Os01g0149500

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Annotated Information

Function

The plant genome contains a large number of disease resistance (R) genes that have evolved through diverse mechanisms. Pit confers race-specific resistance against the fungal pathogen Magnaporthe grisea, and is a member of the nucleotide-binding site leucine-rich repeat (NBS-LRR) family of R genes.

Cultivars carrying a single-locus resistance often break down because of an M. oryzae race shift toward strains that escape host recognition by adaptive selection pressure (Bonman et al. 1992; Babujee and Gnanamanickam 2000).Therefore, large-scale cultivation of rice varieties with Pit should also aVect the diversity of blast fungus races. Although Noda et al. (1999) revealed that 86% of 129 isolates of M. oryzae from Vietnam were virulent to Pit, our Pit genotyping using FMs suggested that two cultivars in Vietnam, Khau Mac Kho (WRC48) and Khau Tan Chiem (WRC52), do not have the functional Pit allele. Therefore, it is interesting to pursue further experiments combining monitoring of the Vietnamese cultivar identity using tK59 with inoculation tests to uncover a deeper and strong evolutionary relationship between cultivars and races. FMs

Expression

   The structure of the predicted Pit protein is typical of NBSLRR proteins (Figures 3a, S1 and S3). Pit contains conserved motifs indicative of an NBS domain, and the putative LRR domain (with 18 imperfect repeats) matches the cytoplasmic LRR consensus sequence. The Pit protein encoded by the Nipponbare allele (PitNpb) differs from that encoded by the K59 allele (PitK59) at four amino acid positions: 143 (R fi G), 176 (M fi I), 720 (A fi T) and 780 (M fi V) (Figures 3a, S1 and S3). Two of the substitutions (R143G and M176I) are located in the Nterminal half of the protein, whereas the others are in the LRR domain.
   A series of chimeric transgenes are used to examine which substitution(s) is important for determining Pit resistance(Figure 3b). First, the LRR region of PitK59 is replaced with that of PitNpb (A720T/M780V), because a single amino acid difference in the LRR domain has been shown to affect resistance. However, the PitK59 (A720T/M780V) lines conferred resistance (‘R + RM’) to 92% of the transgenic Nipponbare plants, suggesting that the two substitutions in the LRR region are not important for Pit function. Next, mutating the corresponding amino acids in PitNpb (R143G and M176I) to analyze the two substitutions in the N-terminal half of PitK59. A 24% reduction in resistance was observed in the transgenic plants as a result. Whereas the M176I mutation, located between the second CC motif (CC2) and the P loop, conferred resistance to 87% of the plants, R143G, located in CC2, conferred resistance to only 55% of the plants. These results suggest that the R143G mutation affected the resistance. Indeed, transgenic populations harboring the R143G mutation [i.e. PitK59 (R143G/M176I), PitK59 (R143G), Kpro-PitNpb and Ubi-PitNpb] obviously showed higher proportions of RS phenotypes (more than 18%) compared with the other populations. To examine whether these RS phenotypes are caused by the lower expression of chimeric transgenes, RT-PCR analysis was performed (Figure 3c). Both PitK59 (R143G/M176I) and PitK59 (R143G) transgenic plants showed higher levels of transgene expression than K59, irrespective of their phenotypes: i.e. transgenic plants with the RS phenotype also expressed the transgenes at high levels. These results further suggest that the R143G mutation may have destabilized the resistance function. However, considering the result that 80% of gPitNpb plants showed the complete susceptible (S) phenotype (Figure 3b), Pit requires another factor for its activity. As noted above, the structures of PitK59 and PitNpb differ in their promoter and 5¢-UTR regions; moreover, the efficiency of translation is controlled by various features (e.g. 5¢-UTR). Therefore, experiments were taken to examine the effects of the promoter and 5¢-UTR from each gene on Pit function using the constructs Npro5¢-PitK59, Npro-PitK59, Kpro-PitNpb and Ubi-PitNpb (Figure 3b). Npro5¢-PitK59, which contained 1.9 kb of the promoter and the 1.3-kb 5¢-UTR of PitNpb fused to the remainder of PitK59, conferred resistance to only 31% of the T0 plants. Similarly, only 30% of T0 plants expressing Npro-PitK59, in which the 1.9-kb promoter of PitNpb was fused directly with the coding sequence and terminator of PitK59, to eliminate possible effects of the 5¢-UTR of PitNpb, exhibited a resistant phenotype. These results negate the possibility that the extended 5¢-UTR of PitNpb affects Pit function. However, surprisingly, Kpro-PitNpb, which carried the PitNpb coding sequence driven by the PitK59 promoter consisting of the 3¢ fragment of Renovator (1040 bp) and the 5¢-UTR (254 bp) of PitK59, conferred resistance to 78% of the transgenic plants (Figure 3b). Finally, 71% of plants overexpressing the PitNpb open-reading frame (ORF) (Ubi-PitNpb) exhibited resistance (Figure 3b). The expression levels of PitK59 or PitNpb in gPitNpb, Npro5¢-PitK59, Npro-PitK59 and Ubi-PitNpb were correlated with their resistance (Figure 3d,e). These results clearly indicate that the promoter activity conferred by the Renovator retrotransposon fragment, rather than the amino acid substitutions, plays an important role in Pit resistance by enhancing its transcription. Notably, the increased resistance displayed by the plants expressing Kpro-PitNpb, compared with those expressing Npro-PitK59, indicates that promoter activity plays a dominant role in PitK59 function.

