A class III WRKY transcription factor in sugarcane was involved in biotic and abiotic stress responses

WRKY transcription factors play significant roles in plant stress responses. In this study, a class III WRKY gene ScWRKY5, was successfully isolated from sugarcane variety ROC22. The ScWRKY5 was a nucleus protein with transcriptional activation activity. The ScWRKY5 gene was constitutively expressed in all the sugarcane tissues, with the highest expression level in the stem epidermis and the lowest in the root. After inoculation with Sporisorium scitamineum for 1 d, the expression level of ScWRKY5 was significantly increased in two smut-resistant varieties (YZ01-1413 and LC05-136), while it was decreased in three smut-susceptible varieties (ROC22, YZ03-103, and FN40). Besides, the expression level of ScWRKY5 was increased by the plant hormones salicylic acid (SA) and abscisic acid (ABA), as well as the abiotic factors polyethylene glycol (PEG) and sodium chloride (NaCl). Transient overexpression of the ScWRKY5 gene enhanced the resistance of Nicotiana benthamiana to the tobacco bacterial pathogen Ralstonia solanacearum, however the transiently overexpressed N. benthamiana was more sensitive to the tobacco fungal pathogen Fusarium solani var. coeruleum. These results provide a reference for further research on the resistance function of sugarcane WRKY genes.

Subcellular localization of ScWRKY5. The Euk-mPLoc 2.0 Server predicted the subcellular localization of the sugarcane ScWRKY5 protein in the nucleus. However, the WoLF PSORT predicted the subcellular localization of the ScWRKY5 protein in the nucleus with the highest probability, followed by in the chloroplast, cytoplasm, and peroxisome. Here, to validate the true location of the ScWRKY5 protein, the fusion expression plasmid pMDC83-ScWRKY5-GFP was constructed, and the subcellular localization expression vector pMDC83-GFP was used as a positive control. Using Agrobacterium-mediated transformation and injecting into N. benthamiana leaves, the ScWRKY5-GFP fusion protein emitted green fluorescence predominantly in the nucleus, whereas the fluorescence of free GFP was detected in almost around all cell space (Fig. 3). These results indicated that the ScWRKY5 protein is localized in the nucleus.
Transcriptional activation activity of ScWRKY5. The Y2HGold-GAL4 yeast two-hybrid system was used to analyze the self-activating activity of the target protein. On the SDO (synthetic dropout medium without tryptophan) plate, the positive control pGADT7 + pGBKT7-p53 and the negative controls pGBKT7 and pGBKT7-ScWRKY5 could grow normally, indicating that the target plasmid pGBKT7-ScWRKY5 had been successfully transferred into the yeast Y2HGold, and tryptophan could be successfully expressed in GAL4-BD combined with the ScWRKY5 protein (Figs. 4 and S2). On the SDO plate with X-α-Gal dye added, the positive control and the yeast cells transformed with pGBKT7-ScWRKY5 all turned blue, suggesting that the MEL1 gene had been activated. Further tests showed that the positive control grew normally and turned blue on the SDO/X plate medium with the addition of the aureobasidin A (AbA), indicating that the reporter genes AUR1-C and MEL1 had been activated. The blue colonies of the yeast cells transformed with pGBKT7-ScWRKY5 proved that the protein of ScWRKY5 exhibited transcriptional activation activity.
Tissue-specific expression of ScWRKY5 in sugarcane. The relative expression level of the ScWRKY5 gene in the sugarcane root, bud, leaf, stem pith, and stem epidermis was detected by quantitative real-time PCR (qRT-PCR) (Fig. 5). The results indicated that this gene was constitutively expressed in all of these tissues. The lowest expression level of ScWRKY5 occurred in the root, while its relative expression level in the stem pith and leaf was 6.61-fold and 4.45-fold higher than that in the root, respectively.

