Protein-protein Interaction and Molecular Dynamics of Iturin A Gene on Effector Proteins of Phytophthora infestans.

AIM AND OBJECTIVES: Phytophthora infestans (Mont.) de Bary, the fungal pathogen causes late blight, which results in devastating economic loss among the Solanaceae. The bacillus lipopeptides show the antagonistic activity against the many plant pathogens, among bacillus lipopeptides reported as the antifungal gene. Hence, to understand the in silico antifungal activity, we have selected gene iturin A (AXN89987) produced by Bacillus spp to check the molecular dynamics study with the effector proteins of the P. infestanse. In this concern, known effector proteins of P. infestans were subjected to the protein-protein interaction followed by simulation. MATERIALS AND METHODS: Iturin A gene was amplified using the soil bacterium Bacillus subtilis with gene-specific primers, cloned into pTZ 57R/T vector and confirmed by sequencing. To get better insights, the protein model was developed for Iturin A using Modeller 9.17, using PDB structure of ID 4MRT (Phosphopantetheine transferase Sfp) and 1QR0 (4'-phosphopantetheinyl moiety of coenzyme A) as a template, it shared the identity 72% and expected P-value: 3e-121, respectively. The model quality was assessed using ProSA and PROCHECK programs. RESULTS: The potency of modelled protein against effector proteins of P. infestans were evaluated in silico using the HADDOCK server and the results showed the high affinity of towards the effector protein Host ATG8 (PDB-5L83). Finally, the simulation was performed to the docked conformation of with Host ATG8 to further understand the stability of the complex using the Desmond program. CONCLUSION: Altogether, the protein-protein interaction and simulation study propose a new methodology and to uncover possible antifungal activity of iturin A against effector proteins of P. infestans.

pH effects on plant calcium fluxes: lessons from acidification-mediated calcium elevation induced by the γ-glutamyl-leucine dipeptide identified from Phytophthora infestans.

Cytosolic Ca2+ ([Ca2+]cyt) elevation is an early signaling response upon exposure to pathogen-derived molecules (so-called microbe-associated molecular patterns, MAMPs) and has been successfully used as a quantitative read-out in genetic screens to identify MAMP receptors or their associated components. Here, we isolated and identified by mass spectrometry the dipeptide γ-Glu-Leu as a component of a Phytophthora infestans mycelium extract that induces [Ca2+]cyt elevation. Treatment of Arabidopsis seedlings with synthetic γ-Glu-Leu revealed stimulatory effects on defense signaling, including a weak enhancement of the expression of some MAMP-inducible genes or affecting the refractory period to a second MAMP elicitation. However, γ-Glu-Leu is not a classical MAMP since pH adjustment abolished these activities and importantly, the observed effects of γ-Glu-Leu could be recapitulated by mimicking extracellular acidification. Thus, although γ-Glu-Leu can act as a direct agonist of calcium sensing receptors in animal systems, the Ca2+-mobilizing activity in plants reported here is due to acidification. Low pH also shapes the Ca2+ signature of well-studied MAMPs (e.g. flg22) or excitatory amino acids such as glutamate. Overall, this work serves as a cautionary reminder that in defense signaling studies where Ca2+ flux measurements are concerned, it is important to monitor and consider the effects of pH.

Structural Basis of Host Autophagy-related Protein 8 (ATG8) Binding by the Irish Potato Famine Pathogen Effector Protein PexRD54.

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8 family-interacting motif (AIM) and perturbs host-selective autophagy. However, the structural basis of this interaction remains unknown. Here, we define the crystal structure of PexRD54, which includes a modular architecture, including five tandem repeat domains, with the AIM sequence presented at the disordered C terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector's AIM sequence, and we established a model of the full-length PexRD54-ATG8CL complex using small angle x-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.

Phytophthora infestans specific phosphorylation patterns and new putative control targets.

In this study we applied biomathematical searches of gene regulatory mechanisms to learn more about oomycete biology and to identify new putative targets for pesticides or biological control against Phytophthora infestans. First, oomycete phylum-specific phosphorylation motifs were found by discriminative n-gram analysis. We found 11.600 P. infestans specific n-grams, mapping 642 phosphoproteins. The most abundant group among these related to phosphatidylinositol metabolism. Due to the large number of possible targets found and our hypothesis that multi-level control is a sign of usefulness as targets for intervention, we identified overlapping targets with a second screen. This was performed to identify proteins dually regulated by small RNA and phosphorylation. We found 164 proteins to be regulated by both sRNA and phosphorylation and the dominating functions where phosphatidylinositol signalling/metabolism, endocytosis, and autophagy. Furthermore we performed a similar regulatory study and discriminative n-gram analysis of proteins with no clear orthologs in other species and proteins that are known to be unique to P. infestans such as the RxLR effectors, Crinkler (CRN) proteins and elicitins. We identified CRN proteins with specific phospho-motifs present in all life stages. PITG_12626, PITG_14042 and PITG_23175 are CRN proteins that have species-specific phosphorylation motifs and are subject to dual regulation.

