BACKGROUND
during the whole life cycle, plants are continuously threatened by different pathogens including bacteria, fungi and virus.
to survive under the pathogen invasion, plants build up their primary defense by using a structural barrier like the cell wall or cuticle, which is a non-host resistance but also can be easily conquered by pathogens.
after the collapse of the primary defense, the secondary defense of plants, a more pronounced defense than the primary one, could be triggered by effector proteins that are secreted by plant pathogens.
therefore, the recognition of effector proteins and signal transduction in the second defense are of great importance in the plant-pathogen interaction study.
recent studies have revealed that besides the quantity of protein synthesis, post-translational modification  of the pre-existing signaling proteins is also critical in the signal transduction cascade to ensure that plants respond to the pathogen invasion in a prompt manner  <cit> .
so far, among the ptms reported in defense signaling, phosphorylation is the most common and intensively studied one.
phosphorylation is a reversible, covalent modification usually occurring on the hydroxyl group of hydroxyl amino acids like serine, threonine and tyrosine, but occasionally on hydroxyl-proline  <cit> .
phosphorylation and dephosphorylation on specific sites of proteins are catalyzed by kinases and phosphatases respectively to alter the protein nature and configuration and ultimately provide modified protein with new functions in enzyme activity, substrate specificity, structure stability or intracellular localization.
phosphorylation is a very abundant modification in plant and animal proteins.
it was also suggested that more than one-third of all proteins are potentially phosphorylated  <cit>  with diverse roles in different metabolic pathways and disease signaling.
therefore, the large number of phosphorylated proteins together with the transient, reversible phosphorylation patterns enables plants to own highly dynamic, complex signaling cascades in defense to the pathogen infection.
since the discovery of protein phosphorylation from parsley cells upon fungal infection in  <dig>  our knowledge about phosphorylation in plant-pathogen signaling pathway has been largely expanded  <cit> .
protein phosphorylation participated in the whole process of plant-pathogen interaction, including the signal perception, early signaling transduction as well as the immune response activation  <cit> .
to sense the pathogen signals, an auto-phosphorylation of the receptor-like kinases  on the kinase domain is required in arabidopsis.
mutation in the phosphosites could abolish or weaken the signaling in downstream genes  <cit> .
in plants, the signals from the upstream elicitor receptors/sensors to the downstream mapk  substrates largely rely on the three-step mapkkk -mapkk -mapk cascade  <cit> .
the signals from receptor kinase could be transmitted and amplified from mapkkk to downstream mapkk, then to mapk via phosphorylating certain sites of the downstream substrates on each step, and eventually convert signals generated at the receptors into cellular responses in plants.
such an mapk signaling cascade plays vital roles in plant defense signaling.
given the importance of protein phosphorylation in plant defense signaling, extensive studies have been carried out with tremendous progress achieved in the past decades.
nevertheless, due to the technical bottlenecks, traditional researches usually studied the kinase-substrate pairs one by one, and the phosphosites are determined through amino acid sites mutation of the substrate proteins, which makes the identification of phosphosites on proteins extremely challenging and tedious.
as a result of the recent development of novel methods in phosphopeptides enrichment and mass spectrometry, high through-put identification of the phosphopeptides and phosphosites in the proteome level have become available.
in  <dig>  a phosphoproteomic survey resulted in the detection of  <dig> phosphosites on  <dig> proteins in human hela cells  <cit> .
villen et al.
reported the identification of  <dig> non-redundant phosphosites from  <dig> proteins from mouse liver  <cit> .
up to now, the phosphositeplus website  has accumulated over  <dig>  literatures describing  <dig>  phosphosites of  <dig>  proteins from various tissues and species  <cit> .
according to p3db database ,  <dig> independent phosphoproteome studies have generated the data of  <dig>  phosphosites in  <dig>  phosphoproteins from arabidopsis, medicago, rice and other  <dig> plant organisms  <cit> .
rice  is one of the most important food crops in the world, providing approximately 21 % of the calories for over half of the global population  <cit> .
bacterial blight  caused by xanthomonas oryzae pv.
oryzae  has become one of the most devastating diseases of rice worldwide as the yield loss can be up to 50 % or more.
meanwhile, rice-xoo system provides an ideal model for studying plant-pathogen cross-talk due to the availability of genome sequences and ample genetic variations of both partners  <cit> .
even though large number of phosphoproteomic studies has documented more phosphosites in different plant species, the role of phosphorylation is poorly understood in plant-bacterial interactions especially in the rice-xoo system.
therefore, large-scale identification of phosphoproteins and phosphosites of rice in response to xoo infection is of great significance to reveal the disease signal transduction pathway, and how the pathogen surpasses rice defense that leads to rice resistance or susceptibility.
here, we report the first study on large scale enrichment of phosphopeptides and identification of phosphosites in rice before and 24 h after xoo infection.
we have successfully identified  <dig> phosphosites on  <dig> representative proteins after 24 h of xoo infection.
a total of  <dig> differentially phosphorylated proteins were identified after xoo infection suggesting that they may be functionally relevant to disease resistance.
current phosphoproteomic study ultimately improved our understanding of signal transduction in rice disease resistance.
to the best of our knowledge, this is the first phosphoproteomic report regarding the rice-xoo interaction.
the information obtained in this study would substantially advance our understanding of the signal transduction in rice disease resistance.
RESULTS
phosphorylation dynamics of rice variety irbb <dig> in response to xoo infection
a bb resistant variety irbb <dig> was used as the starting material in this study due to its good performance against bb .
our infection assay found that the lesion area of irbb <dig> was only around 7 % when the xoo strain zhe <dig> was inoculated for 10 days, while irbb <dig>  a bb susceptible variety, showed over 35 % lesion area under the same condition , suggesting irbb <dig> is highly resistant to bb.
to gain a global view of the phosphorylation dynamics of irbb <dig> in response to bb, western blot analysis was conducted for the leaf total protein samples at different time points after zhe <dig> inoculation.
for each sample, equal amount of total protein  was loaded for the assay.
as shown in fig.
1d, multiple bands were detected in all the samples and phosphorylation signal intensity of several bands have been changed during the inoculation of xoo, suggesting protein phosphorylation plays important roles in rice disease resistance.
interestingly sample collected after 24 h of inoculation showed more intense phosphorylation signal than protein samples from other time points in western blot analysis and it also indicated the further exploration of phosphosites is worth studying from 24 h protein sample.fig.
