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Paul R J Birch

Publications and source records attributed to Paul R J Birch.

18 recordsLinked to original sources
PubMed2006

The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana.

The RXLR cytoplasmic effector AVR3a of Phytophthora infestans confers avirulence on potato plants carrying the R3a gene. Two alleles of Avr3a encode secreted proteins that differ in only three amino acid residues, two of which are in the mature protein. Avirulent isolates carry the Avr3a allele, which encodes AVR3aKI (containing amino acids C19, K80 and I103), whereas virulent isolates express only the virulence allele avr3a, encoding AVR3aEM (S19, E80 and M103). Only the AVR3aKI protein is recognized inside the plant cytoplasm where it triggers R3a-mediated hypersensitivity. Similar to other oomycete avirulence proteins, AVR3aKI carries a signal peptide followed by a conserved motif centered on the consensus RXLR sequence that is functionally similar to a host cell-targeting signal of malaria parasites. The interaction between Avr3a and R3a can be reconstructed by their transient co-expression in Nicotiana benthamiana. We exploited the N. benthamiana experimental system to further characterize the Avr3a-R3a interaction. R3a activation by AVR3aKI is dependent on the ubiquitin ligase-associated protein SGT1 and heat-shock protein HSP90. The AVR3aKI and AVR3aEM proteins are equally stable in planta, suggesting that the difference in R3a-mediated death cannot be attributed to AVR3aEM protein instability. AVR3aKI is able to suppress cell death induced by the elicitin INF1 of P. infestans, suggesting a possible virulence function for this protein. Structure-function experiments indicated that the 75-amino acid C-terminal half of AVR3aKI, which excludes the RXLR region, is sufficient for avirulence and suppression functions, consistent with the view that the N-terminal region of AVR3aKI and other RXLR effectors is involved in secretion and targeting but is not required for effector activity. We also found that both polymorphic amino acids, K80 and I103, of mature AVR3a contribute to the effector functions.

Algal Proteins↗
PubMed2006

Comparative genomics reveals what makes an enterobacterial plant pathogen.

The bacterial family Enterobacteriaceae contains some of the most devastating human and animal pathogens, including Escherichia coli, Salmonella enterica and species of Yersinia and Shigella. These are among the best-studied of any organisms, yet there is much to be learned about the nature and evolution of interactions with their hosts and with the wider environment. Comparative and functional genomics have fundamentally improved our understanding of their modes of adaptation to different ecological niches and the genes that determine their pathogenicity. In addition to animal pathogens, Enterobacteriaceae include important plant pathogens, such as Erwinia carotovora subsp. atroseptica (Eca), the first plant-pathogenic enterobacterium to be sequenced. This review focuses on genomic comparisons between Eca and other enterobacteria, with particular emphasis on the differences that exemplify or explain the plant-associated lifestyle(s) of Eca. Horizontal gene transfer in Eca may directly have led to the acquisition of a number of determinants that mediate its interactions, pathogenic or otherwise, with plants, offering a glimpse into its evolutionary divergence from animal-pathogenic enterobacteria.

Enterobacteriaceae↗
PubMed2005

GenomeDiagram: a python package for the visualization of large-scale genomic data.

UNLABELLED: We present GenomeDiagram, a flexible, open-source Python module for the visualization of large-scale genomic, comparative genomic and other data with reference to a single chromosome or other biological sequence. GenomeDiagram may be used to generate publication-quality vector graphics, rastered images and in-line streamed graphics for webpages. The package integrates with datatypes from the BioPython project, and is available for Windows, Linux and Mac OS X systems. AVAILABILITY: GenomeDiagram is freely available as source code (under GNU Public License) at http://bioinf.scri.ac.uk/lp/programs.html, and requires Python 2.3 or higher, and recent versions of the ReportLab and BioPython packages. SUPPLEMENTARY INFORMATION: A user manual, example code and images are available at http://bioinf.scri.ac.uk/lp/programs.html.

Chromosome Mapping↗
PubMed2005

Trafficking arms: oomycete effectors enter host plant cells.

Oomycetes cause devastating plant diseases of global importance, yet little is known about the molecular basis of their pathogenicity. Recently, the first oomycete effector genes with cultivar-specific avirulence (AVR) functions were identified. Evidence of diversifying selection in these genes and their cognate plant host resistance genes suggests a molecular "arms race" as plants and oomycetes attempt to achieve and evade detection, respectively. AVR proteins from Hyaloperonospora parasitica and Phytophthora infestans are detected in the plant host cytoplasm, consistent with the hypothesis that oomycetes, as is the case with bacteria and fungi, actively deliver effectors inside host cells. The RXLR amino acid motif, which is present in these AVR proteins and other secreted oomycete proteins, is similar to a host-cell-targeting signal in virulence proteins of malaria parasites (Plasmodium species), suggesting a conserved role in pathogenicity.

