OsBGLU19 and OsBGLU23 regulate disease resistance to bacterial leaf streak in rice

Journal of Integrative Agriculture 2019, 18(6): 1199–1210 Available online at www.sciencedirect.com

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RESEARCH ARTICLE

OsBGLU19 and OsBGLU23 regulate disease resistance to bacterial leaf streak in rice LI Bei-bei*, LIU Ying-gao*, WU Tao, WANG Ji-peng, XIE Gui-rong, CHU Zhao-hui, DING Xin-hua State Key Laboratory of Crop Biology/Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, Shandong Agricultural University, Tai’an 271018, P.R.China

Abstract β-Glucosidase belongs to the glycoside hydrolase I family, which is widely present in multiple species and responds to various biotic and abiotic stresses. In rice, whether β-glucosidase is involved in the interaction between plants and microorganisms is not clear. In this study, we found that the expression of several genes encoding β-glucosidases, including OsBGLU19 and OsBGLU23, were upregulated after inoculation with Xanthomonas oryzae pv. oryzicola (Xoc) and downregulated after inoculation with X. oryzae pv. oryzae (Xoo). The respective insertion mutants of OsBGLU19 and OsBGLU23, bglu19 and bglu23, were more susceptible to Xoc infection. The expression of OsAOS2, a key gene in the jasmonic acid signal pathway, was dramatically downregulated after inoculation with Xoc in the bglu19 and bglu23 mutants. Simultaneously, the expression of downstream disease resistance-related genes, such as OsPR1a, OsPR5 and a key transcription factors OsWRKY72 were obviously downregulated. The resistance mediated by OsBGLU19 and OsBGLU23 to bacterial leaf streak is related to disease resistance-related genes above mentioned. Keywords: β-glucosidase, OsBGLU19, OsBGLU23, Oryza sativa, bacterial leaf streak

Caused by fungi, bacteria, nematodes and viruses, plant

1. Introduction Rice (Oryza sativa) is one of the major subsistence crops, being an essential part of the diets and livelihoods of over 3.5 billion people worldwide. Rice production is affected by a variety of factors, including biotic and abiotic stresses.

diseases seriously reduce crop production and threaten global food security (Talbot 2003; Azizi et al. 2016). As early as 2002, the whole genome sequencing of rice was completed, which provided convenience in the mining of disease resistance genes (Yu et al. 2002). Compared to the use of pesticides and other methods, cultivating resistant varieties is the most economical and environmentally friendly way to prevent disease outbreaks (Hu et al. 2008; Miah et al. 2013).

Received 25 September, 2018 Accepted 17 October, 2018 Correspondence DING Xin-hua, Tel: +86-538-8245569, E-mail: [email protected] * These authors contributed equally to this study. © 2019 CAAS. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). doi: 10.1016/S2095-3119(18)62117-3

Bacterial leaf streak (BLS) of rice caused by Xanthomonas oryzae pv. oryzicola (Xoc), and rice bacterial blight (BB) caused by X. oryzae pv. oryzae (Xoo), are two major bacterial diseases of rice, which limit production in many rice cultivating areas (Niñoliu et al. 2006). To date, more than 40 genes involved in the resistance against Asian Xoo strains have been mapped (Kim et al. 2015; Hutin et al. 2016),

