Seeds of rice line Taipei 309 (TP309), susceptible to R. solani were available with the authors. Loc_Os11g47510, rice chitinase gene showing highest expression among other chitinase genes of the QTL qSBR11-1 was validated by qRT-PCR analysis after infection with R. solani and displayed antifungal activity during bioassay was previously cloned and reported by the authors (Richa et al., 2016).

Two different set of primers specific to Loc_Os11g47510 (from Tetep and TP309) and HptII gene were designed using Primer 3 primer designing tool (Supplementary Table 1). Primers specific for LOC_Os11g47510 gene had sequence of restriction sites BamHI and XbaI in the forward and reverse primers, respectively, whereas those for HptII gene had SacII restriction enzyme sites in both the forward and reverse primers. LOC_Os11g47510 of Tetep was amplified from a clone which was already confirmed by PCR and restriction digestion (Richa et al., 2016), LOC_Os11g47510 allele from TP309 was also amplified using same set of primers whereas HptII gene was PCR amplified from pCAMBIA1305.1. LOC_Os11g47510 was cloned at corresponding sites in pRT100 vector and confirmed. LOC_Os11g47510 allele from TP309 was cloned in pJET1.2 vector and sequence confirmed. The HptII gene was cloned into the SacII site of the pBS(SK+) vector and clones were confirmed by PCR and restriction digestion. Subsequently, the final construct for rice transformation was developed by cloning the entire PCR amplified fragment (CaMv35S + Loc_Os11g47510 + nos terminator) using primers specific to 35S promoter and nos terminator having SmaI restriction enzyme sites in both forward and reverse primers from pRT100 vector. This fragment was cloned at the corresponding restriction enzyme sites in modified pBS(SK+) vector (already having HptII gene). PCR reaction was carried out by using Finnzymes Phusion^® High-fidelity DNA polymerase using standard protocol from manufacturer (Thermo Scientific) (Supplementary Figures 1, 2). The LOC_Os11g47510 DNA sequences from Tetep and TP309 were aligned using MULTALIN tool¹ (Supplementary Figure 3).

Mature seeds of TP309 were dehusked manually and thoroughly washed with Teepol detergent followed by three washes with sterilized distil water. Sterilization of the seeds was performed by following method: seeds were dipped in 70% ethanol and gently stirred for 1 min followed by three washing with sterile water. Then 5% sodium hypochlorite treatment was given for 20 min and washing was given thrice with sterile distilled water. Finally 10 min sterilization with 0.2% HgCl₂ was done and traces of HgCl₂ was removed by washing with distilled water for five times. Sterile seeds were then drained free of water and blotted dried on a filter paper. The sterilized seeds were inoculated on callus induction media (CIM); Murashige and Skoog (MS) medium containing sucrose (30.0 g/l) + proline (500 mg/l) + casein hydrolysate (400 mg/l) + 2,4-D (2 mg/l) + agar (8.0 g/l) by placing 25 seeds per plate and incubated in dark at 28°C. Twenty-days-old embryogenic calli were excised and about 30 of them were closely arranged in a circle at the center of petriplate containing osmoticum medium (MS + 30.0 g/l sucrose + 0.2 M mannitol + 0.2 M sorbitol) supplemented with agar (8.0 g/l). The calli were ready for bombardment after 4 h of osmoticum treatment.

TP309 calli were transformed using biolistic approach (Sanford et al., 1987). Intact plasmid constructs were bombarded using helium driven particle delivery system (PDS 1000, Bio-Rad). DNA were precipitated and coated to gold particles following the procedure recommended by Sanford et al. (1987). The calli were placed in the center of petridish containing osmotic medium (MS media with 8% bacteriological agar, 30 g/l mannitol and 30 g/l sorbitol). These calli were bombarded with the DNA-coated gold particles and were then maintained at 28°C under dark condition. The distance from the stopping screen to target was 9 cm, and the rupture disk strength was 1100 psi under a vacuum of 25 mm of Hg. After 16 h of bombardment, calli were transferred to the selection medium; CIM + Hygromycin (50 mg/l) and incubated in dark at 28°C. After 15 days, the proliferating calli were again transferred to the fresh selection medium for two more rounds of selection of 15 days each. After a total of 45 days of selection, healthy and proliferating calli were transferred to the regeneration media.

