Rapid and selective surveillance of Arabidopsis thaliana genome annotations with Centrifuge

Summary: Centrifuge is a user-friendly system to simultaneously access Arabidopsis gene annotations and intra- and inter-organism sequence comparison data. The tool allows rapid retrieval of user-selected data for each annotated Arabidopsis gene providing, in any combination, data on the following features: predicted protein properties such as mass, pI, cellular location and transmembrane domains; SWISS-PROT annotations; Interpro domains; Gene Ontology records; verified transcription; BLAST matches to the proteomes of A.thaliana, Oryza sativa (rice), Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens. The tool lends itself particularly well to the rapid analysis of contigs or of tens or hundreds of genes identified by high-throughput gene expression experiments. In these cases, a summary table of principal predicted protein features for all genes is given followed by more detailed reports for each individual gene. Centrifuge can also be used for single gene analysis or in a word search mode. Availability: http://centrifuge.unil.ch/ Contact: [email protected]

Promoter analysis of members of a plant defense-related LRR-RLK gene cluster in Arabidopsis thaliana

M.Sc. (Biochemistry)A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies

Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation

The function of most lncRNA is unknown. Here, the authors show that transcriptional read-through at the Arabidopsis SVALKA locus produces a cryptic lncRNA that overlaps with the neighboring cold-responsive CBF1 gene and limits CBF1 expression via an RNA polymerase II collision-based mechanism

IDENTIFYING HIGHLY CONSERVED PATHOGENICITY GENES IN CHESTNUT BLIGHT AND POWDERY MILDEW FUNGI AS TARGETS FOR NOVEL FORMS OF HOST RESISTANCE

A bioinformatic search of the genomes of chestnut blight fungus, Cryphonectria parasitica (Cp), and the Arabidopsis powdery mildew fungus, Golovinomyces cichoracearum (Gc), yielded six suspected pathogenicity genes with homologues in both species. Deletion of these genes by homologous gene replacement was attempted in Cp, with one success, TG4. The TG4-knockout strain showed changes in phenotype and reduced fungal virulence against chestnut. TG4 appears to be a promising target for host-induced gene silencing (HIGS) in transgenic American chestnut. The use of homologues from genetically tractable species like Cp can help overcome the obstacles to performing reverse genetics on intractable, biotrophic fungi such as Gc. Experiments underway involving the silencing and ectopic overexpression of the Gc homologues of the target genes provide a rapid method to study Cp genes, including to screen additional candidate genes as future targets for HIGS

Proteome analysis of Phytophthora cinnamomi, the causal agent of Dieback

Phytophthora cinnamomi is a nasty pathogen that causes significant environmental and agricultural destruction. In this thesis, proteomic analysis was used to obtain a biochemical snapshot of the organism to understand its contributors to pathogenicity. The mode of action of phosphite, the only chemical used to combat dieback disease was also elucidated as there is a growing pressure of chemical resistance

Host-pathogen interactions in the spring dead spot of bermudagrass pathosystem

Ophiosphaerella herpotricha, O. korrae and O. narmari are the causal agents of spring dead spot of bermudagrass (Cynodon spp.). These pathogens colonize roots of susceptible bermudagrass causing necrosis and death of plants. Bermudagrass mortality is likely due to weakening of rhizomes and stolons by means of nutrient depletion and reduced function of rotted roots that enhance cold temperature sensitivity. Limited information is available regarding host-pathogen interactions in this pathosystem. Although categorized as necrotrophs, the strategy of pathogenesis and genetic information of these pathogens has remained unknown. Additionally, the underlying genetics of root colonization by these pathogens has not been fully elucidated. Therefore, the goal of this research was to use a bioinformatic approach to elucidate the genes expressed by Ophiosphaerella spp. during colonization, and to identify gene(s) from bermudagrasses that determine host susceptibility and tolerance. This study produced the first report of draft genomes of eleven Ophiosphaerella isolates. Candidate necrotrophic effector genes were identified in their genomes, which were also found to be upregulated in planta. This might imply that Ophiosphaerella-induced necrosis is the result of pathogen-associated molecular pattern-triggered immunity (PTI). Expression profiling analysis of roots of susceptible bermudagrass cultivar 'Tifway' infected with O. herpotricha demonstrated activation of PTI mediated by jasmonic acid potentially resulting in necrosis. The tolerant 'U3' biotype showed activation of basal defense response mediated by salicylic acid. This salicylic acid-mediated signaling could be involved in enhanced resistance to nutrient starvation and cold tolerance that allows the host to withstand pathogen infection. Future experiments are required to functionally characterize the roles of these bermudagrass candidate genes in the host-pathogen interaction, which suggest a symbiotic relationship. The results presented will serve as valuable genomic resources for future studies in these plant-pathogen interactions and population genetics in the spring dead spot of bermudagrass pathosystem. Moreover, this will enhance traditional breeding efforts to incorporate better host plant tolerance to the SDS fungi in bermudagrass cultivars