Expression patterns of
ScWRKY5 under different stresses. The qRT-PCR method was used to analyze the expression patterns of the ScWRKY5 gene under different exogenous stresses (Fig. 6). Under 5 mM SA stress for 3 h, the gene expression level of ScWRKY5 was 3.74-fold higher than that of the control (Fig. 6A). Within 24 h of 100 μM MeJA treatment, the gene expression level of ScWRKY5 was relatively stable (Fig. 6A). www.nature.com/scientificreports/ Under 100 μM ABA stress, the gene expression level of ScWRKY5 was significantly increased at 3 h, being 4.65fold higher than that of the control, following which it dropped to the control level at 24 h (Fig. 6A). Under 250 mM NaCl stress, the gene expression level of ScWRKY5 was stabilized from 0.5 h to 6 h and then rose rapidly to a peak at 24 h, at which point it was 1.62-fold higher than that of the control (Fig. 6B). Under 25% PEG 8000 stress, the transcription of ScWRKY5 remained unchanged at 0.5-6 h, but was increased to a maximum at 24 h which was 1.56-fold higher than that of the control (Fig. 6B). In addition, the transcripts of ScWRKY5 was detected in the interaction between six different sugarcane varieties and S. scitamineum (Fig. 6C). In the smut-resistant varieties, the expression level of ScWRKY5 was significantly increased in YZ01-1413 and LC05-136 after inoculation with the S. scitamineum for 1 day post-inoculation (dpi), which were 8.58-and 20.32-fold higher than that in the control group, respectively. However, the expression level of ScWRKY5 did not change significantly within 3 dpi in the smut-resistant variety Yacheng05-179. Among the three smut-susceptible varieties, the expression level of ScWRKY5 was significantly decreased in YZ03-103 and FN40 from 1 to 3 dpi, while it was significantly decreased in ROC22 at 1 dpi and returned to the control level at 3 dpi. These results revealed that the transcripts of ScWRKY5 could be induced by ABA, NaCl, PEG, SA, and S. scitamineum stresses.

Transient overexpression of ScWRKY5 in N. benthamiana leaves. The transient overexpression of
A. tumefaciens cells carrying ScWRKY5 in N. benthamiana leaves was performed. The transcript of the ScWRKY5 gene was successfully detected in the N. benthamiana leaves using a semi-quantitative PCR technology (Figs. 7A and S3). The DAB (3,3′-diaminobenzidine) test showed that the staining color in the 35S::ScWRKY5 leaves was nearly the same as that of the control (Fig. 7B). The qRT-PCR results (Fig. 7C) indicated that in the 35S::ScWRKY5 leaves, the expression levels of the hypersensitive response (HR) marker gene NtHSR203, and JA pathway related genes NtPR2 and NtPR3 were all increased, which were 3.56-, 2.30-, and 7.00-fold higher than that of the control group, respectively. But the HR marker gene NtHSR515, the SA pathway related gene NtPR-1a/c, and the ET synthesis-dependent genes NtEFE26 and NtAccdeaminase were all decreased, which were 0.20-, 0.13-, 0.14-, and 0.22-fold lower than that of the control group, respectively. In addition, the expression level of HR marker gene NtHSR201 was relatively stable. Phenotype analysis showed that there was no obvious symptom in the 35S::00 and 35S::ScWRKY5 leaves at one-day after inoculation with R. solanacearum. However, the leaves were yellow and the allergic spots were more serious in the 35S::00 group after seven days (Fig. 8A). The qRT-PCR results indicated that the expression level of the NtPR2 gene was increased by the overexpression of the ScWRKY5 gene at one-day post inoculation, Figure 4. The ScWRKY5 transactivation activity test. SDO (SD/-Trp), synthetic dropout medium without tryptophan; SDO/X (SD/-Trp/X-α-Gal), synthetic dropout medium without tryptophan, but plus 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside; SDO/X/A (SD/-Trp/X-α-Gal/AbA), synthetic dropout medium without tryptophan but plus 5-bromo-4-chloro-3-indoxyl-α-D-galactopyranoside and aureobasidin A. pGADT7-T + pGBKT7-p53, positive control; pGBKT7, negative control. www.nature.com/scientificreports/ which was 124.46-fold higher than that of the control group, and was repressed after seven days. The expression levels of the NtHSR201, NtHSR203, NtHSR515, NtPR-1a/c, NtPR3, and NtEF26 were repressed at one-day post inoculation, and recovered to the control level after seven days. The expression level of the NtAccdeaminase remained unchanged at one and seven days post inoculation (Fig. 8B). The N. benthamiana leaves overexpressing the ScWRKY5 gene did not show symptoms after one day of inoculation with F. solani var. coeruleum. Conversely, the leaves turned yellow and wilted severely after seven days of inoculation, which was higher than that of the control group (Fig. 8C). The DAB results showed that as the infection time increased, the brown color of the tobacco darkened, and the leaves overexpressing the ScWRKY5 gene were darker than the control group after seven days. (Fig. 8C). The qRT-PCR showed that, compared with the control group, the expression level of NtHSR515 and NtPR2 was decreased in the 35S::ScWRKY5 leaves after inoculation for one day, and the expression level of NtPR-1a/c was stable at the control level. Instead, the expression levels of NtHSR201, NtHSR203, NtPR3, NtEFE26, and NtAccdeaminase were significantly increased in the 35S::ScWRKY5 leaves, which were 1.55-, 2.54-, 22.17-, 1381.62-, and 3.55-fold higher than that of the control group, respectively. After seven days of infection, only the expression level of NtPR-1a/c was decreased, while www.nature.com/scientificreports/ the expression of NtHSR515 and NtPR2 was stable at the control level, and the expression of the other five genes was still increased (Fig. 8D).

Discussion
WRKY is one of the largest transcription factors in plants and plays an important role in the plant response to biotic and abiotic stresses. As more WRKY transcription factors are being identified from different plants, their structural characteristics, origin, evolution, and gene functions are being increasingly studied 3,18 . However, there are only limited studies on WRKY genes in sugarcane [30][31][32][33][34] . In this study, the ScWRKY5 gene was isolated from sugarcane ROC22. The ScWRKY5 protein was clustered with the amino acid sequences from S. spontaneum in the class III WRKY family 34 (Fig. S1). The physical and chemical properties of proteins are of great significance to their molecular biological functions. We predicted that the sugarcane ScWRKY5 was a hydrophilic protein with poor functional stability. Although ScWRKY5 belongs to the class III WRKY family, it only has one zincfinger-like motif CX 7 CX 24 HX 1 C, which is the same as that of tomato SlWRKY80 35 . As reported, only one of 81 tomato WRKY proteins has this zinc-finger-like motif (CX 7 CX 24 HX 1 C) variation 35 , suggesting that this type of structural variation in zinc finger is uncommon. Therefore, it is necessary to further assess whether the variant zinc structure of ScWRKY5 will cause the appearance or loss of some specific functions. The location of proteins in cells is important for determining their biological functions 36 . The subcellular localization analysis of 164 NtWRKY transcription factors in common tobacco showed that 118 NtWRKYs (including most of the class I and class II WRKY family members) were located in the nucleus, and 77.4% of class III WRKYs were located in the cytoplasm 37 . In this study, the ScWRKY5-GFP fusion protein emitted green fluorescence predominantly in the nucleus (Fig. 3) which is consistent with the result predicted by the Euk-mPLoc 2.0 Server, suggesting that the ScWRKY5 protein may play the role of a nucleoprotein in sugarcane. However, for the small probability that ScWRKY5 protein was predicted to locate in the chloroplast, cytoplasm, or peroxisome by WoLF PSORT software, further subcellular localization test is needed in conjunction with promoter analysis of the ScWRKY5 gene itself as well as a western blot to check the integrity of the fusion protein in the tissues infiltrated.
