Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001     

Development of PCR markers for the<Emphasis Type="Italic"> Pl5/Pl8</Emphasis> locus for resistance to<Emphasis Type="Italic"> Plasmopara halstedii</Emphasis> in sunflower,<Emphasis Type="Italic"> Helianthus annuus</Emphasis> L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.Communicated by C. Möllers     

The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans

Key message

Ph-3 is the first cloned tomato gene for resistance to late blight and encodes a CC-NBS-LRR protein.

Abstract

Late blight, caused by Phytophthora infestans, is one of the most destructive diseases in tomato. The resistance (R) gene Ph-3, derived from Solanum pimpinellifolium L3708, provides resistance to multiple P. infestans isolates and has been widely used in tomato breeding programmes. In our previous study, Ph-3 was mapped into a region harbouring R gene analogues (RGA) at the distal part of long arm of chromosome 9. To further narrow down the Ph-3 interval, more recombinants were identified using the flanking markers G2-4 and M8-2, which defined the Ph-3 gene to a 26 kb region according to the Heinz1706 reference genome. To clone the Ph-3 gene, a bacterial artificial chromosome (BAC) library was constructed using L3708 and one BAC clone B25E21 containing the Ph-3 region was identified. The sequence of the BAC clone B25E21 showed that only one RGA was present in the target region. A subsequent complementation analysis demonstrated that this RGA, encoding a CC-NBS-LRR protein, was able to complement the susceptible phenotype in cultivar Moneymaker. Thus this RGA was considered the Ph-3 gene. The predicted Ph-3 protein shares high amino acid identity with the chromosome-9-derived potato resistance proteins against P. infestans (Rpi proteins).     

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta 2 using a highly representative rice BAC library

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta ² (closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2?±?1.3 and 8.0?±?2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta ² region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a “two-step walking” method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta ² . The ratio of physical to genetic distances (>?1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta ² region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.     

Fine mapping of the sunflower resistance locus Pl ARG introduced from the wild species Helianthus argophyllus

Downy mildew, caused by Plasmopara halstedii, is one of the most destructive diseases in cultivated sunflower (Helianthus annuus L.). The dominant resistance locus Pl _ARG originates from silverleaf sunflower (H. argophyllus Torrey and Gray) and confers resistance to all known races of P. halstedii. We mapped Pl _ARG on linkage group (LG) 1 of (cms)HA342 × ARG1575-2, a population consisting of 2,145 F₂ individuals. Further, we identified resistance gene candidates (RGCs) that cosegregated with Pl _ARG as well as closely linked flanking markers. Markers from the target region were mapped with higher resolution in NDBLOS_sel × KWS04, a population consisting of 2,780 F₂ individuals that does not segregate for Pl _ARG . A large-insert sunflower bacterial artificial chromosome (BAC) library was screened with overgo probes designed for markers RGC52 and RGC151, which cosegregated with Pl _ARG . Two RGC-containing BAC contigs were anchored to the Pl _ARG region on LG 1.     

Fine genetic and physical mapping of the CRb gene conferring resistance to clubroot disease in Brassica rapa

The use of clubroot resistance (CR) genes is an effective and economical approach for controlling Plasmodiophora brassicae, the causal agent of clubroot disease in Chinese cabbage (Brassica rapa) and other Brassica crops. In a previous study, we identified and mapped the CRb locus on chromosome A03 of B. rapa in the doubled-haploid (DH) line ‘CR Shinki DH line’ of Chinese cabbage. In this study, CRb, a dominant gene conferring resistance to pathotype 4 of P. brassicae, was finely mapped in combination with bulked segregant analysis and bioinformatics analysis (BIA). Using 1,486 highly susceptible individuals and 2,896 individuals from two separate F₂ populations of ‘702-5’ (B. rapa ssp. chinensis) ×  ‘CR Shinki DH line,’ the CRb locus was narrowed to a region of approximately 0.14 cM between two flanking markers, TCR79 and TCR108. The sequences of seven newly developed markers linked to CRb were landed on bacterial artificial chromosome (BAC) of the reference B. rapa ‘Chiifu-401-42’ by BIA, and a physical map consisting of three BAC clones was constructed. The CRb locus was defined as an interval of approximately 83.5 kb on a BAC clone (KBrB085J21). The target interval contained one Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) gene, one NBS–LRR gene, and several putative regulatory genes in the B. rapa genome. The CRb gene was tightly linked to two other CR genes, CRa and CRb ^Kato . These results provide useful information for isolation of the CRb gene and tightly linked molecular markers for breeding CR in B. rapa.     

