As previously stated by Lv et al. (2014), the near-isogenic line Bph15-NIL (XF07-151) was found to possess the resistance gene Bph15 in the Taichung Native 1 (TN1) background. This particular line, Bph15-NIL, was employed as a control in the study due to its demonstrated resistance. In contrast, the varieties TN1 and Nipponbare (NIP) were selected as susceptible rice controls. The BPH insects, which were reared on the TN1 variety, were utilized for plant feeding during the research conducted at Wuhan University in China.

The wrky71 mutants were generated using CRISPR-Cas9 technology (Ma et al., 2015). The target sequence of WRKY71 was designed and used to generate sgRNA expression cassettes driven by the OsU6a promoter. Using an Agrobacterium-mediated technique, the resulting structure was transformed into Bph15-NIL plants. The confirmation of T₀ transgenic plants was achieved through genomic targeting by directly sequencing PCR products. The identification of homozygous mutant plants was conducted using PARMS (Penta-primer Amplification Refractory Mutation System) genotyping technology, and these plants were subsequently cultivated for bioassay purposes (Lv et al., 2019).

To construct plasmids for the overexpression of OsWRKY71, a 1047 bp coding sequence (CDS) fragment of OsWRKY71 (accession no. AY676927) was amplified from Bph15-NIL ( Supplementary Table S1 ). The PCR product was subsequently introduced into the binary vector pCXUN (accession no. FJ905215). Using an Agrobacterium-mediated technique, the resulting structure was transformed into Nipponbare and Bph15-NIL plants. The T₂ homozygous plants were grown for the following bioassay.

A progeny test was conducted to obtain the rice resistance scores. The seedling stage of rice varieties was evaluated for resistance against BPH, with each cultivar or line being replicated a minimum of three times. Eighteen seeds were collected from individual plants and planted in plastic cups, or fifty seeds were randomly planted in plastic boxes. At the three-leaf stage, eight nymphs of BPH (second- or third-instar) were introduced to infest the seedlings. The plant condition was assessed and assigned a resistance score ranging from 0 to 9, with 9 indicating the highest susceptibility and 0 indicating the highest resistance level, as explained in previous research (Huang et al., 2001).

The assessment of BPH insect performance on rice encompassed the utilization of assays to quantify BPH weight gain and weight gain rate. BPH was introduced to seedlings at the four-leaf stage after the planting of each line’s seeds in a plastic cup with a diameter of 10 cm. Newly emerged female BPH insects were weighed and enclosed in parafilm sachets, which were then affixed to the leaf sheath of a seedling at the four-leaf stage. The BPH instars were allowed to feed on the rice crops for 48 hours. After this feeding period, each insect was removed from the sachet and reweighed. The BPH weight gain was determined by measuring the difference between the initial and final weights of the BPH insect. Furthermore, we computed the weight gain rate, which is determined by dividing the weight gained by the insect’s initial weight before feeding. For each cultivar or line, a minimum of three replicates, each containing 10 insects, were utilized.

Stems of three-leaf stage Bph15-NIL and wrky71 mutant wrky71-5-9 were harvested for 0 (uninfested control), 3, and 24 HAI (hours after BPH infestation). All treatments were terminated at the same time, as previously described (Lv et al., 2014). The Bph15-NIL samples were designated as R0, R3, and R24, while the wrky71-5-9 samples were labeled as S0, S3, and S24. The RNA-seq analysis was conducted using an Illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd.).

Using the Illumina paired-end RNA-seq technique, transcriptome sequencing was performed, resulting in a total of 305 million paired-end reads with a length of 2×150 bp. Following the application of Cutadapt, the reads underwent filtration and purification, resulting in 42 Gbp of reads with high quality. Subsequently, the cleaned reads were aligned to the rice reference genome using HISAT2. The estimation of transcript expression levels was conducted using the StringTie and Ballgown software, and the expression abundance was determined by calculating the FPKM values. The DESeq2 software was employed for performing differential expression analysis between distinct groups. Genes displaying a p-value below 0.05 and an absolute fold change exceeding 2 were categorized as differentially expressed genes (DGEs). Furthermore, the DGEs underwent subsequent analysis to ascertain the enrichment of functions associated with Gene Ontology (GO) and KEGG pathways. The bioinformatics analysis was carried out utilizing the OmicStudio tools accessible at https://www.omicstudio.cn/tool.

