The blast resistance gene Pi37 encodes a nucleotide binding site leucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1

The resistance (R) gene Pi37, present in the rice cultivar St. No. 1, was isolated by an in silico map-based cloning procedure. The equivalent genetic region in Nipponbare contains four nucleotide binding site-leucine-rich repeat (NBS-LRR) type loci. These four candidates for Pi37 (Pi37-1, -2, -3, and -4) were amplified separately from St. No. 1 via long-range PCR, and cloned into a binary vector. Each construct was individually transformed into the highly blast susceptible cultivar Q1063. The subsequent complementation analysis revealed Pi37-3 to be the functional gene, while -1, -2, and -4 are probably pseudogenes. Pi37 encodes a 1290 peptide NBS-LRR product, and the presence of substitutions at two sites in the NBS region (V239A and I247M) is associated with the resistance phenotype. Semiquantitative expression analysis showed that in St. No. 1, Pi37 was constitutively expressed and only slightly induced by blast infection. Transient expression experiments indicated that the Pi37 product is restricted to the cytoplasm. Pi37-3 is thought to have evolved recently from -2, which in turn was derived from an ancestral -1 sequence. Pi37-4 is likely the most recently evolved member of the cluster and probably represents a duplication of -3. The four Pi37 paralogs are more closely related to maize rp1 than to any of the currently isolated rice blast R genes Pita, Pib, Pi9, Pi2, Piz-t, and Pi36.     

The identification of Pi50(t), a new member of the rice blast resistance Pi2/Pi9 multigene family

The deployment of broad-spectrum resistance genes is the most effective and economic means of controlling blast in rice. The cultivar Er-Ba-Zhan (EBZ) is a widely used donor of blast resistance in South China, with many cultivars derived from it displaying broad-spectrum resistance against blast. Mapping in a set of recombinant inbred lines bred from the cross between EBZ and the highly blast-susceptible cultivar Liangjiangxintuanheigu (LTH) identified in EBZ a blast resistance gene on each of chromosomes 1 (Pish), 6 (Pi2/Pi9) and 12 (Pita/Pita-2). The resistance spectrum and race specificity of the allele at Pi2/Pi9 were both different from those present in other known Pi2/Pi9 carriers. Fine-scale mapping based on a large number of susceptible EBZ?×?LTH F(2) and EBZ?×?LTH BC(1)F(2) segregants placed the gene within a 53-kb segment, which includes Pi2/Pi9. Sequence comparisons of the LRR motifs of the four functional NBS-LRR genes within Pi2/Pi9 revealed that the EBZ allele is distinct from other known Pi2/Pi9 alleles. As a result, the gene has been given the designation Pi50(t).     

Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance

The rice blast resistance gene Pikm was cloned by a map-based cloning strategy. High-resolution genetic mapping and sequencing of the gene region in the Pikm-containing cultivar Tsuyuake narrowed down the candidate region to a 131-kb genomic interval. Sequence analysis predicted two adjacently arranged resistance-like genes, Pikm1-TS and Pikm2-TS, within this candidate region. These genes encoded proteins with a nucleotide-binding site (NBS) and leucine-rich repeats (LRRs) and were considered the most probable candidates for Pikm. However, genetic complementation analysis of transgenic lines individually carrying these two genes negated the possibility that either Pikm1-TS or Pikm2-TS alone was Pikm. Instead, it was revealed that transgenic lines carrying both of these genes expressed blast resistance. The results of the complementation analysis and an evaluation of the resistance specificity of the transgenic lines to blast isolates demonstrated that Pikm-specific resistance is conferred by cooperation of Pikm1-TS and Pikm2-TS. Although these two genes are not homologous with each other, they both contain all the conserved motifs necessary for an NBS-LRR class gene to function independently as a resistance gene.     

