Identification, Phylogeny, and Transcript of Chitinase Family Genes in Sugarcane

Chitinases are pathogensis-related proteins, which play an important role in plant defense mechanisms. The role of the sugarcane chitinase family genes remains unclear due to the highly heterozygous and aneuploidy chromosome genetic background of sugarcane. Ten differentially expressed chitinase genes (belonging to class I~VII) were obtained from RNA-seq analysis of both incompatible and compatible sugarcane genotypes during Sporisorium scitamineum challenge. Their structural properties and expression patterns were analyzed. Seven chitinases (ScChiI1, ScChiI2, ScChiI3, ScChiIII1, ScChiIII2, ScChiIV1 and ScChiVI1) showed more positive with early response and maintained increased transcripts in the incompatible interaction than those in the compatible one. Three (ScChiII1, ScChiV1 and ScChiVII1) seemed to have no significant difference in expression patterns between incompatible and compatible interactions. The ten chitinases were expressed differentially in response to hormone treatment as well as having distinct tissue specificity. ScChiI1, ScChiIV1 and ScChiVII1 were induced by various abiotic stresses (NaCl, CuCl2, PEG and 4 °C) and their involvement in plant immunity was demonstrated by over-expression in Nicotiana benthamiana. The results suggest that sugarcane chitinase family exhibit differential responses to biotic and abiotic stress, providing new insights into their function.

Phylogenetic analysis of chitinase gene family. Among the 17 differentially expressed chitinase unigenes, a total of 9 members was predicted to have full-length sequences with open reading frames (ORFs). The assembled sequence of ScChiVII1 based on homologous cloning method according to the predicted S. bicolor chitinase gene (XM_002460419.1) was added. To study the phylogenetic relationships of the chitinase family genes in sugarcane, a multiple alignment analysis was performed. The 10 genes with ORF structures were classified into seven types (class I ~ VII) based on the similarity of their amino acid sequences with 21 biotic stress resistance-related chitinases of other plant species from NCBI 17,18 . As shown in Fig. 1, they were segregated into two branches, one comprising classes III and V, and the other one including the classes I, II, IV, VI and VII. The 10 sugarcane chitinase genes were named by classification system of the chitinase in the phylogenetic tree and described as ScChiI1 (gi32815041), ScChiI2 (gi34957207), ScChiI3 (Sugarcane_Unigene_BMK.68059), ScChiII1 (gi35992663), ScChiIII1 Unigene ID Yacheng05-179 log 2 fold change (T/CK) * ROC22 log 2 fold change (T/CK) * BLAST annotation 24 hpi 48 hpi 120 hpi 24  Sequence analysis of chitinase gene family. In order to gain insight into the diversification among the above 10 chitinases from sugarcane and 21 from other plant species, several features of the predicted proteins were analyzed. The typical domains of chitinase, including chitin binding domain (CBD), chitinase domains of glycoside hydrolase family 18 and family 19, were shown in Fig. 2. In addition, the signal peptide, isoelectric point (pI) and the number of amino acids (aa) were also presented in Fig. 2. We found that not all chitinases contained signal peptide at their N-termini, such as ScChiI3 and O. Sativa chitinase (294979698) in class I and Momordica charantia chitinase (AAM18075.1) in class V. The length of the ORFs in sugarcane chitinases ranged from 238 aa to 325 aa. The average ORF length was 291 aa. The isoelectric point (pI) in different members was not identical in the same class, as some were acidic and others were basic. The