agronomy Molecular Mapping of a New Brown Planthopper Resistance Gene Bph43 in Rice ( Oryza sativa L.)

: Brown planthopper (BPH) has become the most devastating insect pests of rice and a serious threat to rice production. To combat newly occurring virulent BPH populations, it is still urgent to explore more new broad-spectrum BPH resistance genes and integrate them into rice cultivars. In the present study, we explored the genetic basis of BPH resistance in IRGC 8678. We identiﬁed and mapped a new resistance gene Bph43 to a region of ~380 kb on chromosome 11. Genes encoding nucleotide-binding domain leucine-rich repeat-containing (NBS-LRR)-type disease resistance proteins or Leucine Rich Repeat family proteins annotated in this region were predicted as the possible candidates for Bph43 . Meanwhile, we developed near isogenic lines of Bph43 (NIL- Bph43 -9311) in an elite restorer line 9311 background using marker-assisted selection (MAS). The further characterization of NIL- Bph43 -9311 demonstrated that Bph43 confers strong antibiosis and antixenosis effects on BPH. Comparative transcriptome analysis revealed that genes related to the defense response and resistance gene-dependent signaling pathway were signiﬁcantly and uniquely enriched in BPH-infested NIL- Bph43 -9311. Our work demonstrated that Bph43 can be deployed as a valuable donor in BPH resistance breeding programs.


Introduction
Rice (Oryza sativa L.), being an important cereal crop in the Asian-Pacific region, accounts for a staple food resource for around four billion people worldwide [1]. Nevertheless, like other plants, the growth of rice is continuously threatened by pathogens and herbivore insects during its entire growth cycle and thus leads to an estimated annual loss of 10-30% of the total rice yield [2,3]. Among 20 kinds of known serious paddy pests, brown planthopper (BPH, Nilaparvata lugens Stål), is a migratory and monophagous one and becomes the most destructive pest of rice in recent years [4,5]. BPH has sharp, elongated mouthparts that penetrate plant cells and sucks the phloem sap from rice leaf sheath, during which process viral diseases are also transmitted [6]. Serious BPH feeding causes the complete dying of rice, leading to a phenomenon called "hopperburn" [7]. In China, the BPH-infested rice area was estimated at over 25 million hectares, resulting in a rice yield loss of 2.7 million tons between 2005 and 2008 [8,9]. In Asia, the economic loss caused by BPH could reach more than 300 million dollars per year [10].
In agricultural practice, BPH management is still depending heavily on chemical pesticides, which not only cause severe environment pollution and food safety concerns, but also induce increased BPH resistance to chemical pesticides [11]. Therefore, breeding and deploying of resistant cultivars have been the most economically effective and environmental-friendly approach to BPH management [12]. Hence, the identification of BPH resistance genes and the dissection of the underlying resistance mechanism are critical for their successful use in rice breeding. To date, around 40 BPH resistance genes have been identified in the traditional cultivated rice varieties and wild rice species, most of which are usually present in clusters on chromosomes 3, 4, 6, and 12 [13]. Among them, Bph14, Bph3/Bph15, Bph26/bph2, bph29, bph7/Bph9/Bph10/Bph21, Bph18/Bph1, Bph32, Bph6, Bph30/Bph40, and Bph37 have been isolated and characterized via a map-based cloning approach [14][15][16][17][18][19][20][21][22][23][24][25], which provide useful targets for marker-assisted selection (MAS) breeding.
However, the resistance of varieties carrying single BPH resistance gene is easy to be quickly broken down due to the rapid adaptation of BPH or evolution of new biotypes [26]. For example, IR26, the first resistant variety with single resistant gene Bph1, was released by the International Rice Research Institute (IRRI) in 1973. However, by 1975, IR26 and other Bph1-containing resistant varieties were incapable of reducing BPH damage due to the development of BPH biotype II [27]. In 1976, IRRI released IR36 and other resistant varieties harboring bph2. However, a few years later, these varieties were also adapted by the new BPH population [28,29]. Therefore, it is still urgent to explore more new broad-spectrum BPH resistance genes and integrate them into rice cultivars to combat the new virulent BPH populations and ultimately achieve durable and broad-spectrum resistance.
IRGC 8678, a Bangladesh indica rice cultivar, was reported to be resistant to BPH biotype 3 [30], but the underlying BPH resistance gene(s) have not been identified yet. In this study, we revealed that IRGC 8678 was highly resistant to the current BPH population of China, the more destructive Bangladesh type. We identified and mapped a new BPHresistant gene, namely Bph43, in IRGC 8678. A highly resistant near-isogenic line (NIL-Bph43-9311) was developed by the introgression of Bph43 into the elite restorer line 9311 through MAS. We also performed comparative transcriptome analysis and explored early defense responsive genes and pathways underlying Bph43. Our work identified a new BPH-resistant gene and demonstrated that Bph43 can be deployed as a valuable donor in BPH resistance breeding programs.

