1,721,074 research outputs found
The Phytophthora infestans avirulence gene PiaAvr4 and its potato counterpart R4
No full textThe potato late blight disease that is caused by the oomycete pathogen Phytophthora infestans is a major threat for potato crops worldwide. In recent years research on oomycete plant pathogens was boosted by the availability of novel genomic tools and resources for several oomycete genera, such as Phytophthora, Hyaloperonospora, Pythium and Aphanomyces. This has led to the identification of genes involved in diverse biological processes such as sporulation, mating, signaling and pathogenesis. One of the approaches that breeders use to obtain late blight resistant potato cultivars is the introgression of resistance traits from wild Solanum species into the cultivated potato Solanum tuberosum. The pathogen, however, is able to circumvent this resistance; it is often lost shortly after introduction of new cultivars. To better understand the mechanisms underlying this loss of resistance it is of utmost importance to gain insight into the characteristics of the cognate avirulence (Avr) genes of the pathogen. According to the gene-for-gene model Avr genes encode effectors that trigger resistance responses mediated by resistance (R) genes. This thesis first describes the identification of a P. infestans Avr gene, in particular the elicitor activity of the encoded effector protein, the domain structure of the effector and its putative sub-cellular localization. In the second part the recognition specificity of the corresponding R gene and the identification of a marker linked to this R gene are described. Chapter 1 summarizes the advances in oomycete genomics in recent years and the tremendous progress that has been made in gene discovery in oomycete plant pathogens. It describes the different oomycete species that have been studied in more detail and assesses which species are suitable model species for research on oomycete-plant interactions. The identification of the P. infestans avirulence gene PiAvr4 is presented in Chapter 2. PiAvr4, which encodes an RXLR-dEER effector protein, was isolated by positional cloning. AFLP markers were used for landing on BACs and cDNA-AFLP markers pinpointed the gene of interest. Transformation of race 4 strains with PiAvr4 resulted in transformants that are avirulent on the R4 differential of the Mastenbroek differential set (clone Ma-R4). Moreover, in planta expression of PiAvr4 resulted in a necrotic response on clone Ma-R4 but not on plants lacking R4 such as Bintje. All together this proves that PiAvr4 is the avirulence gene that corresponds to the R gene present in clone Ma-R4. In many identified avirulence proteins one or a few amino acid changes in the protein abolish avirulence function. In case of PiAvr4, race 4 strains have frame shift mutations in the open reading frame, resulting in a truncated protein that is not functional as avirulence factor. Effectors within the RXLR-dEER family are rapidly evolving. The selective pressure is targeted predominantly on the C-terminal region of these proteins. Despite this selective pressure the majority of these proteins carry motifs that can be distinguished using Hidden Markov Models searches. They are named W, Y and L motifs after the conserved tryptophan (W), tyrosine (Y) and leucine (L) residues, respectively. As described in Chapter 3 PiAvr4 carries three W motifs and a single Y motif. The motifs together with their flanking regions were tested for activity on Ma-R4 plants. Agroinfection of constructs carrying the W2 motif in combination with either the W1 or W3 motif resulted in a necrotic response. Moreover, we showed that the PiAvr4 homolog PmirAvh4, isolated from Phytophthora mirabilis was also able to elicit a necrotic response on the Ma-R4 potato clone. For several Phytophthora RXLR-dEER effectors it was demonstrated that these proteins are targeted into the host cell and that the RXLR-dEER motif is required for translocation. In Chapter 4 we investigated whether PiAvr4 and IPI-O, like other RXLR-dEER effectors, are also targeted into the host cell. A race 4 P. infestans isolate was transformed with constructs encoding either PiAvr4 or IPI-O fused to a monomeric red fluorescent protein (mRFP) at the C-terminus. Fluorescence microscopy of these transformants showed no specific mRFP fluorescence in free living, non-sporulating mycelium. However, in germinating cysts, the tips of germ tubes and appressoria showed mRFP fluorescence, and during infection of etiolated potato plantlets localized fluorescence was visible at the haustorial neck. Haustoria are highly specialized infection and feeding structures that are in close contact with the plant cell and have a putative role in delivering effector proteins into the host cell. In order to monitor the development of the infection a novel experimental set-up was developed. In this method etiolated in vitro grown potato plantlets are inoculated with P. infestans, which has the advantage that there is no autofluorescence of chlorophyll that masks the mRFP fluorescence and thus disturbs the microscopic analysis in green plant tissues. The lack of chlorophyll does not seem to interfere with infection; zoospores