OsBSK1-2, an Orthologous of AtBSK1, Is Involved in Rice Immunity

The brassinosteroid-SIGNALING KINASE (BSK) belongs to the receptor-like cytoplasmic kinase XII subgroup. BSK1 regulates development and immunity in Arabidopsis. However, the function of rice (Oryza sativa) BSK1 is largely unknown. Here, we report that the expression level of OsBSK1-2 is induced after a chitin or fagellin22 (flg22) treatment. Silencing OsBSK1-2 in rice results in compromised responses to chitin- or flg22-triggered immunity and resistance to Magnaporthe oryzae, but does not alter the plant’s architecture nor reduce plant responses to brassinosteroid signaling. Our study reveals that OsBSK1-2 functions as a major regulator in rice plant immunity.


INTRODUCTION
Receptor-like cytoplasmic kinases (RLCKs) are a subgroup of the receptor-like kinase family, but unlike the receptor-like kinases, which have an extracellular receptor domain, a transmembrane domain, and an intracellular kinase domain (Shiu and Bleecker, 2001), RLCKs just have a typical catalytic kinase, and they localize to the cytoplasm. Several RLCKs are predicted as regulators of plant development and immunity (Vij et al., 2008).
Receptor-like cytoplasmic kinases VII is an important gene subfamily in plant. In Arabidopsis, BOTRYTIS INDUCED KINASE 1 (BIK1) was first isolated as an early-induced kinase in response to infection by Botrytis cinerea (Veronese et al., 2006). BIK1 was then revealed to play an essential role in regulating immune responses mediated by pattern recognition receptors (PRRs), including flagellin-sensitive-2 (FLS2), EF-Tu receptor (EFR), and PEP-RECEPTOR1 (PEPR1) (Zhang et al., 2010;Eckardt, 2011;Liu et al., 2013). The orthologs of BIK1, PBS1, PBL1, PBL2 and PBL27, were also identified as regulators for pattern-triggered immunity (PTI) (Zhang et al., 2010;Lin et al., 2013;Liu et al., 2013;Yamaguchi et al., 2013a,b;Shinya et al., 2014). Other members of the RLCK VII subfamily have also been characterized as regulators in rice immunity. For examples, OsRLCK185, which is phosphorylated by OsCERK1 upon recognition of chitin and then dissociates from the OsCERK1 complex to activate MAPK cascades, is involved in an immune response (Yamaguchi et al., 2013a,b). OsRLCK57, OsRLCK107, OsRLCK118 and OsRLCK176, four homologs of BIK1, positively regulate immune responses by contributing to the expression of the immune receptor XA21 (Zhou et al., 2016).
Recently, brassinosteroid (BR)-signaling kinase1 (BSK1), a member of RLCK-XII, which belongs to another RLCK subfamily, was reported to play vital roles in plant immunity. BSK1, first isolated as a phosphorylation substrate of the BR receptor kinase BRI1, plays critical role in BR signaling (Tang et al., 2008). In Arabidopsis, 12 BSK members were predicted (BSK1-12). Biochemical assays revealed that BRI1 targets many BSKs for phosphorylation, including BSK1, BSK3, BSK5, BSK6 and BSK11, suggesting that these BSKs might have redundant functions in response to BRs (Tang et al., 2008;Sreeramulu et al., 2013). Moreover, the bsk1 mutant showed compromised resistance to various pathogens and responses to PTI. BSK1 associates with the PRR FLAGELLIN SENSING2 (FLS2) to activate downstream signal transduction in plant immunity (Shi et al., 2013). In rice, a member of the RLCK-XII subgroup, OsBSK3, plays a positive role in BR signaling, which can be phosphorylated by OsBRI1 and then associates with BRI1 SUPPRESSOR1 to transduce the BR signal (Zhang et al., 2016). However, the biological roles of their functions in rice immunity are poorly known.
In this study, we report that the enrichment of the transcripts levels of OsBSK1-2 was induced by both flg22 and chitin. Silencing OsBSK1-2 reduced the transcriptional expression of the pathogenesis-related genes, Os04g10010 and OsPR10b, in response to the treatment of flg22 or chitin and compromised plant resistance to Magnaporthe oryzae. However, plants with reduced OsBSK1-2 expression levels did not alter plant architecture or BR signaling. Thus, our study reveals that OsBSK1-2 functions as a positive regulator in rice immunity.

