Sugarcane calcineurin B-like (CBL) genes play important but versatile roles in regulation of responses to biotic and abiotic stresses

Free calcium ions are common second messengers in plant cells. The calcineurin B-like protein (CBL) is a special calcium sensor that plays an important role in plant growth and stress response. In this study, we obtained three CBL genes (GenBank accession nos. KX013374, KX013375, and KX013376) from sugarcane variety ROC22. The open reading frames of ScCBL genes ranged from 642 to 678 base pairs in length and encoded polypeptides from 213 to 225 amino acids in length. ScCBL2-1, ScCBL3-1, and ScCBL4 were all located in the plasma membrane and cytoplasm. ScCBL2-1 and ScCBL3-1 expression was up-regulated by treatment with salicylic acid (SA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2), polyethylene glycol (PEG), sodium chloride (NaCl), or copper chloride (CuCl2). ScCBL4 expression was down-regulated in response to all of these stresses (abscisic acid (ABA), SA, MeJA, and NaCl) except for H2O2, calcium chloride (CaCl2), PEG, and CuCl2. Expression in Escherichia coli BL21 cells showed that ScCBLs can enhance tolerance to NaCl or copper stress. Overexpression of ScCBLs in Nicotiana benthamiana leaves promoted their resistance to infection with the tobacco pathogen Ralstonia solanacearum. The results from the present study facilitate further research regarding ScCBL genes, and in particular, their roles in the response to various stresses in sugarcane.

have been identified in Sorghum biocolor 13,14 . In addition, CBL genes have been investigated in Brassica napus 15 , Solanum melongena 16 , and other plant species 17,18 . CBL proteins contain a classical EF-hand helix-loop-helix motif with a 12-residue loop 19 . In EF-hand motifs, the Ca 2+ -binding sites are located at residues 1 (X), 3 (Y), 5 (Z), 7 (Y), 9 (X), and 12 (Z) 10,19 . Different CBL proteins have different degrees of variation in the EF-hand structure, but the number of EF-type regions and the distance between them is the same in all known CBL proteins 11 .
The function of CBL genes has been studied in A. thaliana, O. sativa, and other plant species. In A. thaliana, AtCBLs play a role in the response to multiple abiotic stresses [20][21][22] . For instance, AtCBL1 functions as a positive regulator in response to salt and drought but as a negative regulator in response to cold 20 . Abscisic acid (ABA) is a signaling molecule that plays a role in the plant response to aging and stress 23 . AtCBL9 is a common element in the ABA signaling and stress-induced ABA biosynthesis pathways 21 . Ten OsCBL genes in rice are expressed in various organs at the adult stage and have also been found to respond to different stress conditions [sodium chloride (NaCl), polyethylene glycol (PEG), and cold] 24 . In addition, OsCBL8 overexpressing transgenic rice seedlings showed more tolerance to salt stress than non-transgenic seedlings 24 . S. bicolor CBL genes are thought to regulate sodium carbonate stress-specific cellular adaptation responses and influence the plant growth and developmental patterns 14 . Analysis of CBL transcripts in Populus euphratica under abiotic stress suggested that seven CBL (PeCBL1, 2, 3, 4, 5, 9, and 10) members may play important roles in responding to specific external stimuli 12 .
Sugarcane (Saccharum spp.) is an economically attractive polyploid C4 grass that is used not only to produce approximately 60% of the world's sugar but also to produce ethanol, a low-carbon-emission fuel 25 . To date, there have been few reports on CBL genes in sugarcane [26][27][28] . Zhang (2013) cloned five CBL genes (GenBank accession Nos. KC800815, KC800816, KC800817, KC800818, and KC800819) from Saccharum hybrid variety GT28 and found that CBL5 and CBL6 may play key roles in adaptation to low temperatures 28 . Using real-time quantitative polymerase chain reaction (qRT-PCR) analyses,  found that SsCBL1 and SsCBL6 play important regulatory roles in response to a variety of stresses (low potassium, drought, and salt) 26 . Yeast two-hybrid assays showed that ScCIPK8 interacts with ScCBL1, ScCBL3, and ScCBL6 27 . In the past 15 years, ROC22 is the most widely cultivated sugarcane in China due to its high yield and high sugar and good ratoon properties. Previous research found that ROC22 can well resist infection by Pokkah boeng disease 29,30 . Lan et al. (2014) found that ROC22 has better drought tolerance compared to five sugarcane varieties 31 . However, a systematic analysis of CBL genes in sugarcane variety ROC22 especially on the view of function differentiation, however, has not yet been reported.
In this study, we successfully cloned three sugarcane ScCBL genes by reverse transcription-PCR (RT-PCR) and subjected the cloned sequences to bioinformatics analysis. The expression patterns of these three ScCBL genes in different sugarcane tissues and under various exogenous stresses were investigated by qRT-PCR. In addition, we assessed the subcellular localization of these ScCBL proteins and analyzed their function by expression in Escherichia coli BL21 and transient expression in N. benthamiana. This study aims to provide useful information about the sequence characteristics of these three ScCBL proteins as well as their expression patterns in response to phytohormones and various stresses. This increased knowledge of ScCBL genes could be applied by sugarcane breeder to develop resistant variety.

