Os01g57340

This Magnaporthe grisea resistance-sh was first discovered by Japanese scholars Imbe and Matsumoto in 1985. It's well known for its moderate resistance for Kyu77-07A in Shin-2.

Annotated Information

Function

 R gene-mediated resistance is one of the most effective mechanisms of immunity against pathogens in plants. To date some components that regulate the primary steps of plant immunity have been isolated, however, the molecular dissection of defense signaling downstream of the R proteins remains to be completed. In addition, R genes are known to be highly variable, however, the molecular mechanisms responsible for this variability remain obscure.To identify novel factors required for R gene-mediated resistance in rice, we used rice insertional mutant lines, induced by the endogenous retrotransposon Tos17, in a forward screen involving the rice blast fungus Magnaporthe oryzae. We inoculated 41,119 mutant lines with the fungus using a high throughput procedure, and identified 86 mutant lines with diminished resistance. A genome analysis revealed that 72 of the 86 lines contained mutations in a gene encoding a nucleotide binding site (NB) and leucine rich repeat (LRR) domain-containing (NLR) protein. A genetic complementation analysis and a pathogenesis assay demonstrated that this NLR gene encodes Pish, which confers resistance against races of M. oryzae containing avrPish. The other 14 lines have intact copies of the Pish gene, suggesting that they may contain mutations in the signaling components downstream of Pish. The genome analysis indicated that Pish and its neighboring three NLR genes are high similar to one another and are tandemly located. An in silico analysis of a Tos17 flanking sequence database revealed that this region is a "hot spot" for insertion. Intriguingly, the insertion sites are not distributed evenly among these four NLR genes, despite their similarity at the sequence and expression levels.
 Gene pyramiding is considered one of the most effective strategies for achieving durable resistance against blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.), although few studies have evaluated the combining effect of the resistance genes. We report the development of pyramided lines with two major blast resistance genes, Pish and Pib, and the evaluation of the combining effect of them. The two genes pyramided lines were selected from the progenies of a cross between one near isogenic line (NIL), which harbours Pish, and another NIL, which harbours Pib, in the genetic background of blast susceptible variety, CO 39. The presence of the resistance genes was confirmed by DNA markers linked to them. To obtain DNA markers for Pish, we genetically mapped the Pish locus. We confirmed the additive effect of Pish and Pib in the pyramided lines by their reaction patterns to blast isolates, suggesting the potential availabilities of the combinations of these genes. In addition, we provide DNA markers linked to Pish for marker aided selection in rice blast resistance breeding.
 A gene analysis to identify the genes accounting for the moderate resistance of the rice variety Shin 2 and some other varieties to the blast fungus strain Kyu 77-07A was The differential varieties, Aichi Asahi (Pi-a) and carried out in the present study. Yashiro-mochi (Pi-ta), were found to be susceptible to the strain, while Ishikari Shirol*.e (Pi-i), Kanto 51 (Pi-k), Tsuyuake (Pi-k'!b), Fukunishiki (Pi-z) and Toride I (Pi-zt) were resistant, and Shin 2 (Pi-ks) and Pi N0.4 (Pi-ta2) moderately resistant. Therefore, Kyu 77-07A was classified as one of the strains belonging to race 102. Howver, the variety Reiho with the resistance gene Pi-ta2 was susceptible to this strain. Based on the knovn facts mentioned above, the objectives 0L the present study were' to determinA- whether Pi-ks is the gene responsible for the moderate resistance of Shin 2' to Kyu 77-07A along with the reason for the different reactions to Kyu 77-07A betweerL Pi N0.4 and Reiho which both have the same resistance gene Pi-ta2. For the gene analysis, the F2 or F3 plants of the crosses were inoculated with Kyu 77-07A and three other fungus strains using the spraying rnethod when the plants were at the four- to five-1eaf stage. Two varieties, Mineyutaka. and Saikai 155 belonging to the Shin 2 type varieties, were used as the representative parents of the varieties susceptible to Kyu 77-07A for the crosses.

