A new,pathogen-inducible gene of Arabidopsis is expressed in an ecotype-specific manner

In this study we describe a novel gene, which was isolated in an attempt to search for specific plant resistance genes of Arabidopsis against isolates of the phytopathogenic bacterium Xanthomonas campestris pv. campestris. The gene was cloned by differential screening of a genomic library of the Xcc 750-resistant ecotype Col-0, using cDNA populations derived from ecotype Col-0 and the Xcc 750-susceptible ecotype Oy-0. The isolated gene, CXc750, is differentially expressed in ecotypes of Arabidopsis thaliana. In addition, although highly expressed in uninfected plants, gene expression increases in response to pathogen attack. CXc750 potentially codes for a small, basic protein of about 10 kDa. The predicted protein product contains a potential signal leader peptide at the amino-terminal end but no ER retention sequence and no further transmembrane domain. This indicates that the gene product is transported to other compartments or out of the cell.The possible function of CXc750 as a member of the plant defense response system is discussed.     

Genetic diversity and historical population structure in the New Zealand mayfly Acanthophlebia cruentata

1. Nucleotide sequences of a 280 base pair region of the cytochrome b gene were used to assess genetic diversity and to infer population histories in the New Zealand mayfly Acanthophlebia cruentata. 2. A hierarchial examination of populations from 19 streams at different spatial scales in the central and northern North Island of New Zealand found 34 haplotypes. A common haplotype was found in all central region streams and unique haplotypes in northern streams. Several central streams had region specific haplotypes with genetically differentiated populations at the 70–100 km scale. 3. Haplotype diversity was high (0.53–0.8) at most sites, but low (0–0.22) in some central sites. amova analyses found significant genetic diversity among regions (69%) and among catchments (58%). Most population pairwise F_ST tests were significant, with non‐significant pairwise tests among sites in the central region and pairs of sites between neighbouring streams. 4. The levels of sequence divergence are interpreted as the result of Pleistocene divergence in multiple refugia, leading to the evolution of regionally unique haplotypes. The low diversity in some central region populations may result from recent colonisation following local extinctions, associated with volcanic events.     

Molecular mapping of the Arabidopsis locus RPP11 which conditions isolate-specific hypersensitive resistance against downy mildew in ecotype RLD

Isolate WELA of the plant pathogenic oomycete fungus Peronospora parasitica causes downy mildew in the Arabidopsis thaliana ecotypes Weiningen (Wei-0) and La-er, whereas ecotypes RLD and Col-0 are resistant. Genetic crosses between resistant RLD and susceptible Wei-0 showed that resistance was inherited in a simple Mendelian fashion as a monogenic dominant trait. The interactions between different isolates of P. parasitica and ecotypes of A. thaliana show race-specific variation and fit a gene-for-gene relationship. The RPP11 resistance gene was mapped by following the co-segregation of the resistance phenotype with RFLP markers in a mapping population of 254 F₃ families derived from RLD x Wei-0 F₂ individuals. Linkage analysis using version 1.9 of the MAPMAKER program placed the RPP11 resistance locus on chromosome III between marker m249 (two recombinants) and marker g2534 (six recombinants). Markers g2534 and g4117 are on YAC EG7H1. Marker g4117 and one end probe (N5) generated from YAC EG7H1 showed no recombinants. The YAC end probe N5, which was generated by plasmid rescue, was used to screen clones in the Eric Ward YAC library and a YAC was fished (EW19B12) which also hybridised with m249. Thus, a YAC contig has been established over the region where the resistance locus maps. Because the YACs were made with ecotype Columbia DNA it is necessary to isolate the equivalent region from RLD in order to clone the resistance locus. To this end a phage -DASH genomic library was prepared from RLD and a contig covering the relevant region of the YACs is currently under construction.     

Protecting evolutionary significant units for the remnant populations of <Emphasis Type="Italic">Berchemiella wilsonii</Emphasis> var. <Emphasis Type="Italic">pubipetiolata</Emphasis> (Rhamnaceae)

Berchemiella wilsonii var. pubipetiolata (Rhamnaceae) is an endangered tree in eastern China. Habitat destruction has resulted in fragmentation of remnant populations and extinction of local populations. AFLP and cpDNA markers were used to determine the population structure of remnant populations of B. wilsonii var. pubipetiolata. Moderate nuclear genomic diversity was found within each of the four remnant populations (H _S = 0.141–0.172), while the cpDNA haplotype diversity in each population ranged from 0.356 to 0.681. Six haplotypes were identified by a combined cpRFLP and cpSSR analysis in a total of 89 individuals. AMOVA revealed significantly AFLP genetic differentiation within and between regions (Φ_SC = 0.196, Φ_CT = 0.396, respectively), and a high cpDNA haplotype differentiation between regions (Φ_CT = 0.849). The results suggest low gene flow between populations of B. wilsonii var. pubipetiolata. Strong genetic divergence between two regional populations as revealed by both AFLP and cpDNA markers provided convincing evidence that two distinct evolutionary lineages existed, and should be recognized as ‘evolutionary significant units’ (ESUs) for conservation concerns.     

