BACKGROUND
plants have developed survival strategies, including the strengthening of plant cell walls, to protect themselves from continuous abiotic and biotic stresses.
as the first battlefield of the plant-pathogen interaction, plant cell walls, which are mainly composed of various polysaccharides such as cellulose and hemicelluloses, provide tensile strength to plant cells and protect them from biotic invasion  <cit> .
during infection, plant pathogens secrete numerous cell-wall degradation enzymes  such as cellulases , pectinases , and endo-β- <dig> -xylanase to degrade the cell wall and allow the pathogen to enter in the cell.
the majority of these cwdes belong to the glycosyl hydrolase  family, based on similarities in amino acid sequence, catalytic domains, protein folds, and overall architecture  <cit> .
recently, cwdes have attracted interest because of their utility in biotechnological processes, enhancing processes such as bread making  <cit>  and animal feed production  <cit> , as well as their role as pathogenicity factors in plant pathogenic microbes  <cit> .
the microbial gh endoxylanase is an important enzyme in the hydrolysis of xylans, catalyzing the hydrolysis of β- <dig> -glycosidic linkages between the xylofuranosyl units in the xylan main chain in both cereals and hardwoods  <cit> .
comprehensive functional studies based on structural, biochemical, and molecular properties of these enzymes have been reported  <cit> .
at the same time, plants secrete a group of proteinaceous xylanase inhibitors to suppress pathogenic xylanases.
these proteins are thought as “defense molecules” that protect plant cells from attack by pathogenic hydrolytic enzymes  <cit> .
two types of plant xylanase inhibitors have been well studied, i.e., xylanase-inhibiting protein -type inhibitors  <cit>  and triticum aestivum xylanase inhibitor -type inhibitors  <cit> .
biochemical analysis revealed that a wheat xip specifically inhibits the expression of family- <dig> and − <dig> xylanases from aspergillus nidulans and aspergillus niger, respectively  <cit> , while a taxi inhibits the expression of the family- <dig> xylanase  of a. niger and bacillus subtilis <cit> .
recently, three rice xips, including osxip, rice xip, and a putative rice xylanase inhibitor , were found to be differentially expressed during various developmental stages and under stress conditions  <cit> .
among these, osxip was predicted to be a class iii chitinase, based on bioinformatics analysis; however, no chitinase activity was detected for this protein.
instead, the protein showed dosage-dependent xylanase inhibitor activity  <cit> .
in this study, we functionally characterized a chitinase-like protein from rice  that is expressed at both the transcriptional and translational levels during fungal pathogen infection.
the recombinant osclp protein has strong chitinase activity and can dissolve cell walls, leading to the release of cytosolic contents.
this is the first report on taxi-like molecule with chitinase activity.
this work provides new insights into the function of osclp in plant defense mechanisms.
RESULTS
sequence analysis and characterization of osclp
from previous apoplastic secretome analysis of the rice blast fungus interaction , an xip, which was highly expressed upon magnaporthe oryzae  infection, was isolated and identified .
we then obtained a full-length rice xylanase inhibitor like chitinase gene using pcr.
the deduced amino acid sequence of the osclp comprises  <dig> amino acids with an expected molecular mass of  <dig>  kda and a pi of  <dig> .
a homology search of the deduced amino acid sequence of this gene using the genbank database revealed that osclp is homologous to taxi-type xylanase inhibitor .
osclp contains an  <dig> amino acid signal sequence at the n-terminus and a protein-protein interaction site  at the c-terminus .
this protein contains a putative xylanase inhibitor i-like domain between tyr <dig> and leu <dig> and is classified as a putative taxi-type inhibitor.
such inhibitors have similar structures to those of the pepsin-like family of aspartic proteases  <cit> .
however, osclp does not contain the catalytic domain gxdxdxe, which is highly conserved in all class iii chitinases of plants, bacteria, and fungi  <cit> .
a comparison of the deduced amino acid sequence of osclp with that of other taxi-type inhibitors showed that osclp shares  <dig> % identity with wheat taxi80s,  <dig> % with wheat taxi-iv,  <dig> % with wheat taxi-i, and  <dig> % with rye taxi-type xylanase inhibitor.