Plant Materials, Mapping Populations, and NIL-Bph43-9311 Construction
IRGC 8678, a Bangladesh indica rice cultivar, was previously found to be resistant to BPH biotype III [30]. We obtained IRGC 8678 from the IRRI and crossed it with a BPHsusceptible Chinese elite indica rice cultivar 9311. The resulted F 1 was self-pollinated to generate F 2 mapping population and the corresponding F 3 families for genetic analysis and gene mapping. At the same time, 9311/IRGC 8678 F 1 plant was backcrossed with 9311 to generate the BC 1 F 1 population. The heterozygous BC 1 F 1 plants showing the resistance to BPH were selected to generate BC 1 F 2 populations for further mapping. The near isogenic lines of Bph43 in 9311 background was developed by the successive backcrossing of the 9311/IRGC 8678 F 1 with 9311. During this process, the gene-linked markers 16-22 and 16-30 flanking the Bph43 locus were used to select plants with heterozygous Bph43 from each backcrossed populations for the next step of backcrossing. Finally, one BC 3 F 2 individual carrying homozygous Bph43 and with the least amount of genetic background of IRGC 8678 was selected and designated as NIL-Bph43-9311. The detailed procedure for the development of mapping populations and NIL-Bph43-9311 is illustrated in Supplementary Figure S1.

BPH Insects and Evaluation of BPH Resistance
The BPH insects used in this study were collected from rice fields and maintained on the susceptible cultivar Taichung Native 1 (TN1) in a greenhouse at Wuhan University as previously described [20].
For gene mapping, a seedling bulk test for BPH resistance evaluation was performed on the F 3 families as previously described [30,31]. Briefly, approximately 20 seeds per line were sown in a plastic box in 15 cm long rows, with 2.5 cm between rows. A total of Agronomy 2022, 12, 808 3 of 13 two rows of IRGC 8678, three rows of 9311, and three rows of TN1 were randomly sown among the F 3 families as controls. The seedlings were thinned to 15 plants per row on 7 days after sowing. At the third-leaf stage, the seedlings were infested with eight 2nd to 3rd instar BPH nymphs per seedling. The plastic box was completely enclosed in a fine mesh cage that allowed the light to shine through but prevented the escape of the BPH insects. When all of the seedlings of susceptible control TN1 died, the plants were examined and each seedling was given a resistance score of 0 (none damage, none of the leaves shrank and the plant was healthy), 1 (very slight damage or one leaf was yellowing), 3 (one to two leaves were yellowing, or one leaf shrank), 5 (one to two leaves shrank, or one leaf shriveled), 7 (three to four leaves shrank or two to four leaves shriveled, and the plant was still alive), or 9 (the plant died) based on the modified Standard Evaluation System for Rice [30,31]. A lower resistance score represents a higher BPH resistance level. The resistance score of each F 2 individual was then quantified with Microsoft Excel (ME) as the weighted average of the scores of its corresponding F 3 families. The experiments were conducted with at least three biologically independent replicates.
To verify the location of Bph43, a total of 320 BC 2 F 2 seeds derived from heterozygous Bph43 BC 2 F 1 plant were sown in a plastic box, surrounded with susceptible 9311 and subjected to the BPH resistance assay. When all of the seedlings of 9311 died, each BC 2 F 2 individual was examined and given a resistance score.
The performance of BPH insects on rice plants was evaluated as previously described [20]. In the two-host choice test, one 9311 and one NIL-Bph43-9311 plants were grown in the same plastic cup (10 cm in diameter, 15 cm in height). At the four-leaf stage, the cup was covered with a light-transmitting nylon mesh, and 20 2nd to 3rd-instar BPH nymphs were released in the cup. The numbers of BPH insects that settled on each plant were recorded at 3, 6, 24, 48, 72, 96, 120, 144, and 168 h after release. The experiments were repeated at least 20 times. The two-host choice test is an indicator of the BPH antixenotic factor.
For BPH weight gain and honeydew excretion assay, newly emerged female adults were weighed, individually enclosed in a pre-weighed Parafilm sachet and attached to the leaf sheath of the rice plant. After 2 days of insect infestation, both the sachets and BPH insects were weighed again. The weight difference of BPH insects was recorded as the weight gain of BPH, and the weight difference of the Parafilm sachet was recorded as honeydew excretion. At least 15 BPH insects were used for analysis.

