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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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Related in: MedlinePlus

Complementation test of the Bph32 gene and characterization of BPH resistance in Bph32 transgenic rice.(a) BPH resistance test of Bph32 transgenic and susceptible wild-type (WT) rice. TN1, susceptible variety (CK); Ka, susceptible WT rice; N65-7-4-3-5, empty-vector transgenic T2:3 line; N65-2-5-2-6 and N65-7-1-1-8, Bph32 transgenic T2:3 lines. (b) BPH resistance scores of Bph32 transgenic T2:3 lines using the modified standard seedbox screening. The data are presented as means ± SD (three replications). (c) RT-PCR analysis showing Bph32 expression in the transgenic T2:3 lines. (d) BPH resistance test of the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) at the mature stage. Magnified views showed the locations of BPH feeding. (e) BPH survival number in the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) from the first to the twelfth days after BPH infestation. (f) Comparison of the honeydew area in TN1, Ka, N65-7-1-1-8 and Ptb33 using the honeydew excretion test. **P < 0.01. One-way ANOVA was used to generate the P value.
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f2: Complementation test of the Bph32 gene and characterization of BPH resistance in Bph32 transgenic rice.(a) BPH resistance test of Bph32 transgenic and susceptible wild-type (WT) rice. TN1, susceptible variety (CK); Ka, susceptible WT rice; N65-7-4-3-5, empty-vector transgenic T2:3 line; N65-2-5-2-6 and N65-7-1-1-8, Bph32 transgenic T2:3 lines. (b) BPH resistance scores of Bph32 transgenic T2:3 lines using the modified standard seedbox screening. The data are presented as means ± SD (three replications). (c) RT-PCR analysis showing Bph32 expression in the transgenic T2:3 lines. (d) BPH resistance test of the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) at the mature stage. Magnified views showed the locations of BPH feeding. (e) BPH survival number in the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) from the first to the twelfth days after BPH infestation. (f) Comparison of the honeydew area in TN1, Ka, N65-7-1-1-8 and Ptb33 using the honeydew excretion test. **P < 0.01. One-way ANOVA was used to generate the P value.

Mentions: To confirm that LOC_Os06g03240 confers BPH resistance, we transformed a susceptible indica variety, Kasalath (Ka), with the cDNA sequence of the Ptb33 Bph32 gene. Six independent transgenic events were detected using genomic southern blotting. Among them, N65-7-1-1-8 contains a single-copy of Bph32 and N65-2-5-2-6 contains a double-copy. Their T2:3 plants were used to measure the levels of resistance of Bph32. As demonstrated in Fig. 2, upon infestation with BPH at the seedling stage, all the wild-type control (Kasalath and TN1) and empty-vector transgenic plants died, whereas the transgenic rice plants expressing the Ptb33 Bph32 gene survived (Fig. 2a,b and c). When infected at the maturing stage, the wild-type plants exhibited leaf wilting, a decrease in seed and grain plumpness, and even death of the whole plant, whereas the Bph32 transgenic plants were all healthy (Fig. 2d).


Bph32 , a novel gene encoding an unknown SCR domain-containing protein, confers resistance against the brown planthopper in rice
Complementation test of the Bph32 gene and characterization of BPH resistance in Bph32 transgenic rice.(a) BPH resistance test of Bph32 transgenic and susceptible wild-type (WT) rice. TN1, susceptible variety (CK); Ka, susceptible WT rice; N65-7-4-3-5, empty-vector transgenic T2:3 line; N65-2-5-2-6 and N65-7-1-1-8, Bph32 transgenic T2:3 lines. (b) BPH resistance scores of Bph32 transgenic T2:3 lines using the modified standard seedbox screening. The data are presented as means ± SD (three replications). (c) RT-PCR analysis showing Bph32 expression in the transgenic T2:3 lines. (d) BPH resistance test of the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) at the mature stage. Magnified views showed the locations of BPH feeding. (e) BPH survival number in the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) from the first to the twelfth days after BPH infestation. (f) Comparison of the honeydew area in TN1, Ka, N65-7-1-1-8 and Ptb33 using the honeydew excretion test. **P < 0.01. One-way ANOVA was used to generate the P value.
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f2: Complementation test of the Bph32 gene and characterization of BPH resistance in Bph32 transgenic rice.(a) BPH resistance test of Bph32 transgenic and susceptible wild-type (WT) rice. TN1, susceptible variety (CK); Ka, susceptible WT rice; N65-7-4-3-5, empty-vector transgenic T2:3 line; N65-2-5-2-6 and N65-7-1-1-8, Bph32 transgenic T2:3 lines. (b) BPH resistance scores of Bph32 transgenic T2:3 lines using the modified standard seedbox screening. The data are presented as means ± SD (three replications). (c) RT-PCR analysis showing Bph32 expression in the transgenic T2:3 lines. (d) BPH resistance test of the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) at the mature stage. Magnified views showed the locations of BPH feeding. (e) BPH survival number in the Bph32 transgenic plants (N65-7-1-1-8) and susceptible wild-type (WT) plants (Ka) from the first to the twelfth days after BPH infestation. (f) Comparison of the honeydew area in TN1, Ka, N65-7-1-1-8 and Ptb33 using the honeydew excretion test. **P < 0.01. One-way ANOVA was used to generate the P value.
Mentions: To confirm that LOC_Os06g03240 confers BPH resistance, we transformed a susceptible indica variety, Kasalath (Ka), with the cDNA sequence of the Ptb33 Bph32 gene. Six independent transgenic events were detected using genomic southern blotting. Among them, N65-7-1-1-8 contains a single-copy of Bph32 and N65-2-5-2-6 contains a double-copy. Their T2:3 plants were used to measure the levels of resistance of Bph32. As demonstrated in Fig. 2, upon infestation with BPH at the seedling stage, all the wild-type control (Kasalath and TN1) and empty-vector transgenic plants died, whereas the transgenic rice plants expressing the Ptb33 Bph32 gene survived (Fig. 2a,b and c). When infected at the maturing stage, the wild-type plants exhibited leaf wilting, a decrease in seed and grain plumpness, and even death of the whole plant, whereas the Bph32 transgenic plants were all healthy (Fig. 2d).

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