Introduction

Chitinases (CHIs) (EC 3.2.1.14) are lytic enzymes which catalyze the degradation of chitin (Henrissat 1991), a major component of cell walls of bacteria and fungi (Henrissat 1991; Chen et al. 2018; Mir et al. 2019). CHIs belong to a large gene family, and exist in microorganisms, plants and animals (Mishra et al. 2015; Xi et al. 2015; Xu et al. 2016; Chen et al. 2018; Filyushin et al. 2019). Based on the sequence similarity of the catalytic domains, CHIs have been classified into either the Glycosyl hydrolase 18 family (GH18) or GH19 (Henrissat 1991). The GH18 genes are found in various organisms, such as microorganisms, plants and animals, while the GH19 genes exist almost in plants (Jiang et al. 2013; Mir et al. 2019). According to the presence or absence of an N-terminal hevein domain and sequence similarity with an archetypal catalytic domain, traditionally plant CHIs are categorized into six classes (Neuhaus et al. 1996; Levorson and Chlan 1997). However, CHI members in each same class exhibit distinct enzyme activity, strongly suggesting that some other parts of CHI sequence also contribute to enzyme activity and thus should be considered in the classification of CHI gene families (Levorson and Chlan 1997; Sasaki et al. 2006).

Plant CHIs belong to a subgroup of pathogenesis-related proteins (PRs), and it is believed that plant CHIs can directly attack chitin from invading pathogens (Hamid et al. 2013; Chen et al. 2018). Also, the chitin fragments produced by plant CHIs can act as elicitors to activate defense responses in interactions of plant with various pathogens (Xu et al. 2016). The defense roles of some plant CHIs have been supported by many studies. For example, in some plant CHIs are up-regulated in response to infection with pathogenic bacteria and fungi (Mir et al. 2019). Further, some CHIs can confer resistance to plant disease. For example, PnCHI1, a CHI from Panax notoginseng, can confer tobacco resistance to F. solani (Bai et al. 2018). Overexpression of NtPR-Q, a member of the PR3 family encoding chitinases, leads to enhanced resistance to Ralstonia solanacearum in Nicotiana tabacum (Tang et al. 2017). Dong et al. (2017) cloned a new CHI EuCHIT2 from Eucommia ulmoides Oliver and found that expression of the CHI confers resistance to Erysiphe cichoracearum DC in tobacco plants. Thus, these data show that plant CHIs play an important role in defense against pathogen infections, and it is important to understand roles of this gene family in defense.

CHI gene families have been widely studied in various plant species. However, for rice (Oryza sativa ssp. japonica), the important monocotyledonous model plant, a genome-wide overview of the CHI gene family members is not yet available. In fact, in earlier work, CHI gene family of this model plant has been investigated (Xu et al. 2007). However, the data in this work were provided with limited information for CHIs. For example, what was identified for CHIs in the work is their open reading frames (ORFs), consequently being lack of the important information about gene structure as well as chromosomal distribution of CHIs, and these ORFs is not available because their nomenclature was so out-of-date that these ORFs could be not retrieved from currently available databases, such as RAP, RGAP (the Rice Genome Annotation Project Database) and NCBI. Furthermore, gene structure, motif and duplication analysis of CHIs was not performed in O. sativa CHIs (OsCHIs) as well as A. thaliana CHIs (AtCHIs). In this study, CHIs from rice have been identified and detailed analysis, including gene structure, conserved motifs, gene chromosome location, gene birth and expression profiling, were performed on OsCHIs and AtCHIs. This genome wide analysis provides the framework for future studies to dissect functions of these genes.

Materials and Methods

Database screening and identification of OsCHIs and AtCHIs

A search for A. thalinan CHIs was performed using keyword ‘CHITINASE’ in The Arabidopsis Information Resource (TAIR) database (https://www.arabidopsis.org/), and 26 CHI genes of A. thaliana were acquired. To identify CHI genes in rice, sequences of 26 A. thaliana CHI proteins were used in BLAST search of the Rice Annotation Project (RAP) database. Meanwhile, we also used keyword ‘CHITINASE’ to search for CHIs in the RAP database. After removing the redundant sequences, we used the NCBI-CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) database to investigate the conserved domain of remaining sequences (Marchler-Bauer et al. 2017). These protein sequences were analyzed in the SMART (https://smart.embl-heidelberg.de/) and Pfam (https://pfam.xfam.org/) databases to confirm the presence of CHI domain. Those proteins containing CHI domain (GH18 domain or GH19 domain) were defined to belong to the CHI family. GH18 domains include cd02876, cd02877, cd02879, smart00636, cd06544, cl10447 and pfam00704, and GH19 domains include cd00325, pfam00182 and cd06921. Protein subcellular localization was predicted by using WoLF PSORT program (https://wolfpsort.org) (Horton et al. 2007). The pI (isoelectric point), molecular mass and GRAVY (grand average hydropathy) values were determined by using the ExPASy-ProtParam tool (https://web.expasy.org/protparam/).

