Map-Based Cloning And Functional Analysis Of Brown Planthopper Resistance Gene Bph3 In Rice (Oryza Sativa L.)

Brown planthopper (Nilaparvata lugens Stal) is a monophagous insect pest of rice, it causes serious damage not only by directly feeding rice phloem sap, which affects rice growth and even results in "hopper burn", but also by transferring various virus diseases, such as viz. rice grassy stunt and rugged stunt. BPH severely damaged rice production, and has become the most serious pest in the Asian rice-growing countries. In rice production practice, chemical pesticides are still the effective way to control BPH damage, but it is expensive and harmful to the environment, and it can also urge BPH to readily develop resistance and emerge as major pest, so exploring new genes from diverse sources and developing new resistant cultivars are the most economical, effective and environment-friendly strategy for BPH management. The identification and cloning of insect resistance genes is not only helpful for elucidating the molecular mechanism, but also speeding up the application in breeding program,In this study, we researched the feeding behavior of BPH on resistance varity Rathu Heenati and susceptible variety 02428. Two methods were used to evaluate the BPH feeding preference on RH and 02428, results showed that BPH prefer feeding on 02428 rather than RH, suggesting RH displays strong antixenosis against BPH. When forcing BPH to feed on RH and 02428, the nymph survival and honeydew excretion on RH were significantly lower than in insects feeding on 02428, indicating RH displays strong antibiosis to BPH. Sectioning of the infested leaf sheaths showed the proportion of insect stylet sheath reached the phloem of vascular bundles in RH was much lower than that in 02428, suggesting that RH inhibits BPH feeding phloem sap. Therefore, the high BPH resistant rice variety RH displays strong antixenosis and antibiosis to BPH. Bph3 was mapped on chromosome 6 in previous study. In our study, a major resistant QTL was detected on the short arm of chromosome 4 using a F2 population derived from the cross between RH and 02428. On the other hand, we constructed a BC2F1 population, two types of plant were selected using markers on chromosome 4 and 6, and the resistant phenotype of two BC2F2 populations suggested that the resistant gene of RH in this study was on chromosome 4 rather than chromosome 6. Then total 21,783 individuals from F2、BC2F2 and BC3F2 were used to fine-map Bph3, Bph3 was finally mapped to the region between two Indel markers RHD9 and RHC10, the physical distance was about 79 kb. There are 12 genes in this region including three lectin receptor-like kinase (LecRKs) genes, and another predicted LecRKs is located just outside the 79 kb interval; these 4 genes form a receptor kinase gene cluster. Receptor kinase genes, as pattern recognition receptors (PRRs), identify elicitors of pathogens in the interaction between plant and pathogens. Therefore, these four OsLecRKs were used as candidate genes to analyze the structures and expression levels. Through QTL analysis, we found that the BPH resistant genes in BP348-e-4-2, Sarinah, Lv-7 and Lv-4 were allelic to Bph3, sequencing analysis showed that resistant varieties BP348-e-4-2、Sarinah、Lv-7、Lv-4、PTB33 and IR72 have identical amino acid sequences for OsLecRK1-OsLecRK3 as in RH, whereas the BPH-susceptible varieties 02428, Nipponbare and 9311 have a number of nucleotide polymorphisms in these genes, causing either amino acid substitutions, protein truncation or reading frame shift. In addition, polymorphisms are also found in OsLecRK4 between the two classes of varieties. Sequencing analysis to several recombinant lines with breakpoints in the OsLecRK gene cluster showed differential resistance to BPH, suggesting that these four genes may have additive effect on BPH resistance, while OsLecRK4 contributes the least. qRT-PCR analysis showed that the expressions of these four genes are all induced by BPH feeding and have difference between RH and 02428. These results above further suggested that OsLecRK1-OsLecRK4 may contribute to RH resistance to BPH, so we tested and verified these four candidate genes through transgenic method.We constructed complementary vectors of single-gene and polygenes, and then transformed them into the susceptible japonica variety Kitaake. The resistant phenotypes to BPH of T2 transgenic plants showed the resistant levels of transgenic plants transforming two genes were higher than that of one gene, but much lower than that of three genes. The result indicted that these four genes have additive effect on BPH resistance. In addition, RNAi plants of the four genes lost BPH resistance, and the reductions in resistance were approximately proportional to the reductions in target gene transcript levels. These observations further suggest that the OsLecRK genes function additively in conferring high BPH resistance in RH. Through homology analysis, the homology of amino acid encoding observations further suggest that the OsLecRK genes function additively in conferring high BPH resistance in RH. Through homology analysis, the homology of amino acid encoding by the four OsLecRKs is 80.8%, phylogenetic tree analysis showed that they all belong to the G-type LecRK family. GFP fusions of all four OsLecRKs were localized in the plasma membrane when transiently expressed in rice leaf sheath protoplasts, In vitro phosphorylation assays revealed that the kinase catalytic domains of OsLecRK1 OsLecRK4 were capable of phosphorylating the myelin basic protein in a Mn2+ or Mg2+ dependent manner, suggesting that they are active serine/threonine kinases. In addition, yeast two-hybrid experiment suggested that the kinase domain of OsLecRK2 and OsLecRK3 can interact with themselves or each other, these results above all suggested that OsLecRKs in our study belong to typical lectin receptor-like kinase. GUS histochemical staining showed enhanced staining at the BPH-pierced sites in the vascular bundles of leaf sheaths, and BPH-pierced vascular bundles stained with aniline blue, showed less callose deposition in 02428 than in RH after infestation. Quantitative RT-PCR analysis showed that the expression of callose synthases and β-1,3-glucanases were similarly induced in RH and 02428 after BPH infestation, suggesting that transcriptional regulation of callose synthases and β-1,3-glucanases genes does not play a major role in Bph3-mediated accumulation of callose. we compared the genomic sequences of this gene cluster between rice collections including both wild and cultivated rice, the sequence of this locus in RH shares, the highest similarity to that in O. nivara, but has not been introgressed into the majority of rice cultivars during rice breeding, so we introgressed it into cultivar "Ningjing3" through marker-assisted selection to breed new rice varieties to BPH, and the NIL R1266 had good resistance to BPH and WBPH, suggesting Bph3 was very usful in the production.The cloning of Bph3 established the foundation for analyzing the functions of these genes and illuminating the molecular mechanism of rice resistance against BPH, and contributed to research the similarity between plant resistance to insect and pathogen. Furthermore, we can breed new resistant rice varieties against BPH through developping new molecular markers tightly linkage to Bph3, which can enhance breeding efficiency through marker-assisted selection.