Isolation and characterization of disease resistance gene homologues from rice cultivar IR64

We initiated a search for disease resistance (R) gene homologues in rice cultivar IR64, one of the most agronomically important rice varieties in the world, with the assumption that some of these homologues would correspond to previously identified disease resistance loci. A family of rice R gene homologues was identified using the Arabidopsis NBS-LRR disease resistance gene RPS2 as a hybridization probe. Because member genes of this rice R gene family exhibit features characteristic of the NBS-LRR class of resistance genes, the family was given the name NRH (for NBS-LRR resistance gene homologues). Three members of the NRH family, NRH1, NRH2, and NRH3, were cloned and studied in detail. In IR64, NRH1 and NRH2 appear to encode full-length polypeptides, whereas NRH3 is prematurely truncated with a stop codon generated by a frameshift. NRH1 maps on chromosome 5, and NRH2 and NRH3 are less than 48kb apart on chromosome 11. Although NRH1, NRH2, and NRH3 map to regions of the rice genome where disease resistance loci to Xanthomonas oryzae pv. oryzae (Xoo) have been identified, susceptible rice varieties transformed with either NRH1 or NRH2 failed to exhibit increased resistance to a set of well-characterized Xoo strains.     

Linked, if not the same, Mi-1 homologues confer resistance to tomato powdery mildew and root-knot nematodes

On the short arm of tomato chromosome 6, a cluster of disease resistance (R) genes have evolved harboring the Mi-1 and Cf genes. The Mi-1 gene confers resistance to root-knot nematodes, aphids, and whiteflies. Previously, we mapped two genes, Ol-4 and Ol-6, for resistance to tomato powdery mildew in this cluster. The aim of this study was to investigate whether Ol-4 and Ol-6 are homologues of the R genes located in this cluster. We show that near-isogenic lines (NIL) harboring Ol-4 (NIL-Ol-4) and Ol-6 (NIL-Ol-6) are also resistant to nematodes and aphids. Genetically, the resistance to nematodes cosegregates with Ol-4 and Ol-6, which are further fine-mapped to the Mi-1 cluster. We provide evidence that the composition of Mi-1 homologues in NIL-Ol-4 and NIL-Ol-6 is different from other nematode-resistant tomato lines, Motelle and VFNT, harboring the Mi-1 gene. Furthermore, we demonstrate that the resistance to both nematodes and tomato powdery mildew in these two NIL is governed by linked (if not the same) Mi-1 homologues in the Mi-1 gene cluster. Finally, we discuss how Solanum crops exploit Mi-1 homologues to defend themselves against distinct pathogens.     

Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and arabidopsis

The presence of a single resistance (R) gene allele can determine plant disease resistance. The protein products of such genes may act as receptors that specifically interact with pathogen-derived factors. Most functionally defined R-genes are of the nucleotide binding site-leucine rich repeat (NBS-LRR) supergene family and are present as large multigene families. The specificity of R-gene interactions together with the robustness of plant-pathogen interactions raises the question of their gene number and diversity in the genome. Genomic sequences from tomato showing significant homology to genes conferring race-specific resistance to pathogens were identified by systematically "scanning" the genome using a variety of primer pairs based on ubiquitous NBS motifs. Over 70 sequences were isolated and 10% are putative pseudogenes. Mapping of the amplified sequences on the tomato genetic map revealed their organization as mixed clusters of R-gene homologues that showed in many cases linkage to genetically characterized tomato resistance loci. Interspecific examination within Lycopersicon showed the existence of a null allele. Consideration of the tomato and potato comparative genetic maps unveiled conserved syntenic positions of R-gene homologues. Phylogenetic clustering of R-gene homologues within tomato and other Solanaceae family members was observed but not with R-gene homologues from Arabidopsis thaliana. Our data indicate remarkably rapid evolution of R-gene homologues during diversification of plant families.     