Algal Proteins↗
PubMed2005

An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm.

The oomycete Phytophthora infestans causes late blight, the potato disease that precipitated the Irish famines in 1846 and 1847. It represents a reemerging threat to potato production and is one of >70 species that are arguably the most devastating pathogens of dicotyledonous plants. Nevertheless, little is known about the molecular bases of pathogenicity in these algae-like organisms or of avirulence molecules that are perceived by host defenses. Disease resistance alleles, products of which recognize corresponding avirulence molecules in the pathogen, have been introgressed into the cultivated potato from a wild species, Solanum demissum, and R1 and R3a have been identified. We used association genetics to identify Avr3a and show that it encodes a protein that is recognized in the host cytoplasm, where it triggers R3a-dependent cell death. Avr3a resides in a region of the P. infestans genome that is colinear with the locus containing avirulence gene ATR1(NdWsB) in Hyaloperonospora parasitica, an oomycete pathogen of Arabidopsis. Remarkably, distances between conserved genes in these avirulence loci were often similar, despite intervening genomic variation. We suggest that Avr3a has undergone gene duplication and that an allele evading recognition by R3a arose under positive selection.

Agrobacterium tumefaciens↗
PubMed2005

Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines.

The perception of downy mildew avirulence (Arabidopsis thaliana Recognized [ATR]) gene products by matching Arabidopsis thaliana resistance (Recognition of Peronospora parasitica [RPP]) gene products triggers localized cell death (a hypersensitive response) in the host plant, and this inhibits pathogen development. The oomycete pathogen, therefore, is under selection pressure to alter the form of these gene products to prevent detection. That the pathogen maintains these genes indicates that they play a positive role in pathogen survival. Despite significant progress in cloning plant RPP genes and characterizing essential plant components of resistance signaling pathways, little progress has been made in identifying the oomycete molecules that trigger them. Concluding a map-based cloning effort, we have identified an avirulence gene, ATR1NdWsB, that is detected by RPP1 from the Arabidopsis accession Niederzenz in the cytoplasm of host plant cells. We report the cloning of six highly divergent alleles of ATR1NdWsB from eight downy mildew isolates and demonstrate that the ATR1NdWsB alleles are differentially recognized by RPP1 genes from two Arabidopsis accessions (Niederzenz and Wassilewskija). RPP1-Nd recognizes a single allele of ATR1NdWsB; RPP1-WsB also detects this allele plus three additional alleles with divergent sequences. The Emco5 isolate expresses an allele of ATR1NdWsB that is recognized by RPP1-WsB, but the isolate evades detection in planta. Although the Cala2 isolate is recognized by RPP1-WsA, the ATR1NdWsB allele from Cala2 is not, demonstrating that RPP1-WsA detects a novel ATR gene product. Cloning of ATR1NdWsB has highlighted the presence of a highly conserved novel amino acid motif in avirulence proteins from three different oomycetes. The presence of the motif in additional secreted proteins from plant pathogenic oomycetes and its similarity to a host-targeting signal from malaria parasites suggest a conserved role in pathogenicity.

Amino Acid Sequence↗
PubMed2005

Elevated amino acid biosynthesis in Phytophthora infestans during appressorium formation and potato infection.

Appressorium formation is believed to be an important event in establishing a successful interaction between the late blight pathogen, Phytophthora infestans, and its host plants potato and tomato. An understanding of molecular events occurring in appressorium development could suggest new strategies for controlling late blight. We used parallel studies of the transcriptome and proteome to identify genes and proteins that are up-regulated in germinating cysts developing appressoria. As a result, five distinct genes involved in amino acid biosynthesis were identified that show increased expression in germinating cysts with appressoria. These are a methionine synthase (Pi-met1), a ketol-acid reductoisomerase (Pi-kari1), a tryptophan synthase (Pi-trp1), an acetolactate synthase (Pi-als1), and a threonine synthase (Pi-ts1). Four of these P. infestans genes were also up-regulated, although to lower levels, during the early, biotrophic phase of the interaction in potato and all five were considerably up-regulated during the transition (48 hpi) to the necrotrophic phase of the interaction. Real-time RT-PCR revealed that expression of potato homologues of the amino acid biosynthesis genes increased during biotrophic and necrotrophic infection phases. Furthermore, we investigated levels of free amino acids in the pre-infection stages and found that in most cases there was a decrease in free amino acids in zoospores and germinating cysts, relative to sporangia, followed by a sharp increase in germinating cysts with appressoria. Amino acid biosynthesis would appear to be important for pathogenicity in P. infestans, providing a potential metabolic target for chemical control.