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however, these genes do not produce disease resistance to bacterial leaf streak. Previous studies considered that the resistance of rice to bacterial leaf streak was controlled by multiple genes or quantitative trait loci (QTLs) belonging to quantitative inheritance (Tang et al. 2000). Current research indicates that the quantitative trait locus, qBlsr5a, on the short arm of chromosome 5 controls the resistance to Xoc in rice (Han et al. 2008). As a potential part of the qBlsr5a locus, overexpression of OsPGIP4, which encodes the polygalacturonate-inhibiting protein 4, enhances rice resistance to Xoc (Feng et al. 2016). GH3-2, a minor resistance QTL that encodes an indole-3-acetic acid (IAA)amino synthetase in rice, suppresses pathogen-induced IAA accumulation to strengthen the resistance to BLS (Fu et al. 2011). Additionally, He et al. (2012) identified a recessive BLS-resistance gene, bls1, in DP3 derived from the wild rice species, Oryza rufipogon Griff. Another resistance locus triggered by transcription activator-like (TAL) effectors was also reported (Triplett et al. 2016). It was a single dominant locus called Xo1, which was identified in Carolina Gold Select rice and conferred strong resistance specific to multiple African Xoc strains (Triplett et al. 2016). In addition to these efforts, several defense-related genes involved in the rice-Xoc interaction have been reported recently. OsWRKY45-1-overexpressing rice showed increased susceptibility to BLS. Conversely, plants that overexpressed OsWRKY45-2, an allele of OsWRKY45-1, were resistant to BLS (Tao et al. 2009). Overexpression of OsDEPG1, a nucleotide-binding site leucine-rich repeat (NBS-LRR) gene, reduced the resistance of rice to Xoc (Guo et al. 2012). In addition, overexpression of OsHSP18.0-CI, a small heat shock protein gene, enhanced the resistance of rice (Ju et al. 2017). Glucoside hydrolase, also known as glucosidase, is widely distributed among a variety of organisms and catalyzes the breakage of glycosidic bonds (Chandrasekar et al. 2014). According to data from the CAZy database (http://www.cazy.org/fam/GH1.html), glucosidases can be divided into 135 families (Xu et al. 2004; Cantarel et al. 2009; Lombard et al. 2014). Glucosidases are involved in the metabolism of glucolipids in mammalian cells and they are related to a variety of human diseases, such as human immunodeficiency virus (HIV), infantile-onset Pompe disease, Gaucher disease, breast cancer and diabetes, suggesting their significantly important role in human health (Gruters et al. 1987; Kishnani et al. 2011; Antu et al. 2014; Zhou X et al. 2017; Nikookar et al. 2018). Microbial glycosidases are involved in the hydrolysis of cellulose, fermentation and defense against abiotic stresses (Van et al. 2010; Mallek and Belghith 2016; Zanphorlin et al. 2016; Plascencia 2017; Fia et al. 2018), and they have been increasingly used in industrial production research.

Similarly, there are plenty of genes that encode glycosidases in plants (Xu et al. 2004; Opassiri et al. 2006; Cao et al. 2017), however, their functions are still not clear. In recent years, β-glycosidases (E.C.3.2.1.21) belonging to the glycoside hydrolase Ⅰ family have been attracting attention, owing to their important influence on various aspects of plant physiology. β-glucosidases degraded the endosperm cell wall during seed germination (Leah et al. 1995) and regulated lignin synthesis during the growth process (Chapelle et al. 2012), as they are closely related to secondary metabolism (Singh et al. 2016; Zhou Y et al. 2017). Additionally, plant β-glucosidases also respond to abiotic stresses. For instance, four glycosidases genes, AtBGLU18, AtBGLU33, AtBGLU1 and AtBGLU19, have been reported to be involved in the Arabidopsis thaliana response to osmotic stress (Lee et al. 2006; Xu et al. 2012; Cao et al. 2017). AtBGLU42 induced by AtMYB72 regulates the secretion of phenolic substances in the root under low iron conditions (Zamioudis et al. 2015). AtBGLU15 is required for the response to nitrogen deficiency or low temperature stresses through its degradation of flavanol 3-O-β-glucoside-7-O-α-rhamnosides (Roepke and Bozzo 2015; Roepke et al. 2017). Glucosidases also take part in plant-insect and plant-microorganism interactions. Morant et al. (2008) summarized that β-glucosidases form hydrogen cyanide, benzoxazinoids (also referred to as hydroxamic acids), saponins or glucosinolates as a part of the two-component defense system in plants against attacking herbivores and pathogens. A root-specific gene with high expression in Arabidopsis, AtPYK10 (also known as AtBGLU23), encoding a glycosidase localized to the body of the endoplasmic reticulum, inhibits excessive immune effects in the interaction between plants and the rhizosphere endophytic fungus, Piriformospora indica (Nakano et al. 2014). Additionally, this gene may also be involved in protecting roots from parasites, as demonstrated by its further increased expression in nematode-infected tissues (Nakano et al. 2014). The glycosidase AtBG1 converts the inactive abscisic acid glucose ester (ABAGE) into active ABA, and responds to abiotic stress by adjusting ABA levels in plants (Lee et al. 2006). The activity of VvBG1, which is involved in the synthesis of ABA, is induced by 3-fold by Botrytis cinerea when the infected berry matures, suggesting that the glycosidase may be involved in the resistance to biotic stress during fruit development and grape ripening (Jia et al. 2016). In rice, there are 40 β-glucosidase genes, of which, only 38 are functional (Esen et al. 2006). Researchers have used the combination of bioinformatics, prokaryotic expression, and preliminary X-ray crystallography analysis to detect the substrate specificity of multiple glycosidases in vitro and to solve protein structures (Esen et al. 2006; Chuenchor et al.