Arsenical resistance in the IncHI2 plasmids

The IncHI2 plasmid R478, like other arsenic resistance IncH plasmids, provides increased levels of resistance to sodium arsenate (up to 100mM) and sodium arsenite (up to 10mM) to the host cell. An arsenic resistance fragment of R478 was cloned and sequenced revealing four arsenic resistance associated gene homologues, arsR, arsB, arsC, and arsH. Two other open reading frames in the cloned fragment were found to be homologues of sulphate transport associated genes. Both the four gene arsenic resistance operons and the two gene sulphate transport operons have been previously shown to be transposon associated. However, no evidence of transposability was found associated with these operons in R478. Both the R478 associated arsenic and sulphate transport operons were shown to be common to all arsenic resistance IncH plasmids examined by Southern hybridisation and PCR analysis.     

The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance?

Chloroquine has been the mainstay of antimalarial chemotherapy but the rapid spread of resistance to this important drug has now compromised its efficacy. The mechanism of chloroquine resistance has not been known but recent evidence from Plasmodium falciparum, the causative agent of the most severe form of human malaria, suggested similarities to the multidrug resistance phenotype (MDR) of mammalian tumour cells which is mediated by a protein molecule termed P-glycoprotein. Two mdr genes (pfmdr1 and pfmdr2) encoding P-glycoprotein homologues have been identified in P. falciparum and one of these (pfmdr1) has several alleles that have been linked to the chloroquine resistance phenotype. In contrast analysis of a genetic cross between chloroquine-resistant and -sensitive P. falciparum has suggested that the genes encoding the known P-glycoprotein homologues are not linked. This review outlines the similarities of the chloroquine resistance phenotype with the MDR phenotype of mammalian tumour cells and explores the possible role of the pfmdr genes.     

New Substrates on the Block: Clinically Relevant Resistances for EmrE and Homologues

Transporters of the small multidrug resistance (SMR) family are small homo- or heterodimers that confer resistance to multiple toxic compounds by exchanging substrate with protons. Despite the wealth of biochemical information on EmrE, the most studied SMR member, a high-resolution three-dimensional structure is missing. To provide proteins that are more amenable to biophysical and structural studies, we identified and partially characterized SMR transporters from bacteria living under extreme conditions of temperature and radiation. Interestingly, these homologues as well as EmrE confer resistance to streptomycin and tobramycin, two aminoglycoside antibiotics widely used in clinics. These are hydrophilic and clinically important substrates of SMRs, and study of their mode of action should contribute to understanding the mechanism of transport and to combating the phenomenon of multidrug resistance. Furthermore, our study of one of the homologues, a putative heterodimer, supports the suggestion that in the SMR family, heterodimers can also function as homodimers.     

The I2 resistance gene homologues in Solanum have complex evolutionary patterns and are targeted by miRNAs

Background

Several resistance traits, including the I2 resistance against tomato fusarium wilt, were mapped to the long arm of chromosome 11 of Solanum. However, the structure and evolution of this locus remain poorly understood.

Results

Comparative analysis showed that the structure and evolutionary patterns of the I2 locus vary considerably between potato and tomato. The I2 homologues from different Solanaceae species usually do not have orthologous relationship, due to duplication, deletion and frequent sequence exchanges. At least 154 sequence exchanges were detected among 76 tomato I2 homologues, but sequence exchanges between I2 homologues in potato is less frequent. Previous study showed that I2 homologues in potato were targeted by miR482. However, our data showed that I2 homologues in tomato were targeted by miR6024 rather than miR482. Furthermore, miR6024 triggers phasiRNAs from I2 homologues in tomato. Sequence analysis showed that miR6024 was originated after the divergence of Solanaceae. We hypothesized that miR6024 and miR482 might have facilitated the expansion of the I2 family in Solanaceae species, since they can minimize their potential toxic effects by down-regulating their expression.

Conclusions

The I2 locus represents a most divergent resistance gene cluster in Solanum. Its high divergence was partly due to frequent sequence exchanges between homologues. We propose that the successful expansion of I2 homologues in Solanum was at least partially attributed to miRNA mediated regulation.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-743) contains supplementary material, which is available to authorized users.     