sugarcane chitinases, including classes I, II, IV, VI and VII members, have a lysozyme-like domain in their structures which may exhibit lysozyme activity. Class I members ScChiI1 and ScChiI2 both contained the N-terminal signal peptide, following the chitin binding domain (CBD) which was rich in cysteines (9) and a glycoside hydrolase family 19 chitinase domain. Though the protein domain of ScChiI3 lacked a signal peptide and the CBD structure and was different from those of ScChiI1 and ScChiI2, they sharing 63.96% amino acid sequence identity. A spacer hinge region, rich in proline (3, 12 and 13) and glycine (5, 6 and 5) residues, was found between the CBD and the glycoside hydrolase family 19 chitinase domains of ScChiI1, ScChiI2 and ScChiI3. Class II chitinase ScChiII1 lacked the CBD and the hinge region, but contained a N-terminal signal peptide and a glycoside hydrolase family 19 chitinase domain (amino acids 34 ~ 223), sharing a high degree of homology (70.82%) with class I members. Like class I protein, class IV chitinase ScChiIV1 consisted of the CBD, hinge region and glycoside hydrolase family 19 chitinase domain. However, there was only 59% identity in the catalytic domain among class I and class IV. The class VI chitinase ScChiVI1, which lacked the duplicated CBDs in its N-terminal region which was different from chitinase (P11218) in Urtica dioica endochitinase 36 , had a signal peptide, a hinge region (1 prolines and 6 glycines) and a glycoside hydrolase family 19 chitinase domain. Class VII chitinase ScChiVII1 lacked the CBD and the hinge region, and its amino acid sequences were 47.57% homology to class I and Class II chitinases. Unlike  Tissue-specific expression of chitinase family genes in sugarcane. qRT-PCR was performed to determine the expression patterns of these putative chitinase genes in different sugarcane above-ground tissues. As shown in Fig. 3, the expression of chitinase genes belonging to classes I, II, III, V and VII was detected in all of the four sugarcane tissues including leaf, bud, stem pith and stem epidermis. Compared with the other three tissues, the chitinase genes with the highest expression levels in stem pith were ScChiI1, ScChiI2, ScChiI3, ScChiIII1, ScChiIII2, ScChiV1 and ScChiVII1. ScChiII1 showed the highest level of transcripts in sugarcane tissues with transcripts most abundant in leaf. Transcripts of ScChiIV1 and ScChiVI1 accumulated to the highest level in bud tissues. These results showed a certain degree of tissue specificity in sugarcane chitinase family genes (Fig. 3).
Accumulation of chitinase gene mRNAs in sugarcane post inoculation with S. scitamineum. qRT-PCR was used to examine the expression patterns of the 10 sugarcane chitinase family genes during sugarcane-smut interaction (Fig. 4). It was seen that all 10 transcripts were induced by infection of S. scitamineum but different patterns were evident.
During the incompatible interaction using Yacheng05-179, one smut resistant sugarcane genotype, early transcriptional elevation of ScChiI1, ScChiIII1, ScChiIII2 and ScChiVI1 was observed at 24 hpi (Fig. 4A). The transcript of ScChiIII1 reached the maximum at 24 hpi, while the maximal accumulation of the other 3 genes was observed at 168 hpi. ScChiI2 and ScChiV1 transcripts decreased at 24 hpi and 48 hpi, but increased to the peak at 120 hpi and again reduced at 168 hpi. Although the ScChiI3 and ScChiIV1 accumulation decreased at initial stage (from 0 hpi to 48 hpi), they gradually elevated at the later stage (from 120 hpi to 168 hpi). ScChiII1 was up-regulated from 48 hpi to 168 hpi. In contrast, ScChiVII1 demonstrated a down-regulation during the incompatible interaction.
