Map-based cloning and characterization of a brown planthopper resistance gene BPH26 from Oryza sativa L. ssp. indica cultivar ADR52

Abstract

The brown planthopper (BPH) is the most serious insect pest of rice in Asia. The indica rice cultivar ADR52 carries two BPH resistance genes, BPH26 (brown planthopper resistance 26) and BPH25. Map-based cloning of BPH26 revealed that BPH26 encodes a coiled-coil-nucleotide-binding-site-leucine-rich repeat (CC-NBS-LRR) protein. BPH26 mediated sucking inhibition in the phloem sieve element. BPH26 was identical to BPH2 on the basis of DNA sequence analysis and feeding ability of the BPH2-virulent biotype of BPH. BPH2 was widely incorporated in elite rice cultivars and was well-cultivated in many Asian countries as a favorable gene resource in rice breeding against BPH. However, BPH2 was rendered ineffective by a virulent biotype of BPH in rice fields in Asia. In this study, we suggest that BPH2 can be reused by combining with other BPH resistance genes, such as BPH25, to ensure durable resistance to BPH.

Figure 1. High-resolution genetic map of BPH26.

(a) Genetic linkage map of 5,349 BC₆F₃ plants showing the relative position of BPH26 with the single-sequence repeat, insertion-deletion, and single nucleotide polymorphism markers on chromosome 12. The numbers under the linkage map indicate the number of recombinants in the adjacent marker intervals. Framework maps are quoted from Harushima et al. (b) Putative genes in the candidate region whose presence is predicted by the Rice Annotation Project Database. A fragment 1A5 shows the genomic DNA of ADR52 which was used for the functional complementation test. (c) The structure of BPH26 on the genomic DNA of ADR52.

Figure 2. Functional complementation test of candidate genes.

T₁ lines that have been PCR validated to harbor the gene were used in the bioassays. The BPH2-avirulent biotype was used in these experiments. (a) The survival number of brown planthopper (BPH) on the three rice lines. The 1A5 line is the transgenic plant with the predicted gene on the DNA fragment 1A5 in the background of Taichung 65. The vector line is the transgenic plant with only vector fragment in the background of Taichung 65. BPH26-NIL is the near isogenic line with BPH26 in the genetic background of Taichung 65. In all, sixty last instar larvae that had been fasted for 24 h were released on each rice line for 5 days. (b) The amount of honeydew excreted by BPH females on the three rice lines. Values represent mean and SE (n = 10). Means labeled with the same letters are not significantly different by Tukey–Kramer test (P < 0.05). (c) The total duration of phloem ingestion on the three rice lines. The feeding behavior of BPH females was measured using an AC-EPG system for 10 h. Values represent mean and SE (n = 10). Means labeled with the same letters are not significantly different by Tukey–Kramer test (P < 0.05). (d) The total number of probes. The feeding behavior of BPH females was measured using an AC-EPG system for 10 h. Values represent mean and SE (n = 10). Means labeled with the same letters are not significantly different by Tukey–Kramer test (P < 0.05).

Figure 3. The deduced amino acid sequence of BPH26.

Typical motifs with rice R proteins are shown in blue letters, and the leucine-rich repeat (LRR) domain is underlined in red.

Figure 4. The comparisons of BPH26 and BPH2.

(a) Gene expression analysis of BPH26 in some rice varieties by using reverse transcriptase-PCR. Actin primers were used in the control amplification. The gels have been run under the same experimental conditions. Full-length gel is presented in Supplementary Figure S7. (b) The feeding abilities of the BPH2-virulent biotype of brown planthopper (BPH) on BPH26-near isogenic line and Norin-PL4 harboring the BPH2 gene. Total of sixty last instar larvae that had been starved for 24 h were released on each rice line for 5 days.

Figure 5. Phylogenic tree of some disease and pest resistance proteins in Oryza species.

Sequences were aligned by MEGA6 program, and a full-length amino acid sequence was used for clustering.

Figure 6. The results of in situ hybridization of BPH26.

Near isogenic lines with BPH26 at the 5- to 6-leaf stages were used for in situ hybridization analysis. Cross-sections of the leaf sheath are shown. (a): antisense probe of BPH26 m-RNA, (b): enlarged photograph of a square in (a), (c): sense probe of BPH26 m-RNA (blank sample), (d): enlarged photograph of (c).