The calli survived after three round of selection in hygromycin containing medium were transferred to the regeneration medium [MS + BAP (2.5 mg/l) + NAA (1.5 mg/l) + agar (8 g/l) + hygromycin (50 mg/l)] and incubated at 28°C under 1000 lux with 16 h light and 8 h dark regime. Entire process of selection and regeneration took about 2–3 months for completion. As the plantlets reached 2 to 3 inch of height with well developed roots, they were acclimatized in solarite and finally transferred to the National Phytotron Facility (NPF), IARI, New Delhi, India and maintained under standard conditions required for rice.

The transgenic plants obtained from the present study were validated using molecular biology tools like PCR, qRT-PCR and Southern blot hybridization. To determine the presence of the transgene, genomic DNA was extracted from leaf tissues of transgenic plants and analyzed using PCR. For PCR validation, one set of primers specific to promoter and LOC_Os11g47510 gene and another set specific for HptII gene were used. The details of different primers used and the annealing temperature used for PCR are given in Supplementary Table 1. For PCR, DNA was denatured at 94°C for 3 min, followed by 35 cycles of amplification [45 s at 94°C; 30 s at 58°C; 45 s at 72°C] and final incubation at 72°C for 10 min. For gene integration analysis, we performed southern hybridization. High quality genomic DNA was isolated by CTAB method. 10 μg gDNA was completely digested with EcoRI restriction enzyme. Digested DNA samples along with positive control were electrophoresed on 0.8% agarose gel in 1× TBE buffer. After electrophoresis, DNA samples were denatured and transferred on to the nylon membrane. 192 bp fragment of the HptII gene was labeled with α-dUTP and used as a probe for hybridization. After that, southern blotting was performed by using standard protocol (Sambrook et al., 1989).

Real-time quantitative Reverse Transcription PCR (qRT-PCR) analysis was performed to analyze the relative expression of the integrated chitinase gene as well as hptII gene in control and transgenic TP309 plants, separately. Total RNA was isolated from the leaf samples of control and transgenic leaves using RNeasy Plant Mini Kit (Qaizen, USA). Quality and quantity of RNA was analyzed by agarose gel (1.0%) electrophoresis and nanodrop (Thermo Scientific), respectively. cDNA was prepared from 1 μg of each of total RNA sample using Protoscript first strand cDNA synthesis kit (New England Biolabs, UK). 18srRNA primer was used as internal control to normalize the data. Specific primers were designed for LOC_Os11g47510, hptII and 18srRNA gene and used for qRT-PCR (Supplementary Table 1). cDNA prepared was used as a template for expression analysis by Light cycler SYBER green I master Kit using Light Cycler^® 480 II PCR system (Roche) using manufacturer’s guidelines. The qRT-PCR cycling conditions were: initial denaturation 95°C; 5 min, 50 cycles of 95°C; 10 s, and annealing temperature 60°C; 15 s, 72°C; 15 s.

Detached leaf bioassay was performed in control and transgenic plants to check phenotypic response of these plants mediated by the candidate gene against R. solani. For detached leaf assay, mycelium of R. solani was inoculated on PDA plates. Equal sized agar plugs containing mycelium of R. solani were inoculated on the surface of detached leaves (transgenic and control plant) and they were then placed on the moist filter paper in the petriplates. Petriplates were then sealed with parafilm to retain the moisture inside the plates. Phenotypic observation was recorded as degree of infection of R. solani at different hpi. Statistical analysis was performed for the three replicates of each of the transgenic plant.