DNA Extraction and Gene Mapping
Total genomic DNA was extracted from fresh rice leaves using modified CTAB protocol [32]. The extracted DNA was dissolved in a 1×TE buffer. According to phenotype of F 2 mapping population, equal amounts of DNA from 11 extremely resistant F 2 plants were mixed to form a resistant bulk DNA pool. Similarly, equal amounts of DNA from 18 extremely susceptible F 2 plants were mixed to form a susceptible bulk DNA pool. The green super rice chip-based bulked segregant analysis method (BSA-chip) was used to map the BPH resistance gene. The DNA pools of the resistant bulk and the susceptible bulk as well as the DNAs of the two parents (9311 and IRGC 8678) were subjected to the green super rice chip GSR 40K array at the Greenfafa (Wuhan, China), according to the Infinium HD Assay Ultra Protocol (available online: http://www.illumina.com/ (accessed on 17 September 2021)).
For the insertion-deletion (InDel) calling and development, the DNAs of the two parents (9311 and IRGC 8678) were deeply (~30×) sequenced on the platform of Hiseq X Ten. Low-quality reads were deleted or trimmed by a base-quality Q score with a Phred scale of <20 and a read size of <60 bp from raw data using Perl scripts. The cleaned data were mapped to the reference genome of Nipponbare retrieved from Rice Genome Annotation Project website (available online: http://rice.plantbiology.msu.edu/pub/data/ Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_7.0/ (accessed on 20 October 2019)) using Burrows-Wheeler Aligner (BWA) software (version 0.5.8c) [33].
InDels of 20 to 40 bp in length between IRGC 8678 and 9311 were extracted using SAMtools (version 0.1.12a) [34]. The upstream and downstream sequences of the candidate InDel loci on the reference genome Nipponbare were obtained and subjected to PCR primer design using Primer 3. The size of PCR products ranged from 160 to 250 bp, so that the polymorphism between IRGC 8678 and 9311 can be easily identified on a 1.5-2% agarose gel. Primers designed in this study are listed in Supplementary Table S1. PCR was performed as previously described [35], with minor modification for different InDel markers. PCR products were separated on a 1.5-2% agarose gel and stained with ethidium bromide.

RNA-Seq and Transcriptome Analysis
The seedlings of NIL-Bph43-9311 and 9311 plants were infested with BPH nymphs (8 s to third-instar nymphs per plant). At 3 h of BPH infestation, leaf sheaths of the rice plants infected by BPH were collected. The samples were referred to as NIL-3 for NIL-Bph43-9311 and 9311-3 for 9311, the number representing 3 h of BPH infestation. At the same time, leaf sheaths of NIL-Bph43-9311 and 9311 without BPH infestation were collected as an undamaged control and referred to as NIL-0 and 9311-0, respectively. Two to four biological replicates per treatment with 15 seedlings per replicate were used for RNA-seq analysis. Total RNA was isolated using an RNAprep Pure Plant Kit (Tiangen Biotech (Beijing) Co., Ltd., Beijing, China) following the manufacturer's instructions. Concentrations of RNA were checked and integrity was verified on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The cDNA library for each sample was constructed using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) and quantified on a 150 bp paired-end run by Agilent2100 and sequenced by illumina novaseq 6000 (Illumina, San Diego, CA, USA). Clean reads were obtained after the removal of adaptors, low-quality reads and reads with >5% unknown nucleotides with Trimmomatic software [36] and mapped on the rice genome of the cultivar Shuhui498 (available online: http://www.mbkbase.org/R498/ (accessed on 29 October 2021)) using the Hisat2 (v2.2.1.0) [37]. Gene counts were obtained by HTseq, and gene expression was determined using the fragments per kilobase of the exon model per million mapped fragments (RPKM) method [38]. Differentially expressed genes (DEGs) were filtered by DESeq2 after significance. p-values were performed at absolute values of log2FC of ≥1 (p < 0.05) [39]. The gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed separately using R based on the hypergeometric distribution [40,41]. The summary of RNA-Seq data is presented in Supplementary Table S2. The Pearson's correlation coefficients for all tested samples are shown in Supplementary Figure S2.

