Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

Structures of the flax-rust effector AvrM reveal insights into the molecular basis of plant-cell entry and effector-triggered immunity.

Fungal and oomycete pathogens cause some of the most devastating diseases in crop plants, and facilitate infection by delivering a large number of effector molecules into the plant cell. AvrM is a secreted effector protein from flax rust (Melampsora lini) that can internalize into plant cells in the absence of the pathogen, binds to phosphoinositides (PIPs), and is recognized directly by the resistance protein M in flax (Linum usitatissimum), resulting in effector-triggered immunity. We determined the crystal structures of two naturally occurring variants of AvrM, AvrM-A and avrM, and both reveal an L-shaped fold consisting of a tandem duplicated four-helix motif, which displays similarity to the WY domain core in oomycete effectors. In the crystals, both AvrM variants form a dimer with an unusual nonglobular shape. Our functional analysis of AvrM reveals that a hydrophobic surface patch conserved between both variants is required for internalization into plant cells, whereas the C-terminal coiled-coil domain mediates interaction with M. AvrM binding to PIPs is dependent on positive surface charges, and mutations that abrogate PIP binding have no significant effect on internalization, suggesting that AvrM binding to PIPs is not essential for transport of AvrM across the plant membrane. The structure of AvrM and the identification of functionally important surface regions advance our understanding of the molecular mechanisms underlying how effectors enter plant cells and how they are detected by the plant immune system.

Microtubules and biotic interactions.

Plant microtubules undergo extensive reorganization in response to symbiotic and pathogenic organisms. During the development of successful symbioses with rhizobia and mycorrhizal fungi, novel microtubule arrays facilitate the progression of infection threads and hyphae, respectively, from the plant surface through epidermal and cortical cells. During viral and nematode infections, plant microtubules appear to be commandeered by the pathogen. Viruses use plant microtubules for intra and intercellular movement, as well as for interhost transmission. Nematodes manipulate spindle and phragmoplast microtubules to enhance mitosis and partial cytokinesis during the development of syncytia and giant cells. Pathogenic bacteria, fungi and oomycetes induce a range of alterations to microtubule arrays and dynamics. In many situations, the pathogen, or the elicitor or effector proteins derived from them, induce depolymerization of plant cortical microtubule arrays. In some cases, microtubule disruption is associated with the plant defence response and resistance. In other cases, microtubule depolymerization increases plant susceptibility to the invading pathogen. The reasons for this apparent inconsistency may depend on a number of factors, in particular on the identity of the organism orchestrating the microtubule changes. Overall, the weight of evidence indicates that microtubules play an important role in both the establishment of functional symbioses and in defence against invading pathogens. Research is beginning to unravel details about the nature of both the chemical and the mechanical signals to which the plant microtubule arrays respond during biotic interactions.

Bioinformatic characterisation of genes encoding cell wall degrading enzymes in the Phytophthora parasitica genome.

BACKGROUND: A critical aspect of plant infection by the majority of pathogens is penetration of the plant cell wall. This process requires the production and secretion of a broad spectrum of pathogen enzymes that target and degrade the many complex polysaccharides in the plant cell wall. As a necessary framework for a study of the expression of cell wall degrading enzymes (CWDEs) produced by the broad host range phytopathogen, Phytophthora parasitica, we have conducted an in-depth bioinformatics analysis of the entire complement of genes encoding CWDEs in this pathogen's genome. RESULTS: Our bioinformatic analysis indicates that 431 (2%) of the 20,825 predicted proteins encoded by the P. parasitica genome, are carbohydrate-active enzymes (CAZymes) involved in the degradation of cell wall polysaccharides. Of the 431 proteins, 337 contain classical N-terminal secretion signals and 67 are predicted to be targeted to the non-classical secretion pathway. Identification of CAZyme catalytic activity based on primary protein sequence is difficult, nevertheless, detailed comparisons with previously characterized enzymes has allowed us to determine likely enzyme activities and targeted substrates for many of the P. parasitica CWDEs. Some proteins (12%) contain more than one CAZyme module but, in most cases, multiple modules are from the same CAZyme family. Only 12 P. parasitica CWDEs contain both catalytically-active (glycosyl hydrolase) and non-catalytic (carbohydrate binding) modules, a situation that contrasts with that in fungal phytopathogens. Other striking differences between the complements of CWDEs in P. parasitica and fungal phytopathogens are seen in the CAZyme families that target cellulose, pectins or ß-1,3-glucans (e.g. callose). About 25% of P. parasitica CAZymes are solely directed towards pectin degradation, with the majority coming from pectin lyase or carbohydrate esterase families. Fungal phytopathogens typically contain less than half the numbers of these CAZymes. The P. parasitica genome, like that of other Oomycetes, is rich in CAZymes that target ß-1,3-glucans. CONCLUSIONS: This detailed analysis of the full complement of P. parasitica cell wall degrading enzymes provides a framework for an in-depth study of patterns of expression of these pathogen genes during plant infection and the induction or repression of expression by selected substrates.