During the compatible interaction using ROC22, a popular genotype which is susceptible to S. scitamineum, transcripts of ScChiI1, ScChiII1 and ScChiIII2 were observed to be elevated as early as 24 hpi, suggesting rapid response to the infection of smut pathogen (Fig. 4B). Their expression values were accumulated to the maximal levels at either 24 hpi or 48 hpi. Transcripts of ScChiI2 and ScChiIV1 maintained almost at the same level after inoculation. ScChiI3 was down-regulated compared with that at 0 hpi. The transcripts of ScChiIII1, ScChiV1, ScChiVI1 and ScChiVII1 peaked at 48 hpi. The data indicated that all genes except ScChiVI1 had the lowest expression level at 168 hpi during the compatible interaction. Gene expression in response to different defense-related signal compounds. Transcript accumulation of chitinase genes in sugarcane plantlets under different phytohormone treatments, including SA, MeJA (methyl jasmonate) and ABA stresses, were examined by qRT-PCR (Fig. 5). The results   revealed that all three signal compounds up-regulated ScChiI2, ScChiIII2 and ScChiV1, while ScChiIII1 was down-regulated. ScChiI1, ScChiIV1 and ScChiVII1 were up-regulated by MeJA and ABA but down-regulated by SA. In addition ScChiVI1 was down-regulated by MeJA and ABA but up-regulated by SA. ScChiI3 was up-regulated by SA and MeJA but suppressed by ABA. ABA treatment down-regulated ScChiII1 while ScChiVII1 was up-regulated. These results suggest that the transcription of individual chitinase genes respond differently to SA, MeJA and ABA. Functional characterization of three chitinase genes, ScChiI1, ScChiIV1 and ScChiVII1, during pathogen infection. Based on the information of differentially expressed chitinase genes post S. scitamineum infection, the full-length cDNA sequences of three chitinase genes, ScChiI1, ScChiIV1 and ScChiVII1, were isolated from sugarcane. The sequence data of ScChiI1, ScChiIV1 and ScChiVII1 were submitted to GenBank under accession number of KF664182, KF664178 and KF664179, respectively. The ORF fragment was recombined into the plant expression vector of pCAMBIA 2300 containing the 35S promoter and the GFP reporter gene. Their subcellular localization was characterized by transient expression of the target gene and GFP in N. benthamiana leaves with Agrobacterium-mediated transformation method 37 . Infiltrated leaves observed under a confocal laser scanning microscope showed that 35S::ScChiI1::GFP, 35S::ScChiIV1::GFP and 35S::ScChiVII1::GFP fusion proteins were located in cytoplasm and plasma membrane, plasma membrane, cytoplasm and plasma membrane, respectively (Fig. 6). In addition, the mock of 35S::GFP was shown in the nucleus, cytoplasm and plasma membrane cells.
Chitinase genes have been reported to be induced not only by biotic but also by abiotic stress 12,38 . The expression patterns of ScChiI1, ScChiIV1 and ScChiVII1 in Yacheng05-179 plantlets were investigated after treatment with 25% PEG (polyethylene glycol), 250 mM NaCl (sodium chloride), 100 μ M CuCl 2 (copper chloride), and low temperature (4 °C) (Fig. 7). This showed induction of high levels of ScChiIV1 transcripts with all four abiotic treatments. PEG, NaCl and CuCl 2 appeared to cause an increase of accumulated ScChiI1 transcripts post stress, while low temperature caused slightly decrease at 12 h. The expression of ScChiVII1 was up-regulated by low temperature and down-regulated by NaCl. In response to PEG and NaCl stresses, the level of ScChiVII1 transcript reduced slightly at 6 h and 12 h, but increased at 12 h and 24 h, respectively.

Transient expression of ScChiI1, ScChiIV1 and ScChiVII1 induces a defense response in N.
benthamiana. To test whether the target genes can induce hypersensitive response (HR) and immunity in plant, ScChiI1, ScChiIV1 and ScChiVII1 genes were transiently over-expressed in N. benthamiana leaves. After 48 h post infiltration, a typical HR symptom with deeper DAB staining was found in the leaves expressing 35S::ScChiI1, 35S::ScChiIV1 and 35S::ScChiVII1, respectively (Fig. 8). The bronzing color after over-expressing ScChiI1 was the darkest. Furthermore, the expression levels of seven immunity associated marker genes including the HR marker genes NtHSR201 and NtHSR203, the JA-associated genes NtPR-1a/c and NtPR2 and NtPR3, and the ethylene synthesis depended genes NtEFE26 and NtAccdeaminase, were increased post 24 h infiltration. These results suggest that ScChiI1, ScChiIV1 and ScChiVII1 were involved in cell death responses.