Genetic differentiation between two color morphs of <Emphasis Type="Italic">Apogon taeniophorus</Emphasis> from southern Japan

The anterior half of the mitochondrial 16S rRNA gene (ca. 610bp) was compared for two color morphs (spotted and lined types) of a dark-striped cardinalfish, previously identified as Apogon taeniophorus. Phylogenetic analyses using maximum-parsimony (MP) and neighbor-joining (NJ) methods, with haplotypes of A. cookii as an outgroup, showed that the haplotypes of each color-morph were reciprocally monophyletic with 100% bootstrap values. In addition, the degree of sequence difference between the two morphs was comparable to that existing between the other clearly distinct congeneric species. These results, together with the differences in coloration and overlapped geographical ranges, indicated that the two color morphs of A. taeniophorus represent two distinct species.     

Mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 and plastid <Emphasis Type="Italic">rbc</Emphasis>L genes of <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> (Gracilariaceae,Rhodophyta)

The mitochondrial cytochrome c oxidase subunit I gene sequence was recently developed for DNA barcoding of red algal species. We determined the 1245 base pairs of the gene from 27 taxa of an agar-producing species, Gracilaria vermiculophylla, and putative relatives and compared the results with rbcL data from the same species. A total of 392 positions (31.5%) were variable, 282 positions (22.6%) were parsimoniously informative, and average sequence divergence was 13% in an ingroup. Within G. vermiculophylla, pairwise divergence of the gene was variable up to 11 bp (0.9%). Seven recognized haplotypes of cox1 tended to be geographically related. In the aligned 1386 bp of rbcL, three haplotypes were recognized. These results suggest that cox1 is a valuable molecular marker within species and will be very useful in haplotype analyses.     

Subspecific relationships and genetic structure in the spotted owl

Hierarchical genetic structure was examined in the three geographically-defined subspecies of spotted owl (Strix occidentalis) to define relationships among subspecies and quantify variation within and among regional and local populations. Sequences (522 bp) from domains I and II of the mitochondrial control region were analyzed for 213 individuals from 30 local breeding areas. Results confirmed significant differences between northern spotted owls and the other traditional geographically defined subspecies but did not provide support for subspecific level differences between California and Mexican spotted owls. Divergence times among subspecies estimated with a 936 bp portion of the cytochrome b gene dated Northern and California/Mexican spotted owl divergence time to 115,000–125,000 years ago, whereas California/Mexican spotted owl divergence was estimated at 15,000 years ago. Nested clade analyses indicated an association between California spotted owl and Mexican spotted owl haplotypes, implying historical contact between the two groups. Results also identified a number of individuals geographically classified as northern spotted owls (S. o. caurina) that contained haplotypes identified as California spotted owls (S. o. caurina). Among all northern spotted owls sampled (n=131), 12.9% contained California spotted owl haplotypes. In the Klamath region, which is the contact zone between the two subspecies, 20.3% (n=59) of owls were classified as California spotted owls. The Klamath region is a zone of hybridization and speciation for many other taxa as well. Analyses of population structure indicated gene flow among regions within geographically defined subspecies although there was significant differentiation among northern and southern regions of Mexican spotted owls. Among all areas examined, genetic diversity was not significantly reduced except in California spotted owls where the southern region consists of one haplotype. Our results indicate a stable contact zone between northern and California spotted owls, maintaining distinct subspecific haplotypes within their traditional ranges. This supports recovery efforts based on the traditional subspecies designation for the northern spotted owl. Further, although little variation was found between California and Mexican spotted owls, we suggest they should be managed separately because of current isolation between groups.     