Expression

Quantitative real-time RT-PCR analysis was carried out to investigate the expression pattern of Pish after infection with various M. oryzae races. The analysis revealed that there were no distinguishable alterations in Pish expression levels at different time points after inoculation with either incompatible or compatible races of the fungus, or after mock treatment. This result is consistent with previous reports that other Pi genes were constitutively expressed and not induced by pathogen challenge [10,11,13-15]. It is likely that most Pi genes are expressed before pathogen invasion and are post-transcriptionally regulated for activation of the signal transduction pathways leading to resistance responses.

Evolution

 Classical genetics and genome analysis have demonstrated that R genes tend to be clustered. The 55-kb region containing the Pish locus contains three other NBS-LRR genes, all oriented in the same direction. Among the proteins encoded by these genes, Npi37-1 exhibits little similarity to the other three, while Npi37-3 shows high similarity to Npi37-2 (91%) and Pish (98%). Intriguingly, the N-terminal half of Npi37-3 is identical to that of Pish and the other half is identical to that of Npi37-2 . These data suggest that at first the ancestral NBS-LRR gene was duplicated to produce Npi37-1 and Npi37-2-pre. This would be followed by a second duplication event in which Npi37-2-pre was duplicated to produce Npi37-2 and Pish. More recently, a crossover and/or duplication presumably occurred between Npi37-2 and Pish, resulting in Npi37-2, Npi37-3, and Pish. Npi37-1 and Pish are identical between the cultivars NB and St. No.1, suggesting that the two paralogs, Npi37-2 and Npi37-3, probably mutated independently in NB and/or St. No.1 after the duplication events described above.

 How did these frequent gene duplications in the Pish region occur? Although R gene loci in general tend to be duplicated, the mechanism for this duplication remains obscure. Gene duplication is sometimes caused by the misrepair of chromosomal double-strand breaks (DSBs), which arise spontaneously during the life of a cell. One possible inducer of DSBs is the endonuclease activity encoded by TEs. In Drosphila melanogaster, it has been reported that DSBs are important triggers of segmental duplication (SD), and the distribution of SDs correlates positively with that of TEs. Here we showed that the Pish locus is one of the hot spots for Tos17 insertionsc. Therefore, the Pish locus was presumably attacked frequently by the Tos17 endonuclease, resulted in DSBs, which might have caused gene duplication. In addition to this effect of endonuclease activity, the insertion of TEs is thought to be important for the molecular evolution of R genes. Actually, many types of TEs have been identified in R gene clusters. Recently, Hayashi and Yoshida reported that the insertion of a retrotransposon Renovator in the promoter region of the blast R gene Pit promoted its expression and reactivation, demonstrating that the insertion of TEs has contributed to R gene evolution.

  However, the disease resistance genes annotated in the report were predicted from their DNA sequence similarity with sequences encoding NBS and/or LRR domains, therefore there was no direct evidence indicating they truly function as R genes or components of defense signaling. Here, we demonstrated that the functional R gene Pish is actually a hot spot of Tos17 insertion in the NB genome, and is the preferred target site among four highly conserved and closely linked NBS-LRR genes, even though the genes are highly similar at both the nucleotide sequence and expression levels. These results suggest that Tos17 inserts most frequently in functional genes within hot spot regions. A search of the FST database indicates that not all NBS-LRR gene loci are hot spots for Tos17 insertion. It is possible that the NBS-LRR gene loci that are hot spots for TE insertions are functional R genes that have not yet been identified. Thus, it may be possible to predict novel functional R genes in the FST database by looking for regions that are hot spots for TE insertions. This possibility should be assessed in the future.

