Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

99th Dahlem conference on infection, inflammation and chronic inflammatory disorders: Caenorhabditis elegans as a model to study tissues involved in host immunity and microbial pathogenesis.

The molecular mechanisms involved in host-microbe interactions during the initial stages of infection are poorly understood. The bacteria-eating nematode Caenorhabditis elegans provides an opportunity to dissect host-microbe interactions in the context of the whole organism, using powerful genomic, genetic and cell-biological tools. Because of the evolutionary conservation of ancient innate host defences and bacterial virulence mechanisms, studies in C. elegans hold great promise to shed light on defences in higher organisms, including mammals. Additionally, C. elegans pathogenesis models provide a platform for the identification of novel classes of anti-infective compounds with therapeutic value.

Common virulence factors for bacterial pathogenicity in plants and animals.

A Pseudomonas aeruginosa strain (UCBPP-PA14) is infectious both in an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin burn model. UCBPP-PA14 exhibits ecotype specificity for Arabidopsis, causing a range of symptoms from none to severe in four different ecotypes. In the mouse model, UCBPP-PA14 is as lethal as other well-studied P. aeruginosa strains. Mutations in the UCBPP-PA14 toxA, plcS, and gacA genes resulted in a significant reduction in pathogenicity in both hosts, indicating that these genes encode virulence factors required for the full expression of pathogenicity in both plants and animals.

Molecular genetics of plant disease resistance.

Plant breeders have used disease resistance genes (R genes) to control plant disease since the turn of the century. Molecular cloning of R genes that enable plants to resist a diverse range of pathogens has revealed that the proteins encoded by these genes have several features in common. These findings suggest that plants may have evolved common signal transduction mechanisms for the expression of resistance to a wide range of unrelated pathogens. Characterization of the molecular signals involved in pathogen recognition and of the molecular events that specify the expression of resistance may lead to novel strategies for plant disease control.

Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans.

Genetic analysis of host-pathogen interactions has been hampered by the lack of genetically tractable models of such interactions. We showed previously that the human opportunistic pathogen Pseudomonas aeruginosa kills Caenorhabditis elegans, that P. aeruginosa and C. elegans genes can be identified that affect this killing, and that most of these P. aeruginosa genes are also important for mammalian pathogenesis. Here, we show that Salmonella typhimurium as well as other Salmonella enterica serovars including S. enteritidis and S. dublin can also kill C. elegans. When C. elegans is placed on a lawn of S. typhimurium, the bacteria accumulate in the lumen of the worm intestine and the nematodes die over the course of several days. This killing requires contact with live bacterial cells. The worms die with similar kinetics when placed on a lawn of S. typhimurium for a relatively short time (3-5 hours) before transfer to a lawn of E. coli. After the transfer to E. coli, a high titer of S. typhimurium persists in the C. elegans intestinal lumen for the rest of the worms' life. Furthermore, feeding for 5 hours on a 1:1000 mixture of S. typhimurium and E. coli followed by transfer to 100% E. coli, also led to death after several days. This killing correlated with an increase in the titer of S. typhimurium in the C. elegans lumen, which reached 10,000 bacteria per worm. These data indicate that, in contrast to P. aeruginosa, a small inoculum of S. typhimurium can proliferate in the C. elegans intestine and establish a persistent infection. S. typhimurium mutated in the PhoP/PhoQ signal transduction system caused significantly less killing of C. elegans.

Cloning the Arabidopsis GA1 Locus by Genomic Subtraction.

Arabidopsis thaliana ga1 mutants are gibberellin-responsive dwarfs. We used the genomic subtraction technique to clone DNA sequences that are present in wild-type Arabidopsis (ecotype Landsberg erecta, Ler) but are missing in a presumptive ga1 deletion mutant, ga1-3. The cloned sequences correspond to a 5.0-kb deletion in the ga1-3 genome. Three lines of evidence indicated that the 5.0-kb deletion in the ga1-3 mutant is located at the GA1 locus. First, restriction fragment length polymorphism mapping showed that DNA sequences within the 5.0-kb deletion map to the GA1 locus. Second, cosmid clones that contain wild-type DNA inserts spanning the deletion in ga1-3 complemented the dwarf phenotype when integrated into the ga1-3 genome by Agrobacterium tumefaciens-mediated transformation. Third, we identified molecular lesions in four additional ga1 alleles within the 5.0-kb region deleted in mutant ga1-3. One of these lesions is a large insertion or inversion located within the most distal intron encoded by the GA1 locus. The three other lesions are all single base changes located within the two most distal exons. RNA gel blot analysis indicated that the GA1 locus encodes a 2.8-kb mRNA. We calculated a recombination rate of 10-5 cM per nucleotide for the GA1 region of the Arabidopsis genome.