Protein sequence alignment and phylogenetic analysis

The ClustalX program was used to perform the multiple sequence alignments of protein sequences, and the alignments were corrected manually. The neighbor-joining method was used to construct the unrooted phylogenetic tree. Bootstrap analyses were performed using 1000 replicates. The phylogenetic trees were displayed using MEGA software version 6.0 (Tamura et al. 2013).

Gene structure of the CHIs and conserved motifs analyses

Gene structure information of CHIs was collected from the annotations from NCBI. The motifs in these CHI protein sequences were identified by using the program MEME (https://meme-suite.org/index.html) (Bailey et al. 2006).

Chromosomal localization and gene duplication

BLASTN was used to position the CHIs on the rice or Arabidopsis chromosomes. Based on close phylogenetic relationships, tandem duplications of CHIs tandemly arrayed at the same chromosomal location were identified. Segmental duplicates were recognized through comparing positions of CHIs in duplicated chromosomal blocks previously identified in the Arabidopsis genomes (https://bioinformatics.psb.ugent.be/beg/research/genome-duplication-polyploidy) or rice genomes (Blanc et al. 2003; Lin et al. 2006).

EST profiling and microarray analysis

For rice, the microarray data from GEO (Gene Expression Omnibus ) database under the accession numbers GSE7256 (infection by Magnaporthe grisea) (Ribot et al. 2008) and GSE10373 (interaction with the parasitic plant Striga hermonthica) (Swarbrick et al. 2008) were used for expression analysis of OsCHIs. For Arabidopsis, the microarray data for various pathogen treatments were downloaded from GEO (series accession numbers GSE5684, GSE5685 and GSE5686). In addition, for genes with more than one probe sets, the median values represented their expression values. The genes which are up- or down-regulated more than 2-fold were considered to be differentially expressed significantly. Finally, the expression pattern images were based on the average log signal values and generated using the Genesis program (Sturn et al. 2002).

Results

Identification and phylogenetic analysis of CHIs in rice and Arabidopsis

A total of 48 OsCHIs were identified in the rice genome, according to multiple searches followed by confirming as encoding CHI proteins (Table 1). In these OsCHI proteins, there were 18 Xylanase inhibitor proteins (XIPs) which belong to GH18 protein because they contain typical GH18 domains and have sequence similarity to GH18. 

Table 1: Chtinase genes identified in rice

¹PL means Protein Length; ²GRAVY means Grand average of hydropathicity; ³ PSORT predictions: E (extracellular), P (plasma membrane), V (vacuolar membrane), CY (cytosol), C (chloroplast), N (nuclear), E.R. (endoplasmic reticulum), M (mitochondrion) and G (Golgi apparatus), Cysk (eytoskeleton)

In A. thaliana, 24 AtCHI genes have been annotated previously. Here, from TAIR database (https://www.arabidopsis.org/) we found 26 AtCHIs (Table 2). The extra two AtCHIs are At4g01040 and At3g47540-2, an alternative form of At3g47540.

Most of these CHIs encode hydrophobic polypeptides (<0) ranged from 125 (Os11g0701600) to 430 (AT4G01040) amino acids residues, with pI values ranged from 4.41 to 12.59. Subcellular location prediction showed that most CHIs identified in this study are localized in the extracellular or Chloroplast. In addition, many CHIs were predicted to be localized to other organelles such as the mitochondrial, plasma or vacuolar membranes, nucleus, golgi apparatus or cytoplasmic. These subcellular localization predictions suggested that the CHIs would function in various aspects.