<dig> the phenotype of irbb <dig> and irbb <dig>  and global phosphorylation dynamics of irbb <dig> under xoo infection.
a and b the phenotype of irbb <dig> and irbb <dig> under xoo infection, respectively.
c the lesion area counted for irbb <dig> and irbb <dig>  d western-blot analysis of irbb <dig> globe phosphorylation dynamics at the different time points under xoo infection
identification of phosphorylation sites, peptides and proteins
to explore the role of protein phosphorylation in rice disease signaling and resistance, a quantitative, non-gel, label-free phophoproteomic study was conducted for the leaf samples of irbb <dig> at the time points of 0 h and 24 h after xoo infection with three biological replicates.
phosphopeptides were enriched from leaf total proteins by tio2-moac  method followed by lc-ms/ms assay.
in the current study, a total of  <dig> and  <dig> phosphopeptides were identified in 0 h and 24 h samples, representing  <dig> and  <dig> proteins, respectively .
in the  <dig> phosphopeptides of 0 h, there were  <dig> phosphosites, including  <dig> serine ,  <dig> threonine  and  <dig> tyrosine  sites.
similarly, in the sample of 24 h, all  <dig> phosphopeptides covered  <dig> serine,  <dig> threonine and  <dig> tyrosine phosphosites, representing a percentage of  <dig>  %,  <dig>  % and  <dig>  % of the all  <dig> phosphosites respectively .
the distribution of phophorylation types in our study is consistent with other reports in rice, triticum aestivum and brachypodium distachyon .
in both 0 h and 24 h samples, most of the peptides carried only one phosphorylation modification; around 10 % peptides carried two phosphorylations, whereas three phosphorylation modifications occurred in less than 1 % of the peptides .fig.
<dig> the distribution of phosphosite types and subcellular localization of phosphoproteins.
a pie chart showing the distribution of phosphoserine, phosphothreonine and phosphotyreosine.
b pie chart showing the number of phosphopeptides carrying multiple phosphosites.
c the subcellular localization distribution of phosphoproteins
conserved phosphorylation motifs analysis of the unique phosphpeptides
by using the motif-x tool   <cit> , the over-presented motifs around the phosphosites were analyzed.
firstly, a  <dig> amino acid  sequence centered by the phosphorylation site were extracted from both 0 h and 24 h phosphopeptides.
after removing the redundant sequences from both datasets, we obtained totally  <dig> unique amino acid sequence extractions, including  <dig> centered by phsophoserine,  <dig> centered by phosphothreonine and  <dig> tyrosine-centered phosphopeptides.
due to the small number of phosphorylated tyrosine sites, no obvious conserved motif was detected in our assay.
intriguingly, at least five types of conserved motifs were significantly enriched around the phosphoserine sites .
was the most common motifs as  <dig> matches were found in our result.
followed were  and  with over  <dig> hits been detected.
there were also more than  <dig> hits of  and  motifs.
nevertheless, to the best of our knowledge,  was not found in any other reports in plants except to this study, which possibly due to the fact that different protein extraction methods and plant tissues were used in different studies.
on the other hand, the very limited phosphosite data accumulation in plants would also be a reason for this phenomenon.
as for phosphothreonine,  was the only conserved motif found in this study.
recent studies have revealed numerous over-presented motifs from plants, and linked them with certain kinase substrates  <cit> .
besides this research,  motif was over-presented in other studies in arabidopsis, rice and wheat  <cit> .
this proline-directed motif could be a potential targets for mapk, snrk <dig> , rlk , agc , cdk , cdpk  and slk  kinases  <cit> .
motif could be recognized by mapkk, camk-ii and protein kinase a  <cit> .
though  has been detected by some researches, its potential kinases remain unknown yet  <cit> .
by far,  is the most common phosphothreonine motif found in plants  <cit> .table  <dig> motif-x analysis of unique phosphopeptides
lower case “s” and “t” indicate phosphoserine and phosphothreonine respectively.
“x” represents any amino acid
fig.
<dig> over-presented amino acid motifs detected from the identified phosphosites by motif-x.
a-e five enriched motifs from phosphoserine.
f enriched motif from phosphothreonine
subcellular localization of phophoproteins
to predict the putative subcellular localization of phosphoproteins, the sequences of both 0 h and 24 h phosphoproteins were used to search against the “eukaryotes” database of cello with default setting   <cit> .
the results showed that all of the 0 h and 24 h phosphoproteins obtained a hit respectively, and both showed very similar cellular compartment distributions .
over 50 % of the phosphoproteins were located in the nuclus, followed by cytoplasm, chloroplast and plasmamembrane localized proteins.
however, other compartments, such as mitochondrial, golgi and er, had less than 20 % of the phosphoproteins in total.
so far, no report regarding the cellular compartments distribution of rice leaf phosphoproteins is available, but several other cases in rice pistil, cotton leaf or physcomitrella patens protoplast showed divergent distribution patterns  <cit> .
the difference in distribution patterns may be due to the differences in species, tissues or methods used for protein extraction.
differentially phosphorylated  peptides and proteins in response to xoo infection
based on the average phosphorylation intensity of three biological replicates,  <dig> dp peptides were screened out with  <dig> fold change or more , including  <dig> up-phosphorylated and  <dig> down-phosphorylated peptides after xoo infection .
in the up-phosphorylated peptides,  <dig> peptides were specifically phosphorylated in 24 h, but not in 0 h, while the other  <dig> showed over  <dig> fold intensity increasing after infection.
we also found  <dig>  of the down-phosphorylated peptides were specifically phosphorylated in 0 h, and the other  <dig> peptides had decreased intensity less than  <dig>  folds.
in addition to the dp peptides, there were  <dig> phosphopeptides showing no significant changes in intensity after xoo infection, suggesting that they may be functionally unrelated to disease resistance.table  <dig> numbers of differentially phosphopeptides and phosphoproteins
athe phosphorylation intensity of 0 h/24 >  <dig> folds, p <  <dig>
bthe phosphorylation intensity of 0 h/24 <  <dig>  folds, p <  <dig>
a database search of the phosphopeptides resulted in the identification of  <dig> corresponding phosphoproteins, among which there were  <dig> dp proteins with  <dig> being up-phosphorylated and  <dig> being down-phosphorylated after xoo infection .
we also found that there were  <dig> and  <dig> proteins that were specifically phosphorylated in 0 h and 24 h respectively.
transcription factor  is a major group of the dp proteins as  <dig> tfs were identified, including  <dig> down-phosphorylated and  <dig> up-phosphorylated .
furthermore, the dp proteins covered  <dig> epigenetic control factors whose function were involved in dna methylation, histone methylation, chromatin condensing etc.
; implying that cross-talk of various ptms plays important roles in the plant disease resistance .
differential phosphorylation pattern usually indicates the regulatory roles of the dp protein in the corresponding biological process.
up to date, numerous high through-put, quantitative studies have been reported investigating the phosphorylation dynamics in seed development, seed germination, fruit ripening, abiotic stress in arabidopsis, maize, rice, soybean, sweet orange and wheat .
previous studies also clearly showed that phosphorylation/dephosphorylation of signaling proteins transmit messages from the pathogen secreted elicitor to the cell nucleus, where the immune reaction could be triggered upon the message reception  <cit> .
nevertheless, few literatures describing plant phosphoproteome to biotic stress are available so far.
only five differentially phosphorylated proteins were found in arabidopsis during the defense response to pseudomonas syringae pv.
tomato dc <dig>  <cit> .
in grape vine,  <dig> proteins were found to be differentially changed in abundance or/and phosphorylation intensity under flavescence dorée phytoplasma infection  <cit> .
benschop et al.
found  <dig> membrane-associated proteins including a number of defense-related proteins were differentially phosphorylated from arabidopsis cells treated with bacterial elicitor flg <dig> or fungal elicitor xylanase  <cit> .
