Characterisation of disease resistance gene-like sequences in Brassica oleracea L.

Several cloned disease resistance genes from a wide range of plant species are known to share conserved regions with similar structural motifs. Degenerate primers based on conserved sequences of the nucleotide binding site of the genes RPS2, N and L6 were used for polymerase chain reaction (PCR) amplification from genomic DNA of two doubled haploid lines of Brassica oleracea. Sequences of amplified products were highly variable, but most of them showed similarity to known disease resistance genes, including RPS5, RPS2 and N, and to disease resistance gene-like sequences (RGLs) from different species. Primers based on B. oleracea sequences amplified five groups of RGLs. Products were mapped through cleaved amplified polymorphic sequence assays onto four different linkage groups of B. oleracea. PCR amplification from cDNA and allele analysis indicated that four locus-specific RGL fragments are expressed in cauliflower. Screening of a B. oleracea bacterial artificial chromosome library (BAC) with four B. oleracea RGL probes identified a small number of clones, suggesting that the four RGLs may not be highly copied. Screening of a BAC library of A. thaliana with the same probes identified clones that mapped onto four different chromosomes. These map positions correspond to known disease resistance loci of A. thaliana. Received: 12 November 1999 / Accepted: 19 June 2000     

Characterization of disease resistance gene-like sequences in near-isogenic lines of tomato

 We examined near-isogenic lines (NILs) carrying either of the tomato mosaic virus (ToMV) resistance genes Tm-1 and Tm-2 for sequences homologous to the isolated disease-resistance genes. DNA fragments were amplified from the genomic DNA of the NILs by the polymerase chain reaction (PCR) using primers designed on the basis of sequences of certain domains conserved among some disease-resistance genes. Of ten PCR products cloned, five were identified as having homology to either of the two classes of disease-resistance genes. The first class encoded proteins containing leucine-rich repeats (LRRs) and a nucleotide-binding site (NBS), such as the RPS2 gene in Arabidopsis and the N gene in tobacco. The second class encoded proteins containing a C-terminal membrane anchor but no NBS, such as the Cf 2 and Cf 9 genes in tomato. In Southern hybridization of the genomic DNAs of the NILs carrying either Tm-1 or Tm-2 and their parental NIL carrying neither of these resistance genes, multiple bands could be detected with most of the clones used as probes. This suggests that the genomes of the NILs contain multiple copies of sequences homologous to some of the known disease-resistance genes. No evidence was obtained to show that the Tm-1 and/or Tm-2 loci encode either class of protein, since no polymorphic band patterns between the NILs were detected by Southern hybridization. Received: 15 August 1997 / Accepted: 2 September 1997     

Characterization of three loci controlling resistance of Arabidopsis thaliana accession Ms-0 to two powdery mildew diseases

Arabidopsis thaliana accession La-er was susceptible, and accession Ms-0 was resistant, to powdery mildew diseases caused by Erysiphe cruciferarum UEA1 and E. cichoracearum UCSC1. The resistance reaction phenotype of A. thaliana Ms-0 to both pathogens was characterized, and the resistance loci were genetically mapped. Growth of E. cruciferarum UEA1 on Ms-0 leaves was arrested after formation of the first appressorium: the underlying host epidermal cell collapsed, and occasionally there was necrosis of one or two host mesophyll cells. Growth of E. cichoracearum UCSC1 on Ms-0 leaves was arrested after emergence of several germ tubes from the conidium, and there was necrosis of host mesophyll cells at the sites of infection. Examination of F₂ progeny of a cross La-er x Ms-0 indicated that two independently-segregating dominant loci were required for resistance to E. cruciferarum UEA1. One locus, named RPW6, was genetically mapped to chromosome 5, in a 5.6 cM interval flanked by pCITf16 and PI. The other locus, named RPW7, mapped to chromosome 3 in a 8.5 cM interval flanked by CDC2A and AFC1. Independent effects of RPW6 and RPW7 on E. cruciferarum UEA1 could be detected by quantitative measurements of growth of mycelium and production of conidia. Resistance to E. cichoracearum UCSC1 mapped to a single locus, named RPW8, at a location on chromosome 3 which we could not distinguish from RPW7. Evidently, RPW7 and RPW8 define either a complex resistance locus, or a common resistance gene with dual specificity.     

Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily

The nucleotide binding site (NBS) is a characteristic domain of many plant resistance gene products. An increasing number of NBS-encoding sequences are being identified through gene cloning, PCR amplification with degenerate primers, and genome sequencing projects. The NBS domain was analyzed from 14 known plant resistance genes and more than 400 homologs, representing 26 genera of monocotyledonous, dicotyle-donous and one coniferous species. Two distinct groups of diverse sequences were identified, indicating divergence during evolution and an ancient origin for these sequences. One group was comprised of sequences encoding an N-terminal domain with Toll/Interleukin-1 receptor homology (TIR), including the known resistance genes, N, M, L6, RPP1 and RPP5. Surprisingly, this group was entirely absent from monocot species in searches of both random genomic sequences and large collections of ESTs. A second group contained monocot and dicot sequences, including the known resistance genes, RPS2, RPM1, I2, Mi, Dm3, Pi-B, Xa1, RPP8, RPS5 and Prf. Amino acid signatures in the conserved motifs comprising the NBS domain clearly distinguished these two groups. The Arabidopsis genome is estimated to contain approximately 200 genes that encode related NBS motifs; TIR sequences were more abundant and outnumber non-TIR sequences threefold. The Arabidopsis NBS sequences currently in the databases are located in approximately 21 genomic clusters and 14 isolated loci. NBS-encoding sequences may be more prevalent in rice. The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicate that they are ancient, diverse and common in plants. Sequence inferences suggest that these genes encode a novel class of nucleotide-binding proteins.     

TMV resistance gene N homologues are linked to Synchytrium endobioticum resistance in potato

 The fungus Synchytrium endobioticum, the causal agent of potato wart disease, is subject to world-wide quarKantine regulations due to the production of persistent resting spores and lack of effective chemical control measures. The selection of Synchytrium-resistant potato cultivars may be facilitated by using markers closely linked with a resistance gene or by transferring a cloned gene for resistance into susceptible cultivars. Sen1, a gene for resistance to Synchytrium endobioticum race 1, was localized on potato chromosome XI in a genomic region which is related to the tobacco genome segment harbouring the N gene for resistance to TMV. Using N as probe, we isolated homologous cDNA clones from a Synchytrium-resistant potato line. The N-homologous sequences of potato identified by RFLP mapping a family of resistance gene-like sequences closely linked with the Sen1 locus. Sequence analysis of two full-length N-homologous cDNA clones revealed the presence of structural domains associated with resistance gene function. One clone (Nl-25) encodes a polypeptide of 61 kDa and harbours a Toll-interleukin like region (TIR) and a putative nucleotide binding site (NBS). The other clone (Nl-27) encodes a polypeptide of 95 kDa and harbours besides the TIR and NBS domains five imperfect leucine-rich repeats (LRRs). Both clones have at their amino terminus a conserved stretch of serine residues that was also found in the N gene, the RPP5 gene from Arabidopsis thaliana and several other resistance gene homologues, suggesting a function in the resistance response. Cloning of the disease resistance locus based on map position and the establishment of PCR-based marker assays to assist selection of wart resistant potato genotypes are discussed. Received: 4 August 1998 / Accepted: 14 August 1998     

Characterisation and genetic mapping of resistance and defence gene analogs in cocoa (Theobroma cacao L.)

