Identification and characterization of Bph14, a gene conferring resistance to brown planthopper in rice

Abstract

Planthoppers are highly destructive pests in crop production worldwide. Brown planthopper (BPH) causes the most serious damage of the rice crop globally among all rice pests. Growing resistant varieties is the most effective and environment-friendly strategy for protecting the crop from BPH. More than 19 BPH-resistance genes have been reported and used to various extents in rice breeding and production. In this study, we cloned Bph14, a gene conferring resistance to BPH at seedling and maturity stages of the rice plant, using a map-base cloning approach. We show that Bph14 encodes a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC-NB-LRR) protein. Sequence comparison indicates that Bph14 carries a unique LRR domain that might function in recognition of the BPH insect invasion and activating the defense response. Bph14 is predominantly expressed in vascular bundles, the site of BPH feeding. Expression of Bph14 activates the salicylic acid signaling pathway and induces callose deposition in phloem cells and trypsin inhibitor production after planthopper infestation, thus reducing the feeding, growth rate, and longevity of the BPH insects. Our work provides insights into the molecular mechanisms of rice defense against insects and facilitates the development of resistant varieties to control this devastating insect.

Fig. 1.

Map-based cloning and complementation tests of the planthopper-resistance gene Bph14. (A) Fine mapping of the Bph14 locus. The Bph14 locus is located within a 34-kb region of chromosome 3, which contains two predicted genes. Numbers under the linkage map indicate the number of recombinants detected between the molecular markers and the Bph14 locus. (B) Structure of Ra and Rb. The three exons in each gene are boxed; the black boxes show the ORFs. (C) BPH-resistance test of the Bph14-transgenic and susceptible wild-type (WT) rice. RI35, resistant parental rice; Kasalath, susceptible WT rice; Ra1–Ra10, Bph14-transgenic T₂ lines. (D) BPH-resistance scores of the Bph14-transgenic rice at the seedling stage. The lower scores indicate the higher resistance to the insect. Data are means ± SD (n = 60 plants). (E) RT-PCR analysis showing the expression of Ra in the transgenic T₂ lines.

Fig. 2.

Molecular characterization of Bph14. (A) Phylogenetic relationships of Bph14 homologs in rice (Os), wheat (Ta), cassava (Me), potato (Sb), tomato (Le), maize (Zm), barley (Hv), and Arabidopsis (At). (Scale bar, 0.1 amino acid substitutions per site.) (B) Time-dependent expression of Bph14 and its alleles in the resistant and susceptible plants after BPH infestation. The mean is based on the average of three biological repeats calculated. (C) Expression analysis of Bph14 in the root, leaf sheath, and leaf blade of rice by RT-PCR. (D) Bph14 subcellular localization. The 35S::GFP (Upper) and 35S::Bph14-GFP (Lower) fusion genes were transiently expressed in onion epidermal cells. The Bph14-GFP fusion protein is localized in the cytoplasm. (E–J) Bph14 promoter–GUS expression pattern in transgenic rice plants. GUS express in the vascular system of root (E), leaf sheath (G), and leaf blade (I). Cross-sections of root (F), leaf sheath (H), and leaf blade (J) indicated that Bph14 is strongly expressed in the parenchyma cells bordering xylem vessels and sieve tubes. X, xylem; P, phloem. (Scale bars: D, 50 μm; F, H, and J, 20 μm.)

Fig. 3.

Characterization of insect resistance in Bph14-transgenic rice. (A) Planthopper-resistance test of the Bph14-transgenic and wild-type rice at the mature stage. Magnified views show the locations of BPH feeding. (B) Settling of BPH in a host choice test. (C) BPH fecundity. (D)Total duration of electronic penetration graph (EPG) waveform types for the BPH over an 8-h recording period. (E) Honeydew excretion on filter paper. The size of the honeydew area and the intensity of the honeydew color correspond to the BPH feeding activity. (F) BPH population growth rate. (G) BPH survival rate. The number of surviving BPHs per plant was significantly lower on resistant plants than on wild-type plants 3 days after infestation (P = 0.0038). (H) Induced callose deposition (red arrows) in the vascular bundle indicated by bright blue fluorescence. X, xylem; P, phloem. (Scale bar, 20 μm.) WT, the susceptible wild-type rice; Ra4–20, the resistant homozygous Bph14-transgenic rice. Data are means ± SD (n = 10). **, P < 0.01.

Fig. 4.

Expression patterns of plant defense-response genes. EDS1, PAD4, PAL, and ICS1 are the SA synthesis-related genes. NPR1 is a key regulator of SA-dependent systemic acquired resistance. LOX and AOS2 are the JA synthesis-related genes. PR1b is pathogen-related gene 1 in rice. EIN2 is the ethylene signaling pathway receptor gene. Rice Actin1 was used as reference control. Expression of genes was quantified relative to the value obtained from 0-h susceptible samples. Solid bars, the wild-type rice; open bars, the resistant homozygous Bph14-transgenic rice (Ra4–20). In all panels, the mean is based on the average of three biological repeats calculated. *, P < 0.05; **, P < 0.01. One-way ANOVA was used to generate the P values.