Plant Growth
All of the transgenic plants were created in the 'Kitaake' background. All of the plants were grown in a controlled rice field in Chengdu, Sichuan, China.

Construction
All of the primers used here are listed in Supplementary  Table S1. To generate RNA interference (RNAi) construction, the unique cDNA sequence of OsBSK1-2 was amplified and cloned into the pCRR8 TM /GW/TOPO R (Invitrogen, Carlsbad, CA, United States) vector to create the pCR8-OsBSK1-2Ri constructs. Then, the RNAi fragments were inserted into the pANDA vector (Miki and Shimamoto, 2004) using LR recombination to create the RNAi construct pANDA-OsBSK1-2Ri (OsBSK1-2Ri).

Generation of Transgenic Rice
OsBSK1-2Ri was introduced into 'Kitaake' plants through Agrobacterium-mediated transformation, and hygromycin was used to select the regenerated plants (Chern et al., 2005).

RNA Extraction and Quantitative Real Time RT-PCR Analyses
Total RNA was prepared from samples collected using Trizol (Invitrogen) according to the manufacturer's instructions. The RNA samples were then subjected to cDNA synthesis system with RT reagent Kit with gDNA Eraser (Takara, Dalian, China). A Quantitative RT-PCR Kit was used to establish the qRT-PCR reactions (Qiagen, Valencia, CA, United States). The gene expression levels were normalized to the transcript level of the reference gene ubiquitin5 (LOC_06g46770) gene. All of the primer pairs used for qRT-PCR, which are listed in Supplementary Table S2, have been tested for efficiency and specificity following the guidelines for successful real time 1 . qRT-PCR analysis were performed follow the MIQe guidelines (Bustin et al., 2009).

Defense Gene Expression Analysis
The 2-3 cm long leaf strips from fully developed leaves of 4-week-old seedlings were incubated for 12 h in ddH 2 O. They were then treated with 1 µM flg22 peptide (Pacific Immunology, San Diego, CA, United States) or 20 µg/ml chitin from shrimp shells (Sigma, St. Louis, MO, United States) according to a method described previously (Felix et al., 1999). The samples were collected after 12 h for further investigation.

Brassinolide (BL) Treatment
To analyze the effects of BL on the lamina joint angles of the transgenic plants, 1 µl 2,4-epiBL (100 ng/µl) was applied to the leaf angles and the lamina joint angles were measured following a method described previously .

Phylogenetic and Molecular Evolutionary Analyses
To identify ortholog(s) of AtBSK1 in the rice genome, the amino acid sequence of AtBSK1 was used as the seed sequence to perform a BLAST algorithm-related search of the rice protein database 2 . The proteins with hit scores more than 1,000 and E-value less than 1.6e −100 , were chosen for further study. Twelve AtBSK1 and five OsBSKs were first aligned using ClusterX software. Then, a phylogenetic tree was constructed using MEGA5.10 software. The evolutionary history was inferred using the Maximum Likelihood method based on the Poisson correction model. The tree with the highest log likelihood (−6702.6606) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured using the number of substitutions per site (Tamura et al., 2011).

Rice Blast Incubation
Magnaporthe oryzae isolates, ZHONG1 and ZB25, were used for the inoculations. Leaf strips from 8-week-old plants were lightly wounded with a mouse ear punch, and 5 µl of spore suspension (5 × 10 5 spores/ml) was added to the wound. The lesion size was measured after incubating for 8-14 days at 28 • C. The DNA was then extracted to calculate the expression level of the Pot2 gene of M. oryzae relative to the genomic Ubiquitin in rice (Park et al., 2012).