Discussion
Many plants contain multiple chitinase isozymes. They have been categorized into seven classes (class I ~ VII) based on their primary structure, substrate specificity, mechanisms of catalysis and sensitivity to inhibitors 17,18 . On the basis of the annotations of O. sativa and Arabidopsis genomic sequences, 37 and 24 chitinases were found in O. sativa and Arabidopsis, respectively 24 . Analysis revealed that each cluster had distinct amino acid characteristics. Krishnaveni et al. 39 had observed three antifungal chitinases, CH1, CH2 and CH3, from S. bicolor. Four cDNAs encoding acidic and basic isoforms of chitinases were isolated from Cladosporium fulvum-infected tomato leaves 40 . We have previously reported cloning and identification of one class III and one class VII chitinases from sugarcane post S. scitamineum inoculation 22,34 . The current study of the sugarcane chitinase family indicated the presence of at least 17 expressed genes induced by smut pathogen.
Chitinase isozymes are a diverse group of enzymes with different characteristics, such as enzymatic activities, primary sequence, pI and cellular localization 41 . Based on the domain architecture of chitinases classes I~VII in sugarcane and other plants, not all chitinases contained a signal peptide, and the CBD structure was absent in ScChi VI1 (class VI). According to the most popular classification system described earlier 17,18,42 , class I chitinases contain three domains: a cysteine-rich N-terminal CBD, a proline-and glycine-rich hinge region and a highly conserved C-terminal catalytic domain. Class II chitinases are generally extracellular which lack the CBD and the hinge region, but their amino acid sequences in the catalytic domain are nearly identical to class I chitinases (more than 65%). Class III lacks CBD and has little sequence identity to the class I and class II catalytic domain, while Class IV contains the CBD, hinge region and catalytic domain, but displays deletion in the catalytic domain. Class V chitinases has little sequence similarity with the other chitinases, but more similar to bacterial chitinases, such as those from Bacillus circulans and Serratia marcescens. Class VI chitinases possess the duplicated CBDs in their N-terminal regions, while Class VII chitinases lack the CBD and the hinge region. In this study, seven types of sugarcane chitinases coincided with the former classification 17,18,42 . In Fig. 1, although ScChiV1 and the chitinase proteins from Momordica charantia (AAM18075.1) and Scientific RepoRts | 5:10708 | DOi: 10.1038/srep10708 N. tabacum (CAA54373.1) were not at the consistent branch of the phylogenic tree, it was assigned to the class V subfamily containing the same domain of glycoside hydrolase family 18. Nearly all sugarcane chitinases, except ScChiI 3, contained the N-terminal targeting domain which may involve in directing them to either the vacuole or the apoplast (Fig. 2). Like other plant species 43 , sugarcane chitinases of classes I, II, IV, VI and VII have the glycoside hydrolase family 19 domain belong to class PR-3 family, and class III and class V possess the glycoside hydrolase family 18 domain belong to PR-8 and PR-11 families, respectively. Chitinases including class I, II, IV, VI and VII were predicted to contain a lysozyme like domain 44 , suggesting that most sugarcane chitinases possess lysozyme activity.
According to previous reports, the only route of invasion of the smut pathogen is via sugarcane buds 45 . Previous studies also revealed that plant chitinases are developmentally regulated, indicating a role in the specific physiological processes 18,46 . In this study, transcripts of sugarcane chitinase genes differently accumulated in the noninfected sugarcane above-ground tissues (Fig. 3). Seven chitinase genes expressed at high expression levels in stem pith, suggesting specific roles in stem pith. ScChiII1 showed the highest expression level in sugarcane and its transcript was most abundant in leaf. Considering the significantly higher expression of ScChiIV1 and ScChiVI1 in sugarcane buds than in other tissues, it suggests that ScChiIV1 and ScChiVI1 may play a positive role in sugarcane smut resistance.