Pit protein encoded by the Nipponbare allele diVers from that encoded by the K59 allele at four amino acid positions (Fig. 1b): R143G, M176I, A720T, and M780V. Inoculation tests using chimeric constructs carrying the various regions of the K59 and Nipponbare alleles suggest that R143G and M176I have a potential eVect in Pit resistance, while A720T and M780V do not (Hayashi and Yoshida 2009). Sequence comparison of Pit alleles between K59 and nine susceptible cultivars revealed that the nucleotide polymorphisms, which were correlated with the resistance phenotypes in the transgenic experiments, were not always used for FMs: arginine at position 143 and methionine at position 176, which potentially aVected the Pit resistance, also existed in the susceptible cultivars of Takanari- and Senshou-types. Methionine at position 780, which had no eVect on the Pit resistance, was unique in the K59-type cultivars (Fig. 1b). These results show that polymorphic allele sequences, including those of susceptible cultivars, are a prerequisite to evaluating candidate nucleotide polymorphisms for FMs.

Evolution

Compared with the non-functional allele PitNpb, the functional allele PitK59 contains four amino acid substitutions, and has the LTR retrotransposon Renovator inserted upstream. Pathogenesis assays using chimeric constructs carrying the various regions of PitK59 and PitNpb suggest that amino acid substitutions might have a potential effect in Pit resistance; more importantly, the upregulated promoter activity conferred by the Renovator sequence is essential for Pit function. It is said that transposon-mediated transcriptional activation may play an important role in the refunctionalization of additional 'sleeping'R genes in the plant genome.

   Many R gene clusters are thought to have evolved via tandem and segmental duplications, rearrangement between the genes in a cluster, and point mutations or the deletion of genes (Meyers et al., 2003). As shown in Figure 7, the Pit locus has evolved through a combination of duplication, point mutations and TE insertions. The barley Pit homolog RGA S-reg19 was mapped to chromosome 3H (Madsen et al., 2003). Intriguingly, this position corresponds to the rice Pit locus, and a barley blast resistance QTL (bbr3H) was also mapped near this region

(Chen et al., 2003). This suggests that at least one copy of the ancestral Pit gene emerged before the diversification of the ancestors of rice and barley. Furthermore, although the genomic structure of this homolog(s) is unknown, barley may have a copy of the functional Pit gene with resistance to the blast fungus. After the emergence of the ancestral Pit gene, a duplication event occurred, and the two paralogs probably mutated independently of each other. The overexpression of NBSt1K59 failed to confer resistance (Figure 2a), suggesting that NBSt1K59 lost its function through amino acid substitutions. Amino acid substitutions were detected throughout the protein, not only in the LRR domain but also in the consensus elements of the NBS domain (Figure S1). NBSt1 has an amino acid substitution (C fi F) that is highly conserved in the RNBS-D motif, suggesting one possible cause of the loss of function. Interestingly, the R143G substitution in the CC domain did not occur in NBSt1K59, suggesting that the substitution was generated in Pit after the tandem duplication of the ancestral gene, and is not involved in the disruption of NBSt1K59 function. The ancestral Pit promoter may have been attenuated by mutations before the duplication of the gene, because NBSt1Npb and PitNpb share high similarity in their promoter sequences (data not shown), and both are expressed at low levels in Nipponbare. The point mutations and promoter attenuation would have suppressed Pit resistance to an undetectable level; however, the insertion of Renovator upstream of Pit reactivated its transcription, although we cannot exclude the possibilities of the removal of a repressor sequence or insulation of the promoter against an upstream repressor (Figure 7). The transposition of Renovator in an ancestor of K59 is likely to be a recent event, because the sequences of its 5¢- and 3¢-LTRs are identical.

As far as we know, allelic genes of functional Pit have not been identiWed. In a previous study, we revealed that Pit of K59-type was activated by transcriptional alteration caused by the Xanking Renovator sequence. Although Kiyosawa et al. suggested identiWcation of Pit in the Tongil variety by inoculation tests, our FMs did not detect Renovator upstream of Pit in Tongil (Hayashi, unpublished data). This may suggest at least three possibilities: (a) Tongil possesses diVerent mechanisms to activate Pit function without the Renovator-Pit system; (b) the line of Tongil we used in this study was diVerent from the line of Kiyosawa et al.’s experiment, and the functional Pit allele was segregated out in our line; and (c) the inoculation tests were misinterpreted in the previous report. The Wrst possibility is attractive because such kinds of genes would be candidates for the novel Pit allelic genes and provide important insights into the evolution of the Pit locus. Therefore, the FMs may indirectly contribute to the identiWcation of allelic Pit genes and subsequent evolutionary studies of the Pit genes.

Labs working on this gene

National Agricultural Research Center, National Agriculture and Food Research Organization (NARO), 1-2-1 Inada, Jo-etsu, Niigata 943 0193, Japan

References

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1.Keiko Hayashi;Hitoshi Yoshida. Refunctionalization of the ancient rice blast disease resistance gene Pit by the recruitment of a retrotransposon as a promoter, The Plant Journal(2009)57,413-425

2.K.Hayashi;N.Yasuda;Y.Fujita;S.Koizumi;H.Yoshida, Identification of the blast resistance gene Pit in rice cultivars using functional markers,Theor Appl Gene(2010)121:1357-1367 3.Genetics of blast resistance Kiyosawa-S Rice Breeding, 1972, (0) : 203-225