Materials of plants, insect populations, and rice blast pathogen strain
Rice (Oryza sativa L.) varieties of Zhonghua 11 (ZH11), Nipponbare (Nip), and Taichung Native 1 (TN1) were developed and maintained at our institute (Shanghai, China). The transgenic lines of osmyc2 were generated by genetically modifying WT Nip plants. It is derived from laboratory preservation. All plants were grown in the fields at the experimental station at Songjiang Shanghai of CEMPS or in a greenhouse at 28 ± 2°C with a 12-h light/12-h dark cycle and 70%–80% relative humidity.
The BPH populations used in this study, which were originally obtained from rice fields in China National Rice Research Institute (Fuyang, China), comprised biotypes 1 and 2 (Sun et al., 2005). They were maintained on the susceptible rice cultivar TN1 plants in a climate-controlled room at 26 ± 2℃, with a 12-h light/12-h dark cycle and 80% relative humidity. The second or third instar nymphs were used for experiments.
The rice blast pathogen (M. oryzae TH12) was kindly provided by Prof. Zuhua He (CAS Center for Excellence in Molecular Plant Sciences, Shanghai, China).
Plant inoculations
Plants were inoculated with the rice blast pathogen under field conditions using previously described punch and injection inoculation methods (Li Y, 2020; Tian et al., 2016). The ZH11 plants , osmyc2 mutant plants and Nip plants were sown in a field in Songjiang Shanghai. To prepare the inoculum, TH12 was grown on complete agar medium in plates for 2 weeks. Spores were collected by flooding the surface of the medium with sterile water. The spore concentration of the resulting suspension was adjusted to 1×105 spores mL-1 before the punch and injection inoculations.
For the punch inoculation, detached rice leaves (6 cm) were cut in three places. Using a transfer pipette, 5 μL TH12 spore suspension was added to each cut site. The inoculated leaves were maintained in culture dishes containing 0.1% 6-benzylaminopuricn (6-BA) sterile water to ensure they did not dry out. The culture dishes were placed in an incubator set at 28°C. The typical rice blast disease symptom is a fusiform lesion with a gray center surrounded by brown necrotic tissue and a light-yellow outer layer. The lesion length was measured using a ruler at 7 days post-inoculation to determine the differences in the resistance of the rice varieties to TH12. The test was replicated 15 times.
Evaluation of BPH resistance
The resistance of individual rice seedlings or a small population of seedlings to BPH was evaluated according to a previously described method that was slightly modified (Du et al., 2009; Guo, et al, 2014; 2020).
To analyze individual plants, a single rice seedling was grown in a 7 cm square pot with a plastic cage (6 cm diameter; 30 cm height). At the 3-leaf stage, 15 second or third instar BPH nymphs were added to each seedling. The test was replicated six times.
To evaluate small populations, approximately 30 seeds per rice variety were sown in rectangular plastic boxes (length × width × height = 30 × 20 × 8.5 cm). At 7 days after sowing, the seedlings were thinned so 28 plants remained per plastic box. At 20 days after sowing, five second or third instar BPH nymphs were added to each seedling. The test was performed in triplicate.
Rice treated with TH12 followed by BPH treatment
10 ‘ZH11’ seeds were sown in a 10 cm diameter plastic pot with a hole at the bottom. At the two- to three-leaf stage, the seedlings were sprayed with 1 mL TH12 spore suspension (1×106 spores mL-1) and 100 first-instar BPH nymphs were released on the seedlings 24 h after.
MeJA treatment
10 ‘ZH11’ seeds were sown in a 10 cm diameter plastic pot with a hole at the bottom. At the two- to three-leaf stage, the seedlings were sprayed with 100μM MeJA and conduct multiple time point sampling. Sampling is repeated three times for each time point.
Sample collection for the RNA-seq analysis
At 45 days after sowing, 1 mL TH12 spore suspension (1×105 spores mL-1) was injected into the leaf sheaths of ZH11 plants. The leaves were collected 0, 12, and 24 h later, with three biological replicates per time-point. A total of 9 samples were collected.