Quantitative Real-Time PCR (qRT-PCR) Analysis
qRT-PCR was used to validate the DEGs analysis results. All of the rice samples used for qRT-PCR verification were prepared as described above. Total RNAs were isolated from rice plants using TRIzol reagent (TaKaRa) and then converted into first-strand cDNA using PrimeScript™ RT reagent kits accompanied with gDNA Eraser (TaKaRa, code no. RR047A) according to the manufacturer's instructions. Gene expression was analyzed by qRT-PCR using SYBR green supermixes and CFX96 Real-Time System (Bio-Rad). Primers are listed in Supplementary Table S1. The expression levels of genes were calculated with the 2 -∆∆C (t) method using CFX Manager Software 2.1. Being stably expressed in rice various organs and developmental stages, OsAct1 (AB047313) was used as the internal control for qRT-PCR [42]. Each experiment was performed in three biological replicates and three technical replicates.

Statistical Analysis
The t-test was used to examine the difference between two groups. One-way ANOVA was used to compare multiple samples. Statistical tests were conducted using the software GraphPad Prism 7.

Genetic Analysis of BPH Resistance
IRGC 8678, which was previously reported to be resistant to BPH biotype III [30], exhibited high resistance to the current BPH population of China, the more destructive Bangladesh type [43]. The average resistance scores of IRGC 8678 were 1.15 in two independent tests ( Figure 1A), suggesting that IRGC 8678 was highly resistant to BPH and might be a useful donor for BPH resistance breeding programs. We further investigated the performance of BPH fed on IRGC 8678 and 9311 plants. Compared to those on 9311 plants, BPH insects fed on IRGC 8678 showed a significantly lower weight gain, which was 0.1808 mg on average ( Figure 1B), indicating a strong antibiosis effect on BPH.
logical replicates and three technical replicates.

Statistical Analysis
The t-test was used to examine the difference between two groups. One-way ANOVA was used to compare multiple samples. Statistical tests were conducted using the software GraphPad Prism 7.

Genetic Analysis of BPH Resistance
IRGC 8678, which was previously reported to be resistant to BPH biotype III [30], exhibited high resistance to the current BPH population of China, the more destructive Bangladesh type [43]. The average resistance scores of IRGC 8678 were 1.15 in two independent tests ( Figure 1A), suggesting that IRGC 8678 was highly resistant to BPH and might be a useful donor for BPH resistance breeding programs. We further investigated the performance of BPH fed on IRGC 8678 and 9311 plants. Compared to those on 9311 plants, BPH insects fed on IRGC 8678 showed a significantly lower weight gain, which was 0.1808 mg on average ( Figure 1B), indicating a strong antibiosis effect on BPH. To explore the genetic basis of BPH resistance in IRGC 8678, we evaluated BPH resistance scores of the F2 mapping population, which showed a continuous distribution with an apparent valley bottom between 7.0 and 7.9 ( Figure 1C). According to a previous BPH resistance-scoring criterion [35], we categorized the plants with resistance scores below 6.99 as resistant plants, whereas those with resistance scores above 7.00 were considered as susceptible ones. The segregation of the resistant to susceptible plants was found to be 122:34, which was in agreement with a 3:1 ratio (122:34; χ 2 c = 0.504 < χ 2 0.05,1 = To explore the genetic basis of BPH resistance in IRGC 8678, we evaluated BPH resistance scores of the F 2 mapping population, which showed a continuous distribution with an apparent valley bottom between 7.0 and 7.9 ( Figure 1C). According to a previous BPH resistance-scoring criterion [35], we categorized the plants with resistance scores below 6.99 as resistant plants, whereas those with resistance scores above 7.00 were considered as susceptible ones. The segregation of the resistant to susceptible plants was found to be 122:34, which was in agreement with a 3:1 ratio (122:34; χ 2 c = 0.504 < χ 2 0.05,1 = 3.84), demonstrating that BPH resistance of IRGC 8678 was controlled by a major dominant gene.