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2008; Seshadri et al. 2009; Opassiri et al. 2010; Sansenya et al. 2010, 2011; Luang et al. 2013; Baiya et al. 2014; Chen et al. 2014; Hua et al. 2015), which promotes research in this field. It has been reported that BGLUs have different expression patterns after low temperature treatment, PEG treatment and salt treatment, suggesting that glucosidases may be related to the resistance to abiotic stresses in rice (Cao et al. 2017). Despite numerous efforts, the role of rice glucosidases still need to be fully elucidated, especially their function in the interactions between rice and pathogens. In this study, we used T-DNA insertional mutant (bglu19 and bglu23) plants to explore the possible functions of OsBGLU19 (LOC_Os05g30250) and OsBGLU23 (LOC_ Os05g30390), which encode different β-glucosidases on chromosome 5, in the rice-Xoc interaction, by combining bioinformatics, genetics and molecular biology methods.

2. Materials and methods 2.1. Sequence acquisition and analysis The coding and protein sequences of OsBGLU12 (LOC_ Os04g39880), OsBGLU19 (LOC_Os05g30250), OsBGLU20 (LOC_Os05g30280), OsBGLU21 (LOC_Os05g30300), OsBGLU22 (LOC_Os05g30350) and OsBGLU23 (LOC_ Os05g30390) were obtained from a database called RiceXPro (http://ricexpro.dna.affrc.go.jp/), using MEGA7, clustalX 2.1 and ENDscript (http://espript.ibcp.fr/ESPript/ ESPript/) for analysis (Robert and Gouet 2014).

2.2. Plant materials and growth condition To explore the function of rice glucosidases, the rice mutants, bglu19 and bglu23, which were T-DNA insertional mutants of OsBGLU19 and OsBGLU23, respectively, were purchased from the Rice Tos17 Insertion Mutant Database (https://tos. nias.affrc.go.jp/), and planted in a greenhouse with 10 h dark and 14 h light cycles at 26°C. The authenticity of the mutants was confirmed by PCR. The primers used are shown in Appendix A.

2.3. Pathogen inoculation and disease assessment The virulent Xoc strain, RS105, used in this study was incubated at 28°C for 2 days on polypeptone-sucrose-agar (PSA) medium, which contains 10 g L–1 polypeptone, 1 g L–1 glutamic acid, 10 g L–1 sucrose and 15 g L–1 agar (Kauffman et al. 1973). The bacterial mass was suspended in sterile 10 mmol L–1 MgCl2 to an OD600=0.5 for inoculation. The fully expanded leaves at the seeding stage were inoculated with the bacterial suspension by a nonneedle pinprick method as described previously (Feng et al. 2016). The leaves were

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collected at different times after inoculation (0, 2, 24, 48 and 72 h) and used to extract RNA for expression analysis. Score lesion length at 14 days post-inoculation (dpi) was measured as an indication of disease level. Statistical analysis was carried out by t-test. The virulent Xoo strain, PXO99, was incubated on PSA medium. It was suspended in sterile 10 mmol L–1 MgCl2 to an OD600=0.5 for inoculation, similar to RS105. The leaves at the top of the adult stage (after 8 weeks) were selected and inoculated by the leaf clipping method (Kauffman et al. 1973). The leaves were collected at different time points after inoculation (0, 0.5, 3, 6, 12 and 24 h) and used to extract RNA for expression analysis. Statistical analysis was carried out by t-test.