 The molecular mechanisms that determine TE integration site specificity in plants are still unknown. Studies of the Ty retrotransposons of yeast have revealed that interactions with bound chromosomal proteins can tether the Ty integration machinery to chromosomes and thereby direct integration to nearby sites . The human immunodeficiency virus (HIV) integrates preferentially into actively transcribed genes at sites with transcription-associated histone modifications. Therefore, it is possible that the insertion of Tos17 is regulated by chromatin structure or through interaction with chromatin binding proteins, rather than being controlled directly by the structures or expression levels of the targeted sequences.

mutation

 To confirm whether the null mutation of Pish(t) caused disruption of the resistance mediated by Pish, we screened for  allelic mutants among the 86 selected mutants from our screening. As expected, most of the mutants (72 of the 86 lines) had mutations caused by Tos17 insertions, deletions, or an unknown insertion in this locus. Because these mutant lines were produced by tissue culture and are derived from a relatively small number of induced calli, some of these mutations are shared in multiple independently regenerated plants. By considering which calli the mutants were derived from and by examining each mutation pattern (i.e., the positions of Tos17 insertions or the deletion sizes in those mutants), we determined that there are 56 independent mutant alleles at the Pish(t) locus among the 72 mutant lines. Of these, 46 alleles were caused by Tos17 insertion. The direction of Tos17 was not always the same and the insertion sites were dispersed evenly at this locus, suggesting that the insertion site within the locus was random rather than depending on specific DNA sequences. Nine independent deletion mutations were detected among thirteen lines. The deletion sizes were diverse, ranging from 24 bp to over 50 kb. One allele in the ND2032/ND2105/ND2452/ND2562 lines contained an unidentified insert of about 2.5-kb. In these mutants, the transcription of Pish(t) was barely detectable or not detected at all.
 On the other hand, 14 of the 86 lines with diminished Pish-mediated resistance contained neither mutations, insertions, nor deletion in this locus. In these mutants, the expression of Pish(t) was no different from in the wild type plants. Therefore, we concluded that they are not pish(t) mutants and designated them ttm (tissue-culture triggered mutation). Although we cannot exclude the possibility that some of the ttm mutants have mutations in unknown R genes that correspond to the blast isolate carrying additional avr genes other than avrPish, others are likely to have mutations in components required for activation of Pish-mediated disease resistance.
 To examine whether Pish(t) confer race-specific resistance, we transformed KM plants with an empty vector control and a construct containing the Pish(t) cDNA under the control of the cauliflower mosaic virus 35 S promoter. We obtained more than five independent transgenic lines for each construct, and used the T1 and T2 generations for the following analyses. Transgenic plants containing the Pish(t) construct were as healthy as KM plants transformed with the empty vector. When infected with a rice blast isolate containing avrPish, three independent transgenic lines expressing Pish(t) exhibited a resistance phenotype, whereas the lines containing the empty vector were susceptible. Thus, expression of the Pish(t) cDNA conferred Pish-mediated resistance on KM. To determine the resistance spectrum of Pish(t), the transgenic lines were inoculated with seven additional rice blast isolates. As expected, the transgenic KM plants containing Pish(t) exhibited the same pattern of resistance specificity as the donor cultivar NB. Thus, we concluded that Pish(t) is the Pish gene.

Labs working on this gene

1.Plant Disease Resistance Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan 2.Division of Genome and Biodiversity Research, National Institute of Agrobiological Sciences, Ibaraki, 305-8602, Japan

References

Please input cited references here. 1. Y. Koide;A. Kawasaki;M. J. Telebanco-Yanoria;A. Hairmansis;N. T. M. Nguyet;J. Bigirimana;D. Fujita;N. Kobayashi;Y. Fukuta

 Development of pyramided lines with two resistance genes, Pish and Pib, for blast disease (Magnaporthe oryzae B. Couch) in rice (Oryza sativa L.)
 Plant Breeding, 2010, 129(6): 670-675

2. Akira Takahashi;Nagao Hayashi;Akio Miyao;Hirohiko Hirochika

 Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging
 BMC Plant Biology, 2010, 10: 175

3. Tokio IMBE;Shohei MATSUMOTO

 Inheritance of Resrstance of Rice Vanetles to the Blast Fungus Strains Virulent to the Variety "Reiho"
 Japanese Journal of Breeding, 1985, 35(0): 332-339