Materials and Methods
plant materials and treatments. The sugarcane variety ROC22 were provided by the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture (Fuzhou, China).
We sampled sugarcane tissues as described by Wang 32 . Selecting three healthy and mature ROC22 sugarcane stalks with uniform growth rates from the field. The bud, stem pith, stem skin, meristem, and the youngest fully expanded viz + 1 leaf with a visible dewlap (the collar between the leaf blade and sheath) were sampled. These tissues were wrapped, frozen in liquid nitrogen, and stored at −80 °C until total RNA extraction. We performed the sampling after abiotic stress treatment as follows 33 : uniform four-month-old cultured ROC22 plantlets were transferred to water for one week and then treated with eight exogenous treatments, including 100 μM ABA, 5 mM salicylic acid (SA), 100 μM methyl jasmonate (MeJA), 50 μM calcium chloride (CaCl 2 ), 10 μM hydrogen peroxide (H 2 O 2 ), 25% PEG 8000, 250 mM NaCl, or 100 mM copper chloride (CuCl 2 ), by root dipping at 28   RNA samples with an OD 260 /OD 280 of between 1.8 and 2.0 were selected and treated with DNase I (Promega, Madison, WI, USA) to remove DNA contamination. First-strand cDNA was synthesized using a Prime-Script TM RT Reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's protocol, and then checked by 1% agarose gel electrophoresis.
isolation of sugarcane ScCBL genes and gateway entry vector construction. The Z. mays sequence (GenBank Acc No. NM_001155706) which derived from ZmCBL3 (GenBank Acc No.EU962348.1) was used as a probe and the NCBI BlastN tool was applied to retrieve homologous EST sequences in the sugarcane genome. The BioEdit Contig Assembly Program (CAP) was employed to assemble one sugarcane CBL sequence (ScCBL2-1). The other two sugarcane CBL sequences (ScCBL3-1 and ScCBL4) were selected from our previous transcriptome data of sugarcane infected with Sugarcane Mosaic virus 35 . The specific primers were designed using Primer 5.0 and the NCBI primer designing tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Table S1).
qRt-pcR analysis. The 7500 qRT-PCR system (Applied Biosystems, San Francisco, CA, USA) was applied to detect and analyze the expression of ScCBL genes in different sugarcane tissues and under various exogenous stresses. The qRT-PCR primers (Table S1) were designed using Beacon Designer 8.12 software. Cullin (CUL) 37 and clathrin adaptor complex (CAC) 37 were employed as the internal controls (Table S1). The 20 μL qRT-PCR reaction contained 10 μL SYBR Green Master Mix, 0.8 μL each of the 10 μM primers, 1.0 μL cDNA templates (20 × diluted cDNA), and 7.4 μL double distilled water. Each qRT-PCR reaction was repeated three times, and the conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The 2 -ΔΔCt method was used to analyze the qRT-PCR data 38 and the statistical analysis was conducted by using Data Processing System v9.50 software (China). Data were expressed as the mean ± standard error (SE). Significance (p < 0.05) was calculated using one-way analysis of variance (ANOVA), followed by Duncan's new multiple range test.