Disease resistance and defence gene analog (RGA/DGA) sequences were isolated in cocoa using a PCR approach with degenerate primers designed from conserved domains of plant resistance and defence genes: the NBS (nucleotide binding site) motif present in a number of resistance genes such as the tobacco N, sub-domains of plant serine/threonine kinases such as the Pto tomato gene, and conserved domains of two defence gene families: pathogenesis-related proteins (PR) of classes 2 and 5. Nucleotide identity between thirty six sequences isolated from cocoa and known resistance or defence genes varied from 58 to 80%. Amino acid sequences translated from corresponding coding sequences produced sequences without stop codons, except for one NBS –like sequence. Most of the RGAs could be mapped on the cocoa genome and three clusters of genes could be observed : NBS-like sequences clustered in two regions located on chromosomes 7 and 10, Pto-like sequences mapped in five genome regions of which one, located on chromosome 4, corresponded to a cluster of five different sequences. PR2-like sequences mapped in two regions located on chromosome 5 and 9 respectively. An enrichment of the genetic map with microsatellite markers allowed us to identify several co-localisations of RGAs, DGAs and QTL for resistance to Phytophthora detected in several progenies, particularly on chromosome 4 where a cluster of Pto-like sequences and 4 QTL for resistance to Phytophthora were observed. Many other serious diseases affect cocoa and the candidate genes, isolated in this study, could be of broader interest in cocoa disease management.     

Identification and characterization of NBS-encoding disease resistance genes in Lotus japonicus

Nucleotide-binding site (NBS) disease resistance genes play an important role in defending plants from a range of pathogens and insect pests. Consequently, NBS-encoding genes have been the focus of a number of recent studies in molecular disease resistance breeding programs. However, little is known about NBS-encoding genes in Lotus japonicus. In this study, a full set of disease resistance (R) candidate genes encoding NBS from the complete genome of L. japonicus was identified and characterized using structural diversity, chromosomal locations, conserved protein motifs, gene duplications, and phylogenetic relationships. Distinguished by N-terminal motifs and leucine-rich repeat motifs (LRRs), 92 regular NBS genes of 158 NBS-coding sequences were classified into seven types: CC-NBS-LRR, TIR-NBS-LRR, NBS-LRR, CC-NBS, TIR-NBS, NBS, and NBS-TIR. Phylogenetic reconstruction of NBS-coding sequences revealed many NBS gene lineages, dissimilar from results for Arabidopsis but similar to results from research on rice. Conserved motif structures were also analyzed to clarify their distribution in NBS-encoding gene sequences. Moreover, analysis of the physical locations and duplications of NBS genes showed that gene duplication events of disease resistance genes were lower in L. japonicus than in rice and Arabidopsis, which may contribute to the relatively fewer NBS genes in L. japonicus. Sixty-three NBS-encoding genes with clear conserved domain character were selected to check their gene expression levels by semi-quantitative RT-PCR. The results indicated that 53 of the genes were most likely to be acting as the active genes, and exogenous application of salicylic acid improved expression of most of the R genes.     

Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides

Current knowledge of resistance (R) genes and their use for genetic improvement in buffalograss (Buchloe dactyloides [Nutt.] Engelm.) lag behind most crop plants. This study was conducted to clone and characterize cDNA encoding R gene-like (RGL) sequences in buffalograss. This report is the first to clone and characterize of buffalograss RGLs. Degenerate primers designed from the conserved motifs of known R genes were used to amplify RGLs and fragments of expected size were isolated and cloned. Sequence analysis of cDNA clones and analysis of putative translation products revealed that most encoded amino acid sequences shared the similar conserved motifs found in the cloned plant disease resistance genes PRS2, MLA6, L6, RPMI, and Xa1. These results indicated diversity of the R gene candidate sequences in buffalograss. Analysis of 5′ rapid amplification of cDNA ends (RACE), applied to investigate upstream of RGLs, indicated that regulatory sequences such as TATA box were conserved among the RGLs identified. The cloned RGL in this study will further enhance our knowledge on organization, function, and evolution of R gene family in buffalo grass. With the sequences of the primers and sizes of the markers provided, these RGL markers are readily available for use in a genomics-assisted selection in buffalograss.     

