A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Two independent approaches converge to the cloning of a new Leptosphaeria maculans avirulence effector gene,AvrLmS‐Lep2

Brassica napus (oilseed rape, canola) seedling resistance to Leptosphaeria maculans, the causal agent of blackleg (stem canker) disease, follows a gene‐for‐gene relationship. The avirulence genes AvrLmS and AvrLep2 were described to be perceived by the resistance genes RlmS and LepR2, respectively, present in B. napus ‘Surpass 400’. Here we report cloning of AvrLmS and AvrLep2 using two independent methods. AvrLmS was cloned using combined in vitro crossing between avirulent and virulent isolates with sequencing of DNA bulks from avirulent or virulent progeny (bulked segregant sequencing). AvrLep2 was cloned using a biparental cross of avirulent and virulent L. maculans isolates and a classical map‐based cloning approach. Taking these two approaches independently, we found that AvrLmS and AvrLep2 are the same gene. Complementation of virulent isolates with this gene confirmed its role in inducing resistance on Surpass 400, Topas‐LepR2, and an RlmS‐line. The gene, renamed AvrLmS‐Lep2, encodes a small cysteine‐rich protein of unknown function with an N‐terminal secretory signal peptide, which is a common feature of the majority of effectors from extracellular fungal plant pathogens. The AvrLmS‐Lep2/LepR2 interaction phenotype was found to vary from a typical hypersensitive response through intermediate resistance sometimes towards susceptibility, depending on the inoculation conditions. AvrLmS‐Lep2 was nevertheless sufficient to significantly slow the systemic growth of the pathogen and reduce the stem lesion size on plant genotypes with LepR2, indicating the potential efficiency of this resistance to control the disease in the field.     

Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Leptosphaeria maculans AvrLm9: a new player in the game of hide and seek with AvrLm4‐7

Blackleg disease of Brassica napus caused by Leptosphaeria maculans (Lm) is largely controlled by the deployment of race‐specific resistance (R) genes. However, selection pressure exerted by R genes causes Lm to adapt and give rise to new virulent strains through mutation and deletion of effector genes. Therefore, a knowledge of effector gene function is necessary for the effective management of the disease. Here, we report the cloning of Lm effector AvrLm9 which is recognized by the resistance gene Rlm9 in B. napus cultivar Goéland. AvrLm9 was mapped to scaffold 7 of the Lm genome, co‐segregating with the previously reported AvrLm5 (previously known as AvrLmJ1). Comparison of AvrLm5 alleles amongst the 37 re‐sequenced Lm isolates and transgenic complementation identified a single point mutation correlating with the AvrLm9 phenotype. Therefore, we renamed this gene as AvrLm5‐9 to reflect the dual specificity of this locus. Avrlm5‐9 transgenic isolates were avirulent when inoculated on the B. napus cultivar Goéland. The expression of AvrLm5‐9 during infection was monitored by RNA sequencing. The recognition of AvrLm5‐9 by Rlm9 is masked in the presence of AvrLm4‐7, another Lm effector. AvrLm5‐9 and AvrLm4‐7 do not interact, and AvrLm5‐9 is expressed in the presence of AvrLm4‐7. AvrLm5‐9 is the second Lm effector for which host recognition is masked by AvrLm4‐7. An understanding of this complex interaction will provide new opportunities for the engineering of broad‐spectrum recognition.     

Utility of the white gene in estimating phylogenetic relationships among mosquitoes (Diptera: Culicidae)

The utility of a nuclear protein-coding gene for reconstructing phylogenetic relationships within the family Culicidae was explored. Relationships among 13 species representing three subfamilies and nine genera of Culicidae were analyzed using a 762-bp fragment of coding sequence from the eye color gene, white. Outgroups for the study were two species from the sister group Chaoboridae. Sequences were determined from clone PCR products amplified from genomic DNA, and aligned following conceptual intron splicing and amino acid translation. Third codon positions were characterized by high levels of divergence and biased nucleotide composition, the intensity and direction of which varied among taxa. Equal weighting of all characters resulted in parsimony and neighboring-joining trees at odds with the generally accepted phylogenetic hypothesis based on morphology and rDNA sequences. The application of differential weighting schemes recovered the traditional hypothesis, in which the subfamily Anophelinae formed the basal clade. The subfamily Toxorhynchitinae occupied an intermediate position, and was a sister group to the subfamily Culicinae. Within Culicinae, the genera Sabethes and Tripteroides formed an ancestral clade, while the Culex-Deinocerites and Aedes- Haemagogus clades occupied increasingly derived positions in the molecular phylogeny. An intron present in the Culicinae- Toxorhynchitinae lineage and one outgroup taxon was absent in the basal Anophelinae lineage and the second outgroup taxon, suggesting that intron insertions or deletions may not always be reliable systematic characters.      

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Position of the reduced mycorrhizal colonisation (<Emphasis Type="Italic">Rmc</Emphasis>) locus on the tomato genome map

Our research aims to investigate the molecular communication between land plants and arbuscular mycorrhizal (AM) fungi in the establishment of symbiosis. We have identified a mutation in the facultative AM host tomato, which we named rmc. Plants that are homozygous for rmc no longer host most AM fungi. The mutation also affects the interaction of tomato with root knot nematode and Fusarium wilt. However, the function/s encoded by the intact Rmc locus is/are unknown. To clone and sequence the gene or genes that comprise the Rmc locus, we have initiated a positional cloning project. In this paper, we report the construction of mapping populations and use of molecular markers from the published genome map to identify the location of Rmc on tomato chromosome 8. Nucleotide binding site-leucine rich repeat resistance genes, reported to reside in the same region of that chromosome, provided insufficient differences to develop cleaved amplified polymorphic sequence markers. Therefore, we were unable to map these sequences in relation to rmc. Our results potentiate future work to identify the Rmc function and to determine the genetic basis for the multiple plant-microbe interaction functions that the rmc mutation has defined.