Subcellular localization assay.
The pFAST-R05-ScCBL2-1-GFP, pFAST-R05-ScCBL3-1-GFP, and pFAST-R05-ScCBL4-GFP vectors were constructed by LR reaction using the LR Clonase TM II Enzyme Mix (Invitrogen) according to the manufacturer's instructions. The three recombinant ScCBL-GFP vectors were transformed into competent Agrobacterium tumefaciens GV3101 cells. The Agrobacterium-mediated transient expression in N. benthamiana leaves was performed according to the method described by Su et al. 39 . After infiltration for 48 h, the subcellular localization of the fusion protein were visualized by laser scanning confocal microscopy (Leica TCS SP5, Wetzlar, Germany).

expression in E. coli BL21 (DE3) cells. Prokaryotic expression vectors were constructed based on
pEZYHb from the pDONR221-ScCBL plasmids by LR reaction. The recombinant pEZYHb-ScCBL plasmids and the empty vector pEZYHb (control) were transformed into competent E. coli BL21 (DE3) cells for the prokaryotic expression experiments.
A spot assay was conducted to characterize the expression of ScCBLs in competent E. coli BL21 cells in response to NaCl and CuCl 2 stresses. When the OD 600 of E. coli BL21 cells containing pEZYHb-ScCBLs or pEZYHb (control) in LB medium (containing 80 μg/mL ampicillin) reached 0.6, 1.0 mM Isopropyl β-D-thiogalactoside (IPTG) was added, and the cells were cultured at 37 °C for another 12 h. The concentration of the cultures was adjusted to OD 600 = 0.6, and the samples were then diluted 10 −3 and 10 −4 in LB medium 40 . Next, 10 μL from each of the 10 −3 and 10 −4 dilutions was spotted on LB agar plates. For the salt tolerance assay, we prepared LB media with 250 mM, 500 mM, or 750 mM of NaCl. For the heavy metal tolerance assay, we added 250 μM, 500 μM, or 750 μM of CuCl 2 to the LB media 36 . All of the plates were incubated at 37 °C overnight and then photographed.

transient assay for ScCBL genes in N. benthamiana leaves.
To understand how ScCBL expression changes in response to pathogen infection and whether the plant hypersensitive reaction is also activated, pEarleyGate 203-ScCBLs overexpressing vectors were constructed using the Gateway cloning technique. Competent Agrobacterium GV3101 cells were transformed with recombinant pEarleyGate 203-ScCBL plasmids. The empty pEarleyGate 203 vector was transformed into Agrobacterium GV3101 cells for use as a control. The cells were then cultured in LB liquid medium (supplemented with 50 μg/mL kanamycin and 35 μg/mL rifampicin) overnight at 28 °C. After incubation, the cells were centrifuged and resuspended in MS liquid medium (containing 200 μM acetosyringone) at an OD 600 of 0.8. After infiltration into the N. benthamiana leaves, the plants were cultured at 24 °C for 24 h (16 h light/8 h darkness) 39 . RT-PCR was exploited to detect whether ScCBL genes have been overexpressed in N. benthamiana, with the RNA of treated leaves and ScCBL genes specific primers (gScCBL2-1, gScCBL3-1, and gScCBL4, Table S1), and the NtEF1-α was treated as control. The treated N. benthamiana leaves were used for the transcriptional analysis of the eight tobacco immunity-associated marker genes (Table S1) 41 .
To analyze the inhibitory effect of ScCBL genes on pathogen infection, Ralstonia solanacearum was cultured to an OD 600 of 0.8 in potato dextrose water (PDW) liquid medium at 28 °C. Then, N. benthamiana leaves that had been infiltrated with pEarleyGate 203-ScCBLs or pEarleyGate 203 for 24 h were infected with R. solanacearum. All of the treated plant materials were cultured at 24 °C (16 h light/8 h darkness) for 7 days and then photographed. DAB and trypan blue staining were utilized to analyze the Agrobacterium-infiltrated leaves as described by Liu et al. 33 .