2.4. RNA extraction, cDNA synthesis and DNA preparation The total RNA of rice leaves was extracted using the OMEGA Plant RNA Extraction Kit (Omega Bio-Tek, China, R6827-02) according to the instructions. The cDNA was obtained by reverse transcription according to the manual using the TOYOBO Reverse Transcription Kit and ReverTra Ace® qPCR RT Master Mix with gDNA Remover (TOYOBO Co., Japan, FSQ-301). Total plant DNA was extracted from rice leaves using the CTAB (cetyltrimethylammonium bromide) method (Xu et al. 2006).

2.5. qRT-PCR and analysis For analysis of gene expression, fluorescent quantitative PCR was performed using the TOYOBO KOD SYBR ® qPCR Mix Kit (Japan) according to the instructions, using the expression level of OsActin (LOC_Os03g50890) to standardize the genes in each qPCR reaction. The primer sequences used in the experiment are shown in Appendix A.

3. Results 3.1. Expression analysis of β-glycosidases induced by rice bacterial leaf streak and rice bacterial blight Transcriptome analysis technology has matured, but it is still necessary to determine the expression of individual genes using qRT-PCR. Cao et al. (2017) used qRT-PCR to detect the tissue-specific expression patterns of 37 rice β-glycosidases and found that these β-glycosidases differ in their transcriptional levels in response to abiotic stresses, such as low temperature and osmotic stresses. It has long been known that β-glycosidases of monocotyledons, such as sorghum and corn, participate in the process of plant resistance to pests and pathogens (Morant et al. 2008).

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Because it is not clear whether rice β-glycosidases can be induced by pathogens, we designed target primers for the 37 β-glycosidases genes in rice to investigate their gene expression over a time-course of pathogen treatment (Appendix B). The expression of the β-glycosidase genes was analyzed after rice was inoculated with two of the most typical rice bacterial pathogens, Xoc strain RS105 and Xoo strain PXO99 (Fig. 1). After inoculation with RS105, most genes were induced and a few were inhibited (Fig. 1-A). Most genes showed increased levels of expression at 2 h after RS105 infection and peaked at 24 or 48 h (Fig. 1-A). In contrast, most genes were down-regulated after inoculation with PXO99, with only a few genes displaying significantly induced expression (Fig. 1-B). Focusing on the glycosidase genes on chromosome 5 (OsBGLU19, OsBGLU20, OsBGLU21, OsBGLU22 and OsBGLU23), we found that OsBGLU19 was significantly inhibited after 2 h, while the other 4 genes did not change significantly during the interaction between rice and Xoc. At 24 and 48 h, these 5 genes were induced to a certain extent, and the expression of OsBGLU23 was significantly higher than other 4 genes (Fig. 1-A). The expression of OsBGLU19, OsBGLU21 and OsBGLU22 decreased over time during infection with PXO99, with OsBGLU19 being the most obvious. OsBGLU20 and OsBGLU23 showed a trend of first decreasing, and then increasing, with OsBGLU23 A OsBGLU1 OsBGLU2 OsBGLU4 OsBGLU5 OsBGLU3 OsBGLU30 OsBGLU9 OsBGLU18 OsBGLU6 OsBGLU14 OsBGLU29 OsBGLU7 OsBGLU27 OsBGLU28 OsBGLU31 OsBGLU8 OsBGLU20 OsBGLU10 OsBGLU11 OsBGLU38 OsBGLU12 OsBGLU26 OsBGLU16 OsBGLU24 OsBGLU13 OsBGLU23 OsBGLU17 OsBGLU22 OsBGLU21 OsBGLU15 OsBGLU19 OsBGLU34 OsBGLU36 OsBGLU25 OsBGLU32 OsBGLU37

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being the most obvious (Fig. 1-B). These data suggest that these 5 genes may be involved in the interaction between rice and pathogenic bacteria, and may affect the resistance of rice.