Molecular Mapping of Bph43
To map the BPH resistance gene in IRGC 8678, we performed a bulked segregant analysis (BSA) analysis. According to the phenotype of F 2 population, 11 extremely resistant plants and 18 extremely susceptible plants were selected to prepare two contrasting bulks, respectively. The green super rice chip GSR 40K was then used to determine the region containing putative BPH resistance genes. It was revealed that there was only one contiguous region ranging from 8.0 to 18.0 Mb on chromosome 11 significantly differentiating between Agronomy 2022, 12, 808 6 of 13 the resistant and susceptible bulks (Figure 2A), suggesting that the BPH-resistant gene in IRGC 8678 is located in this region. We designed this gene as Bph43.

Verification of Bph43 in BC2F2 Populations
To confirm the location of Bph43, BC2F1 plants that were heterozygous between In-Del markers 16-22 and 16-30 were selected to produce BC2F2 generations. BPH resistance evaluation revealed 236 and 84 seedlings were resistant and susceptible to BPH, respectively, and the segregation of the resistant to susceptible plants was in agreement with a 3:1 ratio (236:84, χ 2 c = 0.267 < χ 2 0.05,1 = 3.84). We further genotyped these surviving resistant 236 BC2F2 plants with InDel markers 16-22 and 16-30. All of them contained either homozygous or heterozygous alleles from the resistant parent IRGC 8678, verifying the existence of Bph43 on the region between markers 16-22 and 16-30.

Development and Characterization of NIL-Bph43-9311
9311, an elite restorer parent for hybrids in China, is highly susceptible to BPH. To improve its BPH resistance, 9311 was used as the recurrent parent to backcross with IRGC 8678 (Supplementary Figure S1). In the process, the flanking markers 16-22 and 16-30 tightly linked to Bph43 locus were used to select the positive progenies for continuous backcrossing. Simultaneously, background selections using rice chip GSR 40K were conducted. One BC3F1 plant containing a heterozygous Bph43 region and the least amount of genetic background of IRGC 8678 was selected to produce BC3F2 populations.  Figure 2B). A further analysis of 600 BC 1 F 2 plants using the flanking markers 78-16 and 78-17 identified 21 recombinants. These recombinants were genotyped with polymorphic InDel markers within the mapping interval ( Figure 2C). According to the genotypes and BPH resistance phenotypes of the recombinants, Bph43 was located between InDel markers 16-22 and 16-30 ( Figure 2D), within intervals of 350 and 380 kb according to R498 and Nipponbare reference genomes, respectively. Putative genes were predicted in the corresponding candidate regions of R498 and Nipponbare reference genomes by using a rice genome annotation project database [44]. A gene cluster encoding putative nucleotide-binding domain leucine-rich repeat-containing (NBS-LRR) proteins and LRR family proteins was identified in Bph43 mapping regions of both R498 and Nipponbare Agronomy 2022, 12, 808 7 of 13 reference genomes (Supplementary Table S3). Interesting, one of them was found to be specifically induced in NIL-Bph43-9311 upon BPH infestation as revealed by RNA-Seq (Supplementary Table S3). As most BPH resistance genes isolated to date encode NBS-LRR proteins, we considered these putative genes encoding NBS-LRR and LRR family proteins as the candidates for Bph43.

Verification of Bph43 in BC 2 F 2 Populations
To confirm the location of Bph43, BC 2 F 1 plants that were heterozygous between InDel markers 16-22 and 16-30 were selected to produce BC 2 F 2 generations. BPH resistance evaluation revealed 236 and 84 seedlings were resistant and susceptible to BPH, respectively, and the segregation of the resistant to susceptible plants was in agreement with a 3:1 ratio (236:84, χ 2 c = 0.267 < χ 2 0.05,1 = 3.84). We further genotyped these surviving resistant 236 BC 2 F 2 plants with InDel markers 16-22 and 16-30. All of them contained either homozygous or heterozygous alleles from the resistant parent IRGC 8678, verifying the existence of Bph43 on the region between markers 16-22 and 16-30.