Results
Identification of CBL genes in sugarcane. Three ScCBL genes were successfully amplified by RT-PCR from sugarcane variety ROC22. According to the homology with AtCBLs ( Figure S1), three ScCBL genes were designated as ScCBL2-1, ScCBL3-1, and ScCBL4, respectively. Basic information about these three genes is shown in Table 1. The length of the ScCBLs open reading frames (ORFs) ranged from 642 to 678 base pairs, and they encoded polypeptides from 213 to 225 amino acids in length. The isoelectric points (pIs) of the polypeptides ranged from 4.77 to 4.82, and the grand average of hydropathicity (GRAVY) of each ScCBL was negative. The molecular weight (MW) of these ScCBLs ranged from 24.31 to 25.85 kDa. phylogenetic analysis of SccBLs. Sequences for CBL proteins identified in A. thaliana 11,42 , O. sativa 11,24 , Zea mays 43 , and S. bicolor 44 were obtained from GenBank (https://www.ncbi.nlm.nih.gov/). A phylogenetic tree analysis was performed and the CBL proteins were grouped into four clades (A-D; see Fig. 1). The three sugarcane CBL proteins fell into two different groups: ScCBL2-1 and ScCBL3-1 were in group A, and ScCBL4 was in group D.

Sequence analysis of the SccBL proteins.
A multiple alignment analysis was performed using the amino acid sequences of the three ScCBLs and ten AtCBLs 11 . As shown in Figure 2, all of these CBL proteins contained more than two EF-hand domains, which are essential for CBL to bind Ca 2+19 . The C-terminal region of all three ScCBL proteins contained an FPSF motif. Note that only ScCBL4 contained an N-terminal MGCVSSK sequence, which is a unique CBL protein domain referred to as the myristoylation domain 45,46 . ScCBL2-1 and ScCBL3-1 had N-terminal tonoplast targeting sequences (TTSs), which may mediate their subcellular localization 47 . The size of the linker regions between the EF-hand loops was absolutely invariant in all three of the proteins: 23 amino acids separated EF1 and EF2, whereas 25 amino acids separated EF2 and EF3, and 32 amino acids separated EF3 and EF4.
Tissue-specific expression of the ScCBL genes. ScCBL expression in different sugarcane tissues (bud, stem pith, leaf, meristem, and stem skin) was detected by qRT-PCR. Figure 3 shows that the three ScCBLs were expressed in all of the tissues tested. ScCBL2-1, ScCBL3-1, and ScCBL4 were expressed at the highest levels in the meristem. ScCBL2-1 and ScCBL4 were expressed at the lowest levels in the stem skin, while ScCBL3-1 had the lowest transcription in the stem pith. expression of the ScCBL genes in response to phytohormones and various abiotic stresses. qRT-PCR analysis showed that the three ScCBL genes exhibited different expression patterns in response to ABA, SA, MeJA, H 2 O 2 , CaCl 2 , PEG, NaCl, or CuCl 2 stress (Fig. 4). When subjected to ABA stress, ScCBL2-1, ScCBL3-1 and ScCBL4 transcription were all inhibited. Under SA, ScCBL2-1 and ScCBL3-1 were expression-induced, but ScCBL4 was down-regulated. As for MeJA, the three genes have the similar expression pattern with that under SA. Handling with H 2 O 2 , the expression of all ScCBLs peaked at 12 h. ScCBL2-1 and ScCBL3-1 expression was inhibited in response to treatment with CaCl 2 . We did not find significant difference, however, in the expression of ScCBL4 between treatment and control. PEG stress did not induce a significant expression level difference of ScCBL4 in compared with the control, but ScCBL2-1 and ScCBL3-1 expression was up-regulated (ScCBL2-1 peaked at 24 h at a value 17.1 times higher than that of the control, and ScCBL3-1 peaked at 6 h at a value 3.0 times higher than that of the control). Treatment with NaCl inhibited ScCBL4 expression but induced ScCBL2-1 and ScCBL3-1 expression, with the highest expression levels (16.7 times and 2.5 times higher than that of the control, respectively) occurring at 12 h. In response to CuCl 2 treatment, ScCBL2-1, ScCBL3-1, and ScCBL4 were up-regulated (ScCBL2-1 expression sharply increased at 48 h to a value 9.3 times higher than that of the control, and ScCBL3-1, and ScCBL4 expression peaked at 12 h at values that were 6.1 and 2.9 times higher, respectively, than that of the control).