3.2. Comparison of the protein structure and substrate binding sites of 6 glycosidases Studies have predicted that the catalytic acid-base sequences of β-glycosidases are W-X-T/I-F/L/I/V/S/MN/A/L/I/D/G-E/Q-P/I/Q and that the nucleophile consensus sequences are V/I/L-X-E-N-G (Esen et al. 2006). To investigate the relationship between protein structure and substrate binding sites, 5 genes including OsBGLU19 (LOC_Os05g30250), OsBGLU20 (LOC_Os05g30280), OsBGLU21 (LOC_Os05g30300), OsBGLU22 (LOC_ Os05g30350) and OsBGLU23 (LOC_Os05g30390), were aligned with OsBGLU12 (LOC_Os04g39880), for which the structure has been preliminarily analyzed (Sansenya et al. 2010, 2011). These 6 glycosidases have the relevant sequences required for acid-base catalysis, and the key amino acid, glutamic acid (E) (Hua et al. 2015). The N-termini of OsBGLU19, OsBGLU21 and OsBGLU22 all have catalytic nucleophile-base sequences, while OsBGLU20 and OsBGLU23 lack this sequence (Fig. 1). B

2 h 24 h 48 h 72 h

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OsBGLU1 OsBGLU2 OsBGLU34 OsBGLU3 OsBGLU4 OsBGLU8 OsBGLU10 OsBGLU9 OsBGLU29 OsBGLU6 OsBGLU12 OsBGLU32 OsBGLU16 OsBGLU36 OsBGLU13 OsBGLU17 OsBGLU11 OsBGLU23 OsBGLU20 OsBGLU26 OsBGLU37 OsBGLU31 OsBGLU5 OsBGLU24 OsBGLU27 OsBGLU7 OsBGLU14 OsBGLU18 OsBGLU19 OsBGLU21 OsBGLU28 OsBGLU30 OsBGLU22 OsBGLU25 OsBGLU15 OsBGLU38

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3.11 2.68 2.25 1.83 1.40 0.97 0.54 0.11 −0.31 −0.74 −1.17 −1.60 −2.03 −2.45 −2.88

Fig. 1 Heatmaps of the expression patterns of 36 rice β-glycosidases, with the exception of OsBGLU33 and OsBGLU35, after inoculation with RS105 (A) and PXO99A (B) as determined by qRT-PCR.

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3.3. Identification of bglu19 and bglu23 insertion mutants

and bglu23 leaves at 0 and 4 dpi, however, the number of bacteria within each bglu19 and bglu23 lesion was significantly higher than within WT lesions from 8 to 12 dpi (Fig. 4-C). In summary, these results strongly suggest that OsBGLU19 and OsBGLU23 are positively regulating rice resistance to BLS.

To investigate the function of the rice glycosidase genes in rice disease resistance, the T-DNA insertion mutants of OsBGLU19 and OsBGLU23 obtained from the Rice Tos17 Insertion Mutant Database were bglu19 (ND8010_0_101_1A (exon)) and bglu23 (T00389T (exon)), respectively. These mutants were confirmed by PCR, which showed that both bglu19 and bglu23 were exon insertion mutants, with bglu19 having an insertion in the 2nd exon and bglu23 having an insertion in the 9th exon (Fig. 2).

3.5. The disease resistance-related genes are downregulated in bglu19 and bglu23 plants after inoculation with RS105 Previous studies have shown that salicylic acid and jasmonic acid are involved in the interaction of rice and BLS (Guo et al. 2014; Ju et al. 2017). We analyzed the pathogenesis-related (PR) genes without inoculation and 24 h after inoculation with RS105. Interestingly, the PR gene expression patterns in bglu19 and bglu23 were not identical. We found that, in the wild-type plants, the disease resistance-related genes OsAOS2, OsJAMyb, OsWRKY72, OsPR1a, OsPR1b and OsPR5 all showed upregulated expression after inoculation (Fig. 5). In bglu19 plants, we found that OsPR1b and OsPR5 had very low expression levels compared to the wild type. By contrast, OsAOS2, OsJAMyb, OsWRKY72 and OsPR1a accumulated higher expression levels when not inoculated but were significantly inhibited after inoculation (Fig. 5). In bglu23 plants, the expression levels of OsAOS2, OsWRKY72, OsPR1a and OsPR5 were downregulated and the expression levels after inoculation were significantly lower than that of the wild