Finding RNA targets for the multidomain cyclophilin AtCyp59 by Genomic SELEX in Arabidopsis thaliana

Die Cyclophiline gehören zu den weitverbreitesten Proteinen in Archeen, Bakterien und Eukarionten und bilden auch die größte beschriebene Proteinfamilie der Spezies A. thaliana. Die Cyclophiline weisen eine peptidyl–prolyl cis–trans Isomerase (PPIase) Aktivität auf und katalysieren die cis–trans Isomerisierung der Prolin vorangehender Peptid-Bindungen. Die Mehrheit der Cyclophiline (PPIasen) ist eher klein, sie beinhalten lediglich eine einzige PPIase Domäne bestehend aus 120 Aminosäuren. Dennoch sind wenige Cyclophilin Proteine mit Mehrfachdomänen bekannt. Zu den höchst komplexen Cyclophilin Vertretern, gehört die A. thaliana Cyp59 (AtCyp59). Das Protein hat eine, in der Evolution von Hefe zu Menschen konservierte, besondere Domänen-Organisation. Diese besteht aus einer N-Terminal PPIase gefolgt von einem RNA-Erkennungs-Motiv (RNA recognition motif, RRM) und der C-Terminus Domäne, welche wiederum geladene Aminosäuren wie auch Serinen oder RS/RD Dipeptid Wiederholungen beinhaltet. Aufgrund ihrer multidomän Struktur könnte AtCyp59 in vielen zellulären Prozessen, wie beispielsweise Splicing, RNA-processing, Proteintransport und Zellreifung etc., involviert sein. Darüberhinaus wurde bereits gezeigt dass AtCyp59 Proteine sich im Nukleus aufhalten und dort mit der C-Terminus Domäne von RNA Polymerase II interagieren. Die SR/RD Domäne hingegen wird zur Bindung an SR Proteine verwendet. Die exakte biologische Funktion des RRM Motives ist immer noch unklar. In dieser Arbeit präsentiere ich ein genomisches SELEX, eine genomweite analyse Methode, die eine Suche des RNA-bindenden Motivs der RRM Domäne von AtCyp59 ermöglicht. Mit Hilfe von random priming haben wir eine genomische Bibliothek von A. thaliana, bestehend aus 50-300nt langen überlappenden Sequenzen, erstellt. In Verbindung mit dem SELEX Verfahren waren wir im Stande eine 7nt lange RNA-bindende Konsensusequenz zu identifizieren, die eine spezifische Bindung an die AtCyp59 und dessen RRM Domäne in vitro und in vivo aufweist. Mutationen an der RRM Domäne so wie auch der Konsensusequenz verhinderten eine Bildung des RNA- Protein Komplexes. Erweiterte bioinformatischen Analysen zeigten, dass dieses Bindung –Motiv ein globales Sequenzmerkmal repräsentiert, welches sich in der Nähe des transnationalen stop-codon bei 70% aller A. thaliana mRNAs befindet. Untersuchungen der Bindungsaktivität zwischen RNA und der AtCyp59 der RRM Domäne wiesen eine Beteiligung der PPIase und RS/RD- Reichen Domäne in die Bindung an die strukturierte RNA. Die präsentierten Daten erlauben uns einen Einblick in die Funktion der RRM Domäne von AtCyp59, eines regulatorischen Proteins welches zwischen 2 wichtigen zellulären Prozessen; Transkription und Splicing steht, zu gewähren.Cyclophilins are ubiquitous proteins found in archea, bacteria, and eukarya, with the largest family described so far in the plant species A. thaliana. Cyclophilins possess peptidyl–prolyl cis–trans isomerase (PPIase) activity, they catalyze cis–trans isomerization of peptide bonds preceding proline. The majority of cyclophilins (PPIases) are small proteins containing only a PPIase domain of about 120 amino acids. However, several multidomain cyclophilins from different organisms have been also described. Among them, the most complex multidomain cyclophilin characterized so far is A. thaliana Cyp59 (AtCyp59). It has a unique, conserved from yeast to human, domain organization, consisting of N-terminal PPIase domain followed by an RNA recognition motif (RRM) and a C-terminal domain enriched in charged amino acids and serines or RS/RD dipeptide repeats. As a consequence of its multidomain organization, AtCyp59 may be involved in several cellular processes such as splicing, RNA processing, protein trafficking and maturation, etc. AtCyp59 has been shown to localize in the nucleus where it interacts with the C-terminal domain of RNA Polymerase II. Via SR/RD domain it interacts with SR proteins. Up-to-date, the exact biological functions of RRM domain are still remaining unclear. Here we present genomic SELEX, the genome-wide screen method allowing us to search for RNA targets of RRM domain of AtCyp59. By random priming we constructed the representative genomic library of A. thaliana, consisting of 50-300nt long overlapping sequences. The library was accordingly used for genomic SELEX, allowing identification of the 7nt-long RNA binding consensus which was shown to bind to RRM domain of AtCyp59 in a sequence specific manner in vitro and in vivo. Mutations in either RRM domain or consensus sequence prevented formation of RNA-protein complex. Bioinformatics analysis has shown that this binding motif represents a global signature located near the translational stop-codon in 70% of A. thaliana mRNAs. Comparative analysis of RNA-binding activity AtCyp59 and its RRM domain indicated contribution of PPIase and RS/RD-rich domains to binding to structured RNAs. Presented data allowed us to shed a light on function of RRM domain of AtCyp59, global regulator protein lying on interconnection between two important cellular processes, transcription and splicing