purification of recombinant osclp
to measure the biochemical activity of osclp, which comprises his-tagged osclp without the signal peptide, we attempted to purify recombinant pqe30::osclp in e. coli using ni2+-affinity resins at various temperatures  and with various final concentrations of iptg .
soluble osclp was obtained when cells were cultured at 18°c with iptg at a final concentration of  <dig>  mm.
the crude and soluble osclp were then separated by  <dig> % sds-page, followed by coomassie brilliant blue  staining .
as shown in figure  <dig>  the protein appeared as one primary band of approximately  <dig> kda on sds-page, which closely matched the calculated mw of  <dig>  kda that was determined after purification of the protein on the ni2+-affinity resins.
the purification of soluble recombinant protein made it possible to investigate the biochemical activity of osclp.
chitinase activity of osclp
bioinformatics analysis revealed that osclp is a putative xip.
to confirm the xylanase inhibitor activity of osclp, we carried out a xylanase inhibition assay using standard fungal endo- <dig> -β-xylanases isolated from t. aviride and a. pullulans.
ten micrograms of purified osclp protein was pipetted onto filter discs, along with  <dig> μmol of endo- <dig> -β-xylanases, on an agar plate containing xylan substrate.
interestingly, osclp did not inhibit xylan degradation, while treatment with xylanase inhibitor  or boiled xylanase had no activity .
previous research has revealed that xip-i and taxi-i can reduce the activity of xylanase, while xip-i did not show chitinolytic activity due to the presence of salt bridges  <cit> .
however, our results show that osclp does not possess xylanase inhibitor activity against endo- <dig> -β-xylanases .
recently, it was reported that an xip identified from coffee, caclxip, plays an important role in the inhibition of asian soybean rust spore germination through the xylanase inhibitor activity, but not the chitinase activity, of this xip  <cit> .
like caclxip, osxip also has xylanase inhibitor activity, although osxip was predicted to be a class iii chitinase  <cit> .
in contrast to caclxip and osxip, which lack chitinase activity, we wanted to confirm that osclp has chitinase activity by performing an in-gel chitinase activity assay.
in this procedure, crude and purified osclp induced by iptg in e. coli, as well as a control with iptg, were separated by  <dig> % sds-page in a gel containing glycol chitin .
as shown in figure 2b, strong chitinase activity, in the position in which osclp was expressed , was detected under a uv filter.
this suggests that the osclp protein displays strong chitinase activity.
however, further functional analysis of osclp will be required, using biochemical and structural analysis approaches, to understand the different roles played by osclp and taxi-type inhibitors.
transcriptional and translational levels of osclp after fungal infection
in previous studies, taxi-type xylanase inhibitor genes were found to be induced by pathogens in wheat  <cit> .
to determine more accurately the induction level of osclp in response to infection by rice blast fungus , we evaluated the transcriptional and translational levels of this gene using semi-quantitative reverse transcription pcr  and western blot analysis.
rt-pcr analysis revealed that the level of osclp transcript rapidly increased within  <dig> h of fungal inoculation in the incompatible  interaction, which displayed few lesion-type symptoms on rice leaves compared to the control and compatible  interactions .
at the protein level, osclp was shown to be highly and rapidly accumulated in the incompatible interaction at  <dig> h, while the level of osclp slowly increased in the compatible interaction at  <dig> h .
these results suggest that osclp plays an important role in plant defense response.
antifungal activity of osclp
the antifungal activity of osclp protein was evaluated in the presence of a pathogen.
purified osclp protein is unstable to maintain inhibitory activity against slow grower m. oryzae.
we therefore selected rhizoctonia solani, which grows more quickly than m. oryzae, to analyze the biochemical activity of osclp.
in this experiment, we applied  <dig>   <dig>  or  <dig> μg of purified osclp protein to r. solani.
treatment with  <dig> μg bsa  or  <dig> μg/ml osclp protein showed little inhibitory activity on the growth of r. solani .
when treated with  <dig> or  <dig> μg osclp protein, however, the growth of r. solani was inhibited .
therefore, osclp displays antifungal activity in a dose-dependent manner.
a close-up view of fungal hyphae after treatment with osclp protein was observed through a light microscope to investigate how osclp protein suppresses r. solani growth.
the release of cytosolic contents occurred within  <dig> minutes of osclp treatment .
these data suggest that osclp might exhibit strong biochemical activity to dissolve fungal cell walls.