Rice blast resistance gene Pikahei-1(t), a member of a resistance gene cluster on chromosome 4, encodes a nucleotide-binding site and leucine-rich repeat protein

Pikahei-1(t) is the strongest quantitative trait locus (QTL) for blast resistance in upland rice cv. Kahei, which has strong field resistance to the rice blast disease. A high-quality bacterial artificial chromosome library was used to fine-map Pikahei-1(t) within ~300 kb on the 31-Mb region on rice chromosome 4. Of the 42 predicted open reading frames, seven resistance gene analogs (RGAs) with the nucleotide-binding site and leucine-rich repeat (NBS-LRR) domain were identified. Among these, RGA1, 2, 3, 5, and 7, but not RGA4 and 6, were found to be expressed in Kahei and monogenic lines containing Pikahei-1(t). Blast inoculation of transgenic rice lines carrying the genomic fragment of each RGA revealed that only RGA3 was associated with blast resistance. On the basis of these results, we concluded that RGA3 is the Pikahei-1(t) and named it Pi63. Pi63 encoded a typical coiled-coil-NBS-LRR protein and showed isolate-specificity. These results suggest that Pi63 behaves like a typical Resistance (R) gene, and the strong and broad-spectrum resistance of Kahei is dependent on natural pyramiding of multiple QTLs. The blast resistance levels of Pi63 were closely correlated with its gene expression levels, indicating a dose-dependent response of Pi63 function in rice resistance. Pi63 is the first cloned R gene in the R gene cluster on rice chromosome 4, and its cloning might facilitate genomic dissection of this cluster region.     

The in silico map-based cloning of Pi36, a rice coiled-coil nucleotide-binding site leucine-rich repeat gene that confers race-specific resistance to the blast fungus

The indica rice variety Kasalath carries Pi36, a gene that determines resistance to Chinese isolates of rice blast and that has been located to a 17-kb interval on chromosome 8. The genomic sequence of the reference japonica variety Nipponbare was used for an in silico prediction of the resistance (R) gene content of the interval and hence for the identification of candidate gene(s) for Pi36. Three such sequences, which all had both a nucleotide-binding site and a leucine-rich repeat motif, were present. The three candidate genes were amplified from the genomic DNA of a number of varieties by long-range PCR, and the resulting amplicons were inserted into pCAMBIA1300 and/or pYLTAC27 vectors to determine sequence polymorphisms correlated to the resistance phenotype and to perform transgenic complementation tests. Constructs containing each candidate gene were transformed into the blast-susceptible variety Q1063, which allowed the identification of Pi36-3 as the functional gene, with the other two candidates being probable pseudogenes. The Pi36-encoded protein is composed of 1056 amino acids, with a single substitution event (Asp to Ser) at residue 590 associated with the resistant phenotype. Pi36 is a single-copy gene in rice and is more closely related to the barley powdery mildew resistance genes Mla1 and Mla6 than to the rice blast R genes Pita, Pib, Pi9, and Piz-t. An RT-PCR analysis showed that Pi36 is constitutively expressed in Kasalath.     

Functional characterization and signal transduction ability of nucleotide-binding site-leucine-rich repeat resistance genes in plants

Pathogen infection in plants is often limited by a multifaceted defense response triggered by resistance genes. The most prevalent class of resistance proteins includes those that contain a nucleotide-binding site-leucine-rich repeat (NBS-LRR) domain. Over the past 15 years, more than 50 novel NBS-LRR class resistance genes have been isolated and characterized; they play a significant role in activating conserved defense-signaling networks. Recent molecular research on NBS-LRR resistance proteins and their signaling networks has the potential to broaden the use of resistance genes for disease control. Various transgenic approaches have been tested to broaden the disease resistance spectrum using NBS-LRR genes. This review highlights the recent progress in understanding the structure, function, signal transduction ability of NBS-LRR resistance genes in different host-pathogen systems and suggests new strategies for engineering pathogen resistance in crop plants.     

Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.     