The stems of three-leaf stage Bph15-NIL, wrky71-5-9, and TN1 were collected after BPH infestation for 0, 3, 6, 12, 24, and 48 HAI. All treatments were terminated simultaneously. The tissue expression profile samples included different rice tissues: stem and leaf of 10 days seedling; stem, sheath, and leaf at the heading stage; stem, sheath, and leaf at 7 days after pollination. The TRIzol reagent (Invitrogen) was employed for the extraction of total RNA. Subsequently, the PrimeScript RT Reagent Kit (Takara) was utilized to perform reverse transcription of 1 μg of total RNA from each sample into cDNA. The qRT-PCR analysis was performed using the QuantStudio™ 5 (Applied Biosystems) and gene-specific primers ( Supplementary Table S1 ) in conjunction with the TB Green (TaKaRa) in the PCR system. The internal control TBP (LOC_Os03g45410) was utilized to standardize the results, and the gene expression levels were determined using the 2^-ΔΔCt method. Three biological replicates were conducted for each gene in the qRT-PCR analysis.

The coding sequences of OsWRKY71 were amplified through PCR utilizing the primers OsWRKY71-YFP ( Supplementary Table S1 ). Subsequently, the sequences were cloned downstream of a ubiquitin promoter, in frame with YFP, within the vector pCAMBIA1300. The resulting construct was designated as OsWRKY71-YFP. To serve as a nucleus marker, a bZIP63-CFP expressing construct was developed and cloned into the pGWB17 vector. The expression constructs were simultaneously introduced into rice protoplasts. The fluorescence was detected using a confocal laser-scanning microscope (FV1000, Olympus).

The data were subjected to statistical analysis using a one-way ANOVA in either Microsoft Excel or SPSS software (version 20).

In our previous study, it was observed that the expression of OsWRKY71 increased in response to BPH feeding in the Bph15 introgression line (Lv et al., 2014). To further investigate the role of OsWRKY71 in the response to BPH, we conducted an analysis of its expression in both the Bph15-NIL and TN1 rice plants at various time points following BPH infestation (0, 3, 6, 12, 24, and 48 HAI). The expression level of OsWRKY71 in the Bph15-NIL was significantly higher than in TN1 at 0, 3, and 6 HAI, but this expression pattern was reversed at 12 and 24 HAI ( Figure 1A ). This suggests that the expression profile of OsWRKY71 in Bph15-NIL differs from that of TN1. Specifically, OsWRKY71 is early induced by BPH in Bph15-NIL. Tissue expression analysis demonstrated that the expression level of OsWRKY71 is higher in the leaf and sheath tissues at the heading stage compared to other tissues ( Figure 1B ). Therefore, OsWRKY71 may play a role in Bph15-mediated BPH resistance.

Inconsistent subcellular localization of OsWRKY71 has been reported in previous studies (Liu et al., 2007; Kim et al., 2016; Ji et al., 2021). The present study reveals a significant colocalization between OsWRKY71 and the nucleus marker bZIP63 in rice protoplasts, thereby providing substantial evidence supporting the nuclear localization of OsWRKY71 ( Figure 2 ). This localization pattern is consistent with the well-established role of transcription factors, which primarily function within the nucleus to regulate gene expression.

To further understand the role of OsWRKY71 in Bph15-mediated resistance, we used CRISPR-Cas9 technology to knockout OsWRKY71 in the Bph15-NIL background. Consequently, we achieved the creation of four distinct homozygous mutant plants, denoted as wrky71-5-9, wrky71-2-1, wrky71-3-3, and wrky71-6-2, within the T₂ generation. Subsequent DNA sequencing analysis unveiled that each mutant exhibited distinct genetic alterations within the targeted region of the OsWRKY71 gene. It was observed that wrky71-5-9 had an insertion of A, wrky71-2-1 had a deletion of 4 base pairs, wrky71-3-3 had a deletion of 5 base pairs, and wrky71-6-2 had a deletion of 13 base pairs ( Figure 3A ). The BPH resistance scores of the wrky71 mutants were evaluated, revealing that wrky71-5-9, wrky71-2-1, wrky71-3-3, and wrky71-6-2 plants were susceptible. Conversely, the Bph15-NIL plants, which possessed intact OsWRKY71, exhibited resistance and survived the BPH attack ( Figure 3B ). The mean resistance score for Bph15-NIL plants was determined to be 3.27, indicating their resistance to BPH. Conversely, the wrky71 mutants exhibited significantly higher resistance scores, ranging from 7.42 to 8.51, thereby suggesting their susceptibility to BPH ( Figure 3C ). Additionally, BPH insects that consumed the wrky71 mutants displayed a greater increase in weight compared to those that consumed Bph15-NIL plants ( Figures 3D, E ). These findings provide evidence that the knockout of OsWRKY71 impairs the resistance of Bph15-NIL plants to BPH, thereby emphasizing the crucial role of OsWRKY71 in Bph15-mediated resistance.