Breaking restricted taxonomic functionality by dual resistance genes

NB-LRR-type disease resistance (R) genes have been used in traditional breeding programs for crop protection. However, functional transfer of NB-LRR-type R genes to plants in taxonomically distinct families to establish pathogen resistance has not been successful. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and B. napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Thus the successful transfer of two R genes at the family level overcomes restricted taxonomic functionality. This implies that the downstream components of R genes must be highly conserved and interfamily utilization of R genes can be a powerful strategy to combat pathogens.     

Development of a Virus-Induced Gene-Silencing System for Functional Analysis of the RPS2-Dependent Resistance Signalling Pathways in Arabidopsis

Virus-induced gene silencing (VIGS) offers a rapid and high throughput technique platform for the analysis of gene function in plants. Although routinely used in some Solanaceous species, VIGS system has not been well established in Arabidopsis thaliana (L.) Heynh. We have recently reported some factors that potentially influence tobacco rattle virus (TRV)-mediated VIGS of phytoene desaturase (PDS) and actin gene expression in Arabidopsis. In this study, we have further established that the Agrobacterium strain used for agro-inoculation significantly affects the VIGS efficiency. Strain GV3101 was highly effective; C58C1 and LBA4404 were invalid, while EHA105 was plant growth stage-dependent for TRV-induced gene silencing. Furthermore, the VIGS procedure optimised for the PDS gene was applied for the functional analysis of the disease resistance gene RPS2-mediated resistance pathway. Silencing of RPS2 led to loss of resistance to the otherwise avirulence strain of Pseudomonas syringae pv. tomato DC3000 carrying the avirulence gene AvrRpt2. Silencing of RIN4, a RPS2 repressor gene, gave rise to conversion of compatible interaction to incompatible. Silencing of NDR1, RAR1 and HSP90, known to be required for the RPS2-mediated resistance, resulted in loss of the resistance, while silencing of EDS1 and SGT1b, which are not required for the RPS2-mediated resistance, caused no change of the resistance. These results indicate that the optimised procedure for the TRV-based VIGS is a potentially powerful tool for dissecting the signal transduction pathways of disease resistance in Arabidopsis. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at An erratum to this article is available at .     

Identification of three putative signal transduction genes involved in R gene-specified disease resistance in Arabidopsis

The RPS5 disease resistance gene of Arabidopsis mediates recognition of Pseudomonas syringae strains that possess the avirulence gene avrPphB. By screening for loss of RPS5-specified resistance, we identified five pbs (avrPphB susceptible) mutants that represent three different genes. Mutations in PBS1 completely blocked RPS5-mediated resistance, but had little to no effect on resistance specified by other disease resistance genes, suggesting that PBS1 facilitates recognition of the avrPphB protein. The pbs2 mutation dramatically reduced resistance mediated by the RPS5 and RPM1 resistance genes, but had no detectable effect on resistance mediated by RPS4 and had an intermediate effect on RPS2-mediated resistance. The pbs2 mutation also had varying effects on resistance mediated by seven different RPP (recognition of Peronospora parasitica) genes. These data indicate that the PBS2 protein functions in a pathway that is important only to a subset of disease-resistance genes. The pbs3 mutation partially suppressed all four P. syringae-resistance genes (RPS5, RPM1, RPS2, and RPS4), and it had weak-to-intermediate effects on the RPP genes. In addition, the pbs3 mutant allowed higher bacterial growth in response to a virulent strain of P. syringae, indicating that the PBS3 gene product functions in a pathway involved in restricting the spread of both virulent and avirulent pathogens. The pbs mutations are recessive and have been mapped to chromosomes I (pbs2) and V (pbs1 and pbs3).     