Transcriptional activity is important for the functional analysis of transcription factors 38 . Since the GAL4 yeast two-hybrid system was firstly discovered 39 , this method has been increasingly used for studying the interactions between WRKY proteins 40 . In the present study, the protein encoded by the ScWRKY5 cDNA showed a self-activating activity (Fig. 4). Similarly, GhWRKY40 not only has a self-activating activity in yeast, but it also acts as a downstream interaction protein of GhMPK20, participating in plant-borne pathogenic signals through the MAPK cascade pathway 41 . Proteins with self-activation activity can be detected in sections to determine the specific location of the self-activation region of the protein, which can be verified in subsequent experiments.
The expression abundance of the WRKY gene in different plant tissues is not consistent [42][43][44] . There are 28 CiWRKY genes detected in the roots, stems and leaves of Caragana intermedia 42 . Different CiWRKY genes are expressed in different tissues, e.g., CiWRKY69-1 had the highest expression level in the roots, while CiWRKY40-1 and CiWRKY30 were mainly expressed in the leaves 42 . Bakshi et al. 20 found that 12 of the 37 Arabidopsis WRKY genes were specifically expressed in the mature region of the root cells and may be involved in regulating the maturation of Arabidopsis root cells. TaWRKY44 was expressed differently in all of the tested organs, including the roots, stems, leaves, pistils, and stamens, with the highest expression level in the leaves and the lowest in the pistil 43 . In this study, ScWRKY5 was expressed constitutively in the sugarcane tissues, with the highest expression www.nature.com/scientificreports/ level in the stem pith, and the lowest level in the root (Fig. 5). These results indicated that WRKY genes may play different roles in the regulation of plant growth and development. WRKY transcription factors can rapidly perceive signals of changes in external environmental conditions and transmit these signals in a timely manner in order for plants to respond to the environment 12,45 . Abiotic stresses, such as salt and drought can induce the expression of a series of stress response genes in the ABA signal transduction pathway, thereby enhancing the plant resistance 46 . Wang et al. 31 found that the expression level of the ScWRKY3 gene was increased under the stresses of NaCl, PEG, and the plant hormone ABA, but it was repressed under the stresses of SA and MeJA. In this study, the expression level of the ScWRKY5 gene was also increased by the stresses of PEG, NaCl, SA, and ABA (Fig. 6). It is speculated that the ScWRKY5 gene may play a role in the tolerance to salt and drought in sugarcane through SA and ABA signaling pathways. Similarly, under drought and salt conditions, the expression level of SlWRKY8 in Solanum lycopersicum is significantly increased, which occurs by directly activating the expression of SlWRKY8 in the SA and ABA synthesis pathways, thereby enhancing the resistance of S. lycopersicum to drought and salt stresses 47 .
Previous studies have shown that the GhWRKY40 gene is induced by the bacteria R. solanacearum upon infection and is up-regulated after treatment with SA, MeJA, and ET 41 . When GhWRKY40 was transiently overexpressed in tobacco leaves, most resistance-related genes, including SA, ET, JA, and HR response genes, were repressed after infection with R. solanacearum, suggesting that the overexpression of GhWRKY40 reduce the resistance to R. solanacearum 41 . Overexpressing AtWRKY28 and AtWRKY75 increased the tolerance to oxalic acid and the resistance to Sclerotinia sclerotiorum by participating in JA/ET-dependent defense signaling pathways in Arabidopsis 48 . The OsWRKY13 gene enhanced the defense ability of rice blast via cross-talk with the SA-dependent defense pathway, SNAC1-mediated abiotic stress defense pathway, and JA signaling pathway 49 . When infected with R. solanacearum, Arabidopsis WRKY27 has a negative regulatory effect on the expression of defense-related genes and exhibited earlier disease symptoms than the mutant WRKY27-1 plant, which had lost WRKY27 50 . Overexpressing GhWRKY44 induced the expression of several defense-related genes in transgenic plants, including PR-1, PR-2, PR-5, and NPR1 in the SA pathway and PR-4 in the JA pathway, and enhanced the resistance of transgenic tobacco to R. solanacearum and Rhizoctonia solani 51 . After infected with Phytophthora nicotianae, the overexpression of SpWRKY1 enhanced the expression level of SA-and JA-associated genes (NtPR1, NtPR2, NtPR4, NtPR5, and NtPDF1.2), suggesting that SpWRKY1 acts as a positive regulator in tobacco defense responses to P. nicotianae 52 . The overexpression of NtPR-Q in tobacco enhanced the expression of SA dependent genes (NtPR1a/c, NtPR2, and NtCHN50), thus enhanced the resistance of plants to R. solanacearum 53 . In transgenic tobacco plants, the symptom of leaf wilting was lighter than that of wild type plants, and the expression levels of HR-associated gene HSR515 and SA-dependent marker genes NPR1 and PR2 were continuously upregulated by the ectopic overexpression of CaC3H14, indicating that CaC3H14 played a positive regulatory role in resistance to R. solanacearum 54 . In this study, the expression level of the NtPR2 gene was increased in the 35S::ScWRKY5 leaves at one-day post inoculation with R. solanacearum (Fig. 8B). Compared with the 35S::ScWRKY5, the leaves were yellow and the allergic spots were more serious in the control group after seven days of inoculation with R. solanacearum (Fig. 8A). These suggested that the transient overexpression of ScWRKY5 may enhance the resistance of N. benthamiana to R. solanacearum by participating in SA-dependent defense signaling pathways. After inoculation with F. solani var. coeruleum, the wilting disease symptoms were greater in 35S::ScWRKY5 leaves than in the control. Furthermore, the expression levels of the HR marker gene NtHSR515, and the SA pathway related genes NtPR-1a/c and NtPR2 were decreased in 35S::ScWRKY5 leaves (Fig. 8D). The results revealed that the transient overexpression of ScWRKY5 may reduce the resistance of N. benthamiana to F. solani var. coeruleum via repressed the expression of HR and SA pathway related genes. The regulation mechanism of the ScWRKY5 gene in disease resistance need to be further verified after stable genetic transformation. In summary, the ScWRKY5 gene may be involved in plant ABA, SA, and ET signal regulatory networks and plays a role in the plant response to biotic and abiotic stresses. However, more WRKY family genes in sugarcane need to be discovered and identified, which is more conducive to the further study of the possible functional differentiation between different family members of WRKY genes.

Methods
Plant materials and methods. In order to analyze the tissue-specific expression level of ScWRKY5, nine 10-month-old ROC22 (the main sugarcane variety in mainland China) plants were randomly selected from the field based on the method of Huang et al. 55 . The sugarcane white root, bud, stem pith, stem epidermis, and + 1 leaf were collected and frozen in liquid nitrogen, and then stored at − 80 °C until use. Each sample contained three biological replicates, and each biological replicate contained three plants.
To detect the expression level of ScWRKY5 in sugarcane inoculated with smut pathogen, six different sugarcane varieties, including three smut-resistant varieties (YZ01-1413, LC05-136, and Yacheng05-179) and three smut-susceptible varieties (ROC22, YZ03-103, and FN40), were provided by the Key Laboratory of Sugarcane Biology and Genetics and Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University 56 . The 10-month-old sugarcane stalks were selected and cultured at 32 °C. After the buds had germinated to 1-2 cm in length, an acupuncture inoculation method with 5 × 10 6 mL −1 (containing 0.01% Tween-20, v/v) smut pathogen suspension was used to inoculate the sugarcane buds, and the control group was inoculated with sterile water (containing 0.01% Tween-20, v/v) 57 . All of the samples were cultured at 28 °C for 16 h in the light and 8 h in the dark. Five single buds were collected at 0, 1, and 3 dpi, frozen in liquid nitrogen, and stored at − 80 °C for future use.