3.4. OsBGLU19 and OsBGLU23 positively regulate the resistance to Xoc in rice X. oryzae has been used as one of the model pathogens for studying the molecular mechanism of the interaction between rice and pathogens (Niñoliu et al. 2006). Osglu19 and Osbglu23 exhibited different expression patterns during pathogen infection, showing their potential roles in the rice-pathogen interaction (Fig. 3). The bglu19 mutant plants displayed a longer lesion length on day 14 after RS105 inoculation, with lesion lengths ranging from 1.7 to 3.4 cm, compared to the average length of 1.44 cm in WT plants (Fig. 4-A and B). The bglu23 mutant plants were more sensitive to RS105 with an average lesion length that was 1.1 cm longer than WT (Fig. 4-A and B). There were no differences in the number of bacteria on WT, bglu19

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Fig. 3 Protein sequence alignments for the 6 rice β-glycosidases, OsBGLU12, OsBGLU19, OsBGLU20, OsBGLU21, OsBGLU22, and OsBGLU23. The region shown includes the 2 catalytic amino acids, which fall in the consensus sequences in the green box, as well as most sugar-binding residues. Glutamate (E) in the sequence is a key amino acid.

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Fig. 4 Knockout mutations of OsBGLU19 or OsBGLU23 enhance susceptibility to the Xoc strain, RS105. A, disease symptoms of plants at 10 days post-inoculation (dpi) with RS105. B, the average lesion length at 14 days after Xoc inoculation, scored for the OsBGLU19 or OsBGLU23 knockout lines (bglu19 and bglu23). C, statistical analysis of bacterial population growth in OsBGLU19 or OsBGLU23 knockout lines (bglu19 and bglu23) and wild-type (WT) inoculated with RS105. Error bars represent the standard deviation of three independent leaves. Asterisks indicate significant differences between WT and mutants based on Student’s t-test (P<0.01).

type (Fig. 5). The expression of OsJAMyb and OsPR1b increased slightly after inoculation, but did not reach wild-type levels. Mei et al. (2006) found that activation of OsPR1a and OsPR5 in transgenic plants with high expression of OsAOS2 enhanced the resistance of rice to Magnaporthe oryzae. We suggested that the resistance mediated by OsBGLU19 and OsBGLU23 to bacterial leaf streak is related to the disease resistance-related genes above mentioned.

4. Discussion As early as 1995, it was concluded that the glycosidase hydrolysis reaction was divided into two steps, including the first nucleophilic catalytic reaction (glycosylation from the substrate to form a glucose-enzyme complex) and the second acid-base catalyzed reaction (release of the glycosyl group from the glucose-enzyme complex) (Davies and Henrissat 1995). Both the acid-base catalytic site (WTTSIEP) and the nucleophilic catalytic site (IQENG) exist in OsBGLU19, while OsBGLU23 only has an acid-

base catalytic site (WTTVGEP) and lacks a nucleophilic catalytic site (IQENG) (Fig. 3). However, the respective mutants, bglu19 and bglu23, exhibited a susceptible phenotype when inoculated with Xoc. Whether these two key conserved sites in these proteins participate in the glycosylation or deglycosylation of the substrate still need further investigation. β-Glucosidases are an important class of hydrolases that are widely distributed across many organisms, as they are vital for the processes of life. There are multiple genes encoding β-glucosidases in higher plants, including 47 genes in Arabidopsis (Xu et al. 2004), 38 genes in rice (Esen et al. 2006), and 26 genes in maize (Gomezanduro et al. 2011), and there may be functional differences or redundancies between them. Studies have found that β-glycosidase is not only involved in the response to abiotic stresses, such as low temperature stress, low nitrogen, salt stress and iron deficiency but also affects the process of plant resistance to biotic stress (Lee et al. 2006; Xu et al. 2012; Roepke and Bozzo 2015; Zamioudis et al. 2015; Cao et al. 2017; Roepke et al. 2017). In Arabidopsis, AtBGLU42 regulates systemic

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Fig. 5 Expression of disease resistance-related genes in wild-type (WT) and OsBGLU19 or OsBGLU23 knockout lines (bglu19 and bglu23). Analysis of the expression levels of 6 genes by qRT-PCR. Error bars represent the standard deviation of three replicates for genes expression.