Internalization of flax rust avirulence proteins into flax and tobacco cells can occur in the absence of the pathogen.

Translocation of pathogen effector proteins into the host cell cytoplasm is a key determinant for the pathogenicity of many bacterial and oomycete plant pathogens. A number of secreted fungal avirulence (Avr) proteins are also inferred to be delivered into host cells, based on their intracellular recognition by host resistance proteins, including those of flax rust (Melampsora lini). Here, we show by immunolocalization that the flax rust AvrM protein is secreted from haustoria during infection and accumulates in the haustorial wall. Five days after inoculation, the AvrM protein was also detected within the cytoplasm of a proportion of plant cells containing haustoria, confirming its delivery into host cells during infection. Transient expression of secreted AvrL567 and AvrM proteins fused to cerulean fluorescent protein in tobacco (Nicotiana tabacum) and flax cells resulted in intracellular accumulation of the fusion proteins. The rust Avr protein signal peptides were functional in plants and efficiently directed fused cerulean into the secretory pathway. Thus, these secreted effectors are internalized into the plant cell cytosol in the absence of the pathogen, suggesting that they do not require a pathogen-encoded transport mechanism. Uptake of these proteins is dependent on signals in their N-terminal regions, but the primary sequence features of these uptake regions are not conserved between different rust effectors.

N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance.

To investigate the role of N-terminal domains of plant disease resistance proteins in membrane targeting, the N termini of a number of Arabidopsis and flax disease resistance proteins were fused to green fluorescent protein (GFP) and the fusion proteins localized in planta using confocal microscopy. The N termini of the Arabidopsis RPP1-WsB and RPS5 resistance proteins and the PBS1 protein, which is required for RPS5 resistance, targeted GFP to the plasma membrane, and mutation of predicted myristoylation and potential palmitoylation sites resulted in a shift to nucleocytosolic localization. The N-terminal domain of the membrane-attached Arabidopsis RPS2 resistance protein was targeted incompletely to the plasma membrane. In contrast, the N-terminal domains of the Arabidopsis RPP1-WsA and flax L6 and M resistance proteins, which carry predicted signal anchors, were targeted to the endomembrane system, RPP1-WsA to the endoplasmic reticulum and the Golgi apparatus, L6 to the Golgi apparatus, and M to the tonoplast. Full-length L6 was also targeted to the Golgi apparatus. Site-directed mutagenesis of six nonconserved amino acid residues in the signal anchor domains of L6 and M was used to change the localization of the L6 N-terminal fusion protein to that of M and vice versa, showing that these residues control the targeting specificity of the signal anchor. Replacement of the signal anchor domain of L6 by that of M did not affect L6 protein accumulation or resistance against flax rust expressing AvrL567 but removal of the signal anchor domain reduced L6 protein accumulation and L6 resistance, suggesting that membrane attachment is required to stabilize the L6 protein.

The role of effectors of biotrophic and hemibiotrophic fungi in infection.

Biotrophic and hemibiotrophic fungi are successful groups of plant pathogens that require living plant tissue to survive and complete their life cycle. Members of these groups include the rust fungi and powdery mildews and species in the Ustilago, Cladosporium and Magnaporthe genera. Collectively, they represent some of the most destructive plant parasites, causing huge economic losses and threatening global food security. During plant infection, pathogens synthesize and secrete effector proteins, some of which are translocated into the plant cytosol where they can alter the host's response to the invading pathogen. In a successful infection, pathogen effectors facilitate suppression of the plant's immune system and orchestrate the reprogramming of the infected tissue so that it becomes a source of nutrients that are required by the pathogen to support its growth and development. This review summarizes our current understanding of the function of fungal effectors in infection.

Phytophthora nicotianae transformants lacking dynein light chain 1 produce non-flagellate zoospores.