Large scale prediction and reverse genetics analysis of programmed cell death genes in Chlamydomonas reinhardtii

Programmed cell death (PCD) refers to any form of cell death that is coordinated by the genome. PCD consists of complex molecular pathways which directly cause the death of a cell. The most well-known form of PCD is an animal-specific process known as apoptosis, of which the underlying molecular pathways are well-characterized. However, despite the observation that PCD occurs ubiquitously throughout the tree of life, little is known regarding the molecular mechanisms of PCD in non-animal systems. In response to a number of different environmental stressors, Chlamydomonas reinhardtii undergoes a form of PCD which exhibits characteristics of apoptosis, including DNA laddering, accumulation of reactive oxygen species, and externalization of phosphatidylserine. The presence of these shared features between C. reinhardtii PCD and apoptosis suggests that similar molecular pathways may underlie the two processes. Despite this, many of the genes required for apoptosis in animals appear to be absent in C. reinhardtii. In the present study, we first employed a large-scale, homology-based, bioinformatics approach to predict the gene products that contribute to C. reinhardtii PCD. From the list of sequences that were obtained by these methods, we selected several entries to study in further detail using a reverse genetic approach. We obtained C. reinhardtii mutant strains, each with an insertional mutation in one of the selected genes, from the Chlamydomonas Library Project (CLiP) and validated that the insert had been mapped accurately in each of the mutant strains. To determine the effects of losing any of these selected genes, we subjected the mutant and parental background strains to a PCD-inducing heat stress. First, we sought to determine if the loss of a single selected gene would affect the ability of cells to undergo PCD in response to stress. Second, we wanted to determine if the loss of one of the selected genes would alter either the timing or intensity of phenotypes characteristic of C. reinhardtii PCD. Our results suggest a role for several of the selected genes in PCD, and future studies will be aimed at further characterizing these roles in more detail