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Bph32 , a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice

View Article: PubMed Central - PubMed

ABSTRACT

An urgent need exists to identify more brown planthopper (Nilaparvata lugens Stål, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as “Bph32”. This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24 h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests.

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Bph32 expression analysis.(a) Comparison of Bph32 expression as measured by RT-PCR in different organs in BPH-resistant 195B (red bar) and BPH-susceptible163B (blue bar). (b) Bph32 promoter-GUS expression pattern in transgenic rice plants. GUS expression profiles in root, culm, leaf blade, leaf sheath, glume, flower, immature seed and germinating seed, respectively (scale bars are as follows: 1–4, 500 μm; 5, 7 and 8, 800 μm; 6, 100 μm). (c) Bph32 subcellular localization. i–ii, Localization of the empty vector. Fluorescence (i) and merged image (ii) of the red fluorescence channel is shown in the top panel. iii–iv, Onion epithelial cells expressing the Bph-ptb33 fusion protein. Fluorescence (iii) and merged (iv) images showing that the Bph32 protein was localized mainly in the plasma membrane. (Scale bar: 100 μm) (d) Comparison of Bph32 expression by RT-PCR after BPH infestation in 195B (red bar) and 163B (blue bar). **P < 0.01; *P < 0.05. One-way ANOVA was used to generate the P value.
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f3: Bph32 expression analysis.(a) Comparison of Bph32 expression as measured by RT-PCR in different organs in BPH-resistant 195B (red bar) and BPH-susceptible163B (blue bar). (b) Bph32 promoter-GUS expression pattern in transgenic rice plants. GUS expression profiles in root, culm, leaf blade, leaf sheath, glume, flower, immature seed and germinating seed, respectively (scale bars are as follows: 1–4, 500 μm; 5, 7 and 8, 800 μm; 6, 100 μm). (c) Bph32 subcellular localization. i–ii, Localization of the empty vector. Fluorescence (i) and merged image (ii) of the red fluorescence channel is shown in the top panel. iii–iv, Onion epithelial cells expressing the Bph-ptb33 fusion protein. Fluorescence (iii) and merged (iv) images showing that the Bph32 protein was localized mainly in the plasma membrane. (Scale bar: 100 μm) (d) Comparison of Bph32 expression by RT-PCR after BPH infestation in 195B (red bar) and 163B (blue bar). **P < 0.01; *P < 0.05. One-way ANOVA was used to generate the P value.

Mentions: To reveal the molecular mechanisms underlying Bph32-mediated BPH resistance, we examined the expression profile of the Bph32 gene. Real-time (RT) PCR analysis showed that Bph32 was expressed in all investigated tissues at the flowering stage, and its expression level was highest in leaf sheaths followed by leaf blades, culms, panicles and roots (Fig. 3a), consistent with the preference of BPH to settle and probe in leaf sheaths at the flowering stage49. Bph32 expression was further analysed in more detail using transgenic plants carrying a Bph32 promoter-driven GUS reporter gene. GUS expression was observed in the root, leaf blade, leaf sheath, culm, glume, flower, immature seed and germinating seed (Fig. 3b), and GUS activity was strongly detected in parenchyma cells and the vascular bundle (Fig. 3b1 and 3b2). Notably, the expression levels of the Bph32 gene in some tissues of the susceptible line were different from that of the resistant line, which may be associated with the different in 5′ regulatory sequences (Figure S1a).


Bph32 , a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice
Bph32 expression analysis.(a) Comparison of Bph32 expression as measured by RT-PCR in different organs in BPH-resistant 195B (red bar) and BPH-susceptible163B (blue bar). (b) Bph32 promoter-GUS expression pattern in transgenic rice plants. GUS expression profiles in root, culm, leaf blade, leaf sheath, glume, flower, immature seed and germinating seed, respectively (scale bars are as follows: 1–4, 500 μm; 5, 7 and 8, 800 μm; 6, 100 μm). (c) Bph32 subcellular localization. i–ii, Localization of the empty vector. Fluorescence (i) and merged image (ii) of the red fluorescence channel is shown in the top panel. iii–iv, Onion epithelial cells expressing the Bph-ptb33 fusion protein. Fluorescence (iii) and merged (iv) images showing that the Bph32 protein was localized mainly in the plasma membrane. (Scale bar: 100 μm) (d) Comparison of Bph32 expression by RT-PCR after BPH infestation in 195B (red bar) and 163B (blue bar). **P < 0.01; *P < 0.05. One-way ANOVA was used to generate the P value.
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Related In: Results  -  Collection