resistance induced by rhizosphere bacteria (Zamioudis et al. 2015). Four myrosinases, AtBGLU34, AtBGLU35, AtBGLU37 and AtBGLU38, hydrolyze glucosinolate to produce chemical defense compounds that were found to be involved in plant resistance to Lepidoptera herbivores (Jander 2006; Wittstock and Burow 2010). In monocot maize, the β-glucosidases, ZmGlu1 and ZmGlu2, which hydrolyze substrates to yield glucose and the bioactive benzoxazinoids, have been reported to be involved in the process of resisting biotic stress (Morant et al. 2008; Cicek and Esen 2015). However, whether β-glucosidases in rice participate in the process of resisting biotic stress has not been reported. This study found that the expression levels of rice OsBGLU19-23 genes were affected during pathogen infection, especially 24 and 48 h post-inoculation with Xoc (Fig. 1-A), implying that these genes may be involved in the interaction between rice and bacterial leaf streak pathogens. The inoculation experiments with bglu19 and bglu23 mutant plants showing a more susceptible phenotype verified this hypothesis (Fig. 4-A–C). Previous studies have found that β-glycosidases can

hydrolyze substrates in vitro to form hormones such as salicylic acid (SA), tuberonic acid (TA), gibberellic acid (GA), abscisic acid (ABA) and cytokinin, participating in the synthesis of chemical defense substances, such as cyanide and sulfide, in vivo (Kiran et al. 2006; Lee et al. 2006; Morant et al. 2008; Wakuta et al. 2010; Xu et al. 2012; Himeno et al. 2013; Hua et al. 2015). Salicylic acid and jasmonic acid are important signaling molecules for plant defense responses, both with existing glycoside-containing derivatives (Chen et al. 1995; Wasternack and Hause 2013). It has been found that the Arabidopsis thaliana glycosyltransferases, UGT74F1 and UGT74F22 (Thompson et al. 2017), transfer glucose to SA to form salicylic acid glucoside and primarily salicylic acid glucose ester, respectively, but the process of deglycosylation has not been reported. Himeno et al. (2013) found that the rice β-glycosidase, Os4bglu12, can remove the sugar group from SAG to form salicylic acid in vitro. Wakuta et al. (2010) found that rice Os4bglu12 and Os4bglu13 enzymes form 12-O-glucosyl-JA in vitro, which is a glycoside derivative of jasmonic acid. The substrate specificity and partial protein structures of 11 glycosidases

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have been explored (Kiran et al. 2006; Lee et al. 2006; Morant et al. 2008; Wakuta et al. 2010; Xu et al. 2012; Himeno et al. 2013; Baiya et al. 2014; Hua et al. 2015), however, the functions of multiple other glycosidase genes have not been addressed. We found that the key gene of jasmonic acid signal pathway, OsAOS2, accumulated higher expression levels when not inoculated and was significantly inhibited after inoculation. The disease resistance-related genes in Fig. 5 play a role in different hormonal signaling, thus implying a complexity signal interaction in disease resistance mediated by Osbglu19 or Osbglu23, such as OsWRKY72 is a ABA- and auxin-inducible genes (Song et al. 2010), while expression levels of OsPR1a and OsPR1b are specifically affected/regulated by signalling molecules including SA, ABA and JA (Agrawal et al. 2001). Whether these two genes are involved in the deglycosylation of hormones or hormones derivatives in rice requires future exploration.

5. Conclusion In this study, pathogen inoculation experiments found that OsBGLU19 and OsBGLU23 positively regulate the disease resistance of rice to bacterial leaf streak. The expression level of OsAOS2 declined after inoculation with RS105 in bglu19 and bglu23 plants and the expression levels of downstream transcription factors OsWRKY72 and pathogenesis-related genes were also reduced. This indicated the participation of these genes in the interaction between rice and Xoc.

Acknowledgements This study was supported by the National Natural Science Foundation of China (31872925), the Natural Science Fund for Outstanding Young Scholars of Shandong Province, China (JQ201807), the Shandong Provincial Natural Science Foundation of China (ZR2016CB26), the funds of the Shandong “Double Tops” Program, China, the Taishan Industrial Experts Program, China (tscy20150621), the National Program of Transgenic Variety Development of China (2016ZX08001-002, 2018ZX0801023B) and the Shandong Modern Agricultural Technology & Industry System, China (SDAIT-17-06). Appendices associated with this paper can be available on http://www.ChinaAgriSci.com/V2/En/appendix.htm

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