ScCBL genes expression in E. coli BL21 (DE3) strain. Bacterial cells overexpressing pEZYHb-ScCBLs
had similar growth to control cells on solid LB medium (control), whereas the cells grown on media containing different concentrations of salt or CuCl 2 showed marked differences in growth (Fig. 6). None of the cells grew on LB plates supplemented with 500 or 750 mM NaCl (Fig. 6(a)). Cells transformed with pEZYHb-ScCBLs, however, exhibited better survival on LB plates supplemented with 250 mM NaCl compared with untransformed cells (Fig. 6(a)). These results indicated that bacterial cells overexpressing pEZYHb-ScCBLs had better tolerance to   Table S3. and the ethylene synthesis-dependent genes NtEFE26 and NtAccdeaminase (Fig. 7(b)). After 24 h of infiltration, the expression levels of Sequences highlighted in dark blue, red, and light blue indicated homology 100%, ≥ 75%, and ≥ 50% homology, respectively. aa, amino acids. All the corresponding GenBank Accession were listed in Table S3.

Discussion
Studies show that plant signal transduction processes under stress are accompanied by changes in cellular calcium concentration 48,49 . As a unique Ca 2+ sensor in plants, CBL plays an important role in signal pathways of plant development and response to various stresses 50 . In this study, we characterized and discussed the possible functions of ScCBL2-1, ScCBL3-1, and ScCBL4 from sugarcane based on the results of bioinformatic analysis and experiments.