In the present study, during S. scitamineum infection (0 hpi ~ 168 hpi), the expression of at least 10 sugarcane chitinases was induced. However they showed different expression patterns in the incompatible/compatible interactions. In Yacheng05-179, four chitinase genes, ScChiI1, ScChiIII1, ScChiIII2 and ScChiVI1, rapidly responded to smut pathogen inoculation at initial stage (from 0 hpi~24 hpi) (Fig. 4), and reached maximal accumulation at 168 hpi. Conversely, in ROC22, almost all the target genes (except ScChiVI1) had lower expression levels at 168 hpi (Fig. 4). These results suggest that sugarcane chitinase genes are pathogen-inducible and are involved in disease resistance. Previously, a class III sugarcane chitinase gene ScChi was shown to be induced after challenge in the incompatible interaction (Yacheng05-179 vs. S. scitamineum) and its expression remained higher than that in a compatible interaction (Liucheng03-182 vs. S. scitamineum) 22 .
In plants, levels of chitinases are regulated by biotic and abiotic stress, such as pathogen infection, cold, drought, heavy metals, salt, and plant hormones 12,22,38 . As reported, SA, JA and ethylene are considered as the defense signal compounds for systemic acquired resistance (SAR) and induced systemic resistance (ISR), two types of plant induced resistance 21 . In plant responses to environmental stress, the reaction of the signaling molecule JA is the fastest, and plays an important part in resistance reaction. JA-related gene expression has been reported to be up-regulated and cause JA accumulation under biotic and abiotic stress 47 . Previous studies suggested that ABA affects plant response to biotic stress mainly via interaction with other stress responsive pathways 48 . In our study, the expression levels of sugarcane chitinase genes could be differentially modulated by SA, MeJA and ABA (Fig. 5). Exogenously applied SA resulted in an increase accumulation of ScChiI2, ScChiI3, ScChiIII2, ScChiV1 and ScChiVI1 transcripts. Application of MeJA increased the expressions of ScChiI1, ScChiI2, ScChiI3, ScChiII1, ScChiIII2, ScChiIV1, ScChiV1 and ScChiVII1. The exogenous application of ABA increased the levels of ScChiI1, ScChiI2, ScChiIII2, ScChiIV1, ScChiV1 and ScChiVII1 transcripts.
Full-length cDNA sequences of three sugarcane chitinase genes, each one of class I chitinase ScChiI1, class IV chitinase ScChiIV1 and class VII chitinase ScChiVII1, were isolated from smut resistant genotype Yacheng05-179. These three genes were pathogen-inducible post S. scitamineum infection (Fig. 4), and were up-regulated by MeJA and ABA but down-regulated by SA (Fig. 5). Protein localization revealed that 35S::ScChiI1::GFP and 35S::ScChiVII1::GFP fusion proteins were located in cytoplasm and plasma membrane, while 35S::ScChiIV1::GFP was located in plasma membrane (Fig. 6). ScChiI1 and ScChiIV1 were up-regulated by PEG, NaCl and CuCl 2 stresses, while ScChiVII1 was not (Fig. 7). ScChiIV1 and ScChiVII1 transcripts were increased under 4 °C low temperature stress, but ScChiI1 was not (Fig. 7). However, all these genes induced defense responses in N. benthamiana by transient expression (Fig. 8). These results suggest that the different sugarcane chitinases have individual functions in response to various environmental stresses.
Although functions of sugarcane chitinases genes are not fully understood, some chitinases in plant species have been shown to inhibit the growth of chitin-containing fungi, both in vitro 49 and in vivo 14,15 . Data were normalized to the GAPDH expression level. All data points were the means ± SE (n = 3). Different lowercase letters indicated a significant difference, as determined by the least-significant difference test (p-value < 0.05). PEG, polyethylene glycol; NaCl, sodium chloride; CuCl 2 , copper chloride.