We also generated overexpression lines of OsWRKY71 in both the Nipponbare and Bph15-NIL backgrounds. However, the overexpression of OsWRKY71 in both backgrounds did not result in significant differences in BPH resistance compared to the control plants. Specifically, the Bph15-NIL-OE plants, which overexpressed OsWRKY71 in the Bph15-NIL background, exhibited BPH resistance scores similar to those of the Bph15-NIL control plants. This suggests that the overexpression of OsWRKY71 did not enhance the resistance provided by Bph15 ( Supplementary Figure S1A ). In a similar vein, the NIP-OE plants, characterized by the overexpression of OsWRKY71 in the Nipponbare background, exhibited susceptibility to BPH and were killed by BPH at the same time as the susceptible Nipponbare plants ( Supplementary Figure S1B ). These observations imply that increasing the expression of the OsWRKY71 gene does not exert any discernible influence on BPH resistance, irrespective of the presence or absence of Bph15. Consequently, it can be deduced that OsWRKY71 does not function as a positive regulator of basal resistance in the absence of Bph15.

To investigate the potential mechanism underlying the impact of the wrky71 mutant on Bph15-mediated resistance to BPH, we conducted an analysis of the transcriptomes of wrky71-5-9 and Bph15-NIL using RNA-seq. The RNA-seq analysis identified a total of 1387 differentially expressed genes (DEGs) among seven comparisons: R0_S0, R3_S3, R24_S24, R0_R3, R0_R24, S0_S3, and S0_S24 ( Supplementary Table S2 ). Significant disparities in gene expression were observed between the wrky71 mutant and Bph15-NIL. Specifically, in the comparisons of R0_S0, R3_S3, and R24_S24, the wrky71 mutant displayed significant alterations in 390, 208, and 153 genes, respectively, when compared to Bph15-NIL. Moreover, the wrky71 mutant displayed 248 and 339 DEGs in the S0_S3 and S0_S24 comparisons, respectively. Conversely, in Bph15-NIL, there were 607 and 529 DEGs in the R0_R3 and R0_R24 comparisons, respectively. Notably, both the R and S comparisons exhibited a higher abundance of up-regulated genes compared to down-regulated genes ( Figure 4A ). Subsequent analysis revealed significant disparities in the majority of the up-regulated and down-regulated genes between the wrky71 mutant and Bph15-NIL ( Figure 4B ). Additionally, hierarchical cluster analysis of 1387 DEGs further confirmed substantial distinctions between the R and S comparisons ( Supplementary Figure S2 ).

The enriched Gene Ontology (GO) terms identified in this study suggest that the response to BPH feeding in both the wrky71 mutant and Bph15-NIL rice involves multiple key biological processes. Specifically, the common GO terms observed in the up-regulated DEGs ( Supplementary Figure S3 ; Table S3_1 ) in the S0_S3, S0_S24, R0_R3, and R0_R24 comparisons include wounding response, chitin response, fungus defense, and salicylic acid (SA) response. These shared GO terms indicate that both the wrky71 mutant and Bph15-NIL rice employ similar defense mechanisms in response to BPH feeding. Significant disparities in enriched GO terms were observed when comparing gene expression between the wrky71 mutant and Bph15-NIL rice. Specifically, the R0_R3 and R0_R24 comparisons revealed enriched GO terms related to water deprivation response, cell wall, autophagy, and trehalose phosphatase. Conversely, the S0_S3 and S0_S24 comparisons exhibited specifically enriched GO terms associated with ethylene (ET) response, jasmonic acid (JA) response, gibberellin metabolic, bacterium defense, virus defense, and herbivore response ( Figure 5 ; Supplementary Tables S3_2-S3_5 ). The enriched GO term “sesquiterpene biosynthetic” in both the R0_R24 and S0_S3 comparisons suggests that this biological process may play a crucial role in conferring resistance to BPH in both the wrky71 mutant and Bph15-NIL rice ( Figure 5 ). These results emphasize the potential contribution of diverse biological processes and signaling pathways in the resistance mechanisms of the wrky71 mutant and Bph15-NIL rice, providing valuable insights for future investigations on BPH resistance in rice.

The KEGG pathway enrichment analysis demonstrated significant enrichment in multiple pathways within the transcript profiles of both the wrky71 mutant and Bph15-NIL plants upon exposure to BPH infestation. Notably, these pathways encompassed plant-pathogen interaction, plant hormone signal, autophagy, et al. ( Supplementary Figure S4 ; Table S4_1 ). Intriguingly, the R0_R3 and R0_R24 comparisons exhibited a higher number of genes involved in these pathways compared to the S0_S3 and S0_S24 comparisons ( Supplementary Figure S5 ; Tables S4_2-S4_5 ). This observation implies the existence of discrete regulatory mechanisms functioning in the two genotypes in response to BPH infestation. Overall, these findings provide insights into the molecular mechanisms of BPH resistance in rice and underscore the importance of OsWRKY71 in governing gene expression during BPH infestation.