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.

Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene.

We have cloned an Arabidopsis thaliana chalcone synthase (CHS) gene on the basis of cross-hybridization with a Petroselinum hortense CHS cDNA clone. The protein sequence deduced from the A. thaliana CHS DNA sequence is at least 85% homologous to the CHS sequences from P. hortense, Antirrhinum majus, and Petunia hybrida. Southern blot analysis indicated that CHS is a single-copy gene in A. thaliana. High-intensity light treatment of A. thaliana plants for 24 h caused a 50-fold increase in CHS enzyme activity and an accumulation of visibly detectable levels of anthocyanin pigments in the vegetative structures of these plants. A corresponding increase in the steady-state level of CHS mRNA was detected after high-intensity light treatment for the same period of time. The accumulation of CHS mRNA in response to high-intensity light was due, at least in part, to an increased rate of transcription of the CHS gene as demonstrated by nuclear runoff experiments.

Caenorhabditis elegans: a model genetic host to study Pseudomonas aeruginosa pathogenesis.

In the past year, a Caenorhabditis elegans-Pseudomonas aeruginosa pathogenesis model has been developed to facilitate the systematic dissection of both host and pathogen genes involved in pathogenic interactions. Analysis of the P. aeruginosa-C. elegans interaction should shed light on the larger question of how organisms interact at the molecular level in antagonistic relationships.

Isolation of new Arabidopsis mutants with enhanced disease susceptibility to Pseudomonas syringae by direct screening.

To identify plant defense components that are important in restricting the growth of virulent pathogens, we screened for Arabidopsis mutants in the accession Columbia (carrying the transgene BGL2-GUS) that display enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) ES4326. Among six (out of a total of 11 isolated) enhanced disease susceptibility (eds) mutants that were studied in detail, we identified one allele of the previously described npr1/nim1/sai1 mutation, which is affected in mounting a systemic acquired resistance response, one allele of the previously identified EDS5 gene, and four EDS genes that have not been previously described. The six eds mutants studied in detail (npr1-4, eds5-2, eds10-1, eds11-1, eds12-1, and eds13-1) displayed different patterns of enhanced susceptibility to a variety of phytopathogenic bacteria and to the obligate biotrophic fungal pathogen Erysiphe orontii, suggesting that particular EDS genes have pathogen-specific roles in conferring resistance. All six eds mutants retained the ability to mount a hypersensitive response and to restrict the growth of the avirulent strain Psm ES4326/avrRpt2. With the exception of npr1-4, the mutants were able to initiate a systemic acquired resistance (SAR) response, although enhanced growth of Psm ES4326 was still detectable in leaves of SAR-induced plants. The data presented here indicate that eds genes define a variety of components involved in limiting pathogen growth, that many additional EDS genes remain to be discovered, and that direct screens for mutants with altered susceptibility to pathogens are helpful in the dissection of complex pathogen response pathways in plants.

Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening.

To discover which components of plant defense responses make significant contributions to limiting pathogen attack, we screened a mutagenized population of Arabidopsis thaliana for individuals that exhibit increased susceptibility to the moderately virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). The 12 enhanced disease susceptibility (eds) mutants isolated included alleles of two genes involved in phytoalexin biosynthesis (pad2, which had been identified previously, and pad4, which had not been identified previously), two alleles of the previously identified npr1 gene, which affects expression of other defense genes, and alleles of seven previously unidentified genes of unknown function. The npr1 mutations caused greatly reduced expression of the PR1 gene in response to PsmES4326 infection, but had little effect on expression of two other defense genes, BGL2 and PR5, suggesting that PR1 expression may be important for limiting growth of PsmES4326. While direct screens for mutants with quantitative pathogen-susceptibility phenotypes have not been reported previously, our finding that mutants isolated in this way include those affected in known defense responses supports the notion that this type of screening strategy allows genetic dissection of the roles of various plant defense responses in disease resistance.

A superfamily of Arabidopsis thaliana retrotransposons.