Sequences and phylogenetic analysis of SccBLs.
In the present study, three CBL genes were isolated from sugarcane. The ScCBL proteins appear to be rather conserved in size and structure. These ScCBL genes were predicted to encode polypeptides ranging from 24.31 to 25.85 kDa. The results are similar to those in A. thaliana and O. sativa, in which most AtCBLs and OsCBLs ranged from 23.5 to 25.9 kDa in size 11 . The sequence comparison results showed that the three ScCBL proteins all contained a C-terminal FPSF motif, and the serine residue in these FPSF motifs could be phosphorylated by the CIPK protein kinase 51,52 . A TTS motif, which mediates www.nature.com/scientificreports www.nature.com/scientificreports/ subcellular localization 47 , was clearly seen in the N-terminus of ScCBL2-1 and ScCBL3-1. The N-terminus of ScCBL4 contained a MGCVSSK sequence, which is a unique CBL protein domain known as the myristoylation domain 45,46 . This domain has been hypothesized to be the ancestral localization domain for CBLs 53 . The TTS motif of ScCBL2-1 and ScCBL3-1 had the same consensus motif as that found in AtCBL2 and AtCBL3, which spanned 19 amino acids 47 . The TTS in AtCBL2 or AtCBL3 was necessary and sufficient for targeting GFP fusion proteins to the tonoplast in A. thaliana mesophyll cells 47 . We thus speculate that ScCBL2-1 and ScCBL3-1 may  www.nature.com/scientificreports www.nature.com/scientificreports/ have the same function to AtCBL2 or AtCBL3 47 . However, we found that ScCBL2-1 and ScCBL3-1 not only located in the plasma membrane, but also in the cytoplasm. Phylogenetic analysis placed the three ScCBL proteins into two clades. The ScCBL members investigated here clustered closely with SoCBL, SsCBL, ZmCBL, SbCBL, and OsCBL orthologs, which indicated that the closer evolutionary relationship between the four species from the gramineae family (Saccharum spp., S. bicolor, Z. may, and O. sativa) compared to those of A. thaliana. In addition,  www.nature.com/scientificreports www.nature.com/scientificreports/ interestingly, we found that CBL family members in A. thaliana (AtCBL1, 4, 5, and 9) and O. sativa (OsCBL1, 4, 5, 7, and 8) that harbor an N-myristoylation motif were distributed into two neighboring subgroups of the phylogenetic tree (C and D), and ScCBL4 fell into subgroup D. This site diversity may have enabled the evolutionary separation of CBL-type membrane-associated and membrane-independent calcium signaling pathways 11 .