Phylogenetic analysis showed that all of OsCHIs and AtCHIs were divided into two distinct clades: the GH18 and GH19. Further, according to phylogenetic relationships, the GH18 class was divided into 5 subclasses designated as Groups 1–5, respectively. For the GH19 class, phylogenetic analysis revealed three subclasses designated as Groups 6–8 (Fig. 1a). Remarkably, Groups 1 and 3 do not include any AtCHIs, suggesting these were acquired for monocotyledonous rice but not for dicotyledonous Arabidopsis. Groups 2 and 7 mainly consist of OsCHIs and only one and three AtCHIs are, respectively, in the two groups, which suggested that these ATCHIs were actually clustered into about 5 groups. In contrast, Group 8, consisting of 13 members, ten of which are AtCHIs, and the other three are OsCHIs. Additionally, Group 4 consists of two CHIs, one from rice and another from Arabidopsis. Group 1 constitutes the largest clades in the CHI phylogeny, containing 21 members. These data indicated that some CHI groups could to some extent be specific for rice and others for Arabidopsis, suggesting that some CHIs have specialized roles in monocotyledons while others in dicotyledons.

Gene structure, conserved domains and motifs of the CHI family genes in rice and Arabidopsis

Table 2: Chitinase genes identified in Arabidopsis

*Notes of PL, GRAVY and PSORT are shown in Table 1

In order to understand the structural diversity of the CHIs, gene structure of each CHI was investigated. Firstly, we compared the exon/intron structure of each CHI and found that most members within the same groups shared very similar exon/intron structure on either intron numbers or exon lengths (Fig. 1b). Further, it was observed that 54% of CHIs in rice are intron less. For example, all but two members of Group 1 and all of Group 3 are intron less. In contrast, none of CHIs in Arabidopsis are intron less, although 75% of these genes only consist of one intron. These observations indicated strikingly distinct CHI gene structural patterns between monocotyledonous rice and dicotyledonous Arabidopsis.

Fig. 1: Phylogenetic relationships, gene structure and motif composition of CHIs in Arabidopsis (At) and rice (Os). (a) The molecular phylogeny (left panel) was constructed by neighbor-joining method. The number at the nodes represent the bootstrap values (>50%) from 1000 replicates. The 8 major groups designated from 1 to 8 are marked with different color backgrounds. (b) The gene structure (5′-UTR/exon/intron/3′-UTR organization) of the CHIs is shown in the middle panel. Light green boxes represent 5′-UTR or 3′-UTR, dark green boxes represent exons and green lines represent introns. And their length in base pairs is also indicated, respectively. Numbers between brackets correspond to the intron phase. (c) A schematic representation of conserved motifs (obtained by MEME) in CHIs is displayed in the panel on the right. Different motifs are diaplayed by different colored boxes

Intron phase was used to assess gene models of OsCHIs and AtCHIs. Introns phase means the position of an intron within a codon and assigned to three different phase classes: phase 0 (before the first base), phase 1 (after the first base) and phase 2 (after the second base). As shown in Fig. 1b, the majority of introns are within phase 1, for OsCHIs (54%) and AtCHIs (65%), while 29% and 25% of introns found in OsCHIs and AtCHIs, respectively, are within phase 2. Phase 0 introns represent only 18% of all OsCHIs introns and only 10% of all AtCHIs introns. Interestingly, most members of each group shared the same or similar intron phase. Further, it was found that within several pairs of putative paralogous genes (At1g05850/At3g16920 in Group 6, Os09g0494200/Os08g0522500 in Group 6, and Os10g0542900/Os10g050543400 in Group 7), not only is phase of two adjacent introns shared, but length of exon between the two introns is highly conserved (Fig. 1b), indicating a close evolutionary relationship of these paralogous genes. In addition, it was observed that there are two alternative mRNA forms in the locus At3g47540 wherein the 3’ end sequences were recruited to generate a new intron, this results in birth of two genes, named At3g47540-1 and At3g47540-2, from the locus.

Fig. 2: Evolution of the Oryza CHIs (OsCHIs). (a) Chromosomal locations. (b) Phylogenetic relationships of the GH18 OsCHIs. The letters T and S on the nodes of the phylogenetic tree indicate the positions where tandem duplication and segmental duplication have occurred, respectively. (c) Hypothetical origins of 21 OsCHIs by tandem duplication and, most likely, retro position. (d) Phylogenetic relationships of the GH19 OsCHIs