Characterization of OsBSK1-1 and OsBSK1-2
Many BR signaling partners have been characterized in rice, and they have functions that are similar to those of their orthologs in Arabidopsis (Tong and Chu, 2012). AtBSK1 was reported to regulate Arabidopsis immunity. We therefore presumed that the rice ortholog(s) of AtBSK1 should function in rice immunity. To test this hypothesis, we identified five OsBSKs, OsBSK1-1 (LOC_Os03g04050), OsBSK1-2 (LOC_Os10g39670), OsBSK2 (LOC_Os10g42110), OsBSK3 (LOC_Os04g58750), and OsBSK4 (LOC_Os03g61010) from rice genome by blast research using the amino acid sequence of AtBSK1 as the seed sequence. The result showed that two proteins, OsBSK1-1 (LOC_Os03g04050) and OsBSK1-2 (LOC_Os10g39670) which share more than 79% similarity with each other in amino acid sequence were sub-grouped with AtBSK1 in phylogenetic and molecular evolutionary analyses (Figure 1).
To determine the expression patterns of OsBSK1-1 and OsBSK1-2, we measured their transcription levels in different developmental stages using quantitative reverse transcription-PCR (qRT-PCR). Both OsBSK1-1 and OsBSK1-2 were predominantly expressed in the four-leaf stage of development, and the transcripts of OsBSK1-2 were more abundant than those of OsBSK1-1 (Figure 2).
Silencing OsBSK1-2 Inhibits Flagellinand Chitin-Triggered Immune Responses in Rice BSK1 associates with FLS2 to regulate plant PTI in Arabidopsis (Shi et al., 2013). To test whether OsBSK1-1 and OsBSK1-2 regulate PTI in rice, we collected leaf strips that were 2-3 cm in length from fully developed leaves of 4-weekold Kitaake seedlings and treated them with flg22 peptide or chitin. Then, we detected the transcription levels of OsBSK1-1 and OsBSK1-2, and found that OsBSK1-2 was induced after the flg22 and chitin treatments, while OsBSK1-1 was not (Figure 3). We then chose OsBSK1-2 for further studies.
FIGURE 1 | Phylogenetic analysis on five OsBSKs and 12 Arabidopsis BSKs. Five OsBSKs and 12 AtBSKs were subjected to the ClustalX analysis with default parameters. Phylogenetic was built using MEGA version5.10. Two orthologous of AtBSK1 were identified as OsBSK1-1 and OsBSK1-2.
FIGURE 2 | Determination of the expression patterns of OsBSK1-1 and OsBSK1-2. qRT-PCR analysis was performed on cDNA synthesized from RNA samples. The RNA samples were extracted from tissues of rice cultivar Kitaake at the development stages as indicated. The expressions of the OsBSK1-1 and OsBSK1-2 were normalized to level of the ubiquitin reference gene. Error bars indicate SD obtained from three technical replicates. The letters indicate significant differences as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis.  -1 (B), respectively, in rice leaves underflg22-and chitin-treatments. The Results from two independent biological experiments were combined with statistical analysis. The letters indicate significant differences determined by a one-way ANOVA followed by post hoc Tukey HSD analysis.

Silencing of OsBSK1-2 Compromises Plant Resistance to M. oryzae
To further investigate the genetic role of OsBSK1-2 in rice immunity, we inoculated the OsBSK1-2Ri transgenic and wild type plants with the M. oryzae isolate ZB25. Ten days postinoculation, the lesion size of OsBSK1-2Ri-1 (12.6 ± 1.7 cM for lesion length and 2.2 ± 0.1 cM for lesion width) and OsBSK1-2Ri-2 (12.2 ± 1.0 cM and 2.3 ± 0.2 cM) plants displayed larger lesions than the Kitaake plants (5.1 ± 0.9 cM and 1.5 ± 0.2 cM) (Figures 5A-C). To determine the fungal biomass in the inoculated leaves precisely, we isolated the total DNA from the infected leaves and quantified it using a DNA-based quantitative PCR method (Park et al., 2012). Relative to the reference rice Ubiquitin gene, the DNA accumulation level of fungal gene Pto2 in OsBSK1-2Ri-1/-2 plants was higher than in Kitaake plants ( Figure 5D). This result revealed that the fungal biomass was more accumulated in the infected OsBSK1-2Ri-1/-2 than in the wild type Kittake leaves. We then inoculated OsBSK1-2Ri and wild type plants with another compatible M. oryzae isolate, ZHONG1, and obtained similar results (Supplementary Figure S2).
To further confirm the results, we inoculated segregants derived from the OsBSK1-2Ri transgenic plants with the M. oryzae isolate ZB25 and determined the resistance. The segregants carrying the silencing constructs showed larger lesions than wild type Kittake, while segregants lacking the silencing constructs had a similar lesion size as the wild type (Supplementary Figure S3). Therefore, we concluded that silencing OsBSK1-2 resulted in compromised rice resistance to M. oryzae.
Silencing of OsBSK1-2 Does Not Alter Plant Architecture or Sensitivity to BR To explore the biological role of OsBSK1-2 in plant development, we analyzed the plant architecture of OsBSK1-2Ri plants, including plant height or lamina joint. There were no obvious differences in plant height or lamina joint between OsBSK1-2Ri and Kittake plants (Figure 6 and Supplementary  Figures S4A,B).
To determine whether OsBSK1-2 regulated plant responses to the hormone BR, we treated the lamina joint of the wild type and OsBSK1-2Ri plants with BR. The lamina joint of OsBSK1-2Ri plants was seriously enlarged after the BL treatment, which was similar to the wild type (Figure 7 and Supplementary Figure S4C). These results suggest that silencing OsBSK1-2 does not alter plant architecture or responses to BRs.   . The BL treatment contained 2,4-epiBL (100 ng), whereas Eth alone was used for the mock treatment. (B) Statistical analysis of the leaf angles of Kitaake and OsBSK1-2Ri after BL and mock-treatments, respectively. Each bar represents the average and SD of 10 leaves. The letters indicate significant differences as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Two independent repeats were performed with similar results produced.