recently, in a study of the rhizobia-root hair infection process in soybean,  <dig> phosphopeptides corresponding to  <dig> phosphoproteins were found to be significantly regulated in response to inoculation with bradyrhizobium japonicum  <cit> .
the large number of dp proteins identified in this study could be valuable candidate proteins to reveal the phosphorylation-mediated plant disease resistance.
gene ontology analysis of dp proteins
the agrigo online software was employed to classify dp proteins based on their gene ontology annotations in the vocabulary of “cellular component”, “biological process” and “molecular function” .
from the “cellular component” perspective, envelope, cell part, macrocellular complex, membrane-enclosed lumen, organelle part and extra cellular region part were over-presented in our dp proteins when the whole-genome encoding proteins was used as a control .
in terms of “molecular function”, enzyme regulator, structural molecule and translation regulator were significantly enriched in dp proteins, while catalytic was less presented than the control .
from the perspective of “biological process”, dp proteins were preferentially cataloged into multicellular organismal process and reproduction, whereas death and multi-organism process were less preferred .fig.
<dig> go analysis of dp proteins  and quantitative rt-pcr analysis of the mrna abundance of the corresponding dp protein genes .
standardized residue was employed for the statistical analysis of go enrichment, where standardized residue , which follows asymptotically a normal distribution  <cit> .
an absolute sr value larger than  <dig>  indicates statistical significance at p <  <dig> .
based on the distribution of each go category in genome, an expected number of dp proteins in each go category could be calculated out.
observed is the number actually occurred in each go category * indicates p <  <dig> ; ** indicates p <  <dig>
mrna abundance of the corresponding dp proteins
previous transcriptomic analysis has revealed that  <dig> genes were differentially expressed in rice bb resistant variety irbb <dig> at the time point of 24 h after xoo infection  <cit> .
in this study, the transcriptomic data was downloaded to investigate the correlation of the mrna transcript abundance with the protein phosphorylation intensity level.
interestingly, among the  <dig> dp proteins, the mrna transcript expression of  <dig> dp proteins remained unchanged after xoo infection  .
moreover, even for those dp proteins whose mrna level were responsive to xoo infection, no clear correlations were found between the mrna abundance and phosphorylation intensity, indicating that the phosphorylation intensity variation detected in our study was majorly due to the occurrence of phosphorylation event in the pre-existing proteins, instead of the quantity change caused by protein synthesis or degradation.
this hypothesis is also supported by our quantitative rt-pcr of  <dig> randomly selected dp protein genes .
our qrt-pcr results showed that the transcription expression level of four genes  were not significantly altered .
for the rest four genes tested, despite their mrna expression level being significantly changed  or extremely changed , we noticed that the variation in the phosphoprotein level was apparently much larger than these in the mrna level.
for example, the mrna expression level of loc_os05g <dig> and loc_os09g <dig> was approximately 40 % and 70 % down-regulated by xoo infection, whereas the phosphorylation was completely removed for both proteins.
taken together, the results above may suggest that the phosphorylation intensity, rather than the quantity, of the proteins essentially regulates the plant disease resistance.
protein-protein interaction  analysis of dp proteins
string  version  <dig> was employed in this study for the potential ppi analysis of the dp proteins   <cit> .
the parameter for confidence score was set to  <dig>  to assure a high reliability, and the yield ppi results were visualized by cytoscape software  <cit> .
when all the  <dig> dp proteins were used as input for the analysis, the yield result displayed a complicated network with  <dig> nodes  and  <dig> edges  .
we found three groups of dp proteins were aggregated, including hdt <dig> in group ii, suggesting intense interactions among these interaction partners.
to gain an in-depth view of the phosphorylation-mediated signaling, we also analyzed the ppi of the kinases and phosphatases of the dp proteins.
as shown in fig.
5b, a network comprising  <dig> nodes  and  <dig> edges was obtained.
interestingly, three pp2cs were centered in the network, suggesting the aba related signaling plays important roles in the plant disease resistance.fig.
<dig> a sub-network of all the dp proteins  and dp kinases and phosphatases  by using string and cytoscape.
the locus id of the abbreviations in  could be seen in additional file 4: table s4
discussion
in this study, a quantitative, ms-based, label-free proteomic analysis identified  <dig> non-redundant phosphopeptides from  <dig> phosphoproteins of rice at both 0 h and 24 h after xoo infection including  <dig> differentially phosphorylated proteins, representing the first phosphoproteomic attempt to explore the phosphorylation events in rice-pathogen cross-talk.
phosphorylation-dependent signaling
through the phosphorylation/dephosphorylation of kinase cascade controlled by kinase or phosphatase, the signals of pathogen infection stimuli could be transmitted to the nucleus, where disease resistant-related proteins will be directly or indirectly phosphorylated or dephosphorylated to initiate the immune response.
phytohormone abscisic acid  signaling is well-known for its roles in response to abiotic stress as well as to biotic stress  <cit> .
a new “pyr/pyl/rcar -pp2c -snrk2”cascade model for aba signaling has been proposed and validated, in which the soluble pyr/pyl/rcar receptors function at the apex of a negative regulatory pathway to directly regulate pp2c phosphatases, which in turn directly regulate snrk <dig> kinases.
in rice, there are at least  <dig> pp2cs have been identified  <cit> .
intriguingly, four pp2c proteins were found differentially phosphorylated in our result, among which ospp2c <dig>  and ospp2c <dig>  were up-phosphorylated by xoo infection .
as negative regulators, pp2cs competitively bind with aba receptors to relieve the inhibition on snrks under stress conditions.
the up-phosphorylation of ospp2c <dig> and ospp2c <dig> probably promote the binding to aba receptors, thus to trigger the aba-dependent signaling in rice defense.
this hypothesis is supported by similar results from wheat, in which two pp2cs were up-phosphorylated by drought stress  <cit> .table  <dig> some examples of the differentially phosphorylated proteins identified in this study
cdpks are directly activated by the binding of ca2+ to the calmodulin-like domain, and activated cdpks regulate downstream components of calcium signaling.
in our result, totally  <dig> cdpks were identified with  <dig> being up-phosphorylated .
oscpk <dig>  is one of the documented down-phosphorylated cdpks in our result.
literature showed that oscpk12-ox seedlings had increased sensitivity to abscisic acid  and increased susceptibility to blast fungus, probably resulting from the repression of ros production and/or the involvement of oscpk <dig> in the aba signaling pathway  <cit> .
the differential phosphorylation pattern of oscpk <dig> upon xoo infection suggested that it is involved in response to multiple pathogen attacks besides blast fungus.
moreover, in agreement with the previous report, dephosphorylation of oscpk <dig> detected in our data probably resulted in an “inactive” status of this negative regulator to eliminate its inhibition effect, thus enhance plant resistance to pathogen attack.
in addition to pp2cs and oscpk <dig>  we totally identified over  <dig> differentially phosphorylated kinases or phosphatases, like lrr transmembrane protein kinase , map2k  etc., suggesting that the signaling of rice-xoo interaction is a very complex event with multiple signaling pathways involvement.