As CHI domain, GH18 or GH19 domain is essential for the chitin hydrolysis. The results showed that these identified CHIs possess only 1–3 CHI domain (s). In addition, some CHIs contain other domains, such as pfam00187 and cl15255. The domain pfam00187 is characteristic of chitin recognition protein, and cl15255 is Src homology2 (SH2) domain, a protein domain that plays important roles in the signal transduction of receptor tyrosine kinase pathways. To define more divergent patterns in the functional domain, the program MEME was used to examine smaller individual motifs in these CHI sequences. Thirty distinct motifs were identified in these CHI sequences. As shown in Fig. 1c, most members of GH18 class (Group 1–5) possess 19 motifs, while most members of GH19 class (Group 6–8) have 13 motifs. Four same motifs are shared by most of GH19 proteins, while different groups of the GH18 class share specifically different motifs. Importantly, most members in each group have common motif compositions, strongly supporting group-dividing results of the phylogenetic analyses. The motif composition conservation among members of the same groups indicated that members in the same group may be functionally conserved.

Genomic organization and expansion of the CHI gene family

First the genomic distribution of the OsCHIs and AtCHIs was examined and observed that the distribution of these CHIs is uneven throughout chromosomes of the Arabidopsis or rice genomes. As shown in Fig. 2a, for rice, chromosome 12 harbors no CHIs, whereas chromosome 11 harbors 12 CHIs, and each of chromosomes 1 and 5 harbors eight CHIs. Other chromosomes have 1–5 CHIs localized on them. For Arabidopsis, chromosome 5 harbors only one CHI gene, whereas chromosome 4 harbors 11 CHIs. Five CHIs were identified on each of chromosomes 2 and 3, and three on chromosomes 1 (Fig. 3a). Further, it was observed that there are some CHIs clusters at rice or Arabidopsis chromosomes (Fig. 2a and 3a), suggesting that these CHIs in the same clusters may be tandemly duplicated genes.

To gain insights into gene duplication in CHIs genes, separate phylogenetic trees were constructed exclusively using the full-length CHI sequences of rice and Arabidopsis (Fig. 2b and Fig. 3b). In rice, the obvious tandem repeats were Os01g0687400/Os01g0691000 and Os01g0860400/Os01g0860500 on chromosome 1 (Fig. 2a and 2b), Os04g0493400/Os04g0494100 on chromosome 4 (Fig. 2a and 2d), Os10g0542900/Os10g0543400 on chromosome 10 (Fig. 2a and 2d), Os10g0416100/Os10g0416500/Os10g0416800 (Fig. 2a, 2b and 2c) and Os05g0247100/Os05g0246100/Os05g0247800/Os05g0248200 on chromosome 5 (Fig. 2a and 2c). These clustered genes were generated by recent tandem duplication because terminal clades generated by them were well-supported in phylogenetic tree, respectively, and did not contain any CHIs on other chromosomes (Fig. 2b and 2d). Another example that may be the result of tandem duplication is Os05g0399300/Os05g0399400/05g0399700 (Fig. 2a and 2d). The largest CHI gene cluster, located on chromosome 11, contains 11 tandemly arrayed members (Fig. 2a and 2c), and another cluster on chromosome 8 contains 3 tandemly arrayed members (Fig. 2a, 2b and 2c). Further, it was observed that the terminal clades containing the two clusters also contain CHIs located at other chromosomes (Fig. 2b and 2c), respectively, suggesting that the two clusters of 

Fig. 3: Evolution of the Arabidopsis CHIs (AtCHIs). (a) Chromosomal locations, (b) phylogenetic relationships of the GH18 AtCHIs, (c) hypothetical origins of nine AtCHIs by tandem duplication, (d) phylogenetic relationships of the GH19 AtCHIs, and (e) hypothetical origins of six AtCHIs by tandem duplication

genes were generated by more ancient tandem duplication. Interestingly, it was observed that the Os05g0247100/Os05g0247500/Os05g0247800/Os05g0248200 cluster may be formed by a single tandem duplication of a two-gene cluster (Fig. 2c).

For Arabidopsis, the largest CHI gene cluster is on chromosome 4 and contains nine tandemly arrayed genes including all CHIs identified on the chromosome but At4g01040 and At4g01700 (Fig. 3a). The clustered genes were also generated by recent tandem duplication because the terminal clade generated by them is a well-supported in phylogenetic tree and not contain any other chromosome CHIs (Fig. 3b and 3c). Another CHI gene cluster is located on chromosome 2. The cluster includes all CHIs identified on the chromosome (Fig. 3d and 3e). However, the clustered genes were generated by more ancient tandem duplication, because the terminal clade containing them also include other chromosome CHIs, such as At1g56680 on chromosome 1, At3g54420 and At3g47540 on chromosome 3.