Amino Acids↗
PubMed2005

Rotting softly and stealthily.

The soft rot erwiniae, which are plant pathogens on potato and other crops world-wide, synthesize and secrete large quantities of plant cell wall degrading enzymes that are responsible for the soft rot phenotype, earning them the epithet 'brute force' pathogens. They have been distinguished from classic 'stealth' pathogens, such as Pseudomonas syringae, which possesses an extensive battery of Type III secreted effector proteins and phytotoxins to manipulate and suppress host defences. However, recent studies, including whole-genome sequencing, are revealing many components of stealth pathogenesis within the soft rot erwiniae (SRE), suggesting that 'stealth' and 'brute force' should not be regarded as mutually exclusive modes of pathogenesis.

Cell Wall↗
PubMed2004

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans.

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.

Algal Proteins↗
PubMed2004

Activation tagging in plants: a tool for gene discovery.

A significant limitation of classical loss-of-function screens designed to dissect genetic pathways is that they rarely uncover genes that function redundantly, are compensated by alternative metabolic or regulatory circuits, or which have an additional role in early embryo or gametophyte development. Activation T-DNA tagging is one approach that has emerged in plants to help circumvent these potential problems. This technique utilises a T-DNA sequence that contains four tandem copies of the cauliflower mosaic virus (CaMV) 35S enhancer sequence. This element enhances the expression of neighbouring genes either side of the randomly integrated T-DNA tag, resulting in gain-of-function phenotypes. Activation tagging has identified a number of genes fundamental to plant development, metabolism and disease resistance in Arabidopsis. This review provides selected examples of these discoveries to highlight the utility of this technology. The recent development of activation tagging strategies for other model plant systems and the construction of new more sophisticated vectors for the generation of conditional alleles are also discussed. These recent advances have significantly expanded the horizons for gain-of-function genetics in plants.

Genes, Plant↗
PubMed2004

Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins.

Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development. We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp. atroseptica SCRI1039. The cluster is similar in content and structural organization to that in E. amylovora. However, eight putative genes of unknown function located within the E. carotovora subsp. atroseptica cluster do not have homologues in the E. amylovora cluster. An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening. This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN. An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E. carotovora subsp. atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

Bacterial Outer Membrane Proteins↗
PubMed2004

High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis.

Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.

DNA, Plant↗
PubMed2003

Characterisation of early transcriptional changes involving multiple signalling pathways in the Mla13 barley interaction with powdery mildew ( Blumeria graminis f. sp. hordei).

Suppression subtractive hybridisation was used to isolate 21 cDNAs ( bmi1- bmi21) up-regulated 1-5 h post-inoculation (hpi) in a barley ( Hordeum vulgare L. cv. Pallas) near-isogenic line (NIL) P11 ( Mla13) challenged with either avirulent or virulent isolates of Blumeria graminis f. sp. hordei. Transcriptional changes at these time-points are crucial for the Mla-mediated hypersensitive response [W.R. Bushnell and Z. Liu (1994) Physiol Mol Plant Pathol 44:389-402]. Seven sequences were up-regulated by 1 hpi, when the pathogen has formed only the primary germ tube. Some transcripts were similar to genes with a role in regulating programmed cell death in animals, including NF kappaB and oxysterol-binding protein. Moreover, bmi7, similar to rice resistance gene Xa21, was rapidly up-regulated in both compatible and incompatible interactions, but was then down-regulated by 5 hpi in the virulent interaction. Only nine of the transcripts were up-regulated in mlo5 resistance in cv. Pallas NIL P22, confirming differential pathway induction between Mla13 and mlo5. However, eight sequences up-regulated in the Mla13 response in P11 were already highly elevated in uninoculated mlo5 mutant P22, suggesting that they may be negatively regulated by wild-type Mlo. Regulation of bmi sequences was investigated using salicylic acid, methyl jasmonate, ethylene, H(2)O(2), abscisic acid, wounding and a glucan elicitor. No single stimulus up-regulated all genes, suggesting either combinations of these stimuli, or additional stimuli, are involved in early Mla13 and mlo5 resistances. Whereas H(2)O(2) up- or down-regulated 17 of the transcripts detected in Northern analyses, salicylic acid stimulated only down-regulation of 5 transcripts.

Abscisic Acid↗
PubMed2003

A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd.

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the Arabidopsis thaliana-recognised (ATR) avirulence genes. We describe the identification of 10 amplified fragment length polymorphism (AFLP) markers that define a genetic mapping interval for the ATR1Nd avirulence allele, the presence of which is perceived by the RPP1Nd resistance gene. Furthermore, we have constructed a P. parasitica (At) bacterial artificial chromosome (BAC) library comprising over 630Mb of cloned DNA. We have isolated 16 overlapping clones from the BAC library that form a contig spanning the genetic interval. BAC sequence-derived markers and a total mapping population of 311 F(2) individuals were used to refine the ATR1Nd locus to a 1cM interval that is represented by four BAC clones and spans less than 250kb of DNA. This work demonstrates that map-based cloning techniques are feasible in this organism and provides the critical foundations for cloning ATR1Nd using such a strategy.