Development and Characterization of NIL-Bph43-9311
9311, an elite restorer parent for hybrids in China, is highly susceptible to BPH. To improve its BPH resistance, 9311 was used as the recurrent parent to backcross with IRGC 8678 (Supplementary Figure S1). In the process, the flanking markers 16-22 and 16-30 tightly linked to Bph43 locus were used to select the positive progenies for continuous backcrossing. Simultaneously, background selections using rice chip GSR 40K were conducted. One BC 3 F 1 plant containing a heterozygous Bph43 region and the least amount of genetic background of IRGC 8678 was selected to produce BC 3 F 2 populations. Finally, one line homozygous for Bph43 locus and morphologically similar to 9311 was selected as NIL-Bph43-9311 (Supplementary Figure S3). Thus, Bph43 has been successfully introgressed into 9311 through MAS.
We further characterized BPH resistance of NIL-Bph43-9311. NIL-Bph43-9311 plants that were homozygous or heterozygous for Bph43 exhibited high resistance to BPH, and no significant difference was detected between Bph43-heterozygous and -homozygous plants in terms of resistance scores ( Figure 3A,B). The results suggested that Bph43 is valuable in hybrid rice breeding. BPH insects fed on NIL-Bph43-9311 plants showed a significantly lower honeydew excretion and weight gain than those of 9311 ( Figure 3C,D), indicating that Bph43 conferred strong antibiosis effects on BPH. In two-host choice tests, many more BPH insects preferred to settle on 9311 compared with on NIL-Bph43-9311 plants ( Figure 3E), suggesting that Bph43 also had a stronger antixenotic effect on BPH.

Comparative Transcriptome Analysis of NIL-Bph43-9311 and 9311 Underlying BPH Infestation
To further understand the molecular mechanism underlying BPH resistance conferred by Bph43, RNA-Seq analysis was performed using the stems of NIL-Bph43-9311 and 9311 at the early stage (3 h) of BPH infestation (Supplementary Table S2 and Supplementary Figure S2). One hundred and ninety constitutive DEGs between NIL-Bph43-9311 and 9311 without BPH infestation were identified (Supplementary Table S4) and significantly enriched in the defense response, defense response signaling pathway (resistance genedependent), plant-type hypersensitive response, and so on (Supplementary Table S5 and Supplementary Figure S4A). At 3 h after BPH infestation, a higher number of DEGs (449 vs. 281 DEGs) were identified in NIL-Bph43-9311 plants than in 9311, with 208 down-and 241 upregulated, respectively (Supplementary Table S4). These DEGs were mainly enriched in the regulation of the jasmonic acid mediated signaling pathway, monoterpene biosynthetic process, terpenoid biosynthetic process, regulation of defense response, and so on (Supplementary Table S5 and Supplementary Figure S4C). Upregulated DEGs in NIL-Bph43-9311 and 9311 upon BPH infestation were further analyzed. In total, there were 47 upregulated DEGs (G2) shared by NIL-Bph43-9311 and 9311, 194 upregulated DEGs (G1) unique to NIL-Bph43-9311, and 183 upregulated DEGs (G3) unique to 9311 (Figure 4A and Supplementary Table S6). We then focused on analyzing DEGs in G1, as these DEGs are putatively involved in the molecular mechanism underlying Bph43-mediated resistance. GO enrichment analysis revealed that G1 was significantly enriched in the root development, defense response and defense response signaling pathway (resistance-gene-dependent), hydrogen peroxide catabolic process, plant-type hypersensitive response, and so on ( Figure 4B and Supplementary Table S6).
Finally, one line homozygous for Bph43 locus and morphologically similar to 9311 was selected as NIL-Bph43-9311 (Supplementary Figure S3). Thus, Bph43 has been successfully introgressed into 9311 through MAS.
We further characterized BPH resistance of NIL-Bph43-9311. NIL-Bph43-9311 plants that were homozygous or heterozygous for Bph43 exhibited high resistance to BPH, and no significant difference was detected between Bph43-heterozygous and -homozygous plants in terms of resistance scores ( Figure 3A,B). The results suggested that Bph43 is valuable in hybrid rice breeding. BPH insects fed on NIL-Bph43-9311 plants showed a significantly lower honeydew excretion and weight gain than those of 9311 ( Figure  3C,D), indicating that Bph43 conferred strong antibiosis effects on BPH. In two-host choice tests, many more BPH insects preferred to settle on 9311 compared with on NIL-Bph43-9311 plants ( Figure 3E), suggesting that Bph43 also had a stronger antixenotic effect on BPH.   Table S6). We then focused on analyzing DEGs in G1, as these DEGs are putatively involved in the molecular mechanism underlying Bph43-mediated resistance. GO enrichment analysis revealed that G1 was significantly enriched in the root development, defense response and defense response signaling pathway (resistance-gene-dependent), hydrogen peroxide catabolic process, plant-type hypersensitive response, and so on (Supplementary Figure 4B and Supplementary Table S6).
It has been proposed that a large number of BPH resistant varieties come from South Asia [27,30,54]. In this study, IRGC 8678, which is originated from Bangladesh and reported to be resistant to BPH biotype III [30], also showed high resistance to the current BPH population of China, the more destructive Bangladesh type ( Figure 1A,B). These results implied that Bph43 in IRGC 8678 could be a potential durable and broad-spectrum resistance gene. Generally, rice may employ three resistance mechanisms against BPH, including antixenosis, antibiosis, and tolerance [55]. We have demonstrated that Bph43 in IRGC 8678 confers strong antibiosis and antixenosis effects on BPH. Meanwhile, Bph43 is a dominant gene, as plants homozygous or heterozygous for Bph43 both exhibited high resistance to BPH at a similar level ( Figure 3A,B). These characteristics make Bph43 a valuable candidate for hybrid rice breeding and production.
Transcriptome analysis is an effective way to explore rice-BPH interactions [43,56]. In this study, we also used RNA-Seq to explore the molecular mechanism underlying Bph43mediated resistance at the early stage of BPH feeding. Genes related to defense pathways were enriched in un-infested NIL-Bph43-9311 plants, suggesting that the plants might be in a primed state. After BPH infestation, NIL-Bph43-9311 initiated more intensive and prompt defense responses. Comparative analysis revealed that there were 194 upregulated DEGs unique to NIL-Bph43-9311 upon BPH infestation, significantly enriched in the root development, defense response and defense response signaling pathway (resistance genedependent), hydrogen peroxide catabolic process, and plant-type hypersensitive response. These genes are putatively involved in BPH resistance mechanism mediated by Bph43.

