu GZ, Pi LY, Walker JC, Ronald PC, Song WY (2002) Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21. J Biol Chem 277:20264–20269.</ref>

<ref name="ref2">Xuewei Chen, Mawsheng Chern, Patrick E. Canlas, Deling Ruan, Caiying Jiang, and Pamela C. Ronald(2010) An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity. Proc Natl Acad Sci USA 107: 8029–8034.</ref>

<ref name="ref3">Fire A, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811.</ref>

<ref name="ref4">Mourrain P, et al. (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–542.</ref>

<ref name="ref5">Century KS, et al. (1999) Developmental control of Xa21-mediated disease resistance in rice. Plant J 20:231–236.</ref>

<ref name="ref6">Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49.</ref>

<ref name="ref7">Lee SW, Han SW, Bartley LE, Ronald PC (2006) Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity. Proc Natl Acad Sci USA 103:18395–18400.</ref>

</references>

==Structured Information==
{{JaponicaGene|
GeneName = 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 = [[:category:Japonica Chromosome 1|Chromosome 1]]|
AP = Chromosome 1:34299150..34301037|
CDS = 34299283..34299494,34300420..34300804|
GCID = <gbrowseImage1>
name=NC_008394:34299150..34301037
source=RiceChromosome01
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008394:34299150..34301037
source=RiceChromosome01
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaa</cdnaseq>|
AA = <aaseq>MGWRWHDDGDDGGRGLGDIPDLAGGGGGGDGERCATRRVVQSRC HTEEVEPGRFVRKCEKTEQLLRDCVGRPSELVESKTENTEEDVTDEMKSGSLSLGFPT NEPFAFPGLRSDIEALEKGLFGSIGSFLDDAERMTNDFLKSFGVPSINERESSSFDGQ PTGRHIGGQPAGRHIEEGTAKDTKQNDYAEFSSKITDV</aaseq>|
DNA = <dnaseqindica>134..345#1271..1655#agaaacgagccggccggattgctctccaagccaaacacggcccagagaggcgagagcccccacaccgccaaacccgacccggaaatcaactgcacacgctccgatcccctctctccagatcgattcggaccccatgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggtatatacatatactccatggcctcgcgcttttccctgatctgttccttcttttcctgcgaggatcccctctttgcatattactgttcgttgttttagtgatgaattggtgataacatgtcatgcgcagttgtgttactattagtcccgtgtatgattgtgagctgtgtatagccgcctctctgtggacgattttcgtgagtctgtcctcgctgatttgatacaaacacgaattcattaagaatggggaatgtgccgtgatgatcgagacgtttgagttctgtggtgaatagtatgatcaaatgtgtgtaataacttgggcttttgaattttggtagctaattagatatggaatttctatccagttttaggtgcccgtgccatcgacatgggttaaaagttattgtgggctattttgcgctcaacttgttaatcaacaaattttggacggaggcagtactttgtaggcatggggtaaatttgtatgatacacttgttgcttgaagatgttcgtgaactcatgggatttctagacacctacaaccaattacattacagtgttattatattctaacgatatgtaaagagcagctgtgttcgtgttgtctacactaaagattcaacaactagtggtcatcatcaaaccgatagcatatcctcattttgcaggattttgaaaactcacatctcgttagcatccttccttttatttcttcacatgcctcatgttaaacttttaagtgcatgaaacccaaatttctgtatttcgtagctccttgtaaccaatgtacaacactatgattagtattctggatagattttgtacttcccaaagtattttggttgttgacaagctattacaataatctgatggataacctaccatacattatttctcctaaccgatcacaataatcttcttctaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaaggatctacagttagctgacgcacctttgggagcagcttgccaattttgtattttgaacatctccatggttgtaattggaaggggaaggatcagtttgactgttttataagcagagtcgtctgaagtctgaagtgttgcgcttataagaacaattgtgtatattactgttttagataagcctgtttgtgttcctcaacaatgagatcaattatggatggtttttttctctgctt</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001050918.1 RefSeq:Os01g0771200]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 1]]
[[Category:Chromosome 1]]

Os01g0771200

2014-06-09T04:03:28Z

Huanghs: /* Function */

Gene Os01g0771200,namely '''XB24''', is a XA21 binding protein gene .'''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.

==Annotated Information==
===Character===
(Don't Delete)
*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, interacts with XA21 binding protein 24 (XB24), a previously undescribed ATPase.

*The rice PRR, XA21, recognizes the PAMP, Ax21 (Activator of XA21-mediated immunity), which is highly conserved in all sequenced genomes of Xanthomonas and in Xylella . Previous studies have shown that the intracellular non-RD cytoplasmic kinase domain of XA21 contains intrinsic kinase activity. Phosphorylation of amino acids Ser-686, Thr-688, and Ser-689 of XA21 is required to stabilize the XA21 protein . 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. Oryzae (Xoo) strain PXO99, which secretes the Ax21 PAMP . 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.

