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.

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^® (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).

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

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 time¹. qRT-PCR analysis were performed follow the MIQe guidelines (Bustin et al., 2009).

The 2–3 cm long leaf strips from fully developed leaves of 4-week-old seedlings were incubated for 12 h in ddH₂O. 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.

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 (Chen et al., 2013).

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 database². 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).

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 × 10⁵ 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).

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).

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-week-old 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.

To analyze whether OsBSK1-2 functions in rice immunity, we generated OsBSK1-2 RNAi transgenic rice lines. Four independent transgenic lines (OsBSK1-2Ri) with reduced expression levels of OsBSK1-2 were obtained (Supplementary Figure S1).

The genes Os04g10010 and OsPR10b function as markers involved in the downstream responses associated with PTI (Park et al., 2012; Chen et al., 2014). We thus examined the transcription levels of these two genes in OsBSK1-2Ri transgenic plants after the flg22 or chitin treatments. In Kitaake plants, the expressions of Os04g10010 and OsPR10b were induced by 17.0 ± 0.9- and 10.3 ± 0.6-fold, respectively, after the chitin treatment (Figure 4). However, the expression levels of Os04g10010 and OsPR10b were induced only about 12.7 ± 0.7- and 5.1 ± 0.8-fold in OsBSK1-2Ri-1 and 10.6 ± 0.7- and 4.2 ± 0.5-fold in OsBSK1-2Ri-2 plants, respectively. Similarly, the expression levels of Os04g10010 and OsPR10b were induced only about 25.6 ± 0.1- and 8.2 ± 0.1-fold in OsBSK1-2Ri-1 and 26.1 ± 2.2- and 8.6 ± 0.8-fold in OsBSK1-2Ri-2 plants, respectively, whereas induced about 41.0 ± 2.1- and 12.4 ± 0.2-fold in the wild type Kitaake after the flg22 treatment (Figure 4). Taken together, these results suggest that the silencing of OsBSK1-2 compromises the base immune response in rice. Thus, OsBSK1-2 might be involved in the regulation of rice PTI.

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 post-inoculation, 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.

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.