miR159 Represses a Constitutive Pathogen Defense Response in Tobacco.

MicroR159 (miR159) regulation of GAMYB expression is highly conserved in terrestrial plants; however, its functional role remains poorly understood. In Arabidopsis (Arabidopsis thaliana), although GAMYB-like genes are constitutively transcribed during vegetative growth, their effects are suppressed by strong and constitutive silencing by miR159. GAMYB expression occurs only if miR159 function is inhibited, which results in detrimental pleiotropic defects, questioning the purpose of the miR159-GAMYB pathway. Here, miR159 function was inhibited in tobacco (Nicotiana tabacum) and rice (Oryza sativa) using miRNA MIM159 technology. Similar to observations in Arabidopsis, inhibition of miR159 in tobacco and rice resulted in pleiotropic defects including stunted growth, implying functional conservation of the miR159-GAMYB pathway among angiosperms. In MIM159 tobacco, transcriptome profiling revealed that genes associated with defense and programmed cell death were strongly activated, including a suite of 22 PATHOGENESIS-RELATED PROTEIN (PR) genes that were 100- to 1,000-fold upregulated. Constitutive expression of a miR159-resistant GAMYB transgene in tobacco resulted in phenotypes similar to that of MIM159 tobacco and activated PR gene expression, verifying the dependence of the above-mentioned changes on GAMYB expression. Consistent with the broad defense response, MIM159 tobacco appeared immune to Phytophthora infection. These findings suggest that the tobacco miR159-GAMYB pathway functions in the biotic defense response, which becomes activated upon miR159 inhibition. However, PR gene expression was not upregulated in Arabidopsis or rice when miR159 was inhibited, suggesting that miR159-GAMYB pathway functional differences exist between species, or factors in addition to miR159 inhibition are required in Arabidopsis and rice to activate this broad defense response.

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.

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.

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.

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.

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.

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.

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.

Identification of a mastigoneme protein from Phytophthora nicotianae.

Tripartite tubular hairs (mastigonemes) on the anterior flagellum of protists in the stramenopile taxon are responsible for reversing the thrust of flagellar beat and for cell motility. Immunoprecipitation experiments using antibodies directed towards mastigonemes on the flagella of zoospores ofPhytophthora nicotianaehave facilitated the cloning of a gene encoding a mastigoneme shaft protein in this Oomycete. Expression of the gene, designatedPnMas2, is up-regulated during asexual sporulation, a period during which many zoospore components are synthesized. Analysis of the sequence of the PnMas2 protein has revealed that, like other stramenopile mastigoneme proteins, PnMas2 has an N-terminal secretion signal and contains four cysteine-rich epidermal growth factor (EGF)-like domains. Evidence from non-denaturing gels indicates that PnMas2 forms large oligomeric complexes, most likely through disulphide bridging. Bioinformatic analysis has revealed thatPhytophthoraspecies typically contain three or four putative mastigoneme proteins containing the four EGF-like domains. These proteins are similar in sequence to mastigoneme proteins in other stramenopile protists including the algaeOchromonas danica,Aureococcus anophagefferensandScytosiphon lomentariaand the diatomsThalassiosira pseudonana and T. weissflogii.

Anti-tropomyosin antibodies co-localise with actin microfilaments and label plasmodesmata.

The actin cytoskeleton and associated actin-binding proteins form a complex network involved in a number of fundamental cellular processes including intracellular trafficking. In plants, both actin and myosin have been localised to plasmodesmata, and thus it is likely that other actin-binding proteins are also associated with plasmodesmata structure or function. A 75-kDa protein, enriched in plasmodesmata-rich cell wall extracts from the green alga Chara corallina, was sequenced and found to contain three peptides with similarity to the animal actin-binding protein tropomyosin. Western blot analysis with anti-tropomyosin antibodies confirmed the identity of this 75-kDa protein as a tropomyosin-like protein and further identified an additional 55-kDa protein, while immunofluorescence microscopy localised the antibodies to plasmodesmata and to the subcortical actin bundles and associated structures. The anti-tropomyosin antibodies detected a single protein at 42.5 kDa in Arabidopsis thaliana extracts and two proteins at 58.5 and 54 kDa in leek extracts, and these localised to plasmodesmata and the cell plate in A. thaliana and to plasmodesmata in leek tissue. Tropomyosin is an actin-binding protein thought to be involved in a range of functions associated with the actin cytoskeleton, including the regulation of myosin binding to actin filaments, but to date no tropomyosin-like proteins have been conclusively identified in plant genomes. Our data suggests that a tropomyosin-like protein is associated with plasmodesmata.