rice disease resistant-related proteins
among the  <dig> dp proteins detected in this study, several proteins are functionally related to rice disease resistance .
for example, osmapk <dig> , a key component in the osrac1-osmapk3/6-rai1-pal1/oswrky <dig> rice immunity signaling cascade, was down-phosphorylated at 24 h. previous studies have revealed that osrac <dig> is a key regulator involved in basal resistance by inducing the ros production or suppressing the ros scavenging.
osrac <dig> could physically bind to osmapk <dig> and post-translationally activate osmapk <dig>  meanwhile, osmapk <dig> could directly phosphorylate rai <dig>  a putative basic helix–loop–helix transcription factor, the overexpression of which substantially enhanced the rice resistance to blast fungus, probably via regulating pal <dig> and oswrky <dig>  <cit> .
though it has been clear that osmapk <dig> acts as a carrier transmitting the phosphorylation from osrac1to rai <dig> in this defense signaling cascade, how osmapk <dig> is phosphorylated remains unknown.
our phosphoproteomic data indicated that the threonine  <dig> and tyrosine  <dig> are two potential phosphosites in osmapk <dig>  which will be further confirmed by our future study.
we also noticed that osmapk <dig> was down-phosphorylated at 24 h, although osmapk <dig> is reported to be a positive regulator in the plant immunity response.
lieberherr et al.
found that the mrna expression of osmapk <dig> started to decrease at 24 h after sphingolipid elicitor treatment, indicating that osmapk <dig> may be involved in the early response to pathogen infection  <cit> .
checking of the phosphorylation intensity of osmapk <dig> at an earlier time point, like  <dig> or 4 h after xoo infection may be necessary to explore its functions in the future.
another differentially phosphorylated protein gene example is rice yellow mottle virus resistance  <dig>  a recessive gene controlling rice resistance to rice yellow mottle virus.
according to our data, rymv <dig> was dephosphorylated in response to the xoo infection, suggesting that rymv <dig> may play a negative role in bacterial disease resistance.
albar et al.
cloned this gene from rice variety giganta through a map-based strategy, and found rymv <dig> is an isoform of the eukaryotic translation initiation factor 4g 4g).
compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein  <cit> .
our evidences indicated that rymv <dig> might be subject to the activation of phosphorylation upon the xoo infection.
however, whether rymv <dig> mediates resistance to rice bacterial blight or not needs to be further studied by genetic analysis and pathogen inoculation assay.
in addition to ossgt <dig> and rymv <dig>  differential phosphorylation occurred on many other potential disease resistance-related proteins, like osmyc <dig>  which is involved in the jasmonic acid mediated signaling  <cit> , oscatc  and pub <dig>  which mediate the process of h2o2-induced cell death in rice .
furthermore, some abiotic stress related proteins, such as osapx  and oshsp <dig>   were also differentially phosphorylated .
these proteins have proven to be involved in rice drought, cold or heat resistance, but their biological function in biotic stress remains to be explored  <cit> .
transcription factors
transcription factors  serve as important internodes in the disease resistant pathway by linking the mapks signal with the downstream transcriptional reprogramming.
overexpression of osbwmk <dig>  a rice mapk kinase, conferred plants enhanced pathogen resistance via directly phosphorylating transcription factors oserebp <dig> and oswrky <dig>  <cit> .
similar cases were reported in other plant species as well .
in our study, at least  <dig> transcription factors were differentially phosphorylated, including  <dig> down-phosphorylated and  <dig> up-phosphorylated, according to the rice transcription factor list released from drtf database   <cit> .
these tfs consist of bzip, bhlh, myb and wrky family members.
intriguingly, rf2a , a bzip domain containing tf was specifically phosphorylated after xoo infecton.
dai et al.
found that overexpression of rf2a could repress the symptoms of rice tungro disease without having any growth penalty.
they suggested that rf2a together with rf2b suppress the rtbv replication via directly binding to its cis element box ii in the promoter .
as indicated in our qrt-pcr results, xoo infection did not alter the mrna abundance of rf2a, but changed the protein status from unphosphorylated to phosphorylated, implying that rf2a plays essential roles in bacterial blight resistance, and phosphorylation might be an important step for the activation of pre-existing rf2a.
in addition to rf2a, many other tfs such as wrky, myb and bhlh were found to be differentially phosphorylated, which suggested that they are good candidate genes for rice bacterial blight resistance.
epi-genetic controlling factors
recent studies demonstrated that chromatin remodeling accomplished through histone modifications is emerging as a key process in the orchestration of plant biotic stress responses and epi-genetic controlling factors are the critical regulators of plant defense to pathogen attack.
not surprisingly, many epi-genetic controlling factors were differentially phosphorylated in this study.
hdt701/oshdt1encoding a histone deacetylase plays versatile roles in plant development and stress response.
li et al.
reported that hdt701/oshdt1expression displayed a circadian rhythm.
elevated oshdt <dig> expression imposed no effects on plant growth in the parent but led to early flowering in the hybrid.
it was suggested that hdt701/oshdt1may be involved in epigenetic control of parental genome interaction for differential gene expression  <cit> .
besides the control of hybrid flowering, its role in rice innate immunity was unraveled in a recent publication  <cit> .
transcription of hdt <dig> could be induced by compatible reaction and repressed by the incompatible reaction after infection by the fungal pathogen magnaporthe oryzae .
more importantly, in the rice hdt <dig> overexpression lines, the global histone h <dig> acetylation level was reduced and plants became more susceptible to the rice pathogens m. oryzae and xoo.
silencing of hdt <dig> imposed an opposite effect on rice in that the resistance to both m. oryzae and xoo was enhanced.
the underlying mechanism could be that hdt <dig> physically bind to and modulate the levels of histone h <dig> acetylation of pattern recognition receptor  and defense-related genes, such as mapk <dig> and wrky <dig>  in our study, we also found that hdt701/oshdt1in a phosphorylated status at 0 h, but the phosphorylation was found to be removed by the xoo infection.
given that phosphorylation modification usually activates protein function, the dephosphorylation of hdt701/oshdt <dig> upon xoo infection suggested that it is a negative regulator of plant defense, which is highly consistent with the conclusion of ding et al.
<cit> .
however, even though the mask of the biological function of hdt701/oshdt1has been unraveled, how hdt701/oshdt <dig> itself is regulated remain unclear.
our data provide good hints that phosphorylation/dephosphorylation might be a key switch turning on/off hdt701in response to xoo infection.
moreover, this kind of phosphorylation switch overriding the epi-genetic regulation may be a very universal model in the plant disease resistance pathway.
in this study, we have identified  <dig> differentially phosphorylated epi-genetic factor proteins, comprising ago , snf <dig> chromatin remodeling complex proteins, histone demethylases, set domain proteins etc., which are functionally related to dna methylation, histone methylation, acetylation and ubiquitination.
CONCLUSIONS
in conclusion,  <dig> and  <dig> phosphosites on  <dig> and  <dig> representative proteins were identified in 0 h and 24 h after xoo infection, respectively.