CONCLUSIONS
previous studies have suggested that xips are associated with defense, as they play a significant role in protecting host plants from pathogen attack  <cit> .
the amount of osclp released in response to pathogen infection increased at both the transcriptional and translational levels during incompatible interactions, suggesting that osclp is associated with plant defense mechanisms.
however, in strong contrast to taxi-i and xip-i type inhibitors, recombinant osclp protein did not exhibit xylanase inhibitor activity against endo- <dig> -β-xylanases secreted from other fungi.
instead, significant chitinase activity was detected in our experiment.
this xylanase inhibitor-like chitinase protein inhibited the growth of the fungal pathogen r. solani.
fungal cell wall structures were dissolved in the presence of osclp.
we therefore suggest a new model for osclp activity: osclp expression is rapidly induced, and this protein is secreted from the host cell, in response to pathogen attack.
the protein then attacks the pathogen by degrading the pathogen’s cell wall .

 plants have developed survival strategies, including the strengthening of plant cell walls, to protect themselves from continuous abiotic and biotic stresses. as the first battlefield of the plant-pathogen interaction, plant cell walls, which are mainly composed of various polysaccharides such as cellulose and hemicelluloses, provide tensile strength to plant cells and protect them from biotic invasion. during infection, plant pathogens secrete numerous cell-wall degradation enzymes such as cellulases, pectinases, and endo-β- -xylanase to degrade the cell wall and allow the pathogen to enter in the cell. the majority of these cwdes belong to the glycosyl hydrolase family, based on similarities in amino acid sequence, catalytic domains, protein folds, and overall architecture. recently, cwdes have attracted interest because of their utility in biotechnological processes, enhancing processes such as bread making and animal feed production, as well as their role as pathogenicity factors in plant pathogenic microbes. the microbial gh endoxylanase is an important enzyme in the hydrolysis of xylans, catalyzing the hydrolysis of β- -glycosidic linkages between the xylofuranosyl units in the xylan main chain in both cereals and hardwoods. comprehensive functional studies based on structural, biochemical, and molecular properties of these enzymes have been reported. at the same time, plants secrete a group of proteinaceous xylanase inhibitors to suppress pathogenic xylanases. these proteins are thought as “defense molecules” that protect plant cells from attack by pathogenic hydrolytic enzymes. two types of plant xylanase inhibitors have been well studied, i.e., xylanase-inhibiting protein -type inhibitors and triticum aestivum xylanase inhibitor -type inhibitors. biochemical analysis revealed that a wheat xip specifically inhibits the expression of family- and − xylanases from aspergillus nidulans and aspergillus niger, respectively, while a taxi inhibits the expression of the family- xylanase of a. niger and bacillus subtilis. recently, three rice xips, including osxip, rice xip, and a putative rice xylanase inhibitor, were found to be differentially expressed during various developmental stages and under stress conditions. among these, osxip was predicted to be a class iii chitinase, based on bioinformatics analysis; however, no chitinase activity was detected for this protein. instead, the protein showed dosage-dependent xylanase inhibitor activity. in this study, we functionally characterized a chitinase-like protein from rice that is expressed at both the transcriptional and translational levels during fungal pathogen infection. the recombinant osclp protein has strong chitinase activity and can dissolve cell walls, leading to the release of cytosolic contents. this is the first report on taxi-like molecule with chitinase activity. this work provides new insights into the function of osclp in plant defense mechanisms.  