Antisense Expression of a NBS-LRR Sequence in Sunflower (Helianthus annuus L.) and Tobacco (Nicotiana tabacum L.): Evidence for a Dual Role in Plant Development and Fungal Resistance

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.     

Functional complementation analysis in potato via biolistic transformation with BAC large DNA fragments

Gene isolation from plants by positional cloning frequently requires several rounds of transformation. To reduce the resources invested and to accelerate the process, we have used large DNA fragments in transformation experiments, followed by analysis of transgenic plants to assess functional complementation. Specifically, the transformation of potato with DNA from the 106 kb BAC plasmid BA87d17 is described. The large fragment was introduced into the potato genome by biolistic transformation, while attempting to clone the R1 gene conferring a race specific resistance to Phytophthora infestans. Thirty-one kanamycin resistant plants were regenerated of which thirteen showed the necrotic lesions typical for the hypersensitive response after infection with the incompatible P. infestans race 4, which carries the avirulence gene Avr1. The successful complementation supported the location of the R1 gene in the BAC insertion of the BA87d17 plasmid. Based on PCR and Southern gel blot analysis, both complete and incomplete integrations of the large construct into the recipient genome were demonstrated.     

Characterization of two recessive genes controlling resistance to all races of bacterial spot in peppers

Bacterial spot, one of the most damaging diseases of pepper, is caused by Xanthomonas euvesicatoria. This pathogen has worldwide distribution and it is particularly devastating in tropical and sub-tropical regions where high temperatures and frequent precipitation provide ideal conditions for disease development. Three dominant resistance genes have been deployed singly and in combination in commercial cultivars, but have been rendered ineffectual by the high mutation rate or deletion of the corresponding cognate effector genes. These genes are missing in race P6, and their absence makes this race virulent on all commercial pepper cultivars. The breeding line ECW12346 is the only source of resistance to race P6 in Capsicum annuum, and displays a non-hypersensitive type of resistance. Characterization of this resistance has identified two recessive genes: bs5 and bs6. Individual analysis of these genes revealed that bs5 confers a greater level of resistance than bs6 at 25°C, but in combination they confer full resistance to P6 indicating at least additive gene action. Tests carried out at 30°C showed that both resistances are compromised to a significant extent, but in combination they provide almost full resistance to race P6 indicating a positive epistatic interaction at high temperatures. A scan of the pepper genome with restriction fragment length polymorphism and AFLP markers led to the identification of a set of AFLP markers for bs5. Allele-specific primers for a PCR-based bs5-marker have been developed to facilitate the genetic manipulation of this gene.     

Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa

Clubroot disease is one of the major diseases affecting Brassicaceae crops, and a number of these crops grown commercially, such as Chinese cabbage (Brassica rapa L. ssp. pekinensis), are known to be highly susceptible to clubroot disease. To provide protection from this disease, plant breeders have introduced genes for resistance to clubroot from the European turnip into susceptible lines. The CRa gene confers specific resistance to the clubroot pathogen Plasmodiophora brassicae isolate M85. Fine mapping of the CRa locus using synteny to the Arabidopsis thaliana genome and partial genome sequences of B. rapa revealed a candidate gene encoding a TIR-NBS-LRR protein. Several structural differences in this candidate gene were found between susceptible and resistant lines, and CRa expression was observed only in the resistant line. Four mutant lines lacking clubroot resistance were obtained by the UV irradiation of pollen from a resistant line, and all of these mutant lines carried independent mutations in the candidate TIR-NBS-LRR gene. This genetic and molecular evidence strongly suggests that the identified gene is CRa. This is the first report on the molecular characterization of a clubroot Resistance gene in Brassicaceae and of the disease resistance gene in B. rapa.     