<dig> proteins were differentially phosphorylated in response to the xoo infection, including several well-known rice disease resistance related proteins.
our data also suggested that phosphorylation/dephosphorylation might be a key switch turning on/off many epi-genetic controlling factors in plant disease resistance pathway.
to the best of our knowledge, this is the first report exploring the cross-talk of rice-pathogen from a view of quantitative phosphoproteome.
the data obtained in this research will not only provide phosphorylation status and sites information for rice proteins, but also shed new light in studying the roles of phosphorylation in plant disease resistance.

 during the whole life cycle, plants are continuously threatened by different pathogens including bacteria, fungi and virus. to survive under the pathogen invasion, plants build up their primary defense by using a structural barrier like the cell wall or cuticle, which is a non-host resistance but also can be easily conquered by pathogens. after the collapse of the primary defense, the secondary defense of plants, a more pronounced defense than the primary one, could be triggered by effector proteins that are secreted by plant pathogens. therefore, the recognition of effector proteins and signal transduction in the second defense are of great importance in the plant-pathogen interaction study. recent studies have revealed that besides the quantity of protein synthesis, post-translational modification of the pre-existing signaling proteins is also critical in the signal transduction cascade to ensure that plants respond to the pathogen invasion in a prompt manner. so far, among the ptms reported in defense signaling, phosphorylation is the most common and intensively studied one. phosphorylation is a reversible, covalent modification usually occurring on the hydroxyl group of hydroxyl amino acids like serine, threonine and tyrosine, but occasionally on hydroxyl-proline. phosphorylation and dephosphorylation on specific sites of proteins are catalyzed by kinases and phosphatases respectively to alter the protein nature and configuration and ultimately provide modified protein with new functions in enzyme activity, substrate specificity, structure stability or intracellular localization. phosphorylation is a very abundant modification in plant and animal proteins. it was also suggested that more than one-third of all proteins are potentially phosphorylated with diverse roles in different metabolic pathways and disease signaling. therefore, the large number of phosphorylated proteins together with the transient, reversible phosphorylation patterns enables plants to own highly dynamic, complex signaling cascades in defense to the pathogen infection. since the discovery of protein phosphorylation from parsley cells upon fungal infection in our knowledge about phosphorylation in plant-pathogen signaling pathway has been largely expanded. protein phosphorylation participated in the whole process of plant-pathogen interaction, including the signal perception, early signaling transduction as well as the immune response activation. to sense the pathogen signals, an auto-phosphorylation of the receptor-like kinases on the kinase domain is required in arabidopsis. mutation in the phosphosites could abolish or weaken the signaling in downstream genes. in plants, the signals from the upstream elicitor receptors/sensors to the downstream mapk substrates largely rely on the three-step mapkkk -mapkk -mapk cascade. the signals from receptor kinase could be transmitted and amplified from mapkkk to downstream mapkk, then to mapk via phosphorylating certain sites of the downstream substrates on each step, and eventually convert signals generated at the receptors into cellular responses in plants. such an mapk signaling cascade plays vital roles in plant defense signaling. given the importance of protein phosphorylation in plant defense signaling, extensive studies have been carried out with tremendous progress achieved in the past decades. nevertheless, due to the technical bottlenecks, traditional researches usually studied the kinase-substrate pairs one by one, and the phosphosites are determined through amino acid sites mutation of the substrate proteins, which makes the identification of phosphosites on proteins extremely challenging and tedious. as a result of the recent development of novel methods in phosphopeptides enrichment and mass spectrometry, high through-put identification of the phosphopeptides and phosphosites in the proteome level have become available. in a phosphoproteomic survey resulted in the detection of phosphosites on proteins in human hela cells. villen et al. reported the identification of non-redundant phosphosites from proteins from mouse liver. up to now, the phosphositeplus website has accumulated over literatures describing phosphosites of proteins from various tissues and species. according to p3db database, independent phosphoproteome studies have generated the data of phosphosites in phosphoproteins from arabidopsis, medicago, rice and other plant organisms. rice is one of the most important food crops in the world, providing approximately 21 % of the calories for over half of the global population. bacterial blight caused by xanthomonas oryzae pv. oryzae has become one of the most devastating diseases of rice worldwide as the yield loss can be up to 50 % or more. meanwhile, rice-xoo system provides an ideal model for studying plant-pathogen cross-talk due to the availability of genome sequences and ample genetic variations of both partners. even though large number of phosphoproteomic studies has documented more phosphosites in different plant species, the role of phosphorylation is poorly understood in plant-bacterial interactions especially in the rice-xoo system. therefore, large-scale identification of phosphoproteins and phosphosites of rice in response to xoo infection is of great significance to reveal the disease signal transduction pathway, and how the pathogen surpasses rice defense that leads to rice resistance or susceptibility. here, we report the first study on large scale enrichment of phosphopeptides and identification of phosphosites in rice before and 24 h after xoo infection. we have successfully identified phosphosites on representative proteins after 24 h of xoo infection. a total of differentially phosphorylated proteins were identified after xoo infection suggesting that they may be functionally relevant to disease resistance. current phosphoproteomic study ultimately improved our understanding of signal transduction in rice disease resistance. to the best of our knowledge, this is the first phosphoproteomic report regarding the rice-xoo interaction. the information obtained in this study would substantially advance our understanding of the signal transduction in rice disease resistance.  phosphorylation dynamics of rice variety irbb in response to xoo infection a bb resistant variety irbb was used as the starting material in this study due to its good performance against bb. our infection assay found that the lesion area of irbb was only around 7 % when the xoo strain zhe was inoculated for 10 days, while irbb a bb susceptible variety, showed over 35 % lesion area under the same condition, suggesting irbb is highly resistant to bb. to gain a global view of the phosphorylation dynamics of irbb in response to bb, western blot analysis was conducted for the leaf total protein samples at different time points after zhe inoculation. for each sample, equal amount of total protein was loaded for the assay. as shown in fig. 1d, multiple bands were detected in all the samples and phosphorylation signal intensity of several bands have been changed during the inoculation of xoo, suggesting protein phosphorylation plays important roles in rice disease resistance. interestingly sample collected after 24 h of inoculation showed more intense phosphorylation signal than protein samples from other time points in western blot analysis and it also indicated the further exploration of phosphosites is worth studying from 24 h protein sample.fig. the phenotype of irbb and irbb and global phosphorylation dynamics of irbb under xoo infection. a and b the phenotype of irbb and irbb under xoo infection, respectively. c the lesion area counted for irbb and irbb d western-blot analysis of irbb globe phosphorylation dynamics at the different time points under xoo infection identification of phosphorylation sites, peptides and proteins to explore the role of protein phosphorylation in rice disease signaling and resistance, a quantitative, non-gel, label-free phophoproteomic study was conducted for the leaf samples of irbb at the time points of 0 h and 24 h after xoo infection with three biological replicates. phosphopeptides were