Identification of SSR markers for a broad-spectrum blast resistance gene Pi20(t) for marker-assisted breeding

The Pi20(t) gene was determined to confer a broad-spectrum resistance against diverse blast pathotypes (races) in China based on inoculation experiments utilizing 160 Chinese Magnaporthe oryzae (formerly Magnaporthe grisea) isolates, among which isolate 98095 can specifically differentiate the Pi20(t) gene present in cv. IR24. Two flanking and three co-segregating simple sequence repeat (SSR) markers for Pi20(t), located near the centromere region of chromosome 12, were identified using 526 extremely susceptible F₂ plants derived from a cross of Asominori, an extremely susceptible cultivar, with resistant cultivar IR24. The SSR OSR32 was mapped at a distance of 0.2 cM from Pi20(t), and the SSR RM28050 was mapped to the other side of Pi20(t) at a distance of 0.4 cM. The other three SSR markers, RM1337, RM5364 and RM7102, co-segregated with Pi20(t). RM1337 and RM5364 were found to be reliable markers of resistance conditioned by Pi20(t) in a wide range of elite rice germplasm in China. As such, they are useful tags in marker-assisted rice breeding programs aimed at incorporating Pi20(t) into advanced rice breeding lines and, ultimately, at obtaining a durable and broad spectrum of resistance to M. oryaze. Wei Li and Cailin Lei contributed equally to this work.     

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.     

A staphylococcal multidrug resistance gene product is a member of a new protein family.

The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates. The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris. The smr gene was subcloned and expressed in S. aureus and E. coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines. Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations. The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr). Four transmembrane segments are predicted for smr gene product. Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment. This Glu 13 is conserved in all members of the family of small membrane proteins. We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation. This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.     

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.     

Development and validation of co-dominant gene based markers for <Emphasis Type="Italic">Pi9</Emphasis>, a gene governing broad-spectrum resistance against blast disease in rice

Rice production and grain quality are severely affected by blast disease caused by the ascomycetous fungus Magnaporthe oryzae. Incorporation of genes that confer broad-spectrum resistance to blast has been a priority area in rice breeding programs. The blast resistance gene Pi9 sourced from Oryza minuta has shown broad spectrum and durable resistance to blast world-wide. In the present study co-dominant gene-based markers were developed for the precise marker-assisted tracking of Pi9 in breeding programs. The developed markers were validated across a diverse set of cultivars including basmati, indica and japonica varieties. Two markers, Pi9STS-1 and Pi9STS-2, effectively differentiated Pi9 donors from all the indicas and commercial basmati varieties tested. However, these markers were monomorphic between Pi-9 donors (IRBL9-W and Pusa 1637) and japonica type varieties. An additional gene-derived CAPS marker Pi91F_ 2R was developed to differentiate Pi9 donors from japonicas and traditional basmati lines. The co-dominant markers developed in the present study will be of immense utility to rice breeders for precise and speedy incorporation of Pi-9 into susceptible rice varieties through marker-assisted selection.     

SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein.

SSC1 is an essential member of the yeast HSP70 multigene family (E. Craig, J. Kramer, and J. Kosic-Smithers, Proc. Natl. Acad. Sci. USA 84:4156-4160, 1987). Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined. Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak. This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences. Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process. The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies. We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell.     

Genetic characterization and fine mapping of the blast resistance locus Pigm(t) tightly linked to Pi2 and Pi9 in a broad-spectrum resistant Chinese variety

The identification and utilization of broad-spectrum resistance genes have been proven the most effective and economical approach to control rice blast disease. To understand the molecular mechanism of broad-spectrum resistance to rice blast, we conducted genetic and fine mapping analysis of the blast resistance gene in a Chinese rice variety: Gumei 4 (GM4) identified with broad-spectrum resistance and used in rice breeding for blast resistance for more than 20 years. Genetic and mapping analysis indicated that blast resistance to nine isolates of different Chinese races in GM4 was controlled by the same dominant locus designated as Pigm(t) that was finely mapped to an approximately 70-kb interval between markers C5483 and C0428 on chromosome 6, which contains five candidate NBS--LRR disease resistance genes. The allelism test showed that Pigm(t) was either tightly linked or allelic to Pi2 and Pi9, two known blast resistance genes. Mapping information also indicated that another blast resistance gene Pi26(t) might also be located at the same region. Candidate genes were identified by sequence analysis of the Nipponbare and Pi9 locus and the corresponding region in GM4. Sequence divergence of candidate genes was observed between GM4 and model varieties Nipponbare and 9311, and Pi9. Our current study provides essential information and new genetic resource for the cloning of functional resistance gene(s) and for marker-assisted selection in rice breeding for broad-spectrum blast resistance.Yiwen Deng and Xudong Zhu contributed equally to this work.