Interaction between sunflower plants and five isolates of Plasmopara halstedii on the level of pathogenicity

The interaction between sunflower plants showing a high level of quantitative resistance and five Plasmopara halstedii (the causal agent of downy mildew) isolates of several races were studied using five single zoosporangium isolates per pathogen isolate. Aggressiveness criteria were analyzed for 25 P. halstedii single zoosporangium isolates. Based on the reaction for the P. halstedii isolates to four sunflower hybrids H1–H4 varying only in their downy mildew resistance genes, there were differences in virulence spectrum in pathogen isolates. Analysis of five single zoosporangium isolates for P. halstedii isolates showed significant variability within pathogen isolate for all aggressiveness criteria but not for all pathogen isolates. The hypothesis explaining the interaction between P. halstedii and its host plant was discussed on the level of pathogenicity.     

The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b

We have used map-based approaches to clone a locus containing two genes, Avr1b-1 and Avr1b-2, required for avirulence of the oomycete pathogen Phytophthora sojae (Kaufmann & Gerdemann) on soybean plants carrying resistance gene Rps1b. Avr1b-1 was localized to a single 60-kb bacterial artificial chromosome (BAC) clone by fine-structure genetic mapping. Avr1b-1 was localized within the 60-kb region by identification of an mRNA that is expressed in a race-specific and infection-specific manner and that encodes a small secreted protein. When the Avr1b-1 protein was synthesized in the yeast Pichia pastoris and the secreted protein infiltrated into soybean leaves, it triggered a hypersensitive response specifically in host plants carrying the Rps1b resistance gene. This response eventually spread to the entire inoculated plant. In some isolates of P. sojae virulent on Rps1b-containing cultivars, such as P7081 (race 25) and P7076 (race 19), the Avr1b-1 gene had numerous substitution mutations indicative of strong divergent selection. In other isolates, such as P6497 (race 2) and P9073 (race 25), there were no substitutions in Avr1b-1, but Avr1b-1 mRNA did not accumulate. Genetic complementation experiments with P6497 revealed the presence of a second gene, Avr1b-2, required for the accumulation of Avr1b-1 mRNA. Avr1b-2 was genetically mapped to the same BAC contig as Avr1b-1, using a cross between P7064 (race 7) and P6497. The Avr1k gene, required for avirulence on soybean cultivars containing Rps1k, was mapped to the same interval as Avr1b-1.     

Production and Characterization of Two Monoclonal Antibodies Specific for Plasmopara halstedii

Sunflower downy mildew, caused by the fungus Plasmopara halstedii, is a potentially devastating disease. We produced two monoclonal antibodies (MAbs) (12C9 and 18E2) by immunizing mice with a partially purified extract of P. halstedii race 1. Both MAbs detected in enzyme-linked immunosorbent assay (ELISA) all races of P. halstedii present in France. No cross-reactions were observed with Plasmopara viticola or with other fungi commonly associated with sunflowers. Both MAbs recognized the same three fungal antigens with molecular masses of 68, 140, and 192 kDa. However, the epitopes on the fungal antigens were distinct and repetitive. Seed homogenates from infected plants were incubated in wells coated with MAb 18E2. This resulted in the trapping of P. halstedii antigens that were identified with biotinylated MAb 12C9. No reactions were seen with seed homogenates from healthy plants. Thus, our results suggest that these MAbs might be used to develop a sandwich ELISA detection system for P. halstedii in infected seeds.     