Biflagellate zoospores of the highly destructive plant pathogens in the genus Phytophthora are responsible for the initiation of infection of host plants. Zoospore motility is a critical component of the infection process because it allows zoospores to actively target suitable infection sites on potential hosts. Flagellar assembly and function in eukaryotes depends on a number of dynein-based molecular motors that facilitate retrograde intraflagellar transport and sliding of adjacent microtubule doublets in the flagellar axonemes. Dynein light chain 1 (DLC1) is one of a number of proteins in the dynein outer arm multiprotein complex. It is a 22 kDa leucine-rich repeat protein that binds to the catalytic motor domain of the dynein gamma heavy chain. We report the cloning and characterization of DLC1 homologues in Phytophthora cinnamomi and Phytophthora nicotianae (PcDLC1 and PnDLC1). PcDLC1 and PnDLC1 are single copy genes that are more highly expressed in sporulating hyphae than in vegetative hyphae, zoospores or germinated cysts. Polyclonal antibodies raised against PnDLC1 locallized PnDLC1 along the length of the flagella of P. nicotianae zoospores. RNAi-mediated silencing of PnDLC1 expression yielded transformants that released non-flagellate, non-motile zoospores from their sporangia. Our observations indicate that zoospore motility is not required for zoospore release from P. nicotianae sporangia or for breakage of the evanescent vesicle into which zoospores are initially discharged.

Cytoskeleton and cell wall function in penetration resistance.

Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.

Characterization of cyclophilin-encoding genes in Phytophthora.

Recent research has shown that cyclophilins, proteins that catalyze the isomerization of peptidyl-prolyl bonds, play a variety of important roles in infection, including facilitating host penetration and colonization and activating pathogen effector proteins within the host cytoplasm. In the current study, bioinformatic analysis of the genomes of three species of plant pathogens in the genus Phytophthora has revealed extensive synteny between the 20 or 21 members of the cyclophilin gene family. In P. infestans, extensive EST studies give evidence of the expression of 14 of the 21 genes. Sequences homologous to 12 of the 14 expressed P. infestans cyclophilins were isolated using PCR and gene-specific primers in the broad host range pathogen, P. nicotianae. Quantitative real-time PCR measurements of transcript levels in P. nicotianae at four stages of asexual development and during infection of resistant and susceptible tobacco plants gave evidence of expression of seven of the P. nicotianae homologs. The most abundantly expressed gene, PnCyPA, has a lower mRNA level in zoospores compared to other stages of asexual development and its expression increases during infection of susceptible plants. Immunocytochemical studies indicate that PnCyPA occurs in the nucleus and cytoplasm of P. nicotianae cells and is secreted from germinated cysts.

Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance.

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

Characterisation of Stramenopile-specific mastigoneme proteins in Phytophthora parasitica.

Mastigonemes, tripartite tubular hairs on the anterior flagellum of Phytophthora zoospores, are instrumental for disease dissemination to new host plants. A previous study showed that PnMas2 was part of the tubular shaft of Phytophthora parasitica mastigonemes. In the current study, genes encoding two related proteins, PnMas1 and PnMas3, were identified in the genome of P. parasitica. PnMas1 interacts with PnMas2 and also occurs along the mastigoneme shaft. RNA-Seq analyses indicate that PnMas1 and PnMas2 genes have similar expression profiles both in vitro and in planta but that PnMas3 is expressed temporally prior to PnMas1 and PnMas2 during asexual development and plant infection. Immunocytochemistry and GFP-tagging document the occurrence of all three PnMas proteins within the specialised compartments of the ER during mastigoneme formation, but only PnMas1 and PnMas2 occur in mature mastigonemes on the flagellar surface. Anti-PnMas1 and anti-PnMas2 antibodies co-labelled two high-molecular-weight (~400 kDa) protein complexes in native gels but anti-PnMas3 antibodies labelled a 65 kDa protein complex. Liquid chromatography-mass spectrometry analysis identified PnMas1 and PnMas2 but not PnMas3 in flagellar extracts. These results suggest that PnMas3 associates with mastigonemes during their assembly within the ER but is not part of mature mastigonemes on the anterior flagellum. Phylogenetic analyses using homologues of Mas genes from the genomes of 28 species of Stramenopiles give evidence of three Mas sub-families, namely Mas1, Mas2 and Mas3. BLAST analyses showed that Mas genes only occur in flagellate species within the Stramenopile taxon.

Flax rust infection transcriptomics reveals a transcriptional profile that may be indicative for rust Avr genes.