DISCUSSION
In Arabidopsis, a total of 12 BSKs have been isolated, and play various function in plant development and immunity (Tang et al., 2008;Sreeramulu et al., 2013). Although five OsBSKs identified in rice share a conserved kinase domain and a C-terminal tetratricopeptide repeat domain, their genetic functions in plant immunity and development might be different.
OsBSK1-1 and OsBSK1-2 are two homologs that also share similar expression patterns in rice developmental stages. However, the transcripts of OsBSK1-2 are more abundant than those of OsBSK1-1 in rice (Figure 2), suggesting that OsBSK1-2 might play more important roles in rice. In agreement, the expression level of OsBSK1-2, but not OsBSK1-1, is obviously induced after flg22 and chitin treatments (Figure 4). Therefore, OsBSK1-1 and OsBSK1-2 might play different roles in plant immunity. OsBSK1-2 is an ortholog of AtBSK1, which also regulate FLS2-mediated immunity as AtBSK1 (Shi et al., 2013). Although, no direct interaction between OsBSK1-2 and OsFLS2 was determined, OsBSK1-2 might still play a similar molecular mechanism as does AtBSK1 in plant immunity because silencing of OsBSK1-2 also compromised the base immune response in rice (Figure 4).
Brassinosteroid-SIGNALING KINASE are important regulators of BR signaling (Tang et al., 2008;Sreeramulu et al., 2013). However, we did not observe the BR-related phenotypes in rice plants in silence of OsBSK1-2. BSK1 is phosphorylated by BRI1 upon BR perception and disassociates from BRI1 to activate BRI1 SUPPRESSOR1 in Arabidopsis (Tang et al., 2008;Kim et al., 2009). BRI1 interacts with and phosphorylates the BSKs, BSK1, BSK3, BSK5, BSK6, BSK8 and BSK11, to activate downstream signaling (Sreeramulu et al., 2013). Nevertheless, knocking out any individual AtBSK in Arabidopsis does not affect plant architecture because of their redundant functions (Sreeramulu et al., 2013). LOC_Os04g58750 (OsBSK3) interacts with OsBRI1 in vivo, and the over-expression of OsBSK3 increases the hypersensitivity of rice plants to the hormone BR (Zhang et al., 2016). Although the reduced expression of OsBSK1-2 does not alter the BR-triggered response in rice plants (Figure 4), it does not rule out the possibility of its involvement in the regulation of BR-mediated signaling because of the possible redundancies of the other four OsBSKs. Thus, it needs to generate double or multiple knock-out mutants for the OsBSKs in rice to determine whether OsBSK1-2 regulates BR-mediated signaling and plant development for the future studies.

AUTHOR CONTRIBUTIONS
JW and XC conceived this study. HS, LZ, CP, DL, XZ, WW, JY, HQ, WM, JCW performed the experiments. JW, MH, WL, SL, and XC analyzed data. JW and XC wrote the manuscript. All authors approved the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2017.00908/ full#supplementary-material FIGURE S1 | Characterization of the transgenic plants silenced for OsBSK1-2. The relative expression levels of OsBSK1-2 (A) and OsBSK1-1 (B), were determined in the transgenic lines and the wild type Kitaake plants by qRT-PCR. All data were normalized to the reference ubiqutin gene. The average and SD for the relative expression of each line is shown. Data were obtained from three technical replicates. The letters indicate significant differences as determined by a one-way ANOVA followed by post hoc Tukey HSD analysis. Three independent biological experiments were performed and similar results were obtained.  Eth solution containing 2,4-epiBL (100 ng) was used for the BL treatment, whereas Eth alone was used for the mock treatment.