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f3: Bph32 expression analysis.(a) Comparison of Bph32 expression as measured by RT-PCR in different organs in BPH-resistant 195B (red bar) and BPH-susceptible163B (blue bar). (b) Bph32 promoter-GUS expression pattern in transgenic rice plants. GUS expression profiles in root, culm, leaf blade, leaf sheath, glume, flower, immature seed and germinating seed, respectively (scale bars are as follows: 1–4, 500 μm; 5, 7 and 8, 800 μm; 6, 100 μm). (c) Bph32 subcellular localization. i–ii, Localization of the empty vector. Fluorescence (i) and merged image (ii) of the red fluorescence channel is shown in the top panel. iii–iv, Onion epithelial cells expressing the Bph-ptb33 fusion protein. Fluorescence (iii) and merged (iv) images showing that the Bph32 protein was localized mainly in the plasma membrane. (Scale bar: 100 μm) (d) Comparison of Bph32 expression by RT-PCR after BPH infestation in 195B (red bar) and 163B (blue bar). **P < 0.01; *P < 0.05. One-way ANOVA was used to generate the P value.
Mentions: To reveal the molecular mechanisms underlying Bph32-mediated BPH resistance, we examined the expression profile of the Bph32 gene. Real-time (RT) PCR analysis showed that Bph32 was expressed in all investigated tissues at the flowering stage, and its expression level was highest in leaf sheaths followed by leaf blades, culms, panicles and roots (Fig. 3a), consistent with the preference of BPH to settle and probe in leaf sheaths at the flowering stage49. Bph32 expression was further analysed in more detail using transgenic plants carrying a Bph32 promoter-driven GUS reporter gene. GUS expression was observed in the root, leaf blade, leaf sheath, culm, glume, flower, immature seed and germinating seed (Fig. 3b), and GUS activity was strongly detected in parenchyma cells and the vascular bundle (Fig. 3b1 and 3b2). Notably, the expression levels of the Bph32 gene in some tissues of the susceptible line were different from that of the resistant line, which may be associated with the different in 5′ regulatory sequences (Figure S1a).

View Article: PubMed Central - PubMed

ABSTRACT

An urgent need exists to identify more brown planthopper (Nilaparvata lugens St&aring;l, BPH) resistance genes, which will allow the development of rice varieties with resistance to BPH to counteract the increased incidence of this pest species. Here, using bioinformatics and DNA sequencing approaches, we identified a novel BPH resistance gene, LOC_Os06g03240 (MSU LOCUS ID), from the rice variety Ptb33 in the interval between the markers RM19291 and RM8072 on the short arm of chromosome 6, where a gene for resistance to BPH was mapped by Jirapong Jairin et al. and renamed as &ldquo;Bph32&rdquo;. This gene encodes a unique short consensus repeat (SCR) domain protein. Sequence comparison revealed that the Bph32 gene shares 100% sequence identity with its allele in Oryza latifolia. The transgenic introgression of Bph32 into a susceptible rice variety significantly improved resistance to BPH. Expression analysis revealed that Bph32 was highly expressed in the leaf sheaths, where BPH primarily settles and feeds, at 2 and 24&thinsp;h after BPH infestation, suggesting that Bph32 may inhibit feeding in BPH. Western blotting revealed the presence of Pph (Ptb33) and Tph (TN1) proteins using a Penta-His antibody, and both proteins were insoluble. This study provides information regarding a valuable gene for rice defence against insect pests.

No MeSH data available.


Related in: MedlinePlus