Analysis of expressed sequence tags derived from pea leaves infected by Peronospora viciae f. sp. pisi

A cDNA library was constructed from field pea leaves infected by the downy mildew pathogen, Peronospora viciae f. sp. pisi, using a suppression subtractive hybridisation approach. The library consists of 399 expressed sequence tags, from which 207 unisequences were obtained after sequence assembly. Of the unisequences, six were shown to be of Peronospora viciae f. sp. pisi origin. The remaining unisequences were subjected to gene ontology analysis and their functions were predicted in silico. Eleven of these unisequences (representing 24 clones) shared significant sequence similarities with Arabidopsis genes known to be involved in downy mildew resistance, including the well‐characterised genes RPP5, RPP6 and RPP27. Expression analysis of five selected unisequences by real‐time PCR indicated that all five were up‐regulated during downy mildew pathogenesis, suggesting a significant role for these genes in the host response to downy mildew infection.     

Ribosomal protein QM/RPL10 positively regulates defence and protein translation mechanisms during nonhost disease resistance

Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10-interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.     

Targeted resistance gene mapping in soybean using modified AFLPs

The soybean [Glycine max (Merr.) L.] linkage group F contains a vital region of clustered genes for resistance to numerous pathogens including the soybean mosaic virus resistance gene, Rsv1. In order to develop new genetic markers that map to this gene cluster, we employed a targeted approach that utilizes the speed and high-throughput of AFLP, but modified it to incorporate sequence information from the highly conserved nucleotide binding site (NBS) region of cloned disease resistance genes. By using a labeled degenerate primer corresponding to the p-loop portion of the NBS region of resistance genes, such as N, L6, and Rps2, we were able to quickly amplify numerous polymorphic bands between parents of a population segregating for resistance to Rsv1. Of these polymorphic bands, bulk segregant analysis revealed four markers that were closely linked to Rsv1. These markers were cloned and used as probes for RFLP analysis. The four clones mapped to within a 6-cM region surrounding Rsv1, the closest being 0.4 cM away from the gene. Sequence analysis showed that all four clones contain the p-loop sequence corresponding to the degenerate primer and that one of the four clones contains an open reading frame sequence which when translated is related to the NBS region of other cloned disease resistance genes. The rapid identification of four markers closely linked to Rsv1 in soybean demonstrates the utility of this method for generating markers tightly linked to important plant disease resistance genes. Received: 25 September 1999 / Accepted: 3 November 1999     

Map positions of 47 Arabidopsis sequences with sequence similarity to disease resistance genes

Map positions have been determined for 42 non-redundant Arabidopsis expressed sequence tags (ESTs) showing similarity to disease resistance genes (R-ESTs), and for three Pto-like sequences that were amplified with degenerate primers. Employing a PCR-based strategy, yeast artificial chromosome (YAC) clones containing the EST sequences were identified. Since many YACs have been mapped, the locations of the R-ESTs could be inferred from the map positions of the YACs. R-EST clones that exhibited ambiguous map positions were mapped as either cleavable amplifiable polymorphic sequence (CAPS) or restriction fragment length polymorphism (RFLP) markers using F₈ (Ler x Col-0) recombinant inbred (RI) lines. In all cases but two, the R-ESTs and Pto-like sequences mapped to single, unique locations. One R-EST and one Pto-like sequence each mapped to two locations. Thus, a total of 47 loci were identified in this study. Several R-ESTs occur in clusters suggesting that they may have arisen via gene duplication events. Interestingly, several R-ESTs map to regions containing genetically defined disease resistance genes. Thus, this collection of mapped R-ESTs may expedite the isolation of disease resistance genes. As the cDNA sequencing projects have identified an estimated 63% of Arabidopsis genes, a very large number of R-ESTs (～95), and by inference disease resistance genes of the leucine-rich repeat-class probably occur in the Arabidopsis genome.     