Alleles↗
PubMed2003

Profiling and quantifying differential gene transcription in Phytophthora infestans prior to and during the early stages of potato infection.

Phytophthora infestans, the causal agent of potato and tomato late blight, produces several different cell types prior to and during the early stages of potato infection. All of these cell types can be easily generated and studied in the absence of the host plant and so form the basis for developmental stage-specific gene discovery. We have used amplified fragment length polymorphism (AFLP)-based mRNA fingerprinting (cDNA-AFLP) to identify 64 transcripts that appeared to be up-regulated in germinating cysts but not in vegetative mycelium. These transcripts included representatives of most major classes of heat shock proteins: hsp60, hsp70, hsp90, and hsp100. Real-time RT-PCR was used to quantify the expression of 18 transcripts originating from germinating cysts, relative to the constitutively expressed actB gene, in vegetative mycelium, germinating cysts, and at three time-points post-inoculation of potato cultivar Bintje (15, 48, and 72h). All of the transcripts were up-regulated in germinating cysts, and 12, including hsp70, hsp80-2, and hsp90, were found also to be up-regulated in planta. This is the first report of the application of real-time RT-PCR to the relative quantification of plant pathogen gene expression during the early stages of infection.

Gene Expression Profiling↗
PubMed2002

Sample sequencing of a selected region of the genome of Erwinia carotovora subsp. atroseptica reveals candidate phytopathogenicity genes and allows comparison with Escherichia coli.

Genome sequencing is making a profound impact on microbiology. Currently, however, only one plant pathogen genome sequence is publicly available and no genome-sequencing project has been initiated for any species of the genus Erwinia, which includes several important plant pathogens. This paper describes a targeted sample sequencing approach to study the genome of Erwinia carotovora subsp. atroseptica (Eca), a major soft-rot pathogen of potato. A large insert DNA (approx. 115 kb) library of Eca was constructed using a bacterial artificial chromosome (BAC) vector. Hybridization and end-sequence data revealed two overlapping BAC clones that span an entire hrp gene cluster. Random subcloning and one-fold sequence coverage (>200 kb) across these BACs identified 25 (89%) of 28 hrp genes predicted from the orthologous hrp cluster of Erwinia amylovora. Regions flanking the hrp cluster contained orthologues of known or putative pathogenicity operons from other Erwinia species, including dspEF (E. amylovora), hecAB and pecSM (E. chrysanthemi), sequences similar to genes from the plant pathogen Xylella fastidiosa, including haemagglutinin-like genes, and sequences similar to genes involved in rhizobacterium-plant interactions. Approximately 10% of the sequences showed strongest nucleotide similarities to genes in the closely related model bacterium and animal pathogen Escherichia coli. However, the positions of some of these genes were different in the two genomes. Approximately 30% of sequences showed no significant similarity to any database entries. A physical map was made across the genomic region spanning the hrp cluster by hybridization to the BAC library and to digested BAC clones, and by PCR between sequence contigs. A multiple genome coverage BAC library and one-fold sample sequencing are an effective combination for extracting useful information from important regions of the Eca genome, providing a wealth of candidate novel pathogenicity genes for functional analyses. Other genomic regions could be similarly targeted.

Chromosomes, Artificial, Bacterial↗
PubMed2000

cDNA-AFLP analysis of differential gene expression in the prokaryotic plant pathogen Erwinia carotovora.

For studies of differential gene expression in prokaryotes, methods for synthesizing representative cDNA populations are required. Here, a technique is described for the synthesis of cDNA from the potato pathogens Erwinia carotovora subsp. atroseptica (Eca) and Erwinia carotovora subsp. carotovora (Ecc) using a combination of short oligonucleotide (11-mer) primers that were known to anneal to conserved sequences in the 3' regions of enterobacterial genes. Specific PCR amplifications with primers designed to anneal to 14 known genes from either Eca or Ecc revealed the presence of the corresponding transcripts in cDNA, suggesting that the cDNA represented a broad genomic coverage. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used to identify differentially expressed genes in Eca, including one that shows significant similarity, at the protein level, to an avirulence gene from Xanthomonas campestris pv. raphani. Northern analysis was used to confirm that differentially amplified cDNA fragments were derived from differentially expressed genes. This is the first report of the use of cDNA-AFLP to study differential gene expression in prokaryotes.

Blotting, Northern↗