Secreted effectors of fungal pathogens are essential elements for disease development. However, lack of sequence conservation among identified effectors has long been a problem for predicting effector complements in fungi. Here we have explored the expression characteristics of avirulence (Avr) genes and candidate effectors of the flax rust fungus, Melampsora lini. We performed transcriptome sequencing and real-time quantitative PCR (qPCR) on RNA extracted from ungerminated spores, germinated spores, isolated haustoria and flax seedlings inoculated with M. lini isolate CH5 during plant infection. Genes encoding two categories of M. lini proteins, namely Avr proteins and plant cell wall degrading enzymes (CWDEs), were investigated in detail. Analysis of the expression profiles of 623 genes encoding predicted secreted proteins in the M. lini transcriptome shows that the six known Avr genes (i.e. AvrM (avrM), AvrM14, AvrL2, AvrL567, AvrP123 (AvrP) and AvrP4) fall within a group of 64 similarly expressed genes that are induced in planta and show a peak of expression early in infection with a subsequent decline towards sporulation. Other genes within this group include two paralogues of AvrL2, an AvrL567 virulence allele, and a number of genes encoding putative effector proteins. By contrast, M. lini genes encoding CWDEs fall into different expression clusters with their distribution often unrelated to their catalytic activity or substrate targets. These results suggest that synthesis of M. lini Avr proteins may be regulated in a coordinated fashion and that the expression profiling-based analysis has significant predictive power for the identification of candidate Avr genes.

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A.

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.

Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack.

BACKGROUND: Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants - after touching the epidermal surface with a microneedle. RESULTS: Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. CONCLUSION: Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the pathogen as it attempts to invade the epidermal cell surface.

Phytophthora cinnamomi.

Phytophthora cinnamomi is one of the most devastating plant pathogens in the world. It infects close to 5000 species of plants, including many of importance in agriculture, forestry and horticulture. The inadvertent introduction of P. cinnamomi into natural ecosystems, including a number of recognized Global Biodiversity Hotspots, has had disastrous consequences for the environment and the biodiversity of flora and fauna. The genus Phytophthora belongs to the Class Oomycetes, a group of fungus-like organisms that initiate plant disease through the production of motile zoospores. Disease control is difficult in agricultural and forestry situations and even more challenging in natural ecosystems as a result of the scale of the problem and the limited range of effective chemical inhibitors. The development of sustainable control measures for the future management of P. cinnamomi requires a comprehensive understanding of the cellular and molecular basis of pathogen development and pathogenicity. The application of next-generation sequencing technologies to generate genomic and transcriptomic data promises to underpin a new era in P. cinnamomi research and discovery. The aim of this review is to integrate bioinformatic analyses of P. cinnamomi sequence data with current knowledge of the cellular and molecular basis of P. cinnamomi growth, development and plant infection. The goal is to provide a framework for future research by highlighting potential pathogenicity genes, shedding light on their possible functions and identifying suitable targets for future control measures. TAXONOMY: Phytophthora cinnamomi Rands; Kingdom Chromista; Phylum Oomycota or Pseudofungi; Class Oomycetes; Order Peronosporales; Family Peronosporaceae; genus Phytophthora. HOST RANGE: Infects about 5000 species of plants, including 4000 Australian native species. Host plants important for agriculture and forestry include avocado, chestnut, macadamia, oak, peach and pineapple. DISEASE SYMPTOMS: A root pathogen which causes rotting of fine and fibrous roots, but which can also cause stem cankers. Root damage may inhibit water movement from roots to shoots, leading to dieback of young shoots. USEFUL WEBSITES: http://fungidb.org/fungidb/; http://genome.jgi.doe.gov/Phyci1/Phyci1.home.html; http://www.ncbi.nlm.nih.gov/assembly/GCA_001314365.1; http://www.ncbi.nlm.nih.gov/assembly/GCA_001314505.1.

Quantifying the plant actin cytoskeleton response to applied pressure using nanoindentation.

Detection of potentially pathogenic microbes through recognition by plants and animals of both physical and chemical signals associated with the pathogens is vital for host well-being. Signal perception leads to the induction of a variety of responses that augment pre-existing, constitutive defences. The plant cell wall is a highly effective preformed barrier which becomes locally reinforced at the infection site through delivery of new wall material by the actin cytoskeleton. Although mechanical stimulation can produce a reaction, there is little understanding of the nature of physical factors capable of triggering plant defence. Neither the magnitude of forces nor the contact time required has been quantified. In the study reported here, mechanical stimulation with a tungsten microneedle has been used to quantify the response of Arabidopsis plants expressing an actin-binding protein tagged with green fluorescent protein (GFP) to reveal the organisation of the actin cytoskeleton. Using confocal microscopy, the response time for actin reorganisation in epidermal cells of Arabidopsis hypocotyls was shown to be 116 ± 49 s. Using nanoindentation and a diamond spherical tip indenter, the magnitude of the forces capable of triggering an actin response has been quantified. We show that Arabidopsis hypocotyl cells can detect a force as small as 4 µN applied for as short a time as 21.6 s to trigger reorganisation of the actin cytoskeleton. This force is an order of magnitude less than the potential invasive force determined for a range of fungal and oomycete plant pathogens. To our knowledge, this is the first quantification of the magnitude and duration of mechanical forces capable of stimulating a structural defence response in a plant cell.