are capable to encyst and to germinate, and the etiolated tissues are readily colonized by P. infestans. The recognition specificity of R4 potato differentials is described in Chapter 5. Initially two different potato clones were developed as R4 differentials; The Mastenbroek differential set, developed in the Netherlands, contains the clone Cebeco44-31-5 (designated as Ma-R4) and the Black differential set, developed in Scotland, contains clone 1563 c (14) (designated as Bl-R4). Virulence assays using several wild type P. infestans strains revealed that the Bl-R4 clone is susceptible to all isolates that are avirulent on clone Ma-R4. Only one single isolate was found to be avirulent on clone Bl-R4, but virulent on Ma-R4. Moreover, in transient expression assays with binary PVX constructs carrying PiAvr4, the Ma-R4 clone but not the Bl-R4 clone responded with an HR. Similar to the R3 locus two different recognition specificities seem to exist for R4. The R3a and R3b genes are located on one locus but whether this is the case for the two R4 genes (named R4Ma and R4Bl, respectively) remains to be determined. Resistance to P. infestans strains carrying PiAvr4 segregates in an 1:1 ratio in two independent potato F1 populations suggesting that R4Ma resistance is determined by a single dominant locus. More in depth studies on the recognition of PiAvr4 by its cognate R protein are hampered by the fact that the resistance gene R4Ma has not yet been identified. In Chapter 6 nucleotide binding site (NBS) profiling was used to generate R4Ma-associated markers. NBS profiling is a biased approach based on PCR amplification of conserved NBS motifs in R genes and R gene homologs. In a bulked segregant analysis, DNA of resistant and susceptible F1 progeny was pooled and used as template for NBS profiling. Several candidate markers were found but eventually one amplified fragment was found to co-segregate with resistance mediated by R4Ma. DNA sequencing of this fragment revealed high similarity to BAC sequences that are mapped to potato chromosome 12. Moreover, the R4Ma marker is homologous to members of the Rx/Gpa2 gene family. Chapter 7 focuses on the secreted effectors of plant pathogenic oomycetes, with special attention to RXLR-dEER effectors, and the role of these proteins in pathogenesis. The RXLR-dEER effector family is rapidly evolving and comprises all secreted oomycete avirulence proteins that are identified up till now. There is now ample evidence that oomycetes utilize the RXLR-dEER domain to deposit effectors inside host cells. Furthermore, this chapter discusses the experimental results described in this thesis in the light of present knowledge on gene-for-gene interactions, effector recognition and late blight resistance. <br/
Phytophthora infestans, een dynamische ziekteverwekker
No full textSamenvatting van de inaugurele rede van Francine Govers op 11 juni 2009. Dit artikel beschrijft de stand van zaken in het onderzoek aan oömyceten en in het bijzonder aan Phytophthora infestans, de veroorzaker van de aardappelziekte. Er wordt ingegaan op ziektebestrijding en resistentieveredeling, de diversiteit van Phytophthora en zijn gastheren en de genetische blauwdruk: het DNA, de genen, de effectoren en de resistentiegenen
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
Footprints of evolution: the dynamics of effector genes in the Phytophthora genome
Get PDFPhytophthora is a genus comprised of over 65 destructive plant pathogenic species that cause severe damages in agriculture, forestry and natural habitats. Economically important pathogens are Phytophthora infestans (causing potato late blight) and Phytophthora sojae (causing soybean root and stem rot). A newly discovered species, Phytophthora ramorum is destroying oak trees along the west-coast of the USA by causing the Sudden Oak Death syndrome. Phytophthora belongs to the oomycetes, which together with phyopathogenic fungi constitute the major groups of plant pathogens. Oomycetes and fungi resemble each other morphologically but belong to different kingdoms, Stramenopila and Fungi, respectively. Convergent evolution has shaped a similar set of weaponry for attacking plants in oomycetes and fungi.Phytophthora secretes a variety of molecules into the plant apoplast presumably to promote infection. These molecules with a potential role in virulence or pathogenicity are named virulence factors or effectors. Despite their intrinsic virulence functions, effectors may cause failure of infection if plants recognize them and initiate defense responses. Effectors that trigger plant defense responses are called avirulence factors or elicitors. This thesis describes the drastic genome rearrangements at an avirulence locus in P. infestans, the characterization of effector gene reservoirs in the fully sequenced P. sojae and P. ramorum genomes, and evolutionary patterns of effector genes.Potato and P. infestans interact accordmg to the gene-for-gene model. As a first step towards unraveling the molecular interaction mechanisms, P. infestans avriulence (Avr) genes have to be isolated. Chapter 2 describes a transcriptional profiling slrategy to identify avirulence associated transcripts. cDNA-AFLP was used for comparing transcripts