Bioinformatics Analysis Reveals Abundant Short Alpha-Helices as a Common Structural Feature of Oomycete RxLR Effector Proteins.

RxLR effectors represent one of the largest and most diverse effector families in oomycete plant pathogens. These effectors have attracted enormous attention since they can be delivered inside the plant cell and manipulates host immunity. With the exceptions of a signal peptide and the following RxLR-dEER and C-terminal W/Y/L motifs identified from the sequences themselves, nearly no functional domains have been found. Recently, protein structures of several RxLRs were revealed to comprise alpha-helical bundle repeats. However, approximately half of all RxLRs lack obvious W/Y/L motifs, which are associated with helical structures. In this study, secondary structure prediction of the putative RxLR proteins was performed. We found that the C-terminus of the majority of these RxLR proteins, irrespective of the presence of W/Y/L motifs, contains abundant short alpha-helices. Since a large-scale experimental determination of protein structures has been difficult to date, results of the current study extend our understanding on the oomycete RxLR effectors in protein secondary structures from individual members to the entire family. Moreover, we identified less alpha-helix-rich proteins from secretomes of several oomycete and fungal organisms in which RxLRs have not been identified, providing additional evidence that these organisms are unlikely to harbor RxLR-like proteins. Therefore, these results provide additional information that will aid further studies on the evolution and functional mechanisms of RxLR effectors.

Characterization of Phytophthora infestans resistance to mefenoxam using FTIR spectroscopy.

Phytophthora infestans (P. infestans) is the causal agent of late blight in potato and tomato. This pathogen devastated the potato crops in Ireland more than a century years ago and is still causing great losses worldwide. Although fungicides controlling P. infestans have been used successfully for almost 100 years, some isolates have developed resistance to most common fungicides. Identification and characterization of these resistant isolates is required for better control of the disease. Current methods that are based on microbiological and molecular techniques are both expensive and time consuming. Fourier Transform Infra-Red spectroscopy (FTIR) is an inexpensive and reagent-free technique that provides accurate results in only a few minutes. In this study the infrared absorption spectra of the sporangia of P. infestans were measured to evaluate the potential of FTIR spectroscopy in tandem with multivariate analysis in order to classify those sporangia into those that were resistant and those that were non-resistant to the phenylamide fungicide mefenoxam. Based on individual measurements, our results show that FTIR spectroscopy enables classification of P. infestans isolates into mefenoxam resistant and mefenoxam non-resistant types with specificity of 81.9% and sensitivity of 75.5%. Using average spectra per leaf, it was possible to improve the classification results to 88% sensitivity and 95% specificity.

Profiling the secretome and extracellular proteome of the potato late blight pathogen Phytophthora infestans.

Oomycetes are filamentous organisms that cause notorious diseases, several of which have a high economic impact. Well known is Phytophthora infestans, the causal agent of potato late blight. Previously, in silico analyses of the genome and transcriptome of P. infestans resulted in the annotation of a large number of genes encoding proteins with an N-terminal signal peptide. This set is collectively referred to as the secretome and comprises proteins involved in, for example, cell wall growth and modification, proteolytic processes, and the promotion of successful invasion of plant cells. So far, proteomic profiling in oomycetes was primarily focused on subcellular, intracellular or cell wall fractions; the extracellular proteome has not been studied systematically. Here we present the first comprehensive characterization of the in vivo secretome and extracellular proteome of P. infestans. We have used mass spectrometry to analyze P. infestans proteins present in seven different growth media with mycelial cultures and this resulted in the consistent identification of over two hundred proteins. Gene ontology classification pinpointed proteins involved in cell wall modifications, pathogenesis, defense responses, and proteolytic processes. Moreover, we found members of the RXLR and CRN effector families as well as several proteins lacking an obvious signal peptide. The latter were confirmed to be bona fide extracellular proteins and this suggests that, similar to other organisms, oomycetes exploit non-conventional secretion mechanisms to transfer certain proteins to the extracellular environment.

Quantitative label-free phosphoproteomics of six different life stages of the late blight pathogen Phytophthora infestans reveals abundant phosphorylation of members of the CRN effector family.

The oomycete Phytophthora infestans is the causal agent of late blight in potato and tomato. Since the underlying processes that govern pathogenicity and development in P. infestans are largely unknown, we have performed a large-scale phosphoproteomics study of six different P. infestans life stages. We have obtained quantitative data for 2922 phosphopeptides and compared their abundance. Life-stage-specific phosphopeptides include ATP-binding cassette transporters and a kinase that only occurs in appressoria. In an extended data set, we identified 2179 phosphorylation sites and deduced 22 phosphomotifs. Several of the phosphomotifs matched consensus sequences of kinases that occur in P. infestans but not Arabidopsis. In addition, we detected tyrosine phosphopeptides that are potential targets of kinases resembling mammalian tyrosine kinases. Among the phosphorylated proteins are members of the RXLR and Crinkler effector families. The latter are phosphorylated in several life stages and at multiple positions, in sites that are conserved between different members of the Crinkler family. This indicates that proteins in the Crinkler family have functions beyond their putative role as (necrosis-inducing) effectors. This phosphoproteomics data will be instrumental for studies on oomycetes and host-oomycete interactions. The data sets have been deposited to ProteomeXchange (identifier PXD000433).