Regulation of catalase activity and gene expression during Phytophthora nicotianae development and infection of tobacco.

Plant defence against pathogen attack typically incorporates an oxidative burst involving elevated levels of reactive oxygen species such as hydrogen peroxide. In the present study, we have used an in-gel assay to monitor the activity of the hydrogen peroxide scavenging enzyme, catalase, during asexual development of Phytophthora nicotianae and during infection of host tobacco plants. In vitro, catalase activity is highest in sporulating hyphae; in planta, catalase activity increases dramatically about 8 h after host inoculation. We have cloned and characterized three catalase genes, designated PnCat1, PnCat2 and PnCat3, from P. nicotianae and identified their homologues in P. infestans, P. sojae and P. ramorum. In all three species, Cat2 is predicted to be targeted to the peroxisome and the other catalases are likely to be cytosolic. Quantitative real-time PCR assessment of catalase transcripts during development and infection indicates that peroxisomal PnCat2 is the gene predominantly expressed, with transcript levels peaking in vitro in sporulating hyphae and in planta increasing dramatically during the first 24 h after inoculation of susceptible tobacco seedlings. Levels of tobacco catalase gene expression are significantly down-regulated in susceptible tobacco 4, 8 and 24 h post-inoculation and in resistant plants at 24 h post-inoculation. Together, our results give evidence that during infection P. nicotianae increases its own peroxisomal catalase levels while concurrently down-regulating host catalase expression. This behaviour is consistent with a role of pathogen catalase in counterdefence and protection against oxidative stress and of pathogen-orchestrated enhanced plant cell death to support necrotrophic pathogen growth and plant colonization.

Characterisation of manganese superoxide dismutase from Phytophthora nicotianae.

Three polypeptides with manganese superoxide dismutase (MnSOD) activity were found in mycelium, zoospores and germinated cysts of Phytophthora nicotianae. Their relative molecular weights in non-denaturing gels were approximately 34.5, 36 and 50 kDa. No evidence for the presence of either iron or copper/zinc SODs was detected at any of the developmental stages examined. The level of activity of the MnSOD polypeptides was similar in mycelia and spores. Degenerate PCR was used to amplify partial genes of two different MnSODs, designated PnMnSODI and PnMnSOD2, from P. nicotianae. Southern blot analysis indicated that there are two PnMnSOD1 genes in the P. nicotianae genome. Full length sequence was obtained for one of these genes, PnMnSOD1a, from a P. nicotianae bacterial artificial chromosome (BAC) library. RNA blots probed with PnMnSOD1 showed similar levels of expression in vegetative and sporulating hyphae, lower levels in germinated cysts and no detectable expression in zoospores. PnMnSOD1a had 96%, 97 % and 99 % amino acid identity with homologous genes from P. ramorum, P. infestans and P. sojae, respectively. The second gene cloned from P. nicotianae, PnMnSOD2, had only 38 % amino acid identity with PnMnSOD1a and was homologous to MnSODs that possessed an N-terminal mitochondrial targeting sequence in Phytophthora species and other eukaryotes. Southern blots indicated that there is one copy of PnMnSOD2 in the P. nicotianae genome. PnMnSOD2 was expressed at similar levels in mycelia and germinated cysts but PnMnSOD2 transcripts were not detectable in zoospores.

Proteomic identification of putative plasmodesmatal proteins from Chara corallina.

Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.