Evolutionary trade-offs at the Arabidopsis WRR4A resistance locus underpin alternate Albugo candida race recognition specificities

The oomycete Albugo candida causes white rust of Brassicaceae, including vegetable and oilseed crops, and wild relatives such as Arabidopsis thaliana. Novel White Rust Resistance (WRR) genes from Arabidopsis enable new insights into plant/parasite co-evolution. WRR4A from Arabidopsis accession Columbia (Col-0) provides resistance to many but not all white rust races, and encodes a nucleotide-binding, leucine-rich repeat immune receptor. Col-0 WRR4A resistance is broken by AcEx1, an isolate of A. candida. We identified an allele of WRR4A in Arabidopsis accession Øystese-0 (Oy-0) and other accessions that confers full resistance to AcEx1. WRR4A^Oy-0 carries a C-terminal extension required for recognition of AcEx1, but reduces recognition of several effectors recognized by the WRR4A^Col-0 allele. WRR4A^Oy-0 confers full resistance to AcEx1 when expressed in the oilseed crop Camelina sativa.     

Sequence,expression divergence,and complementation of homologous <Emphasis Type="Italic">ALCATRAZ</Emphasis> loci in <Emphasis Type="Italic">Brassica napus</Emphasis>

The genomic era provides new perspectives in understanding polyploidy evolution, mostly on the genome-wide scale. In this paper, we show the sequence and expression divergence between the homologous ALCATRAZ (ALC) loci in Brassica napus, responsible for silique dehiscence. We cloned two homologous ALC loci, namely BnaC.ALC.a and BnaA.ALC.a in B. napus. Driven by the 35S promoter, both the loci complemented to the alc mutation of Arabidopsis thaliana, yet only the expression of BnaC.ALC.a was detectable in the siliques of B. napus. Sequence alignment indicated that BnaC.ALC.a and BolC.ALC.a, or BnaA.ALC.a and BraA.ALC.a, possess a high level of similarity. The understanding of the sequence and expression divergence among homologous loci of a gene is of due importance for an effective gene manipulation and TILLING (or ECOTILLING) analysis for the allelic DNA variation at a given locus. S. Hua and I. H. Shamsi contributed equally to this work.     

High KIR diversity in Amerindians is maintained using few gene-content haplotypes

Interaction between killer cell immunoglobulin-like receptors (KIR) and cognate HLA class I ligands influences the innate and adaptive immune response to infection. The KIR family varies in gene content and allelic polymorphism, thereby, distinguishing individuals and populations. KIR gene content was determined for 230 individuals from three Amerindian tribes from Venezuela: the Yucpa, Bari and Warao. Gene-content haplotypes could be assigned to 212 individuals (92%) because only five different haplotypes were present—group A and four group B. Six different haplotype combinations accounted for >80% of individuals. Each tribe has distinctive genotype frequencies. Despite few haplotypes, all 14 KIR genes are at high frequency in the three tribes, with the exception of 2DS3. Each population has an even frequency of group A and B haplotypes. Allele-level analysis of 3DL1/S1 distinguished five group A haplotypes and six group B haplotypes. The high frequency and divergence of the KIR haplotypes in the Amerindian tribes provide greater KIR diversity than is present in many larger populations. An extreme case being the Yucpa, for whom two gene-content haplotypes account for >90% of the population. These comprise the group A haplotype and a group B haplotype containing all the KIR genes, except 2DS3, that typify the group B haplotypes. Here is clear evidence for balancing selection on the KIR system and the biological importance of both A and B haplotypes for the survival of human populations.Electronic Supplementary Material Supplementary material is available for this article at     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Novel single nucleotide polymorphisms and haplotypes within the bovine<Emphasis Type="Italic"> CXCR2</Emphasis> gene

The objectives of this study were to identify single nucleotide polymorphisms (SNPs) and resulting haplotypes in the bovine CXCR2 gene. A 311-bp segment of the bovine CXCR2 gene was amplified and sequenced. Five SNPs at positions 612, 684, 777, 858, and 861 were expressed in both Holstein and Jersey dairy cattle. Four SNPs resulted in synonymous substitutions, while a non-synonymous switch at position 777 (GC) resulted in a glutamine to histidine substitution at amino acid residue 245. Strong linkage disequilibrium was exhibited for both breeds among all five loci (P<0.001). Both allele and genotype frequencies differed significantly with respect to breed at four of the five loci (P<0.001). The five polymorphisms generated ten distinct haplotypes. Six haplotypes were common between the two breeds, while Holsteins and Jerseys each uniquely expressed two haplotypes. Of the six common haplotypes, two represented 83% of the Jersey population; whereas four of these haplotypes represented 95% of the Holstein population.     

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.     