sequence analysis and characterization of osclp from previous apoplastic secretome analysis of the rice blast fungus interaction, an xip, which was highly expressed upon magnaporthe oryzae infection, was isolated and identified. we then obtained a full-length rice xylanase inhibitor like chitinase gene using pcr. the deduced amino acid sequence of the osclp comprises amino acids with an expected molecular mass of kda and a pi of. a homology search of the deduced amino acid sequence of this gene using the genbank database revealed that osclp is homologous to taxi-type xylanase inhibitor. osclp contains an amino acid signal sequence at the n-terminus and a protein-protein interaction site at the c-terminus. this protein contains a putative xylanase inhibitor i-like domain between tyr and leu and is classified as a putative taxi-type inhibitor. such inhibitors have similar structures to those of the pepsin-like family of aspartic proteases. however, osclp does not contain the catalytic domain gxdxdxe, which is highly conserved in all class iii chitinases of plants, bacteria, and fungi. a comparison of the deduced amino acid sequence of osclp with that of other taxi-type inhibitors showed that osclp shares % identity with wheat taxi80s, % with wheat taxi-iv, % with wheat taxi-i, and % with rye taxi-type xylanase inhibitor. purification of recombinant osclp to measure the biochemical activity of osclp, which comprises his-tagged osclp without the signal peptide, we attempted to purify recombinant pqe30::osclp in e. coli using ni2+-affinity resins at various temperatures and with various final concentrations of iptg. soluble osclp was obtained when cells were cultured at 18°c with iptg at a final concentration of mm. the crude and soluble osclp were then separated by % sds-page, followed by coomassie brilliant blue staining. as shown in figure the protein appeared as one primary band of approximately kda on sds-page, which closely matched the calculated mw of kda that was determined after purification of the protein on the ni2+-affinity resins. the purification of soluble recombinant protein made it possible to investigate the biochemical activity of osclp. chitinase activity of osclp bioinformatics analysis revealed that osclp is a putative xip. to confirm the xylanase inhibitor activity of osclp, we carried out a xylanase inhibition assay using standard fungal endo- -β-xylanases isolated from t. aviride and a. pullulans. ten micrograms of purified osclp protein was pipetted onto filter discs, along with μmol of endo- -β-xylanases, on an agar plate containing xylan substrate. interestingly, osclp did not inhibit xylan degradation, while treatment with xylanase inhibitor or boiled xylanase had no activity. previous research has revealed that xip-i and taxi-i can reduce the activity of xylanase, while xip-i did not show chitinolytic activity due to the presence of salt bridges. however, our results show that osclp does not possess xylanase inhibitor activity against endo- -β-xylanases. recently, it was reported that an xip identified from coffee, caclxip, plays an important role in the inhibition of asian soybean rust spore germination through the xylanase inhibitor activity, but not the chitinase activity, of this xip. like caclxip, osxip also has xylanase inhibitor activity, although osxip was predicted to be a class iii chitinase. in contrast to caclxip and osxip, which lack chitinase activity, we wanted to confirm that osclp has chitinase activity by performing an in-gel chitinase activity assay. in this procedure, crude and purified osclp induced by iptg in e. coli, as well as a control with iptg, were separated by % sds-page in a gel containing glycol chitin. as shown in figure 2b, strong chitinase activity, in the position in which osclp was expressed, was detected under a uv filter. this suggests that the osclp protein displays strong chitinase activity. however, further functional analysis of osclp will be required, using biochemical and structural analysis approaches, to understand the different roles played by osclp and taxi-type inhibitors. transcriptional and translational levels of osclp after fungal infection in previous studies, taxi-type xylanase inhibitor genes were found to be induced by pathogens in wheat. to determine more accurately the induction level of osclp in response to infection by rice blast fungus, we evaluated the transcriptional and translational levels of this gene using semi-quantitative reverse transcription pcr and western blot analysis. rt-pcr analysis revealed that the level of osclp transcript rapidly increased within h of fungal inoculation in the incompatible interaction, which displayed few lesion-type symptoms on rice leaves compared to the control and compatible interactions. at the protein level, osclp was shown to be highly and rapidly accumulated in the incompatible interaction at h, while the level of osclp slowly increased in the compatible interaction at h. these results suggest that osclp plays an important role in plant defense response. antifungal activity of osclp the antifungal activity of osclp protein was evaluated in the presence of a pathogen. purified osclp protein is unstable to maintain inhibitory activity against slow grower