Scientific RepoRts | 5:10708 | DOi: 10.1038/srep10708 When compared with wild-type plants, in many cases, transgenic plants constitutively expressing chitinases showed enhanced resistance to fungal infection or delayed development of disease symptoms 50,51 . The transgenic Musa acuminata expressing the O. sativa chitinase gene exhibited resistance to black leaf streak disease caused by the pathogenic fungus, Mycosphaerella fijiensis 26 . In our previous work, a close relationship between the expression of sugarcane class III chitinase gene ScChi (KF664180) and plant immunity was demonstrated from inoculation experiments and the validation of in vitro antibacterial activity. There was also a report of smut resistance improvement in sugarcane varieties ROC22 and ROC10 by introduction of a β -1,3 glucanase together with the modified class I chitinase gene from N. tabacum 52 . From the characteristics of the 10 sugarcane chitinase genes obtained here, the possible contribution of all these genes for plant defense against pathogen attack is suggested. However, the conclusive validation and precise functional determination of these genes by genetic transformation into sugarcane is still in progress.  The transcripts analysis of the immunity associated marker genes, including the hypersensitive response marker genes NtHSR201 and NtHSR203, the jasmonate associated genes NtPR-1a/c and NtPR2 and NtPR3, and the ethylene synthesis depended genes NtEFE26 and NtAccdeaminase. NtEF1-α was used to normalize the transcript levels. Mock: the Agrobacterium strain carrying 35S::00. All data points are the means ± SE (n = 3). Different lowercase letters indicate a significant difference, as determined by the least-significant difference test (p-value < 0.05).

Plant materials and inoculation with
Scientific RepoRts | 5:10708 | DOi: 10.1038/srep10708 of spores 37 to eliminate the effect of wounding. At 0 hpi, 24 hpi, 48 hpi, 120 hpi and 168 hpi, one biological replicate consisting of five buds for each group were excised, immediately frozen in liquid nitrogen and then stored at − 80 °C.

Tissue distribution study.
For tissue distribution study, one biological replicate with six healthy 10 month old plant of Yacheng05-179 was selected. The samples were collected from the youngest fully expanded leaf (+1 leaf) with a visible dewlap (the collar between the leaf blade and sheath), buds, stem pith and stem epidermis. These samples were fixed in liquid nitrogen and kept at − 80 °C until RNA extraction 37 . Abiotic stress treatments. To investigate the expression of sugarcane chitinase family genes in response to stress factors, 4 month old tissue cultured plantlets of Yacheng05-179 were grown in water for one week and then exposed to various chemical stimuli 37
According to the results of previous researches by Kirubakaran et al. 28 , Rahul et al. 33 and Singh et al. 17 , along with the information from bioinformatic analysis and their expression profile under the stresses of MeJA, ABA and SA indicated in this study, three out of ten chitinase genes, ScChiI1, ScChiIV1 and ScChiVII1 were chosen for further study. Based on the sequences of the above predicted chitinase genes, the primers used to clone the target genes were designed. Amplification of ScChiI1 (gi32815041) was performed with primers ScChiI1: FW-ACATACATAGTTGCTTGCYTTGC and RV-CCTTTTGCTTTATTCATTGCTC on first-strand cDNA template of Yacheng05-179 under 4 °C low temperature treatment for 24 h. ScChiIV1 (Sugarcane_Unigene_BMK.56580) and ScChiVII1 were amplified with primers ScChiIV1: FW-GCACCGCAGCAACGAA and RV-CGGAGCCATGCAAGGAG, ScChiVII1: FW-AAGATGAAGCGGAAGACG and RV-GCTAAAACAGACCCATTGTG, on first-strand cDNA template of Yacheng05-179 post 48 h S. scitamineum inoculation. These PCR products were gel-purified, cloned into the pMD18-T vector (TaKaRa, China) and sequenced (Shenggong, China).