Linkage disequilibrium mapping of a<Emphasis Type="Italic"> Verticillium dahliae</Emphasis> resistance quantitative trait locus in tetraploid potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) through a candidate gene approach

We have used the linkage disequilibrium mapping method to test for an association between a candidate gene marker and resistance to Verticillium dahliae in tetraploid potato. A probe derived from the tomato Verticillium resistance gene (Ve1) identified homologous sequences (StVe1) in potato, which in a diploid population map to chromosome 9, in a position analogous to that of the tomato resistance gene. When a molecular marker closely linked (1.5 cM) to the homologues was used as a candidate gene marker on 137 tetraploid potato genotypes (mostly North American cultivars), the association between the marker and resistance was confirmed (P<0.001). The amount of phenotypic variation in resistance explained by the allele of the STM1051 marker was greater than 10% and 25% in two subpopulations that were inferred from coancestry data matrix. Cloning of homologues from the highly resistant potato cv. Reddale indicates that the resistance quantitative trait locus (QTL) comprises at least an eleven-member family, encoding plant-specific leucine-rich repeat proteins highly similar to the tomato Ve genes. The sequence analysis shows that all homologues are uninterrupted open reading frames and thus represent putative functional resistance genes. This is the first time that the linkage disequilibrium method has been used to find an association between a resistance gene and a candidate gene marker in tetraploid potato. We have shown that it is possible to map QTL directly on already available potato cultivars, without developing a new mapping population.Communicated by F. SalaminiAn erratum to this article can be found at     

The broad-spectrum potato cyst nematode resistance gene (Hero) from tomato is the only member of a large gene family of NBS-LRR genes with an unusual amino acid repeat in the LRR region

The Hero gene of tomato is a broad spectrum resistance gene that confers a high level of resistance to all pathotypes of the potato cyst nematodes Globodera rostochiensis and partial resistance to G. pallida. The gene was identified by map-based cloning, sequencing and complementation analysis of two susceptible tomato lines with an array of 13 overlapping cosmids spanning a total distance of 135 kb. Hero encodes a protein with a nucleotide-binding site (NBS) and a leucine-rich-repeat (LRR) domain and is a member of a gene family of 14 highly homologous genes, which are clustered within a continuous 118-kb region. The isolated Hero gene displayed resistance to various G. rostochiensis pathotypes and partial resistance to G. pallida pathotype Pa2/3 in transgenic tomato lines. None of the Hero homologues conferred resistance to G. rostochiensis pathotypes. Hero can be distinguished from its homologues by the length of a compound hexanucleotide microsatellite, which codes for a charged and repetitive amino acid domain within the LRR. We propose that the expansion of this microsatellite may be involved in the evolution of the Hero resistance gene.     

Genetic mapping of the Tsw locus for resistance to the Tospovirus Tomato spotted wilt virus in Capsicum spp. and its relationship to the Sw-5 gene for resistance to the same pathogen in tomato

The Tsw gene conferring dominant resistance to the Tospovirus Tomato spotted wilt virus (TSWV) in Capsicum spp. has been tagged with a random amplified polymorphic DNA marker and mapped to the distal portion of chromosome 10. No mapped homologues of Sw-5, a phenotypically similar dominant TSWV resistance gene in tomato, map to this region in C. annuum, although a number of Sw-5 homologues are found at corresponding positions in pepper and tomato. The relationship between Tsw and Sw-5 was also examined through genetic studies of TSWV. The capacity of TSWV-A to overcome the Tsw gene in pepper and the Sw-5 gene in tomato maps to different TSWV genome segments. Therefore, despite phenotypic and genetic similarities of resistance in tomato and pepper, we infer that distinct viral gene products control the outcome of infection in plants carrying Sw-5 and Tsw, and that these loci do not appear to share a recent common evolutionary ancestor.     