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques.

The actin cytoskeleton is a dynamic but well-organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact-eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub-apical region whereas in the extreme apex in growing hyphae, waves of fine F-actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

Characterization of three novel desaturases involved in the delta-6 desaturation pathways for polyunsaturated fatty acid biosynthesis from Phytophthora infestans.

Phytophthora infestans is the causative agent of potato blight that resulted in the great famine in Ireland in the nineteenth century. This microbe can release large amounts of the C20 very long-chain polyunsaturated fatty acids arachidonic acid (ARA; 20:4Δ(5, 8, 11, 14)) and eicosapentaenoic acid (EPA; 20:5Δ(5, 8, 11, 14, 17)) upon invasion that is known to elicit a hypersensitive response to their host plant. In order to identify enzymes responsible for the biosynthesis of these fatty acids, we blasted the recently fully sequenced P. infestans genome and identified three novel putatively encoding desaturase sequences. These were subsequently functionally characterized by expression in Saccharomyces cerevisiae and confirmed that they encode desaturases with Δ12, Δ6 and Δ5 activity, designated here as PinDes12, PinDes6 and PinDes5, respectively. This, together with the combined fatty acid profiles and a previously identified Δ6 elongase activity, implies that the ARA and EPA are biosynthesized predominantly via the Δ6 desaturation pathways in P. infestans. Elucidation of ARA and EPA biosynthetic mechanism may provide new routes to combating this potato blight microbe directly or by means of conferring resistance to important crops.

Structures of Phytophthora RXLR effector proteins: a conserved but adaptable fold underpins functional diversity.

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ∼44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.

Identification of appressorial and mycelial cell wall proteins and a survey of the membrane proteome of Phytophthora infestans.

Proteins embedded in the cell wall and plasma membrane of filamentous oomycetes and fungi provide a means by which these organisms can interact with their local environment. However, cell wall and membrane proteins have often proved difficult to isolate using conventional proteomic techniques. Here we have used liquid chromatography tandem mass spectrometry (LC-MS/MS) to facilitate rapid and sensitive quantification of the cell wall proteome. We report the use of LC-MS/MS to identify differentially regulated proteins from the cell walls of three different lifecycle stages of the oomycete plant pathogen Phytophthora infestans: non-sporulating vegetative mycelium, sporulating mycelium, and germinating cysts with appressoria. We have also used quantitative real-time RT-PCR to confirm that the transcripts corresponding to some of these proteins, namely those identified in cell walls of germinating cysts with appressoria, accumulate differentially throughout the lifecycle. These proteins may, therefore, be important for pre-infective development and early pathogenicity. Up to 31 covalently and non-covalently bound cell wall-associated proteins were identified. All of the proteins identified in germinating cysts with appressoria, and several of those from mycelial fractions, were classified as putative effector or pathogen-associated molecular pattern (PAMP) molecules, including members of the CBEL family, the elicitin family, the crinkler (CRN) family and two transglutaminases. Thus, the cell wall of P. infestans may represent an important reservoir for surface-presented, apoplastic effectors or defence activation molecules. Proteins predicted to be cell surface proteins included IPI-B like proteins, mucins, cell wall-associated enzymes and annexin family members. Additionally we identified up to 27 membrane-associated proteins from Triton X-114 phase partitioned mycelial membrane preparations, producing the first inventory of oomycete membrane-associated proteins. Four of these proteins are small Rab-type G-proteins and several are associated with secretion.

Cloning, expression and biochemical characterization of mitochondrial and cytosolic malate dehydrogenase from Phytophthora infestans.

The genes of the mitochondrial and cytosolic malate dehydrogenase (mMDH and cMDH) of Phytophthora infestans were cloned and overexpressed in Escherichia coli as active enzymes. The catalytic properties of these proteins were determined: both enzymes have a similar specific activity. In addition, the natural mitochondrial isoenzyme was semi-purified from mycelia and its catalytic properties determined: the recombinant mitochondrial isoform behaved as the natural enzyme. A phylogenetic analysis indicated that mMDH, present in all stramenopiles studied, can be useful to study the relationships between these organisms. MDH with the conserved domain MDH_cytoplasmic_cytosolic is absent in some stramenopiles as well as in fungi. This enzyme seems to be less related within the stramenopile group. The Phytophthora cMDHs have an insertion of six amino acids that is also present in the stramenopile cMDHs studied, with the exception of Thalassiosira pseudonana cMDH, and is absent in other known eukaryotic cMDHs.