We describe a superfamily of Arabidopsis thaliana retrotransposable elements that consists of at least ten related families designated Ta1-Ta10. The Ta1 family has been described previously. Two genomic clones representing the Ta2 and Ta3 elements were isolated from an A. thaliana (race Landsberg erecta) lambda library using sequences derived from the reverse transcriptase region of Ta1 as hybridization probes. Nucleotide sequence analysis showed that the Ta1, Ta2 and Ta3 families share greater than 75% amino acid identity in pairwise comparisons of their reverse transcriptase and RNase H genes. In addition to Ta1, Ta2 and Ta3, we identified seven other related retrotransposon families in Landsberg erecta, Ta4-Ta10, using degenerate primers and the polymerase chain reaction to amplify a highly conserved region of retrotransposon-encoded reverse transcriptase. One to two copies of elements Ta2-Ta10 are present in the genomes of the A. thaliana races Landsberg erecta and Columbia indicating that the superfamily comprises at least 0.1% of the A. thaliana genome. The nucleotide sequences of the reverse transcriptase regions of the ten element families place them in the category of copia-like retrotransposons and phylogenetic analysis of the amino acid sequences suggests that horizontal transfer may have played a role in their evolution.

The structure, distribution and evolution of the Ta1 retrotransposable element family of Arabidopsis thaliana.

The Ta1 elements are a low copy number, copia-like retrotransposable element family of Arabidopsis thaliana. Six Ta1 insertions comprise all of the Ta1 element copies found in three geographically diverse A. thaliana races. These six elements occupy three distinct target sites: Ta1-1 is located on chromosome 5 and is common to all three races (Col-0, Kas-1 and La-0). Ta1-2 is present in two races on chromosome 4 (Kas-1 and La-0), and Ta1-3, also located on chromosome 4, is present only in one race (La-0). The six Ta1 insertions share greater than 96% nucleotide identity, yet are likely to be incapable of further transposition due to deletions or nucleotide changes that alter either the coding capacity of the elements or conserved protein domains required for retrotransposition. Nucleotide sequence comparisons of these elements and the distribution of Ta1 among 12 additional A. thaliana geographical races suggest that Ta1-1 predated the global dispersal of A. thaliana. As the species spread throughout the world, two additional transposition events occurred which gave rise first to Ta1-2 and finally to Ta1-3.

Haplotypic divergence coupled with lack of diversity at the Arabidopsis thaliana alcohol dehydrogenase locus: roles for both balancing and directional selection?

We designate a region of the alcohol dehydrogenase locus (Adh) of the weedy crucifer, Arabidopsis thaliana, as "hypervariable" on the basis of a comparison of sequences from ecotypes Columbia and Landsberg. We found eight synonymous and two replacement mutations in the first 262 nucleotides of exon 4, and an additional two mutations in the contiguous region of intron 3. The rest of the sequence (2611 bp) has just three mutations, all of them confined to noncoding regions. Our survey of the hypervariable region among 37 ecotypes of A. thaliana revealed two predominant haplotypes, corresponding to the Columbia and Landsberg sequences. We identified five additional haplotypes and 4 additional segregating sites. The lack of haplotype diversity is presumably in part a function of low rates of recombination between haplotypes conferred by A. thaliana's tendency to self-fertilize. However, an analysis in 32 ecotypes of 12 genome-wide polymorphic markers distinguishing Columbia and Landsberg ecotypes indicated levels of outcrossing sufficient at least to erode linkage disequilibrium between dispersed markers. We discuss possible evolutionary explanations for the coupled observation of marked divergence within the hypervariable region and a lack of haplotype diversity among ecotypes. The sequence of the region for closely related species argues against the possibility that one allele is the product of introgression. We note (1) that several loss of function mutations (both naturally and chemically induced) map to the hypervariable region, and (2) the presence of two amino acid replacement polymorphisms, one of which causes the mobility difference between the two major classes of A. thaliana Adh electrophoretic alleles. We argue that protein polymorphism in such a functionally significant part of the molecule may be subject to balancing selection. The observed pattern of extensive divergence between the alleles is consistent with this explanation because balancing selection on a particular site maintains linked neutral polymorphisms at intermediate frequencies.

Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance.

We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.