Chemogenomic Study of Carboplatin in Saccharomyces cerevisiae: Inhibition of the NEDDylation Process Overcomes Cellular Resistance Mediated by HuR and Cullin Proteins

The use of carboplatin in cancer chemotherapy is limited by the emergence of drug resistance. To understand the molecular basis for this resistance, a chemogenomic screen was performed in 53 yeast mutants that had previously presented strong sensitivity to this widely used anticancer agent. Thirty-four mutants were responsive to carboplatin, and from these, 21 genes were selected for further studies because they have human homologues. Sixty percent of these yeast genes possessed human homologues which encoded proteins that interact with cullin scaffolds of ubiquitin ligases, or whose mRNA are under the regulation of Human antigen R (HuR) protein. Both HuR and cullin proteins are regulated through NEDDylation post-translational modification, and so our results indicate that inhibition of this process should sensitise resistant tumour cells to carboplatin. We showed that treatment of a tumour cell line with MLN4924, a NEDDylation inhibitor, overcame the resistance to carboplatin. Our data suggest that inhibition of NEDDylation may be a useful strategy to resensitise tumour cells in patients that have acquired carboplatin resistance.     

Resistance gene homologues in melon are linked to genetic loci conferring disease and pest resistance

Genomic and cDNA fragments with homology to known disease resistance genes (RGH fragments) were cloned from Cucumis melo using degenerate-primer PCR. Fifteen homologues of the NBS-LRR gene family have been isolated. The NBS-LRR homologues show high divergence and, based on the partial NBS-fragment sequences, appear to include members of the two major subfamilies that have been described in dicot plants, one that possesses a TIR-protein element and one that lacks such a domain. Genomic organization of these sequences was explored by DNA gel-blot analysis, and conservation among other Cucurbitaceae was assessed. Two mapping populations that segregate for several disease and pest resistance loci were used to map the RGH probes onto the melon genetic map. Several NBS-LRR related sequences mapped to the vicinity of genetic loci that control resistance to papaya ringspot virus, Fusarium oxysporum race 1, F. oxysporum race 2 and to the insect pest Aphis gossypii. The utility of such markers for breeding resistant melon cultivars and for cloning the respective R-genes is discussed.     

Identification of genes conferring arsenic resistance to Escherichia coli from an effluent treatment plant sludge metagenomic library

The majority of bacteria elude culture in the laboratory. A metagenomic approach provides culture-independent access to the gene pool of the whole bacterial community. A metagenomic library was constructed from an industrial effluent treatment plant sludge containing about 1.25 Gb of microbial community DNA. Two arsenic-resistant clones were selected from the metagenomic library. Clones MT3 and MT6 had eight- and 18-fold higher resistance to sodium arsenate in comparison with the parent strain, respectively. The clones also showed increased resistance to arsenite but not to antimony. Sequence analysis of the clones revealed genes encoding for putative arsenate reductases and arsenite efflux pumps. A novel arsenate resistance gene ( arsN ) encoding a protein with similarity to acetyltransferases was identified from clone MT6. ArsN homologues were found to be closely associated with arsenic resistance genes in many bacterial genomes. ArsN homologues were found fused to putative arsenate reductases in Methylibium petroleiphilum PM1 and Anaeromyxobacter dehalogenans 2CP-C and with a putative arsenite chaperone in Burkholderia vietnamiensis G4. ArsN alone resulted in an approximately sixfold higher resistance to sodium arsenate in wild-type Escherichia coli W3110.     

Broad taxonomic characterization of Verticillium wilt resistance genes reveals an ancient origin of the tomato Ve1 immune receptor

Plant‐pathogenic microbes secrete effector molecules to establish themselves on their hosts, whereas plants use immune receptors to try and intercept such effectors in order to prevent pathogen colonization. The tomato cell surface‐localized receptor Ve1 confers race‐specific resistance against race 1 strains of the soil‐borne vascular wilt fungus Verticillium dahliae which secrete the Ave1 effector. Here, we describe the cloning and characterization of Ve1 homologues from tobacco (Nicotiana glutinosa), potato (Solanum tuberosum), wild eggplant (Solanum torvum) and hop (Humulus lupulus), and demonstrate that particular Ve1 homologues govern resistance against V. dahliae race 1 strains through the recognition of the Ave1 effector. Phylogenetic analysis shows that Ve1 homologues are widely distributed in land plants. Thus, our study suggests an ancient origin of the Ve1 immune receptor in the plant kingdom.     