The locations of CHIs were also compared in duplicated chromosomal blocks previously identified in rice and Arabidopsis. It was found in the rice and Arabidopsis genomes for chromosomal segments (or duplicate blocks) that contain CHIs. In rice, one block contains Os01g0287600, while its duplicate block includes Os05g0138200 at the same position (Table 3, Fig. 2d). This suggests that Os01g0287600/Os05g0138200 might be the results of a segmental duplication event. Likewise, Os08g0522500/Os09g0494200 might be the results of another segmental duplication. However, none of other CHI gene-containing blocks has a CHI gene in its duplicate block. Following the same procedures, however, we did not found any segmental duplication occurred in AtCHIs.

These data showed that tandem duplication explains the birth of relatively large proportion of the CHI family genes in rice and Arabidopsis, whereas segmental duplication plays a very limited role in increasing CHI number in the two plants.

Differential expression of CHIs in response to biotic stresses

It has been reported that plant CHIs exhibit basal expression level under normal conditions. So the change of gene expression caused by pathogen infection can provide important hints for the gene function. The hemi bio-trophic fungi Magnaporthe grisea causes severe loss to rice yield. The microarray data was used to analyze the expression response of OsCHIs against this pathogen. The data analysis revealed that only eight OsCHIs were significantly up-regulated (more than 2-fold) at 3 dpi or/and 4 dpi, while none of genes were significantly down-regulated (Fig. 4), suggesting that a limited number of OsCHIs is involved in defense to the pathogen.

Fig. 4: Expression profiles of OsCHIs showing differential expression in response to various biotic stress treatments. The expression profile image is generated based on the fold-change log values in the treated sample when compared with its mock-treated control sample and is displayed according to the order in the corresponding phylogenetic tree. G1-G8 means Group1-Group8 in Fig. 1. The color scale for fold-change values is shown at the bottom

The obligate root hemi-parasite S. hermonthica also cause severe loss to rice yield, and so far there have been no reports investigating roles of plant CHIs in defense to any parasitic plants. Here, we tried to investigate the response of OsCHIs to S. hermonthica by using microarray data from a study in which the gene expression profiling was analysed in roots of susceptible (IAC165) and highly resistant (Nipponbare) cultivars after infection with this parasitic plant (Swarbrick et al. 2008). The results showed that most OsCHI were significantly differential expressed either in the susceptible cultivar or in the highly resistant one as compared to control (Fig. 4), indicating that many OsCHIs play roles in defense to infection by S. hermonthica.

In Arabidopsis, it was investigated that expression patterns of the AtCHIs in response to the obligate bio-trophs oomycte pathogen Erysiphe orontii, necrotrophs fungal pathogens Botrytis cinerea and the bacterial pathogen Pseudomonas syringae, a hemi-biotroph. The most members in the Group 7 and 8 are clearly up-regulated, indicating their roles in defense to these pathogens (Fig. 5). Interestingly, although E. orontii, B. cinerea and P. syringae were three different type of pathogen, expression patterns of these CHIs appear a similar trend in response to the three pathogens. For example, some genes (At2G43590, At2G43580, At3G54420, At2G43570 and so on) in Group 8 were highly expressed in all of the three pathogen infection. Further, it was found that protein products of these CHIs were predicted to be located in the extracellular (Table 2), suggesting that these CHIs may function as secretory proteins to directly attack the pathogens as CHIs can degrade the cell wall of pathogens. Thus, member of Group 7 and 8 may function actually as PR protein and play important roles in defense to pathogen infection. And these CHIs are selected as resistance candidate genes for further studying in future.

Discussion

Table 3: Duplicate blocks in the rice (Oryza sativa ssp. Japonica) genome that contains Chitinase genes (CHIs)

Fig. 5: Expression profiles of AtCHIs showing differential expression in response to various biotic stress treatments. The expression profile image is generated based on the fold-change log values in the treated sample when compared with its mock-treated control sample and is displayed according to the order in the corresponding phylogenetic tree. G1-G8 means Group1-Group8 in Fig. 1. The color scale for fold-change values is shown at the bottom

Plant CHIs belong to large gene family, and some of these play essential roles in plant defense against pathogens, and thus it is important to unravel their function for application on genetic analysis and breeding. To date, genome-wide identification of CHI family has been performed in many plant species. However, for rice, a genome-wide overview of the CHI family members is not yet available. In fact, in an earlier work, 37 ORFs of OsCHIs has been investigated in this model plant (Xu et al. 2007). However, these ORFs cannot be retrieved from currently available databases. In the study, first a genome-wide survey was performed which provided new data that O. sativa, the important monocotyledonous model plant, have 48 CHIs (OsCHIs) in its genome.