in P. infestans strains with different virulence phenotypes. A large number of avirulence-associated TDFs (Transcript Derived Fragments) were cloned and sequenced, and EST and genome databases were mined to generate more sequence data. To identify promising candidates, bioinformatic predictions such as the presence of signal peptides, number of cysteine residues and putative virulence functions were used as important selection criteria. Chapter 3 describes how a combination of transcriptional profiling, and genetic and physical mapping resulted in the characterisation of a complex avirulence locus. Four avirulence associated TDFs were shown to be derived from one gene, named p't3.4. Genetic mapping and physical mapping placed pi3.4 at the Avr3b-Avr10-Avr11 locus on linkage group VIIl. Comparative genomic hybridization (CGH) revealed that this A^rlocus belongs to one of the six loci in the genome that show copy number variation (CNV)- An amplified pi3.4 gene cluster is present in the avirulent haplotype but absent in the virulent haplotype. OnIy the 3' half of the pi3.4 gene is amplified, and this amplification was found to provide diverse modules for assembly of novel full length genes. Among the proteins secreted by Phytophthora. elicitins are produced most abundantly. Elicitins show elicitor activity by causing a hypersensitive response in tobacco. In Chapter 4 and Chapter 5, the diversity and genome organization of elicitin genes in four Phytophthora species are described. Elicitins were found to be encoded by a large complex gene family, and they belong to one of the most highly conserved groups of proteins in the Phytophthora genus. Many elicitin (ELI) and elicitin-like (ELL) genes are clustered in the genome. Phylogeny construction indicated that the complex elicitin gene family existed before the ancestral Phytophthora species gave rise to the current Phytophthora species. Molecular phylogeny classified elicitin family members into 17 different clades, namely 4 ELI clades and 13 ELL clades. Based on expression patterns and bioinformatic predictions, different clades are proposed to possess distinct functions.The two fully sequenced Phytophthora species, P. sojae and P. ramorum, differ in their genome sizes, sexual behavior and host specificity. Comparative genomics was carried out to gain insight into the evolution of effector genes. In Chapter 6 the genome organization of potential effector genes is described. Overall co-linearity was found between large genomic regions in P. sojae and P. ramorum. However, insertions, deletions and expansions revealed some hotspots for genome rearrangements, and such rearrangement hotspots often harbor genes associated with virulence. Contrasting evolutionary patterns were found for neighboring gene families, for example, families encoding extracellular enzymes showed more rearrangements than those encoding intracellular enzymes. Also genes encoding host specific elicitors showed more rearrangements than those encoding general elicitors.In Chapter 7 the whole reservoir of secreted proteins present in the proteome was revealed by bioinformatic-predictions. A large secretome comprised of overthousand proteins was found in both P. sojae and P. ramorum. The majority of secreted protein encoding genes form fami!ies and many of them are clustered in the genome. Comparison between secretomes of P. sojae and P. ramorum showed that different families are evolving at a different pace. The most rapidly evolving families include the surface anchored proteins, mating associated factors and 'RXLR-DEER' proteins. They may piay important roles in either host-pathogen interactions or in reproduction.In Chapter 8 the base compositions of P. sojae and P. ramorum were calculated and compared. This information is usefui for analyzing basic genome features. The coding regions of Phytophthora clearly show high GC3 (3rd position codon usage) and this preference causes codon bias in Phytophthora genes. Evolutionary forces such as selective pressure and mutation bias were found to drive codon bias in Phytophthora. The higher GC3 value of highly expressed genes in different Phytophthora species is indicative for selection pressure, whereas lineage specific GC increase of non-coding regions is reminiscent of whole genome mutation bias. The most widespread groups of mobile elements were retrieved from the genomes and they show a codon bias that is similar to the genes of the host Phytophthora.Finally, the evolutionary implications of the findings presented in this thesis are discussed in Chapter 9. For pathogenic organisms, genes encoding effectors are instrumental for interaction with their hosts. As a result, the evolutionary pace of effector genes is in general faster than that of average genes. The results presented in this thesis demonstrate that comparative genomics is a powerful toot to discover these genes and to point out promising candidates responsible for the process of pathogenesis. The ongoing P. infestans genome sequencing project will provide new resources for fundamental and applied research, and with the b!ueprint of P. infestans in hand, late bight research will gain momentum
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