Cloning and linkage mapping of resistance gene homologues in apple

Apple (Malus x domestica Borkh.) sequences sharing homology with known resistance genes were cloned using a PCR-based approach with degenerate oligonucleotide primers designed on conserved regions of the nucleotide-binding site (NBS). Sequence analysis of the amplified fragments indicated the presence of at least 27 families of NBS-containing genes in apple, each composed of several very similar or nearly identical sequences. The NBS-leucine-rich repeat homologues appeared to include members of the two major groups that have been described in dicot plants: one possessing a toll-interleukin receptor element and one lacking such a domain. Genetic mapping of the cloned sequences was achieved through the development of CAPS and SSCP markers using a segregating population of a cross between the two apple cultivars Fiesta and Discovery. Several of the apple resistance gene homologues mapped in the vicinity, or at least on the same linkage group, of known loci controlling resistance to various pathogens. The utility of resistance gene-homologue sequences as molecular markers for breeding purposes and for gene cloning is discussed.Communicated by H. Nybom     

A barley gene family homologous to the maize rust resistance gene <Emphasis Type="Italic">Rp1-D</Emphasis>

Many characterized plant disease resistance genes encode proteins which have conserved motifs such as the nucleotide binding site. Conservation extends across different species, therefore resistance genes from one species can be used to isolate homologous regions from another by employing DNA sequences encoding conserved protein motifs as probes. Here we report the isolation and characterization of a barley (Hordeum vulgare L.) resistance gene analog family consisting of nine members homologous to the maize rust resistance gene Rp1-D. Five barley Rp1-D homologues are clustered within approximately 400 kb on chromosome 1(7H), near, but not co-segregating with, the barley stem rust resistance gene Rpg1; while others are localized on chromosomes 3(3H), 5(1H), 6(6H) and 7(5H). Analyses of predicted amino-acid sequences of the barley Rp1-D homologues and comparison with known plant disease resistance genes are presented.     

Pressure inactivation of tetrameric lactate dehydrogenase homologues of confamilial deep-living fishes

Summary The susceptibility to inactivation by hydrostatic pressure of the tetrameric (Fig. 1) muscletype (M₄) lactate dehydrogenase homologues (LDH, EC 1.1.1.27;l-lactate: NAD⁺ oxidoreductase) from six confamilial macrourid fishes was compared at 4 °C. These marine teleost fishes occur over depths of 260 to 4815 m. The pressures necessary to half-inactivate the LDH homologues are related to the pressures which the enzymes are exposed to in vivo (Table 1); higher hydrostatic pressures are required to inactivate the LDH homologues of the deeper-occurring macrourids. The resistance of the LDH homologues to inactivation by pressure is affected by protein concentration (Fig. 3). After an hour of incubation at pressure, the percent remaining activity approaches an asymptotic value (Fig. 2). The inactivation of the macrourid LDH homologues by pressure was not fully reversible. Assuming that inactivation by pressure was due to dissociation of the native tetramer to monomers, apparent equilibrium constants (K _eq) were calculated. Volume changes (V) were calculated over the range of pressures for which plots inK _eq versus pressure were linear (Fig. 4). The V of dissociation values of the macrourid homologues range from –219 to –439 ml mol^–1 (Table 1). Although the hydrostatic pressures required to inactivate the LDH homologues of the macrourid fishes are greater than those which the enzymes are exposed to in vivo, the pressure-stability of these enzymes may reflect the resistance of these enzymes to pressure-enhanced proteolysis in vivo.