===Function===

*'''XB24 ATPase 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 ( '''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 the GST-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<ref name="ref2"/>.

*'''XB24 Possesses ATPase Activity.''' Because XB24 contains aC-terminal ATPasemotif and the residue serine (Ser) 154 is a predicted key site for this motif, we tested whether it indeed possesses intrinsic ATPase activity.Wepurified Ntap-XB24 andNtap-XB24S154A(containing a single amino acid change of Serine 154 by Alanine) from Ntap-Xb24 and Ntap-XB24S154A transgenic plants, respectively, and performed the ATP hydrolysis assay. As shown in Fig. 2A, Ntap-XB24 displayed significant ATP hydrolysis activity, whereas Ntap-XB24S154A had only negligible ATPase activity.We also found that E. coli-produced recombinant protein His-XB24 possesses ATPase activity and that the S154A mutant completely abolished the ATPase activity of XB24 '''(Fig. S4)'''. Taken together, these results show that the XB24 protein possesses an ATPase activity and that amino acid S154 is essential for its ATPase activity.

*'''Silencing of Xb24 Enhances Xa21-Mediated Resistance.''' To investigate the biological function of XB24, we 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 （'''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<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 ('''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<ref name="ref2"/>.

*'''Overexpression of XB24 Compromises XA21-Mediated Resistance.''' To investigate the involvement of XB24 in the XA21-mediated signaling, the author created construct Ubi-Xb24 to overexpress XB24 using the maize Ubi-1 promoter. They introduced the Ubi-Xb24 construct directly into an Xa21 (in the TP309 genetic background)line by Agrobacterium-mediated transformation using mannose selection<ref name="ref6"/> 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 with PXO99.They found that all of the five lines have longer disease lesion lengths compared with the wild-type Xa21 plants. 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.These results demonstrate that overexpression of XB24 compromises XA21-mediated resistance<ref name="ref2"/>.

*'''Overexpression of Xb24 Causes XA21 Instability Following Ax21 Recognition.''' To gain insight into the mechanism of XB24-mediated regulation of XA21 function, they tested whether XB24 affects the amount of the XA21 protein after Xoo inoculation. As shown in '''Fig. 6 A and B''', without Xoo inoculation (Mock treatment). Overexpression of XB24(Xa21/Ntap-Xb24) caused no significant 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<ref name="ref2"/>.

[[File:1HF1.jpg|frame|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.]]

[[File:Fig. 2B.png|frame|Figure 2.An ATPase activity is associated with XB24 and effects XA21 auto-
phosphorylation. (A) ATPase activity assay on purified Ntap-XB24 and Ntap-
XB24S154Aprotein 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 auto-
radiogram. (Lower) Quantitative results (mean + SD) from three independ-
ent 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.”|left]]

[[File:Fig. 3.png|frame|Figure 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.|right]]

[[File:Fig. 4.png|frame|Figure 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.]]

[[File:1HF5.jpg|frame|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.]]

[[File:1HF6.jpg|frame|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).]]

===Expression===
*The XB24 cDNAis expressed from a unique rice gene, Os01g56470, 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.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.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).

*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.

== Other associations with XA21 ==

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'''(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)'''.

'''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. Wenext 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''', we 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.

'''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 '''(Fig. S9)''', 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.

(Don't Delete)
'''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
described superfamilies of ATPases or HSPs. 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)'''.TheATPasemotif in the F1,V1, andA1complexes 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, we 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 ofXB24-type ATPases in other species.

==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.

==References==
<references>

<ref name="ref1">Liu GZ, Pi LY, Walker JC, Ronald PC, Song WY (2002) Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21. J Biol Chem 277:20264–20269.</ref>

<ref name="ref2">Xuewei Chen, Mawsheng Chern, Patrick E. Canlas, Deling Ruan, Caiying Jiang, and Pamela C. Ronald(2010) An ATPase promotes autophosphorylation of the pattern recognition receptor XA21 and inhibits XA21-mediated immunity. Proc Natl Acad Sci USA 107: 8029–8034.</ref>

<ref name="ref3">Fire A, et al. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811.</ref>

<ref name="ref4">Mourrain P, et al. (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–542.</ref>

<ref name="ref5">Century KS, et al. (1999) Developmental control of Xa21-mediated disease resistance in rice. Plant J 20:231–236.</ref>

<ref name="ref6">Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49.</ref>

<ref name="ref7">Lee SW, Han SW, Bartley LE, Ronald PC (2006) Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity. Proc Natl Acad Sci USA 103:18395–18400.</ref>