m. oryzae. we therefore selected rhizoctonia solani, which grows more quickly than m. oryzae, to analyze the biochemical activity of osclp. in this experiment, we applied or μg of purified osclp protein to r. solani. treatment with μg bsa or μg/ml osclp protein showed little inhibitory activity on the growth of r. solani. when treated with or μg osclp protein, however, the growth of r. solani was inhibited. therefore, osclp displays antifungal activity in a dose-dependent manner. a close-up view of fungal hyphae after treatment with osclp protein was observed through a light microscope to investigate how osclp protein suppresses r. solani growth. the release of cytosolic contents occurred within minutes of osclp treatment. these data suggest that osclp might exhibit strong biochemical activity to dissolve fungal cell walls.  previous studies have suggested that xips are associated with defense, as they play a significant role in protecting host plants from pathogen attack. the amount of osclp released in response to pathogen infection increased at both the transcriptional and translational levels during incompatible interactions, suggesting that osclp is associated with plant defense mechanisms. however, in strong contrast to taxi-i and xip-i type inhibitors, recombinant osclp protein did not exhibit xylanase inhibitor activity against endo- -β-xylanases secreted from other fungi. instead, significant chitinase activity was detected in our experiment. this xylanase inhibitor-like chitinase protein inhibited the growth of the fungal pathogen r. solani. fungal cell wall structures were dissolved in the presence of osclp. we therefore suggest a new model for osclp activity: osclp expression is rapidly induced, and this protein is secreted from the host cell, in response to pathogen attack. the protein then attacks the pathogen by degrading the pathogen’s cell wall. 
these data suggest that osclp, which has chitinase activity, may play an important role in plant defenses against pathogens. the expression of this gene was examined at the transcriptional/translational levels during compatible and incompatible interactions, and the biochemical activity of this protein was also examined. however, recombinant osclp did not display inhibitory activity against endo- -β-xylanase enzymes from aureobasidium pullulans or trichoderma viride. the transcription and translation of osclp were highly induced when rice was exposed to pathogens in an incompatible interaction. taken together, our results demonstrate that osclp may have antifungal activity. instead, an in-gel activity assay revealed strong chitinase activity. sequence alignment revealed that the deduced amino acid sequence of osclp shares a high degree of similarity with that of other plant taxi-type xips. this protein may directly inhibit pathogen growth by degrading fungal cell wall components through chitinase activity. during rice blast fungal attack, plant xylanase inhibitor proteins that inhibit fungal xylanase activity are believed to act as a defensive barrier against fungal pathogens. to understand the role of xips in rice, a xylanase inhibitor was cloned from rice.
 during rice blast fungal attack, plant xylanase inhibitor proteins that inhibit fungal xylanase activity are believed to act as a defensive barrier against fungal pathogens. to understand the role of xips in rice, a xylanase inhibitor was cloned from rice. the expression of this gene was examined at the transcriptional/translational levels during compatible and incompatible interactions, and the biochemical activity of this protein was also examined.  sequence alignment revealed that the deduced amino acid sequence of osclp shares a high degree of similarity with that of other plant taxi-type xips. however, recombinant osclp did not display inhibitory activity against endo- -β-xylanase enzymes from aureobasidium pullulans or trichoderma viride. instead, an in-gel activity assay revealed strong chitinase activity. the transcription and translation of osclp were highly induced when rice was exposed to pathogens in an incompatible interaction. in addition, exogenous treatment with osclp affected the growth of the basidiomycete fungus rhizoctonia solani through degradation of the hyphal cell wall. these data suggest that osclp, which has chitinase activity, may play an important role in plant defenses against pathogens.  taken together, our results demonstrate that osclp may have antifungal activity. this protein may directly inhibit pathogen growth by degrading fungal cell wall components through chitinase activity. antifungal activitychitinaseoryza sativaxylanase inhibitor 