Conclusions
Due to the rapid adaptation of BPH or evolution of new biotypes, it is still urgent to explore and utilize more new broad-spectrum BPH resistance genes. In the current study, we explored the genetic basis of BPH resistance of rice cultivar IRGC 8678. We identified and mapped a new resistance gene Bph43 to a region of~380 kb on chromosome 11. Bph43 was successfully introgressed into elite restorer line 9311 through MAS. The resulted near isogenic lines NIL-Bph43-9311 had strong antibiosis and antixenosis effects on BPH. Comparative transcriptome analysis revealed genes related to the defense response and resistance gene-dependent signaling pathway were significantly and uniquely enriched in Bph43 plants and might be the underlying molecular mechanism for BPH resistance. Our work provides a valuable BPH resistance gene for rice breeding programs.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/agronomy12040808/s1, Table S1: Primers used in this study. Table  S2: Summary of RNA-Seq data. Table S3: Candidate genes predicted in the reference genomes and their expression analysis by RNA-Seq. Table S4: Differentially expressed genes (DEGs) in response to BPH feeding between NIL-Bph43-9311 and 9311. Table S5: Gene ontology (GO) annotations of DEGs in response to BPH feeding between NIL-Bph43-9311 and 9311. Table S6: Go annotations of upregulated DEGs in response to BPH feeding between NIL-Bph43-9311 and 9311. Figure S1: Strategy used to develop NIL-Bph43-9311. Figure S2: FPKM box diagram of all tested samples and heatmap of Pearson's correlation coefficients for all tested samples. Figure S3: Genetic background assay of NIL-Bph43-9311 with rice chip GSR 40K array. Figure S4: Enriched GO annotations of DEGs in response to BPH feeding between NIL-Bph43-9311 and 9311.