Microscopic study was performed to study the growth of R. solani mycelium in transgenic as well as NT-TP309 leaves. Leaves were placed over wet filter paper in the petriplates and inoculated with agar plugs containing R. solani mycelia. At 24, 48 and 72 hpi, leaves were fixed on filter papers soaked with 1:1 (v/v) Ethanol: Acetic acid for 24 h and later transferred on to filter paper soaked in lacto glycerol [1:1:1 (v/v) ratio of lactic acid:glycerol:water). Samples were then stained with aniline blue (0.1% w/v) in 0.1 M K3PO4 and excess dye was removed using 0.1 M K3PO4. Leaves were mounted on to microscopic slides in 10% glycerol and observed under light microscope.

Standard calibration curves were established to measure the fungal biomass in the R. solani inoculated samples in T₁ generation. Pure R. solani fungal gDNA was used for serial dilutions to obtain 0.002, 0.02, 0.2, 2 and 20 ng/μl of gDNA. pUC19 plasmid DNA (Thermo Fisher Scientific) was also diluted to obtain 0.001, 0.01, 0.1, 1, and 10 ng/μl DNA. These diluted DNA were mixed separately with a constant amount of 10 ng of genomic DNA isolated from uninfected TP309 leaves. Calibration curves were generated by plotting CT values (averaged from three technical repeats) obtained by qRT-PCR against the corresponding logarithmic R. solani and pUC19 DNA amounts. For the absolute quantification of fungal DNA in inoculated leaf samples, 20 ng/μl genomic DNA was used as a template. Two qPCR reactions were run separately by using the (Rhizoctonia Map Kinase gene) RPMK1_F and RPMK1_R and M13-F and M13-R pair of primers. CT values obtained were then used to calculate the absolute amount of target DNA in a given reaction. For data normalization, 0.1 ng of pUC19 DNA was mixed with all the transgenic and NT plants DNA.

Chitinase gene from rice line Tetep, present on the locus LOC_Os11g47510, which was showing higher relative expression in qRT-PCR as well as highest chitinase activity in in vitro expression study was selected for plant transformation experiments (Richa et al., 2016). The putative recombinant pBS(SK+) plasmids with LOC_Os11g47510 gene cassette (CaMv 35S promoter + LOC_Os11g47510 gene+ nos terminator) were screened for gene integration by PCR using primers specific to this region and also by restriction digestion using BamHI (Figure 1). The PCR reaction (1.6 kb promoter and gene fragment along with nos terminator) and restriction digestion confirmed (∼1.2 kb gene fragment) the presence of gene cassette and gene, respectively (Figure 1 and Supplementary Figure 2). This particular gene cassette was cloned in a modified pBS(SK+) vector which is having hptII gene for plant selection and this modified plant transformation vector was used for genetic transformation experiments and referred to as pShB-510.

Genetic transformation of rice line TP309 was performed with gene cassette containing DR gene Loc_Os11g47510 using biolistic approach. For transformation, healthy calli of TP309 were selected and used for bombardment using particle gun. Sixteen hour post bombardment, the calli were transferred to the selection media containing 50 mg/l hygromycin. At the end of three selection cycles of 15 days each, out of 428 and 400 calli initially used for bombardment in two different set (batches) of experiments, 148 and 112 transformed calli, respectively, were obtained. The proliferating, healthy calli were then transferred to the regeneration medium supplemented with combination of auxin and cytokinin (NAA & BAP) along with hygromycin for selection (Supplementary Figure 4). Successfully regenerated plants from individual calli from each experiment were treated as an event and subjected to hardening. Finally 51 plants from nine events were transferred to the pots and grown in National Phytotron Facility (NPF, IARI, New Delhi, India) and seeds were obtained from eight out of the nine events. Regeneration efficiency was determined for different batches by calculating the percentage of regenerated calli from total number of calli subjected to regeneration after third selection cycle. The overall regeneration frequency was found to be around 55.3%, however, overall transformation efficiency achieved was 6.25% which was validated by molecular analysis (Supplementary Table 2).

