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The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes

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Abstract

The blast resistance gene Pik-p, mapping to the Pik locus on the long arm of rice chromosome 11, was isolated by map-based in silico cloning. Four NBS-LRR genes are present in the target region of cv. Nipponbare, and a presence/absence analysis in the Pik-p carrier cv. K60 excluded two of these as candidates for Pik-p. The other two candidates (KP3 and KP4) were expressed in cv. K60. A loss-of-function experiment by RNAi showed that both KP3 and KP4 are required for Pik-p function, while a gain-of-function experiment by complementation test revealed that neither KP3 nor KP4 on their own can impart resistance, but that resistance was expressed when both were introduced simultaneously. Both Pikp-1 (KP3) and Pikp-2 (KP4) encode coiled-coil NBS-LRR proteins and share, respectively, 95 and 99% peptide identity with the two alleles, Pikm1-TS and Pikm2-TS. The Pikp-1 and Pikp-2 sequences share only limited homology. Their sequence allowed Pik-p to be distinguished from Pik, Pik-s, Pik-m and Pik-h. Both Pikp-1 and Pikp-2 were constitutively expressed in cv. K60 and only marginally induced by blast infection.

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Acknowledgments

We are grateful to Dr K. Shimamoto for his provision of the panda vector. Financial support was provided by the National 973 project and the National Transgenic Research Projects.

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Correspondence to Qinghua Pan.

Additional information

Communicated by B. Keller.

B. Yuan and C. Zhai contributed equally to this work.

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Fig. S1 Presence/absence analysis of

KP1 andKP2. a The structure of KP1 and KP2 as predicted by FGENESH. Fragments a, c indicate the two full-length sequences, and b, d the internal fragments. b Amplicon profiling following separation by agarose gel electrophoresis. Nip, cv. Nipponbare. Black box indicates exon, and line indicates intron. (PPT 202 kb)

Fig. S2

In silico map-based cloning ofPik-p. a The structure of Pik-p. Exons are shaded, and introns shown as a line, UTRs as lightlyshaded boxes. Priming sites for the amplification of full-length cDNAs are indicated. b The strategy used to clone KP3 and KP4. The solid line represents the ligation product of the four overlapping PCR fragments. c The strategy used to clone KP3+4. d The construct of KP3+4 (Pikp-1 + Pikp-2). (PPT 57 kb)

Fig. S3 Loss-of-function analysis of

Pik-p by RNAi. a, b Phenotypes of T0 RNAi cv. K60 plants containing the KP3 RNAi1 and KP4 RNAi1 constructs, respectively. The dotted arrows indicate individuals where blast resistance was suppressed, and the solid one that in which resistance was not suppressed. (PPT 2620 kb)

Fig. S4 Gain-of-function analysis of

Pik-p by transformation with Pikp-1 + Pikp-2. Phenotypes of T0 cv. Q1063 plants were derived from the construct KP3+4 (Pikp-1+Pikp-2). Resistant individuals indicated by the solid arrows, susceptible ones by dotted arrows.(PPT 1887 kb)

Fig. S5 Allelic variation at

Pikp-1. An alignment of the predicted peptide sequences from Pikp-1-K60, Pikp-1-Q1063, Pikm1-TS and Pikm5-NP. The sequence identities between Pikp-1-K60/Pikm1-TS, Pikp-1-K60/Pikp-1-Q1063 and Pikp-1-K60/Pikm5-NP were 95, 98 and 59%, respectively. The allele-specific SNP is marked by a red star. (PPT 163 kb)

Fig. S6 Allelic variation at

Pikp-2 alleles. An alignment of the predicted peptide sequences from Pikp-2-K60, Pikp-2-Q1063, Pikm2-TS, and Pikm6-NP. The sequence identities between Pikp-2-K60 / Pikm2-TS, Pikp-2-K60 / Pikp-2-Q1063, and Pikp-2-K60 / Pikm6-NP were 99%, 100% and 76%, respectively. The allele-specific SNP imarked by a red star. (PPT 80 kb)

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Yuan, B., Zhai, C., Wang, W. et al. The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes. Theor Appl Genet 122, 1017–1028 (2011). https://doi.org/10.1007/s00122-010-1506-3

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