Sequence analysis of chitinase genes. ORF analysis was performed with the ORF Finder (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html). The pI was calculated with the ProtParam tool (http://www. expasy.ch/tools/protparam.html). SignalP 4.0 Server (http://www.cbs.dtu.dk/services/SignalP/), NCBI Conserved Domains (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart. embl-heidelberg.de/) programs were employed to scan for the signal peptides and the motifs on the primary structure of the deduced protein sequences. Subcellular location of the putative proteins was predicted with PSORT Prediction (http://psort.hgc.jp/form.html). ClustalW software was used to perform multiple alignment of sugarcane chitinases with other previously published plant chitinases 17,18 . Based on this alignment, a phylogenetic tree was constructed according to the neighbor-joining (NJ) method (1,000 bootstrap replicates) using the MEGA 5.05 program.
Transcript level analysis. Expression patterns of sugarcane chitinase family genes in different tissues and their response to biotic and abiotic stress were analyzed by qRT-PCR, which followed the instructions of the SYBR Green Master (ROX) (Roche, China) on a 7500 real time PCR system (Applied Biosystems, USA). The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene (Table 2) was used as an internal control. According to sequences of ScChiI1 ~ ScChiVII1, the specific primers (Table 2) were designed using the Beacon Designer 8.12 program. The qRT-PCR reaction system (20 μ L) contained 10 μ L FastStart Universal SYBR Green PCR Master (ROX), 1.0 μ L of first-strand cDNA (10 × diluted) and 0.5 μ M of each primer. PCR with distilled water as template was performed as control. The qRT-PCR reaction condition was held at 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. At the end of the PCR reaction, a melting curve was established. Each qRT-PCR was conducted in triplicate. The 2 −Ct method was adopted to analyze the qRT-PCR results 53 . For calculating gene expression level during developmental stages, the tissue exhibiting the lowest expression level was served as control. For the abiotic stress treatments, unstressed sample was used as control. During the biotic stress, gene expression profile was calculated by the expression level of the inoculated sample of S. scitamineum minus the level of the mock at each corresponding time point to eliminate any effect of wounding. Data points in qRT-PCR time course were plotted as means ± SE of three replicates.

The role of three chitinase genes in response to pathogen infection. Subcellular location assay
with Agrobacterium-mediated transformation was followed from Su et al. 22 . ORF fragments of ScChiI1, ScChiIV1 and ScChiVII1 were inserted into the vector of pCAMBIA 2300-GFP and transformed into the competent cells of A. tumefaciens strain EHA105, respectively. The subcellular localization of the fusion protein was visualized using a confocal laser scanning microscope Leica TCS SP5 (Germany) equipped with 10 × lense.
As reported, cell death presented at the infected site is the most efficient method to restrict pathogen growth and development 54 . The stimulation of reactive oxygen species (ROS) and defense-related hormones, induction of R gene expression and ion fluxes are the common response of cell death 55,56 . For the transient expression of the target gene in N. benthamiana, overexpression vectors pCAMBIA 1301-ScChiI1, pCAMBIA 1301-ScChiIV1 and pCAMBIA 1301-ScChiVII1 were constructed to analyze their defense responses. Agrobacterium strain EHA105 carrying the recombinant vector was transiently expressed in N. benthamiana leaves. Each treatment was carried out in three replicates. DAB (3,3'-diaminobenzidinesolution) was used to stain H 2 O 2 produced in agroinfiltrated leaves 22 . The leaves were incubated in 1.0 mg/mL DAB-HCl solution in the dark overnight and destained by boiling in 95% ethanol for 5 min. The bronzing color of the leaves for H 2 O 2 detection was photographed. qRT-PCR analysis of the expression of seven immunity associated marker genes were conducted post 24 infiltration, including the hypersensitive response marker genes NtHSR201 and NtHSR203, the jasmonate associated genes NtPR-1a/c, NtPR2 and NtPR3, and the ethylene synthesis depended genes NtEFE26 and NtAccdeaminase ( Table 2) 22 . NtEF1-α ( Table 2) was used to normalize the transcript levels.