To confirm the presence and integration of the candidate gene, LOC_Os11g47510 in putative transgenic plants, PCR and Southern blot hybridization was performed using genomic DNA isolated from healthy and normal looking putative transgenic plants (Figures 2A,B,C). PCR amplification using two primer sets: one set specific to hptII and another set specific to promoter region and transgene was performed. PCR results revealed that out of 35 plants, only 12 plants belonging to eight events were positive for both transgene and hptII gene.

Further, copy number of the integrated gene LOC_Os11g47510 was analyzed by Southern hybridization in all eight PCR positive putative transformed rice plants (Figure 2C). Hybridization was performed using DIG labeled HptII specific DNA probe in this study. Single and double insertions were observed in the putative transgenics and no insertion was found in the non-transgenic TP309 (NT) plant confirming the integration of the transgene in transgenics. Different positioning of the transgene in the autoradiogram confirmed that they are independent events. T1 seeds of the putative transformants were screened on hygromycin containing media and raised in the phytotron. Their transgenic nature was also confirmed by molecular analysis (Figure 2D).

Expression study of the gene LOC_Os11g47510 in transgenic plants was performed using qRT-PCR technique. All four Southern blot positive transgenic plants along with control (NT) TP309 plant were screened with the primers specific to Loc_Os11g47510 transcribed region. 18S rRNA gene specific primers were used as an internal control in the present experiment for data normalization. The results obtained indicated varying level of transgene expression, ranging from 3.7- to 9.5-folds in different transgenic events (Figure 3). The expression of hptII in NT and transgenic plants revealed no amplification in NT-TP309, but varying levels of expression of four transgenic lines (Supplementary Figure 5).

Detached leaf bioassay experiments using transgenic and NT-TP309 plants confirmed the transgene mediated resistance response in transgenic plants in comparison to NT plants (Figure 4 and Supplementary Figure 6). At 36 hpi, size of the lesions developed on transgenics was comparatively smaller than control NT-TP309 plants. After 72 hpi, almost whole leaf of NT-plant was infected with sheath blight pathogen R. solani, whereas the infection on the transgenics was restricted to small lesions.

Observations of R. solani infected leaves revealed that lesion development begins at 24 hpi in NT-TP309 leaves whereas no lesion was observed in transgenic TP309 leaves. Mycelia of R. solani grew and colonized surface of the leaves in both transgenic and NT plants. After colonization, hyphal branches were seen and they either penetrated rice cells directly or formed lobate appressorium and invaded leaf tissue via epidermal intercellular spaces as well as through stomata and trichomes (Figure 5A). Usually, R. solani hyphae tends to grow in bunch and ultimately form infection cushions. Similarly we observed penetration peg or infection cushion in NT leaves at 24 hpi, and after 24 hpi number of infection cushions increased with increase in time (at 48 and 72 hpi), whereas in case of transgenic rice leaves infection cushion was observed only at 72 hpi (Supplementary Figure 6). The size of the hyphal mat at the site of colonization and also lesion size was comparatively less in the transgenic lines. We also recorded the number of infection cushions per microscopic field in both transgenic and NT leaves at each time interval. At 72 hpi, the number of infection cushions per microscopic field in transgenic plant leaves was only 2 as compared to 8.3 in NT plant leaves (Figure 5B).

Absolute quantification of the fungal biomass in case of transgenic as well as NT rice leaves inoculated with R. solani was analyzed by using qPCR. The results obtained revealed that with progress in time interval, the amount of the fungal DNA has increased in both NT as well as in transgenic leaves; however, the amount of fungal DNA found in the case of NT leaves was much higher than the amount in transgenic plants (Figure 5C and Supplementary Figure 7).