</references>

==Structured Information==
{{JaponicaGene|
GeneName = 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 = [[:category:Japonica Chromosome 1|Chromosome 1]]|
AP = Chromosome 1:34299150..34301037|
CDS = 34299283..34299494,34300420..34300804|
GCID = <gbrowseImage1>
name=NC_008394:34299150..34301037
source=RiceChromosome01
preset=GeneLocation
</gbrowseImage1>|
GSID = <gbrowseImage2>
name=NC_008394:34299150..34301037
source=RiceChromosome01
preset=GeneLocation
</gbrowseImage2>|
CDNA = <cdnaseq>atgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaa</cdnaseq>|
AA = <aaseq>MGWRWHDDGDDGGRGLGDIPDLAGGGGGGDGERCATRRVVQSRC HTEEVEPGRFVRKCEKTEQLLRDCVGRPSELVESKTENTEEDVTDEMKSGSLSLGFPT NEPFAFPGLRSDIEALEKGLFGSIGSFLDDAERMTNDFLKSFGVPSINERESSSFDGQ PTGRHIGGQPAGRHIEEGTAKDTKQNDYAEFSSKITDV</aaseq>|
DNA = <dnaseqindica>134..345#1271..1655#agaaacgagccggccggattgctctccaagccaaacacggcccagagaggcgagagcccccacaccgccaaacccgacccggaaatcaactgcacacgctccgatcccctctctccagatcgattcggaccccatgggttggcgttggcacgacgacggcgatgacggcggccgcggcctgggcgacatccccgacctcgccggcggcggcggaggcggagatggggagcgctgcgccacgcgccgggtggtgcagtctcggtgccacacggaggaggtggagcccggccgcttcgtccgcaagtgcgagaagaccgagcagctcctccgcgactgcgtcggcaggtatatacatatactccatggcctcgcgcttttccctgatctgttccttcttttcctgcgaggatcccctctttgcatattactgttcgttgttttagtgatgaattggtgataacatgtcatgcgcagttgtgttactattagtcccgtgtatgattgtgagctgtgtatagccgcctctctgtggacgattttcgtgagtctgtcctcgctgatttgatacaaacacgaattcattaagaatggggaatgtgccgtgatgatcgagacgtttgagttctgtggtgaatagtatgatcaaatgtgtgtaataacttgggcttttgaattttggtagctaattagatatggaatttctatccagttttaggtgcccgtgccatcgacatgggttaaaagttattgtgggctattttgcgctcaacttgttaatcaacaaattttggacggaggcagtactttgtaggcatggggtaaatttgtatgatacacttgttgcttgaagatgttcgtgaactcatgggatttctagacacctacaaccaattacattacagtgttattatattctaacgatatgtaaagagcagctgtgttcgtgttgtctacactaaagattcaacaactagtggtcatcatcaaaccgatagcatatcctcattttgcaggattttgaaaactcacatctcgttagcatccttccttttatttcttcacatgcctcatgttaaacttttaagtgcatgaaacccaaatttctgtatttcgtagctccttgtaaccaatgtacaacactatgattagtattctggatagattttgtacttcccaaagtattttggttgttgacaagctattacaataatctgatggataacctaccatacattatttctcctaaccgatcacaataatcttcttctaggccctctgaactggtggaatcaaaaactgaaaatactgaagaagacgtcacagatgaaatgaaaagcgggtcactatctcttggttttccgaccaatgagccctttgcatttcctggacttcgcagtgacatagaagctcttgagaaaggccttttcgggagcattggtagctttctggatgatgctgagaggatgaccaatgatttcttgaagtcttttggtgtcccttccatcaatgaaagggagtcgagctcatttgatggacaacctacaggcaggcacattggtggacaacctgcaggcaggcacattgaggaaggtactgcaaaggacactaaacagaacgactacgcagaattcagcagcaagattacagatgtgtaaggatctacagttagctgacgcacctttgggagcagcttgccaattttgtattttgaacatctccatggttgtaattggaaggggaaggatcagtttgactgttttataagcagagtcgtctgaagtctgaagtgttgcgcttataagaacaattgtgtatattactgttttagataagcctgtttgtgttcctcaacaatgagatcaattatggatggtttttttctctgctt</dnaseqindica>|
Link = [http://www.ncbi.nlm.nih.gov/nuccore/NM_001050918.1 RefSeq:Os01g0771200]|
}}
[[Category:Genes]]
[[Category:Japonica mRNA]]
[[Category:Oryza Sativa Japonica Group]]
[[Category:Japonica Genes]]
[[Category:Japonica Chromosome 1]]
[[Category:Chromosome 1]]