enriched from leaf total proteins by tio2-moac method followed by lc-ms/ms assay. in the current study, a total of and phosphopeptides were identified in 0 h and 24 h samples, representing and proteins, respectively. in the phosphopeptides of 0 h, there were phosphosites, including serine, threonine and tyrosine sites. similarly, in the sample of 24 h, all phosphopeptides covered serine, threonine and tyrosine phosphosites, representing a percentage of  %,  % and  % of the all phosphosites respectively. the distribution of phophorylation types in our study is consistent with other reports in rice, triticum aestivum and brachypodium distachyon. in both 0 h and 24 h samples, most of the peptides carried only one phosphorylation modification; around 10 % peptides carried two phosphorylations, whereas three phosphorylation modifications occurred in less than 1 % of the peptides.fig. the distribution of phosphosite types and subcellular localization of phosphoproteins. a pie chart showing the distribution of phosphoserine, phosphothreonine and phosphotyreosine. b pie chart showing the number of phosphopeptides carrying multiple phosphosites. c the subcellular localization distribution of phosphoproteins conserved phosphorylation motifs analysis of the unique phosphpeptides by using the motif-x tool, the over-presented motifs around the phosphosites were analyzed. firstly, a amino acid sequence centered by the phosphorylation site were extracted from both 0 h and 24 h phosphopeptides. after removing the redundant sequences from both datasets, we obtained totally unique amino acid sequence extractions, including centered by phsophoserine, centered by phosphothreonine and tyrosine-centered phosphopeptides. due to the small number of phosphorylated tyrosine sites, no obvious conserved motif was detected in our assay. intriguingly, at least five types of conserved motifs were significantly enriched around the phosphoserine sites. was the most common motifs as matches were found in our result. followed were and with over hits been detected. there were also more than hits of and motifs. nevertheless, to the best of our knowledge, was not found in any other reports in plants except to this study, which possibly due to the fact that different protein extraction methods and plant tissues were used in different studies. on the other hand, the very limited phosphosite data accumulation in plants would also be a reason for this phenomenon. as for phosphothreonine, was the only conserved motif found in this study. recent studies have revealed numerous over-presented motifs from plants, and linked them with certain kinase substrates. besides this research, motif was over-presented in other studies in arabidopsis, rice and wheat. this proline-directed motif could be a potential targets for mapk, snrk, rlk, agc, cdk, cdpk and slk kinases. motif could be recognized by mapkk, camk-ii and protein kinase a. though has been detected by some researches, its potential kinases remain unknown yet. by far, is the most common phosphothreonine motif found in plants.table motif-x analysis of unique phosphopeptides lower case “s” and “t” indicate phosphoserine and phosphothreonine respectively. “x” represents any amino acid fig. over-presented amino acid motifs detected from the identified phosphosites by motif-x. a-e five enriched motifs from phosphoserine. f enriched motif from phosphothreonine subcellular localization of phophoproteins to predict the putative subcellular localization of phosphoproteins, the sequences of both 0 h and 24 h phosphoproteins were used to search against the “eukaryotes” database of cello with default setting. the results showed that all of the 0 h and 24 h phosphoproteins obtained a hit respectively, and both showed very similar cellular compartment distributions. over 50 % of the phosphoproteins were located in the nuclus, followed by cytoplasm, chloroplast and plasmamembrane localized proteins. however, other compartments, such as mitochondrial, golgi and er, had less than 20 % of the phosphoproteins in total. so far, no report regarding the cellular compartments distribution of rice leaf phosphoproteins is available, but several other cases in rice pistil, cotton leaf or physcomitrella patens protoplast showed divergent distribution patterns. the difference in distribution patterns may be due to the differences in species, tissues or methods used for protein extraction. differentially phosphorylated peptides and proteins in response to xoo infection based on the average phosphorylation intensity of three biological replicates, dp peptides were screened out with fold change or more, including up-phosphorylated and down-phosphorylated peptides after xoo infection. in the up-phosphorylated peptides, peptides were specifically phosphorylated in 24 h, but not in 0 h, while the other showed over fold intensity increasing after infection. we also found of the down-phosphorylated peptides were specifically phosphorylated in 0 h, and the other peptides had decreased intensity less than folds. in addition to the dp peptides, there were phosphopeptides showing no significant changes in intensity after xoo infection, suggesting that they may be functionally unrelated to disease resistance.table numbers of differentially phosphopeptides and phosphoproteins athe phosphorylation intensity of 0 h/24 >  folds, p <  bthe phosphorylation intensity of 0 h/24 <  folds, p <  a database search of the phosphopeptides resulted in the identification of corresponding phosphoproteins, among which there were dp proteins with being up-phosphorylated and being down-phosphorylated after xoo infection. we also found that there were and proteins that were specifically phosphorylated in 0 h and 24 h respectively. transcription factor is a major group of the dp proteins as tfs were identified, including down-phosphorylated and up-phosphorylated. furthermore, the dp proteins covered epigenetic control factors whose function were involved in dna methylation, histone methylation, chromatin condensing etc.; implying that cross-talk of various ptms plays important roles in the plant disease resistance. differential phosphorylation pattern usually indicates the regulatory roles of the dp protein in the corresponding biological process. up to date, numerous high through-put, quantitative studies have been reported investigating the phosphorylation dynamics in seed development, seed germination, fruit ripening, abiotic stress in arabidopsis, maize, rice, soybean, sweet orange and wheat. previous studies also clearly showed that phosphorylation/dephosphorylation of signaling proteins transmit messages from the pathogen secreted elicitor to the cell nucleus, where the immune reaction could be triggered upon the message reception. nevertheless, few literatures describing plant phosphoproteome to biotic stress are available so far. only five differentially phosphorylated proteins were found in arabidopsis during the defense response to pseudomonas syringae pv. tomato dc. in grape vine, proteins were found to be differentially changed in abundance or/and phosphorylation intensity under flavescence dorée phytoplasma infection. benschop et al. found membrane-associated proteins including a number of defense-related proteins were differentially phosphorylated from arabidopsis cells treated with bacterial elicitor flg or fungal elicitor xylanase. recently, in a study of the rhizobia-root hair infection process in soybean, phosphopeptides corresponding to phosphoproteins were found to be significantly regulated in response to inoculation with bradyrhizobium japonicum. the large number of dp proteins identified in this study could be valuable candidate proteins to reveal the phosphorylation-mediated plant disease resistance. gene ontology analysis of dp proteins the agrigo online software was employed to classify dp proteins based on their gene ontology annotations in the vocabulary of “cellular component”, “biological process” and “molecular function”. from the “cellular component” perspective, envelope, cell part, macrocellular complex, membrane-enclosed lumen, organelle part and extra cellular region part were over-presented in our dp proteins when the whole-genome encoding proteins was used as a control. in terms of “molecular function”, enzyme regulator, structural molecule and translation regulator were significantly enriched in dp proteins, while catalytic was less presented than the control. from the perspective of “biological process”, dp proteins were preferentially cataloged into multicellular organismal process and reproduction, whereas death and multi-organism process were less preferred.fig. go analysis of dp proteins and quantitative rt-pcr analysis of the mrna abundance of the corresponding dp protein genes. standardized residue was employed for the statistical