RNA-Seq Analysis of the Expression of Genes Encoding Cell Wall Degrading Enzymes during Infection of Lupin (Lupinus angustifolius) by Phytophthora parasitica.

RNA-Seq analysis has shown that over 60% (12,962) of the predicted transcripts in the Phytophthora parasitica genome are expressed during the first 60 h of lupin root infection. The infection transcriptomes included 278 of the 431 genes encoding P. parasitica cell wall degrading enzymes. The transcriptome data provide strong evidence of global transcriptional cascades of genes whose encoded proteins target the main categories of plant cell wall components. A major cohort of pectinases is predominantly expressed early but as infection progresses, the transcriptome becomes increasingly dominated by transcripts encoding cellulases, hemicellulases, ß-1,3-glucanases and glycoproteins. The most highly expressed P. parasitica carbohydrate active enzyme gene contains two CBM1 cellulose binding modules and no catalytic domains. The top 200 differentially expressed genes include ß-1,4-glucosidases, ß-1,4-glucanases, ß-1,4-galactanases, a ß-1,3-glucanase, an α-1,4-polygalacturonase, a pectin deacetylase and a pectin methylesterase. Detailed analysis of gene expression profiles provides clues as to the order in which linkages within the complex carbohydrates may come under attack. The gene expression profiles suggest that (i) demethylation of pectic homogalacturonan occurs before its deacetylation; (ii) cleavage of the backbone of pectic rhamnogalacturonan I precedes digestion of its side chains; (iii) early attack on cellulose microfibrils by non-catalytic cellulose-binding proteins and enzymes with auxiliary activities may facilitate subsequent attack by glycosyl hydrolases and enzymes containing CBM1 cellulose-binding modules; (iv) terminal hemicellulose backbone residues are targeted after extensive internal backbone cleavage has occurred; and (v) the carbohydrate chains on glycoproteins are degraded late in infection. A notable feature of the P. parasitica infection transcriptome is the high level of transcription of genes encoding enzymes that degrade ß-1,3-glucanases during middle and late stages of infection. The results suggest that high levels of ß-1,3-glucanases may effectively degrade callose as it is produced by the plant during the defence response.

Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi.

Polygalacturonases (PGs) are secreted by fungal pathogens during saprophytic and parasitic growth, and their degradation of pectin in the plant cell wall is believed to play a major role in tissue invasion and maceration. In this study, PG activity was demonstrated in culture filtrates of the oomycete plant pathogen, Phytophthora cinnamomi. A P. cinnamomi pg gene fragment amplified using degenerate primers based on conserved regions in fungal and plant PGs was used to isolate 17 complete P. cinnamomi pg genes and pseudogenes from a genomic library and partial sequence for another two genes. Gel blotting of genomic DNA indicated that there may be even more pg genes in the P. cinnamomi genome. P. cinnamomi pg gene sequences were expressed in PG-deficient yeast and found to confer PG activity, thereby confirming their functional identity. The predicted mature P. cinnamomi PGs fall into subgroups that exhibit large differences in the extent of N-glycosylation, isoelectric points, and N- and C-terminal structure. Evidence for birth-and-death and reticulate evolution in the P. cinnamomi pg gene family was obtained, and some codons for surface exposed residues in the P. cinnamomi PGs were shown to have been subject to diversifying selection. Contrary to accepted phylogenies for other proteins, phylogenetic analysis of the P. cinnamomi PGs revealed a closer relationship with PGs from true fungi than with those from plants.

Transient fusion and selective secretion of vesicle proteins in Phytophthora nicotianae zoospores.

Secretion of pathogen proteins is crucial for the establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp requires transient fusion of the large peripheral vesicles. This is the first report of selective protein secretion via transient fusion outside mammalian cells. Selective secretion is likely to be an important aspect of plant infection by this destructive pathogen.