Truong et al. (2003) indicated that the rice had at least 7 family of CHIs and only 19 OsCHIs were investigated. In this study, 47 OsCHIs, together with 26 AtCHIs, were clustered into 8 groups, and the group classification was strongly supported by gene structure and motif compositions. In addition, Os06g0726200, being basal to a large clade be generated by Groups 6–8 in the phylogenetic tree (Fig. 1a), was not classified into any groups, which is strongly supported by the motif compositions because its motif compositions are distinctly different from those of any group members (Fig. 1c). The phylogenetic analysis also showed that the 26 ATCHIs were actually clustered into 5 groups (Fig. 1a), which was generally consistent with results of previous research (Passarinho and Vries 2002; Xu et al. 2007). Furthermore, it is noted that more number of CHIs were found in rice compared to Arabidopsis, which may be attributed due to large genome size of rice with 12 chromosomes whereas Arabidopsis has only 5 chromosomes. Moreover, OsCHIs or AtCHIs are unevenly distributed in rice or Arabidopsis chromosomes, respectively, and expansion of the CHI family is caused by tandem duplication, instead of segmental duplication, in genomes of the two model plants, which is similar to those reported in Brassica rapa, B. juncea, Camelina sativa and P. trichocarpa (Jiang et al. 2013; Chen et al. 2018; Mir et al. 2019).

CHI can serve as a defense-related enzyme that inhibits fungal growth due to its function in breaking down chitin. However, present study data showed that most members of OsCHI family exhibit down-regulating expression in response to the fungi M. grisea, suggesting these OsCHIs play limited role in the defense. This was supported by results from a RNA-sequencing analysis. In the analysis, 21 identified DEGs (differentially expressed genes) related to CHI were identified in Dongdao-4, a widely grown Japonica-type rice cultivar, after infection with M. grisea, but only 1 DEG was up-regulated while other 20 DEGs were down-regulated (Tian et al. 2018). Contrastly, most members of OsCHI gene family were up-regulated in response to S. hermonthica, a parasitic plant, indicating roles for these OsCHIs in the defense. The plant CHI has been receiving attention concerning its roles in defense to pathogens as well as insects, and to date there have been no reports investigating the roles of CHIs in interactions of host plants with parasitic plants. Thus, this is an unexpected finding and it is valuable to study furthermore.

For AtCHIs, CHIs in the Group 7 and 8 were highly up-regulated in response to the infection with three pathogens. Among these CHIs, AT3G54420, a Group 8 member, has been reported to be transcriptionally induced by infection with a hemibiotrophic pathogen (Gerhardt et al. 1997). Lin et al. (2008) showed that AT5G24090 and AT3G12500, two other Group 8 CHIs, were induced in Arabidopsis plant leaves by the necrotrophs B. cinerea infection (Lin et al., 2008). Similarly, in present investigation, AT3G12500 was significantly induced by B. cinerea, and At5g24090 was down-regulated in response to the hemibiotrophic pathogen P. syringae but up-regulated 48 h after infection by B. cinerea. In Beta vulgaris plants, one CHI, its protein with 53% identity to At3g54420-encoded protein (NP_191010.1), is up-regulated after the necrotrophic pathogen infection (Nielsen et al. 1994). Another CHI, whose protein sequence have 70% identity to NP_191010.1, is induced in Phaseolus vulgaris roots infected with a hemi-biotrophic pathogen (Lange et al. 1996). Further, data showed that the expression of At3g54420 was remarkably up-regulated in response to all the three types of pathogen. Protein subcellular localization prediction showed that these CHIs in Group 7 and 8, which highly expressed in all of the three pathogen infection, are associated with the secretory pathway. It has been reported that apoplastic CHI proteins, which are induced by pathogen infection, can directly inhibit the pathogen growth in the intercellular space as CHIs can catalyze the degradation of chitin (De et al. 1997). Thus, members of Group 7 and 8 may function actually as PR protein and play important roles in defense to pathogen infection, which offer an insight into the defense role of specific CHIs in the large CHI family. And these CHIs were considered as potential resistance candidate genes, and should be further studied for improving plant resistance.