analysis of go enrichment, where standardized residue, which follows asymptotically a normal distribution. an absolute sr value larger than indicates statistical significance at p < . based on the distribution of each go category in genome, an expected number of dp proteins in each go category could be calculated out. observed is the number actually occurred in each go category * indicates p < ; ** indicates p <  mrna abundance of the corresponding dp proteins previous transcriptomic analysis has revealed that genes were differentially expressed in rice bb resistant variety irbb at the time point of 24 h after xoo infection. in this study, the transcriptomic data was downloaded to investigate the correlation of the mrna transcript abundance with the protein phosphorylation intensity level. interestingly, among the dp proteins, the mrna transcript expression of dp proteins remained unchanged after xoo infection. moreover, even for those dp proteins whose mrna level were responsive to xoo infection, no clear correlations were found between the mrna abundance and phosphorylation intensity, indicating that the phosphorylation intensity variation detected in our study was majorly due to the occurrence of phosphorylation event in the pre-existing proteins, instead of the quantity change caused by protein synthesis or degradation. this hypothesis is also supported by our quantitative rt-pcr of randomly selected dp protein genes. our qrt-pcr results showed that the transcription expression level of four genes were not significantly altered. for the rest four genes tested, despite their mrna expression level being significantly changed or extremely changed, we noticed that the variation in the phosphoprotein level was apparently much larger than these in the mrna level. for example, the mrna expression level of loc_os05g and loc_os09g was approximately 40 % and 70 % down-regulated by xoo infection, whereas the phosphorylation was completely removed for both proteins. taken together, the results above may suggest that the phosphorylation intensity, rather than the quantity, of the proteins essentially regulates the plant disease resistance. protein-protein interaction analysis of dp proteins string version was employed in this study for the potential ppi analysis of the dp proteins. the parameter for confidence score was set to to assure a high reliability, and the yield ppi results were visualized by cytoscape software. when all the dp proteins were used as input for the analysis, the yield result displayed a complicated network with nodes and edges. we found three groups of dp proteins were aggregated, including hdt in group ii, suggesting intense interactions among these interaction partners. to gain an in-depth view of the phosphorylation-mediated signaling, we also analyzed the ppi of the kinases and phosphatases of the dp proteins. as shown in fig. 5b, a network comprising nodes  and edges was obtained. interestingly, three pp2cs were centered in the network, suggesting the aba related signaling plays important roles in the plant disease resistance.fig. a sub-network of all the dp proteins and dp kinases and phosphatases by using string and cytoscape. the locus id of the abbreviations in could be seen in additional file 4: table s4 discussion in this study, a quantitative, ms-based, label-free proteomic analysis identified non-redundant phosphopeptides from phosphoproteins of rice at both 0 h and 24 h after xoo infection including differentially phosphorylated proteins, representing the first phosphoproteomic attempt to explore the phosphorylation events in rice-pathogen cross-talk. phosphorylation-dependent signaling through the phosphorylation/dephosphorylation of kinase cascade controlled by kinase or phosphatase, the signals of pathogen infection stimuli could be transmitted to the nucleus, where disease resistant-related proteins will be directly or indirectly phosphorylated or dephosphorylated to initiate the immune response. phytohormone abscisic acid signaling is well-known for its roles in response to abiotic stress as well as to biotic stress. a new “pyr/pyl/rcar -pp2c -snrk2”cascade model for aba signaling has been proposed and validated, in which the soluble pyr/pyl/rcar receptors function at the apex of a negative regulatory pathway to directly regulate pp2c phosphatases, which in turn directly regulate snrk kinases. in rice, there are at least pp2cs have been identified. intriguingly, four pp2c proteins were found differentially phosphorylated in our result, among which ospp2c and ospp2c were up-phosphorylated by xoo infection. as negative regulators, pp2cs competitively bind with aba receptors to relieve the inhibition on snrks under stress conditions. the up-phosphorylation of ospp2c and ospp2c probably promote the binding to aba receptors, thus to trigger the aba-dependent signaling in rice defense. this hypothesis is supported by similar results from wheat, in which two pp2cs were up-phosphorylated by drought stress.table some examples of the differentially phosphorylated proteins identified in this study cdpks are directly activated by the binding of ca2+ to the calmodulin-like domain, and activated cdpks regulate downstream components of calcium signaling. in our result, totally cdpks were identified with being up-phosphorylated. oscpk is one of the documented down-phosphorylated cdpks in our result. literature showed that oscpk12-ox seedlings had increased sensitivity to abscisic acid and increased susceptibility to blast fungus, probably resulting from the repression of ros production and/or the involvement of oscpk in the aba signaling pathway. the differential phosphorylation pattern of oscpk upon xoo infection suggested that it is involved in response to multiple pathogen attacks besides blast fungus. moreover, in agreement with the previous report, dephosphorylation of oscpk detected in our data probably resulted in an “inactive” status of this negative regulator to eliminate its inhibition effect, thus enhance plant resistance to pathogen attack. in addition to pp2cs and oscpk we totally identified over differentially phosphorylated kinases or phosphatases, like lrr transmembrane protein kinase, map2k etc., suggesting that the signaling of rice-xoo interaction is a very complex event with multiple signaling pathways involvement. rice disease resistant-related proteins among the dp proteins detected in this study, several proteins are functionally related to rice disease resistance. for example, osmapk, a key component in the osrac1-osmapk3/6-rai1-pal1/oswrky rice immunity signaling cascade, was down-phosphorylated at 24 h. previous studies have revealed that osrac is a key regulator involved in basal resistance by inducing the ros production or suppressing the ros scavenging. osrac could physically bind to osmapk and post-translationally activate osmapk meanwhile, osmapk could directly phosphorylate rai a putative basic helix–loop–helix transcription factor, the overexpression of which substantially enhanced the rice resistance to blast fungus, probably via regulating pal and oswrky. though it has been clear that osmapk acts as a carrier transmitting the phosphorylation from osrac1to rai in this defense signaling cascade, how osmapk is phosphorylated remains unknown. our phosphoproteomic data indicated that the threonine and tyrosine are two potential phosphosites in osmapk which will be further confirmed by our future study. we also noticed that osmapk was down-phosphorylated at 24 h, although osmapk is reported to be a positive regulator in the plant immunity response. lieberherr et al. found that the mrna expression of osmapk started to decrease at 24 h after sphingolipid elicitor treatment, indicating that osmapk may be involved in the early response to pathogen infection. checking of the phosphorylation intensity of osmapk at an earlier time point, like or 4 h after xoo infection may be necessary to explore its functions in the future. another differentially phosphorylated protein gene example is rice yellow mottle virus resistance a recessive gene controlling rice resistance to rice yellow mottle virus. according to our data, rymv was dephosphorylated in response to the xoo infection, suggesting that rymv may play a negative role in bacterial disease resistance. albar et al. cloned this gene from rice variety giganta through a map-based strategy, and found rymv is an isoform of the eukaryotic translation initiation factor 4g 4g). compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. our evidences indicated that rymv might be subject to the activation of phosphorylation upon the xoo infection. however, whether rymv mediates resistance to rice bacterial blight or not needs to be further studied by genetic analysis and pathogen inoculation assay. in addition to ossgt and rymv differential