Survey of resistance gene analogs in <Emphasis Type="Italic">Solanum caripense</Emphasis>, a relative of potato and tomato,and update on <Emphasis Type="Italic">R</Emphasis> gene genealogy

The resistance (R) proteins of the TIR- and non-TIR (or CC-) superfamilies possess a nucleotide binding site (NBS) domain. Within an R gene, the NBS is the region of highest conservation, suggesting an essential role in triggering R protein activity. We compared the NBS domain of functional R genes and resistance gene analogs (RGA) amplified from S. caripense genomic DNA via PCR using specific and degenerate primers with its counterpart from other plants. An overall high degree of sequence conservation was apparent throughout the P-loop, kinase-2 and kinase-3a motifs of NBS fragments from all plants. Within the non-TIR class of R genes a prominent sub-class similar to the potato R1 gene conferring resistance to late blight, was detected. All non-TIR-R1-like R gene fragments that were sequenced possessed an intact open reading frame, whereas 22% of all non-TIR-non-R1-like fragments and 59% of all TIR-NBS RGA fragments had an interrupted reading frame or contained transposon-specific sequence. The non-TIR-R1-like fragments had high similarity to Solanaceae R genes and low similarity to RGAs of other plant species including A. thaliana and the cereals. It is concluded that appearance of the non-TIR-R1-like NBS domain represents a relatively recent evolutionary development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana

Thirty-six phytohormone-affected mutants of Arabidopsis thaliana (L.) Heynh. and their parental ecotypes were tested for resistance/susceptibility to Botrytis cinerea Pers.; Fr. and ability to develop Trichoderma-mediated induced systemic resistance (ISR). Ecotype Colombia-0 (Col-0) was relatively resistant to B. cinerea, and Trichoderma harzianum Rifai T39 application at sites spatially separated (roots) from the B. cinerea inoculation (leaves) resulted in reduction of grey mold symptoms. Ecotypes Wassilewskija-4, Nossen-0 and Landsberg-0 had low levels of basal resistance to B. cinerea and were unable to express ISR. Mutants derived from ISR-non-inducible ecotypes displayed ISR-non-inducible phenotypes, whereas the ISR inducibility of mutants derived from the ISR-inducible genotype Col-0 varied according to the type of mutant. Thus, salicylic acid (SA)-impaired mutants derived from Col-0 were ISR-inducible, while ethylene/jasmonic acid (ethylene/JA)-impaired mutants of the same origin were ISR-non-inducible. SA-impaired mutants retained basal level of resistance to B. cinerea, while most ethylene/JA-impaired mutants were highly susceptible. Abscisic acid- and gibberellin-impaired mutants were highly susceptible to B. cinerea and showed ISR-non-inducible phenotypes irrespective of their lines of origin. Auxin-resistant mutants derived from Col-0 were ISR-inducible; mutant originating from Landsberg-0 and mutants which were resistant to both auxin and ethylene were ISR-non-inducible. Most of the arabidopsis genotypes which were unable to express Trichoderma-mediated ISR against B. cinerea exhibited enhanced susceptibility to this pathogen. T. harzianum treatments enhanced the growth of arabidopsis plants regardless of genotype or ISR inducibility.     

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)⁺-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses.The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5 non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5 sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3 sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site.This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.Abbreviations AK aspartate kinase - HSDH homoserine dehydrogenase - ID intermediate domain - Tp transit peptide     

Phyllosphere bacterial communities of trichome-bearing and trichomeless Arabidopsis thaliana leaves

This study aimed to investigate whether the presence of trichomes as conspicuous physical attributes of the leaf surface affects the microbial community composition on Arabidopsis thaliana leaves. The A. thaliana ecotype Col-0 and its trichomeless gl1 mutant were grown in growth cabinets under climate-controlled conditions. The gl1 mutant showed a similar wax composition as the Col-0 wild type with slightly reduced amounts of C₂₉, C₃₁ and C₃₃ alkanes by GC/MS and GC/FID analyses. 120 bacterial isolates representing 39 bacterial genera were obtained from A. thaliana Col-0 leaf surfaces. Phylogenetic analysis of nearly full-length 16S rRNA sequences from 29 selected isolates confirmed their affiliation to the Proteobacteria (Alpha-, Beta-, Gamma-), Actinobacteria, Bacteroidetes and Firmicutes. The bacterial diversity on A. thaliana ecotype Col-0 and its gl1 mutant, devoid of trichomes, were further compared by denaturing gradient gel electrophoresis (DGGE). Banding patterns and sequencing of representative DGGE bands revealed the presence of phylotypes related to Sphingomonas (Alphaproteobacteria), Methylophilus (Betaproteobacteria) and Dyadobacter (Bacteroidetes) which are common phyllosphere inhabitants. Furthermore, wildtype and trichomeless mutant plants were exposed to outdoor conditions for 4–5 weeks. The DGGE gels showed only minor differences between the two plant lines, thus suggesting that trichomes per se do not affect bacterial diversity on Arabidopsis leaves under the experimental conditions tested.