Positional cloning of rp2 QTL associates the P450 genes CYP6Z1, CYP6Z3 and CYP6M7 with pyrethroid resistance in the malaria vector Anopheles funestus

Pyrethroid resistance in Anopheles funestus is threatening malaria control in Africa. Elucidation of underlying resistance mechanisms is crucial to improve the success of future control programs. A positional cloning approach was used to identify genes conferring resistance in the uncharacterised rp2 quantitative trait locus (QTL) previously detected in this vector using F6 advanced intercross lines (AIL). A 113 kb BAC clone spanning rp2 was identified and sequenced revealing a cluster of 15 P450 genes and one salivary protein gene (SG7-2). Contrary to A. gambiae, AfCYP6M1 is triplicated in A. funestus, while AgCYP6Z2 orthologue is absent. Five hundred and sixty-five new single nucleotide polymorphisms (SNPs) were identified for genetic mapping from rp2 P450s and other genes revealing high genetic polymorphisms with one SNP every 36 bp. A significant genotype/phenotype association was detected for rp2 P450s but not for a cluster of cuticular protein genes previously associated with resistance in A. gambiae. QTL mapping using F6 AIL confirms the rp2 QTL with an increase logarithm of odds score of 5. Multiplex gene expression profiling of 15 P450s and other genes around rp2 followed by individual validation using qRT–PCR indicated a significant overexpression in the resistant FUMOZ-R strain of the P450s AfCYP6Z1, AfCYP6Z3, AfCYP6M7 and the glutathione-s-transferase GSTe2 with respective fold change of 11.2, 6.3, 5.5 and 2.8. Polymorphisms analysis of AfCYP6Z1 and AfCYP6Z3 identified amino acid changes potentially associated with resistance further indicating that these genes are controlling the pyrethroid resistance explained by the rp2 QTL. The characterisation of this rp2 QTL significantly improves our understanding of resistance mechanisms in A. funestus.     

Genetic and physical localization of an anthracnose resistance gene in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Anthracnose of alfalfa, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. An improved understanding of the genetic and molecular mechanisms underlying host resistance will facilitate the development of resistant alfalfa cultivars, thus providing the most efficient and environmentally sound strategy to control alfalfa diseases. Unfortunately, cultivated alfalfa has an intractable genetic system because of its tetrasomic inheritance and out-crossing nature. Nevertheless, the model legume Medicago truncatula, a close relative of alfalfa, has the potential to serve as a surrogate to map and clone the counterparts of agronomically important genes in alfalfa—particularly, disease resistance genes against economically important pathogens. Here we describe the high-resolution genetic and physical mapping of RCT1, a host resistance gene against C. trifolii race 1 in M. truncatula. We have delimited the RCT1 locus within a physical interval spanning ∼200 kb located on the top of M. truncatula linkage group 4. RCT1 is part of a complex locus containing numerous genes homologous to previously characterized TIR-NBS-LRR type resistance genes. The result presented in this paper will facilitate the positional cloning of RCT1 in Medicago.     

Construction of a BAC library of <Emphasis Type="Italic"> Rosa rugosa </Emphasis>Thunb. and assembly of a contig spanning<Emphasis Type="Italic"> Rdr1</Emphasis>, a gene that confers resistance to blackspot

A BAC library to serve as a general tool for the physical mapping and positional cloning of rose genes has been constructed from Rosa rugosa DNA. With 27,264 clones the library contains 5.2 genome equivalents. The library was used to assemble a contig of BAC clones spanning Rdr1, a locus that confers resistance to blackspot. For this purpose fine-scale mapping of the target locus was achieved by bulked segregant analysis using 816 AFLP primer combinations. The target region around Rdr1 comprises about 400 kb and is covered by a minimum of six BAC clones. Furthermore, the detection of at least five resistance gene analogs of the TIR-NBS-LRR family on the contig indicates the presence of a cluster of resistance genes around Rdr1. These results will not only allow the isolation and identification of Rdr1 in the near future, but also provide the tools for the physical mapping and positional cloning of other horticulturally interesting genes in roses.     

Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin

Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce PC-toxin. Susceptibility to milo disease is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10⁻³ to 10⁻⁴. A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family are the Pc genes that condition susceptibility.