Total RNA extraction and cDNA first-strand synthesis. Total RNA of the sugarcane samples was extracted using the TRIzol method 60 . RQ1 RNase-Free DNase reagent was used to remove the DNA from the total RNA of the sample. The cDNA of the mature leaves of sugarcane ROC22, a template for ScWRKY5 gene cloning, was reverse-transcribed according to the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Shanghai, China). Referring to the instruction manual of the Prime-Script RT Reagent Kit (TaKaRa Biotechnology, Dalian, China), the first-strand cDNA of the sugarcane tissues and different materials under various stresses was reversed and used to quantitatively detect the expression level of the target gene. The obtained RNA and cDNA were tested by 1.0% agarose gel electrophoresis.
Cloning, sequencing and bioinformatics analysis of ScWRKY5. From our previous transcriptome data of sugarcane 61 , a gene encoding a predictable WRKY transcription factor ScWRKY5 was screened. The National Center of Biotechnology Information (NCBI) online software (https ://www.ncbi.nlm.nih.gov/tools / prime r-blast /) was used to design specific cloning amplification primers (Table S2). The cDNA of the mature leaves of ROC22 was used as a template, and a PCR reaction system was prepared according to the ExTaq DNA reagent instructions. The PCR program was set as 94 °C pre-denaturation for 4 min; followed by 35

Subcellular localization analysis.
The Gateway entry vector primer pair ScWRKY5-Gate-F/R (Table S2) was designed based on the sequences of the ScWRKY5 ORF (without the stop codon) and the entry vector pDONR-221. After PCR amplification, the gel-purified product was subjected to the entry vector pDONR-221 by BP reaction to obtain a positive plasmid pDONR-221-ScWRKY5. Subsequently, after Ssp I single digestion and purification, ScWRKY5 was subjected to the subcellular localization vector pMDC83 72 by LR reaction. After sequencing, the recombinant subcellular localization vector 35S::ScWRKY5::GFP was obtained. The positive recombinant plasmid 35S::ScWRKY5::GFP was transformed into A. tumefaciens GV3101 strain. The Agrobacterium cells were collected and diluted to OD 600 = 0.8 with MS blank medium. A 1.0-mL sterile syringe was used to transfer the bacterial solution (containing 200 μM acetylsyringone) into the 5-8 leaves of N. benthamiana 73,74 . After culturing at 28 °C for 16 h in the light and 8 h in the dark for 2 d, the injected leaves were removed, and 1.0 μM 4′, 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. A Leica TCS SP8 laser confocal microscope [Leica Microsystems (Shanghai) Trading Co., Ltd., Mannheim, Germany] with a 10 × lens, 488 nm green fluorescence excitation wavelength, and 458 nm chromatin DAPI filter was used to observe the subcellular localization 75,76 . Analysis of ScWRKY5 transcriptional self-activating activity. The target gene ScWRKY5 was constructed on the pGBKT7 vector, and the recombinant plasmid pGBKT7-ScWRKY5 was digested with enzymes and verified by sequencing analysis. A primer pair ScWRKY5-BD-F/R (Table S2) containing a specific restriction site was designed based on the sequences of ScWRKY5 ORF (without the stop codon) and the DNA-binding domain (BD) vector pGBKT7. The pMD19-T-ScWRKY5 plasmid was used as a template for PCR amplification. The gel-purified product and the pGBKT7 vector plasmid were digested with the Nde I and EcoR I enzymes, (Table S2) and then purified. Subsequently, the ScWRKY5 gene was ligated to the vector pGBKT7 by using T4 DNase, and transformed into DH5α competent cells. After bacterial liquid PCR detection, enzyme digestion verification, and sequencing comparison, the pGBKT7-ScWRKY5 recombinant plasmid was obtained.