The study revealed a significant down-regulation of the sesquiterpene synthase gene OsSTPS2 in the R3_S3 and R24_S24 comparisons ( Figure 6 ). Subsequent analysis using qRT-PCR demonstrated a notable decrease in expression levels of OsSTPS2 in both the wrky71 mutant and TN1 compared to Bph15-NIL at all five time points ( Figure 7 ). Terpenes, a class of secondary metabolites, possess diverse functions, including defense against herbivores. Previous research has demonstrated the differential expression of OsSTPS2 in rice cultivars RH (carrying Bph3) and KD (sensitive to BPH), and its involvement in BPH antixenosis resistance (Kamolsukyunyong et al., 2013; Kamolsukyeunyong et al., 2021). This suggests that OsSTPS2 may participate in BPH resistance conferred by OsWRKY71 in Bph15-NIL plants.

The excretion of proteins associated with resistance and the plant’s resistance to BPH are influenced by the trafficking pathway that relies on the presence of EXO70. Specifically, the functions of OsEXO70E1 and OsEXO70H3 are involved in Bph6-mediated BPH resistance (Guo et al., 2018; Wu et al., 2022). Our investigation led to the discovery of the OsEXO70J1 gene (LOC_Os08g13570), a member of the EXO70 family, which exhibited significant up-regulation in the R0_R3 and R0_R24 comparisons, but down-regulation in the R3_S3 comparison ( Figure 6 ; Supplementary Figure S6 ). Additionally, the results of qRT-PCR analysis showed that the expression levels of OsEXO70J1 were reduced in both the wrky71 mutant and TN1 cultivars compared to Bph15-NIL at the onset of BPH infestation ( Figure 7 ). This suggests that OsEXO70J1 may participate in BPH resistance conferred by OsWRKY71 in Bph15-NIL plants.

Interestingly, the down-regulation of three R genes, namely Xa46(t) (LOC_Os11g37540), Pigm (LOC_Os06g17900), and RGA2 (LOC_Os04g30530), was observed in the R_S comparisons ( Figure 6 ). It was found that BPH feeding did not induce the up-regulation of these genes in any of the rice lines. However, the expression levels of these genes were significantly reduced in the wrky71 mutant compared to Bph15-NIL. Additionally, the expression level of RGA2 was also decreased in TN1 when compared to Bph15-NIL plants ( Figure 7 ). These findings suggest that OsWRKY71 may be involved in controlling the expression of these three R genes and contribute to disease resistance in rice. It is also suggested that the R gene RGA2 may have a dual function in both disease and BPH resistance.

The influence of the SA and JA pathways on plant responses to insects is of significant importance. However, our previous study suggested that the SA signaling pathway may not be triggered in Bph15-mediated resistance (Lv et al., 2014). In this current investigation, we focused on the JA pathway and found that the expression levels of the AOS2 gene, which is involved in JA synthesis, were increased in both the wrky71 mutant and TN1 compared to Bph15-NIL. Furthermore, the transcript levels of the JA repressor JAZ4 were lower in the wrky71 mutant compared to Bph15-NIL and TN1 ( Figure 7 ). The results indicate that OsWRKY71 may inhibit the JA pathway in Bph15-NIL plants. Hence, the Bph15-mediated BPH resistance may not be associated with the SA and JA signaling pathways.

The present study observed an increase in the expression of NPR1 and five PR genes (PR1a, PR4, PR4b, PR10a, and RSOsPR10) in all three rice lines. However, the expression levels of these genes were higher in the wrky71 mutant compared to Bph15-NIL plants ( Figure 7 ). This suggests that these genes may be involved in a common defense mechanism in both resistant and susceptible rice varieties. These results are consistent with our previous study (Lv et al., 2014). It was observed that the transcript levels of OsWRKY28, OsWRKY62, and OsWRKY76, members of the OsWRKY IIa subfamily, exhibited a significant increase in all three rice lines. However, in the wrky71 mutant, their expression levels were even higher compared to Bph15-NIL plants ( Figure 7 ). This suggests that in the absence of Bph15, these genes may act as promoters of BPH resistance, but in the presence of Bph15, they could exert an inhibitory effect. These findings are consistent with the previous studies conducted by Chujo et al. (2013) and Shi et al. (2023).