Arabidopsis mutations at the RPS2 locus result in loss of resistance to Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

We isolated and characterized two Arabidopsis thaliana mutants that fail to mount a hypersensitive defense response (HR) when infiltrated with phytopathogenic Pseudomonas strains carrying the avirulence (avr) gene avrRpt2 but still mount an HR when infiltrated with strains carrying other avr genes. One of these mutants was isolated using a method we developed that enriches for Arabidopsis seedlings that survive vacuum-infiltration with a bacterial strain carrying an avr gene. Genetic analysis showed that the phenotypes of both mutants resulted from mutations at a single locus, RPS2. In contrast to the wild type, both rps2 mutants failed to limit the growth of Pseudomonas strains carrying avrRpt2. Heterozygous RPS2/rps2 plants displayed a phenotype intermediate between those of RPS2/RPS2 and rps2/rps2 homozygotes. These experiments show that the wild-type allele at the rps2 locus, RPS2, encodes a component of a signal transduction pathway that responds to a signal generated by avrRpt2 and that RPS2 is required for the elicitation of an HR. RPS2 was mapped near the restriction fragment length polymorphism marker PG11 on chromosome IV.

Mode of action of the Arabidopsis thaliana phytoalexin camalexin and its role in Arabidopsis-pathogen interactions.

The virulent Arabidopsis thaliana pathogen Pseudomonas syringae pv. maculicola strain ES4326 (Psm ES4326) and other gram-negative bacteria are sensitive to camalexin (3-thiazol-2'-yl-indole), the Arabidopsis phytoalexin. Furthermore, Psm ES4326 is unable to degrade camalexin or to become tolerant to it. Apparently, Psm ES4326 is a successful pathogen even though it elicits synthesis of a host phytoalexin to which it is sensitive. Assays of membrane integrity revealed that, like other phytoalexins, camalexin disrupts bacterial membranes, suggesting that camalexin toxicity is a consequence of membrane disruption. A screen for camalexin-resistant mutants of Psm ES4326 yielded only partially resistant mutants, which displayed partial resistance in both killing and membrane integrity assays. These mutants were also resistant to low concentrations of tetracycline and nalidixic acid, suggesting that they were affected in components of the outer membrane. The mutants were not distinguishable from Psm ES4326 in virulence assays. Camalexin was toxic to Arabidopsis cells growing in tissue culture. However, comparison of the extent of cell death associated with disease symptoms in infected leaves of wild-type Arabidopsis and a camalexin-deficient mutant suggested that camalexin does not contribute significantly to cell death in infected tissue.

Developmental regulation of nodule-specific genes in alfalfa root nodules.

We have cloned alfalfa nodule-specific cDNAs that code for leghemoglobin (Lb), glutamine synthetase (GS), and three unidentified nodulins. Hybrid-select translation of nodule RNA followed by 2-D gel electrophoresis showed that the Lb-specific cDNA corresponded to at least four Lb species of 12 kDa. One of the unidentified cDNA clones (N-32/34) corresponded to at least five polypeptides of 32-34 kDa; a second unidentified cDNA clone (N-14) corresponded to an individual polypeptide of 14 kDa. The in vitro translation product(s) of the RNA hybrid selected by the third unidentified cDNA clone (N-22) formed a single band at 22 kDa on a one-dimensional gel. Northern and dot blot analyses of RNA isolated from wild-type nodules and from defective nodules elicited by a variety of Rhizobium meliloti mutants showed that 1) RNAs corresponding to the Lb, nodule-specific GS, and three unidentified nodulins were coordinately expressed during the course of nodule development, and 2) all five nodulins were expressed in Fix- nodules that contained infection threads and bacteroids but were not expressed in nodules that lacked infection threads and intracellular rhizobia.

Specific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude bacterial cell extracts.

Crude bacterial cell extracts prepared from an Escherichia coli lacIq strain were shown to protect specific nucleotides in the lac operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lacI- strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA "footprinting" experiments to define regions of DNA which bind to unknown regulatory proteins.

A dual-labeling method for identifying differentially expressed genes: use in the identification of cDNA clones that hybridize to RNAs whose abundance in tomato flowers is potentially regulated by gibberellins.

A method for identifying cDNA clones that hybridize to differentially expressed RNAs is described. Briefly, the RNA population in which the RNAs of interest are more abundant is used as a template for the synthesis of 35S-labeled cDNAs and another RNA population in which the RNAs of interest are less abundant is used as a template for the synthesis of 32P-labeled cDNAs. The labeled cDNAs are pooled and hybridized to plaque or colony lifts constructed from a cDNA library. Clones that hybridize to RNAs that are differentially expressed are identified using differential autoradiography/fluorography to discriminate between the 32P and 35S isotopes. We have used this method to identify cDNA clones that hybridize to mRNAs that are more abundant in the flowers of wild-type tomato than in the flowers of mutants that have low endogenous levels of gibberellins.