Organization of an Arabidopsis thaliana gene cluster on chromosome 4 including the RPS2 gene, in the Brassica nigra genome

 Genetic and physical maps, consisting of a large number of DNA markers for Arabidopsis thaliana chromosomes, represent excellent tools to determine the organization of related genomes such as those of Brassica. In this paper we report the chromosomal localization and physical analysis by pulsed-field gel electrophoresis (PFGE) of a well-defined gene complex of A. thaliana in the Brassica nigra genome (B genome n=8). This complex is approximately 30 kb in length in A. thaliana and contains a cluster of six genes including ABI1 (ABA-responsive), RPS2 (resistance against Pseudomonas syringae, a bacterial disease), CK1 (casein kinase I), NAP (nucleosome-assembly protein), X9 and X14 (both of unknown function). The Arabidopsis chromosomal complex was found to be duplicated and conserved in gene number at different levels in the Brassica genome. Linkage group B1 had the most-conserved arrangement carrying all six genes tightly linked. Group B4 had an almost complete complex except for the absence of RPS2. Other partial complexes of fewer members were found on three other chromosomes. Our studies demonstrate that by this approach it is possible to identify ancestrally related chromosome segments in a complex and duplicated genome, such as the genome of B. nigra, permitting one to draw conclusions as to its origin and evolution. Received: 11 July 1997 / Accepted: 9 October 1997     

Silencing of copine genes confers common wheat enhanced resistance to powdery mildew

Powdery mildew, caused by the biotrophic fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a major threat to the production of wheat (Triticum aestivum). It is of great importance to identify new resistance genes for the generation of Bgt‐resistant or Bgt‐tolerant wheat varieties. Here, we show that the wheat copine genes TaBON1 and TaBON3 negatively regulate wheat disease resistance to Bgt. Two copies of TaBON1 and three copies of TaBON3, located on chromosomes 6AS, 6BL, 1AL, 1BL and 1DL, respectively, were identified from the current common wheat genome sequences. The expression of TaBON1 and TaBON3 is responsive to both pathogen infection and temperature changes. Knocking down of TaBON1 or TaBON3 by virus‐induced gene silencing (VIGS) induces the up‐regulation of defence responses in wheat. These TaBON1‐ or TaBON3‐silenced plants exhibit enhanced wheat disease resistance to Bgt, accompanied by greater accumulation of hydrogen peroxide and heightened cell death. In addition, high temperature has little effect on the up‐regulation of defence response genes conferred by the silencing of TaBON1 or TaBON3. Our study shows a conserved function of plant copine genes in plant immunity and provides new genetic resources for the improvement of resistance to powdery mildew in wheat.     

Transfer of penicillin resistance from Streptococcus oralis to Streptococcus pneumoniae identifies murE as resistance determinant

Beta‐lactam resistant clinical isolates of Streptococcus pneumoniae contain altered penicillin‐binding protein (PBP) genes and occasionally an altered murM, presumably products of interspecies gene transfer. MurM and MurN are responsible for the synthesis of branched lipid II, substrate for the PBP catalyzed transpeptidation reaction. Here we used the high‐level beta‐lactam resistant S. oralis Uo5 as donor in transformation experiments with the sensitive laboratory strain S. pneumoniae R6 as recipient. Surprisingly, piperacillin‐resistant transformants contained no alterations in PBP genes but carried murE_Uo5 encoding the UDP‐N‐acetylmuramyl tripeptide synthetase. Codons 83–183 of murE_Uo5 were sufficient to confer the resistance phenotype. Moreover, the promoter of murE_Uo5, which drives a twofold higher expression compared to that of S. pneumoniae R6, could also confer increased resistance. Multiple independent transformations produced S. pneumoniae R6 derivatives containing murE_Uo5, pbp2x_Uo5, pbp1a_Uo5 and pbp2b_Uo5, but not murM_Uo5 sequences; however, the resistance level of the donor strain could not be reached. S. oralis Uo5 harbors an unusual murM, and murN is absent. Accordingly, the peptidoglycan of S. oralis Uo5 contained interpeptide bridges with one L‐Ala residue only. The data suggest that resistance in S. oralis Uo5 is based on a complex interplay of distinct PBPs and other enzymes involved in peptidoglycan biosynthesis.