phosphorylation occurred on many other potential disease resistance-related proteins, like osmyc which is involved in the jasmonic acid mediated signaling, oscatc and pub which mediate the process of h2o2-induced cell death in rice. furthermore, some abiotic stress related proteins, such as osapx and oshsp were also differentially phosphorylated. these proteins have proven to be involved in rice drought, cold or heat resistance, but their biological function in biotic stress remains to be explored. transcription factors transcription factors serve as important internodes in the disease resistant pathway by linking the mapks signal with the downstream transcriptional reprogramming. overexpression of osbwmk a rice mapk kinase, conferred plants enhanced pathogen resistance via directly phosphorylating transcription factors oserebp and oswrky. similar cases were reported in other plant species as well. in our study, at least transcription factors were differentially phosphorylated, including down-phosphorylated and up-phosphorylated, according to the rice transcription factor list released from drtf database. these tfs consist of bzip, bhlh, myb and wrky family members. intriguingly, rf2a, a bzip domain containing tf was specifically phosphorylated after xoo infecton. dai et al. found that overexpression of rf2a could repress the symptoms of rice tungro disease without having any growth penalty. they suggested that rf2a together with rf2b suppress the rtbv replication via directly binding to its cis element box ii in the promoter. as indicated in our qrt-pcr results, xoo infection did not alter the mrna abundance of rf2a, but changed the protein status from unphosphorylated to phosphorylated, implying that rf2a plays essential roles in bacterial blight resistance, and phosphorylation might be an important step for the activation of pre-existing rf2a. in addition to rf2a, many other tfs such as wrky, myb and bhlh were found to be differentially phosphorylated, which suggested that they are good candidate genes for rice bacterial blight resistance. epi-genetic controlling factors recent studies demonstrated that chromatin remodeling accomplished through histone modifications is emerging as a key process in the orchestration of plant biotic stress responses and epi-genetic controlling factors are the critical regulators of plant defense to pathogen attack. not surprisingly, many epi-genetic controlling factors were differentially phosphorylated in this study. hdt701/oshdt1encoding a histone deacetylase plays versatile roles in plant development and stress response. li et al. reported that hdt701/oshdt1expression displayed a circadian rhythm. elevated oshdt expression imposed no effects on plant growth in the parent but led to early flowering in the hybrid. it was suggested that hdt701/oshdt1may be involved in epigenetic control of parental genome interaction for differential gene expression. besides the control of hybrid flowering, its role in rice innate immunity was unraveled in a recent publication. transcription of hdt could be induced by compatible reaction and repressed by the incompatible reaction after infection by the fungal pathogen magnaporthe oryzae. more importantly, in the rice hdt overexpression lines, the global histone h acetylation level was reduced and plants became more susceptible to the rice pathogens m. oryzae and xoo. silencing of hdt imposed an opposite effect on rice in that the resistance to both m. oryzae and xoo was enhanced. the underlying mechanism could be that hdt physically bind to and modulate the levels of histone h acetylation of pattern recognition receptor and defense-related genes, such as mapk and wrky in our study, we also found that hdt701/oshdt1in a phosphorylated status at 0 h, but the phosphorylation was found to be removed by the xoo infection. given that phosphorylation modification usually activates protein function, the dephosphorylation of hdt701/oshdt upon xoo infection suggested that it is a negative regulator of plant defense, which is highly consistent with the conclusion of ding et al.. however, even though the mask of the biological function of hdt701/oshdt1has been unraveled, how hdt701/oshdt itself is regulated remain unclear. our data provide good hints that phosphorylation/dephosphorylation might be a key switch turning on/off hdt701in response to xoo infection. moreover, this kind of phosphorylation switch overriding the epi-genetic regulation may be a very universal model in the plant disease resistance pathway. in this study, we have identified differentially phosphorylated epi-genetic factor proteins, comprising ago, snf chromatin remodeling complex proteins, histone demethylases, set domain proteins etc., which are functionally related to dna methylation, histone methylation, acetylation and ubiquitination.  in conclusion, and phosphosites on and representative proteins were identified in 0 h and 24 h after xoo infection, respectively. proteins were differentially phosphorylated in response to the xoo infection, including several well-known rice disease resistance related proteins. our data also suggested that phosphorylation/dephosphorylation might be a key switch turning on/off many epi-genetic controlling factors in plant disease resistance pathway. to the best of our knowledge, this is the first report exploring the cross-talk of rice-pathogen from a view of quantitative phosphoproteome. the data obtained in this research will not only provide phosphorylation status and sites information for rice proteins, but also shed new light in studying the roles of phosphorylation in plant disease resistance. 
in particular, we found that phosphorylation/dephosphorylation might be a key switch turning on/off many epi-genetic controlling factors, including hdt in response to xoo infection, suggesting that phosphorylation switch overriding the epi-genetic regulation may be a very universal model in the plant disease resistance pathway. bacterial blight caused by xanthomonas oryzae pv. this research represents a substantial advance in understanding the rice phosphoproteome as well as the mechanism of rice bacterial blight resistance. it has been clear that phosphorylation plays essential roles in plant disease resistance. here, we report the first study on large scale enrichment of phosphopeptides and identification of phosphosites in rice before and 24 h after xoo infection. rice phosphoproteomebacterial blightpost-translational modificationissue-copyright-statement© the author 2015 the phosphosites identified in this study would be a big complementation to our current knowledge in the phosphorylation status and sites of rice proteins. we have successfully identified and phosphosites on and representative proteins in 0 h and 24 h after xoo infection, respectively. a total of differentially phosphorylated proteins, including transcription factors, kinases, epi-genetic controlling factors and many well-known disease resistant proteins, are identified after xoo infection suggesting that they may be functionally relevant to xoo resistance. however, the role of phosphorylation is poorly understood in rice-xoo system.
 rice is a major crop worldwide. bacterial blight caused by xanthomonas oryzae pv. oryzae has become one of the most devastating diseases for rice. it has been clear that phosphorylation plays essential roles in plant disease resistance. however, the role of phosphorylation is poorly understood in rice-xoo system. here, we report the first study on large scale enrichment of phosphopeptides and identification of phosphosites in rice before and 24 h after xoo infection.  we have successfully identified and phosphosites on and representative proteins in 0 h and 24 h after xoo infection, respectively. a total of differentially phosphorylated proteins, including transcription factors, kinases, epi-genetic controlling factors and many well-known disease resistant proteins, are identified after xoo infection suggesting that they may be functionally relevant to xoo resistance. in particular, we found that phosphorylation/dephosphorylation might be a key switch turning on/off many epi-genetic controlling factors, including hdt in response to xoo infection, suggesting that phosphorylation switch overriding the epi-genetic regulation may be a very universal model in the plant disease resistance pathway.  the phosphosites identified in this study would be a big complementation to our current knowledge in the phosphorylation status and sites of rice proteins. this research represents a substantial advance in understanding the rice phosphoproteome as well as the mechanism of rice bacterial blight resistance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords rice phosphoproteomebacterial blightpost-translational modificationissue-copyright-statement© the author 2015 