Differential responses of ScCBLs to phytohormones and abiotic stresses. Several studies have shown
that CBL genes play an important role in the plant stress response 12,21,43,54 . ABA, SA and MeJA, which are phytohormones, play an important role in the response of plants to adverse environmental conditions 55 . H 2 O 2 is a ROS molecule that mediates signaling functions 56 . Through the interaction with CIPK, CBL protein regulates the production of H 2 O 2 in the presence of NADPH oxidase, so as to maintain the positive feedback mechanism of stress tolerance 57 . In this study, when subjected to ABA stress, the three ScCBLs genes were all down-regulated. In Arabidopsis, AtCBL2 and AtCBL3 were also not obviously altered by ABA 47 . Under SA and MeJA, ScCBL2-1 and ScCBL3-1 were up-regulated while ScCBL4 was inhibited. PsCBL which is orthologues to AtCBL2, was up-regulated in response to SA 18,58 . And the expression of ScCBLs was induced under H 2 O 2 . We guess that the up-regulated ScCBLs were in response to the regulation of exogenous H 2 O 2 . Besides, CaCl 2 stress did not induce any significant change in ScCBL4 expression, whereas ScCBL2-1 and ScCBL3-1 were down-regulated. Recent biophysical evidence has indicated that Ca 2+ does not stimulate the interaction between CBL2 and CIPK14, even though Ca 2+ is required for kinase activation through CBL 59,60 . Different ScCBL genes showed various expression patterns in response to CaCl 2 stress, so the interaction between these ScCBL genes and Ca 2+ needs to be further investigated. We also found that ScCBL2-1 and ScCBL3-1 were up-regulated in response to PEG, NaCl, and CuCl 2 , while ScCBL4 was induced by CuCl 2 , and inhibited by NaCl. Studies have shown that ZmCBL4 can significantly improve the salt tolerance of transgenic Arabidopsis 43 . AtCBL2 and AtCBL3 were marginally induced by dehydration 47 . From all the above, we deduced that ScCBL genes have different expression patterns in response to various stresses. We thus speculate that the same or different expression patterns among family genes may be caused by the functional divergence during evolution, which is accordance with the previous research that a homologous pattern resulted from genome duplication, and it caused the gain or loss of function as part of fine-tuning cellular function due to new functionalization in the course of genome evolution 61 . prokaryotic expression of ScCBLs under nacl and cucl 2 . Previous studies showed that CBL1, CBL4, CBL9, and CBL10 play important roles in the response to high salt stress 46 , for example, ZmCBL4 can significantly improve the salt tolerance of transgenic A. thaliana 43 . AtCBL10 is mainly induced by salt 62 . At lower concentration of salt (250 mM NaCl), however, ScCBL-transformed bacterial cultures showed better survival compared with the untransformed cells. These results suggested that ScCBL genes can enhance cell tolerance to low concentrations of salt. Studies have shown that excessive Cu 2+ can cause oxidative stress, leading to lipid peroxidation, which destroys cell membrane structure 63 . Ca 2+ can connect phosphates, phospholipids, and protein carboxyl groups on cell membranes, increase the hydrophobicity of cell membranes, and at the same time, reduce membrane permeability and enhance membrane stability 64 . In this study, ScCBLs-overexpressing and control bacterial cells had similar growth on solid LB medium (control). Besides, interestingly, in our studies, under metal stress conditions (CuCl 2 ), recombinant ScCBLs cells exhibited dramatically better survival compared with nonrecombinant cells. These results suggested that ScCBL Ca 2+ sensors can enhance tolerance to CuCl 2 . transient expression of ScCBLs response to R. solanacearum. Ethylene is thought to act as an internal signal regulator during plant growth and development, and can respond to external adverse conditions including biotic and abiotic stresses 65 . In addition, Ca 2+ signaling plays a critical role in the response to biotic and abiotic stimuli 66 . Fagerstedt et al. found that an increase in the concentration of Ca 2+ ions can activate the CBL-CIPK system and cause ethylene-responsive gene activation 67 . In the present study, we found that ethylene synthesis-dependent immunity-associated marker gene (NtAccdeaminase) was up-regulated when transiently overexpressed ScCBLs in N. benthamiana leaves (Fig. 7). Moreover, since CBL proteins can function as Ca 2+ sensor relays 7 , we can hypothesize that ScCBL genes may take part in the ethylene synthesis pathway and play a role in the response to external stressors 67 . Reactive oxygen species (ROS) act as signaling molecules to regulate development and stress responses 68 . As a relatively stable active oxygen, H 2 O 2 , plays different roles in plant responses to external stresses 69 . A previous study showed that, in plants, attempted infection by microbial pathogens is often accompanied by rapid cell death in and around the initial infection site and that this response is associated with restricted pathogen growth and represents a form of PCD 70 . In this study, to investigate changes in ScCBLs expression in response to pathogen infection, we injected R. solanacearum into N. benthamiana containing 35 S::ScCBLs and a control construct. Then, we used DAB staining and trypan blue staining to detect hydrogen peroxide (H 2 O 2 ) accumulation and cell necrosis in the leaves. We observed darker DAB staining compared with the control leaves after overexpression of ScCBL in N. benthamiana leaves and inoculation with R. solanacearum (Fig. 8). Besides, we also observed more intense trypan blue staining of cells in N. benthamiana leaves overexpressing ScCBL genes after inoculation with R. solanacearum compared with control leaves. This result suggested that overexpression of ScCBL genes can effectively promote resistance to infections in tobacco plants.

conclusion
Three CBL genes (ScCBL2-1, ScCBL3-1, and ScCBL4) in sugarcane that encode proteins harboring EF-hand motifs were cloned and identified. These ScCBL genes were constitutively expressed in the sugarcane bud, stem pith, leaf, meristem, and stem skin. And they showed different expression patterns in response to stimulation with phytohormones and various abiotic stresses. Overexpression of ScCBL genes enhanced E. coli BL21 cell growth under conditions of NaCl or CuCl 2 stress. Additionally, transient overexpression of ScCBL genes in N. benthamiana leaves resulted in different expression levels of tobacco immunity-associated marker genes, as well as increased resistance to infection with R. solanacearum. The findings from this study of ScCBLs may serve as a basis for the elucidation of the mechanisms underlying sugarcane immunity.