RNA isolation and RNA-seq analysis
Total RNA was isolated from the 9 prepared samples using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA was used to prepare libraries, which were sequenced on the BGISEQ-500 analyzer at BGI (Shenzhen, China). The clean reads were assembled de novo as contigs using the software of SOAPdenovo. All the reads were then realigned onto contigs according to the paired-end reads overlap relationship, and all these contigs were joined into scaffold sequences. Finally, the intra-scaffold gaps were filled using the paired-end extracted reads. Those sequences that could not be extended in either direction were termed unigenes and for differentially expressed genes (DEG) analysis.
RT-PCR and qRT-PCR assays
Total RNA was isolated from the collected samples using the TRIzol reagent (Invitrogen). The RNA served as the template for synthesizing cDNA using the First Strand cDNA Synthesis Kit (Toyobo). The RT-PCR analysis was performed to examine the transcripts of target genes. The qRT-PCR analysis was performed using the SYBR Green Real-time PCR Master Mix Kit (Toyobo). A rice actin gene (Os03g50885) was used as the internal control for both the RT-PCR and qRT-PCR analyses. Details regarding all primers are listed in Supplementary Table S1.
Determination the content of eriodictyol, naringenin and quercetin
A standard curve of eriodictyol, naringenin and quercetin (Manufacturer) was made as follows: 50 ng/ml, 10 ng/ml, 5 ng/ml, 1 ng/ml, 0.5 ng/ml, and 0.1 ng/ml. Rice seeds of ZH11 were hydroponically cultured for 3-4 days for accelerating germination before transplanting into pots. One month after sowing, ZH11 plants treated with TH12 or treated with MeJA, 50 mg of the sturdy flag leaf on the same part of the plant was sampled, grinded in 300 μL acetonitrile and shaked for 1 hour. 20 mg of sodium chloride was added, vortexed for 1 minute, and centrifuged at 14000 rpm for 10 minutes. The supernatant was centrifuged for another 5 minutes and then the supernatant was taken. The sample was placed in a weighing bottle, and the content was determined using a UPLC-QTRAP 5500 instrument.
Dual luciferase analysis
The plasmid PHB-35S-MYC2-Nos was transformed into A. tumefaciens strain GV3101 to act as an effector. The reporter construct was prepared by cloning the OsF3'H promoter into the pGreenII0800-LUC vector, and subsequently co-transformed
with the helper plasmid pSoup19 into A. tumefaciens strain GV3101 to act as the reporter. The bacterial cultures from the experimental and control groups were infiltrated into opposite ends of the same tobacco leaf (N. benthamiana). The leaves were collected after 3 d under long-day white-light conditions and infiltrated with 150 lg/ml luciferin solution. Images were captured using a CCD camera 5 min later, and quantification was performed using the Dual-Luciferase Reporter Assay System according to the instructions (Promega, Madison, WI, USA).Three biological repeats were measured for each sample.
Electrophoretic mobility shift assay
Expression and purification of DNA-binding regions of OsMYC2. The Cy5-labelled OsF3H probe was amplified using two rounds of PCR. The DNA probes and proteins were co-incubated in the reaction buffer, purified, and incubated with the Cy5-labeled probe at 37°C for 20 min in electrophoretic mobility shift assay (EMSA) buffer (25 mM Hepes (pH 7.5), 40 mM potassium chloride, 3 mM dithiothreitol, 10% glycerol, 0.1 mM EDTA, 0.5 mg/ml bovine serum albumin, 0.5 mg/ml poly-glutamate). After incubation, the reaction mixture was electrophoresed on a 6% native polyacrylamide gel, and then labeled DNA was detected using a Starion FLA-9000 instrument (Fujifilm, Tokyo, Japan).
Subcellular localization
The CDSs of the indicated genes were amplified by PCR and inserted into pC1300-35S-eGFP. The proteins fused with eGFP were transiently expressed in N. benthamiana as described above. After staining with DAPI to visualize the nucleus, the GFP and DAPI signals were examined 48 h later using a confocal microscope (LSM 880; Zeiss).