Expression patterns of ScWRKY5 in sugarcane tissues and under various stresses. The 15-fold diluent cDNA of different sugarcane tissues (root, bud, leaf, stem pith, and stem epidermis) and samples treated with ABA, SA, MeJA, PEG, NaCl, and S. scitamineum was used as the qRT-PCR template. Based on the ScWRKY5 gene sequence, the specific qRT-PCR primer pair ScWRKY5-QF/R (Table S2) was designed on the NCBI online software. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank Accession No. CA254672) was used as the internal reference gene (Table S2). An ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) was used for qRT-PCR detection, and a quantitative reaction system was prepared according to the SYBR Green PCR Master Mix Kit instructions (Roche, Shanghai, China). Three technical replicates were set up for each sample, and sterile water was used as a template in the negative control. The relative expression of the target gene was calculated using 2 -△△Ct method 76 . The significance level of the data was analyzed using DPS 9.50 software, and graphs were plotted using Origin 8.0 software. In the smut pathogen infection test, the relative expression of the target gene was calculated with reference to the method of Su et al. 77 .  www.nature.com/scientificreports/ GV3101. The Agrobacterium strain GV3101 carrying the recombinant plasmid was cultured in an Luria-Bertani (LB) liquid medium containing 50 μg/mL kanamycin and 35 μg/mL rifampicin at 200 rpm and 28 °C. Agrobacterium cells cultured overnight were collected by centrifugation, resuspended in MS liquid medium containing 200 μM acetylsyringone to OD 600 = 0.8, and then the Agrobacterium cells were injected into 8-leaf-stage N. benthamiana leaves on the plate. The control was the Agrobacterium strain GV3101 cell carrying the pEarleyGate 203 empty vector 73,74 . After one day, the leaves were collected for qRT-PCR and DAB histochemical analysis. The tobacco bacterial pathogen R. solanacearum and fungal pathogen F. solani var. coeruleum were cultivated in potato dextrose water (PDW), diluted to OD 600 = 0.5 with 10 mM magnesium chloride (MgCl 2 ) solution, and inoculated into the overexpressing N. benthamiana leaves for one day. All of the treated plants were kept for a week under 28 °C with a light period of 16 h/8 h of darkness to track the changes in leaf symptoms and analyze the relative transcription levels of nine tobacco immune-related marker genes. The qRT-PCR was used to analyze the expression levels of the target gene and some tobacco immune marker-related genes in N. benthamiana, including the HR marker genes NtHSR201, NtHSR203 and NtHSR515, SA-related gene NtPR-1a/c, NtPR2 and NtPR3, and ET synthesis-dependent genes NtEFE26 and NtAccdeaminase (Table S2) 78,79 . NtEF1-α (GenBank Accession No. D63396) was used as an internal reference (Table S2) 78,79 .

Conclusions
In the present study, a ScWRKY5 gene was isolated from sugarcane ROC22 and was found to encode a protein with a special zinc finger structure belonging to the class III WRKY family. ScWRKY5 was a nucleoprotein with self-activating activity. Through the prediction of the upstream promoter sequence of the S. spontaneum WRKY (Sspon.03g0003780-2c), as a homologue gene of ScWRKY5, several cis-acting elements involved in photosystem, hormone signaling substances, and abiotic stresses were found. The ScWRKY5 gene was constitutively expressed in different sugarcane tissues, and its expression level was increased under the stresses of SA, ABA, PEG, and NaCl. After infected by S. scitamineum for 1 d, the expression level of the ScWRKY5 gene was increased in two smut-resistant varieties (YZ01-1413 and LC05-136), while it was decreased in three smut-susceptible varieties (ROC22, YZ03-103, and FN40). Transient overexpression of the ScWRKY5 gene enhanced the defense ability of N. benthamiana to the tobacco bacterial pathogen R. solanacearum, but reduced the resistance response to the tobacco fungal pathogen F. solani var. coeruleum. Here, the potential function of ScWRKY5 in sugarcane was showed in a mechanism-flow chart (Fig. 9). These results may provide references for the excavation and functional identification of ScWRKY family genes in sugarcane.