Laminarin-triggered defence responses are geographically dependent in natural populations of Solanum chilense.

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions, was directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species.

The protein phosphatase 2A catalytic subunit StPP2Ac2b enhances susceptibility to Phytophthora infestans and senescence in potato.

The serine/threonine protein phosphatases type 2A (PP2A) are involved in several physiological responses in plants, playing important roles in developmental programs, stress responses and hormone signaling. Six PP2A catalytic subunits (StPP2Ac) were identified in cultivated potato. Transgenic potato plants constitutively overexpressing the catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were developed to determine its physiological roles. The response of StPP2Ac2b-OE plants to the oomycete Phytophthora infestans, the causal agent of late blight, was evaluated. We found that overexpression of StPP2Ac2b enhances susceptibility to the pathogen. Further bioinformatics, biochemical, and molecular analyses revealed that StPP2Ac2b positively regulates developmental and pathogen-induced senescence, and that P. infestans infection promotes senescence, most likely through induction of StPP2Ac2b expression.

Hydrophilic Interaction Liquid Chromatography Coupled to Mass Spectrometry and Multivariate Analysis of the De Novo Pyrimidine Pathway Metabolites.

In this study, we describe the optimization of a Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC-MS) method for the evaluation of 14 metabolites related to the de novo synthesis of pyrimidines (dnSP) while using multivariate analysis, which is the metabolic pathway for pyrimidine nucleotide production. A multivariate design was used to set the conditions of the column temperature, flow of the mobile phase, additive concentration, gradient rate, and pH of the mobile phase in order to attain higher peak resolution and ionization efficiency in shorter analysis times. The optimization process was carried out while using factorial fractional designs, Box-Behnken design and central composite design while using two zwitterionic columns, ZIC-p-HILIC and ZIC-HILIC, polymeric, and silica-based columns, respectively. The factors were evaluated while using resolution (R), retention factor (k), efficiency of the column (N), and peak height (h) as the response variables. The best optimized conditions were found with the ZIC-p-HILIC column: elution gradient rate 2 min., pH 7.0, temperature 45 °C, mobile phase flow of 0.35 mL min-1, and additive (ammonium acetate) concentration of 6 mM. The total analysis time was 28 min. The ZIC-p-HILIC LC-MS method yielded satisfactory results for linearity of calibration curves, limit of detection (LOD), and limit of quantification (LOQ). The method has been shown to be appropriate for the analysis of dnSP on samples of tomato plants that were infected with Phytophthora infestans.

Genetic Structure and Subclonal Variation of Extant and Recent U.S. Lineages of Phytophthora infestans.

The oomycete Phytophthora infestans is an important plant pathogen on potato and tomato crops. We examined the genetic structure of extant 20th and 21st century U.S. lineages of P. infestans and compared them with populations from South America and Mexico to examine genetic relationships and potential sources of lineages. US-23, currently the most prevalent lineage detected in the United States, shared genetic similarity primarily with the BR-1 lineage identified in the 1990s from Bolivia and Brazil. Lineages US-8, US-14, and US-24, predominantly virulent on potato, formed a cluster distinct from other U.S. lineages. Many of the other U.S. lineages shared significant genetic similarity with Mexican populations. The US-1 lineage, dominant in the mid-20th century, clustered with US-1 lineages from Peru. A survey of the presence of RXLR effector PiAVR2 revealed that some lineages carried PiAVR2, its resistance-breaking variant PiAVR2-like, or both. Minimum spanning networks developed from simple sequence repeat genotype datasets from USABlight outbreaks clearly showed the expansion of US-23 over a 6-year time period and geographic substructuring of some lineages in the western United States. Many clonal lineages of P. infestans in the United States have come from introductions from Mexico, but the US-23 and US-1 lineages were most likely introduced from other sources.

Metabolomics of Solanum lycopersicum Infected with Phytophthora infestans Leads to Early Detection of Late Blight in Asymptomatic Plants.

Tomato crops suffer attacks of various pathogens that cause large production losses. Late blight caused by Phytophthora infestans is a devastating disease in tomatoes because of its difficultly to control. Here, we applied metabolomics based on liquid chromatography⁻mass spectrometry (LC-MS) and metabolic profiling by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in combination with multivariate data analysis in the early detection of late blight on asymptomatic tomato plants and to discriminate infection times of 4, 12, 24, 36, 48, 60, 72 and 96 h after inoculation (hpi). MALDI-MS and LC-MS profiles of metabolites combined with multivariate data analysis are able to detect early-late blight-infected tomato plants, and metabolomics based on LC-MS discriminates infection times in asymptomatic plants. We found the metabolite tomatidine as an important biomarker of infection, saponins as early infection metabolite markers and isocoumarin as early and late asymptomatic infection marker along the post infection time. MALDI-MS and LC-MS analysis can therefore be used as a rapid and effective method for the early detection of late blight-infected tomato plants, offering a suitable tool to guide the correct management and application of sanitary defense approaches. LC-MS analysis also appears to be a suitable tool for identifying major metabolites of asymptomatic late blight-infected tomato plants.

A genome-scale metabolic model of potato late blight suggests a photosynthesis suppression mechanism.

BACKGROUND: Phytophthora infestans is a plant pathogen that causes an important plant disease known as late blight in potato plants (Solanum tuberosum) and several other solanaceous hosts. This disease is the main factor affecting potato crop production worldwide. In spite of the importance of the disease, the molecular mechanisms underlying the compatibility between the pathogen and its hosts are still unknown. RESULTS: To explain the metabolic response of late blight, specifically photosynthesis inhibition in infected plants, we reconstructed a genome-scale metabolic network of the S. tuberosum leaf, PstM1. This metabolic network simulates the effect of this disease in the leaf metabolism. PstM1 accounts for 2751 genes, 1113 metabolic functions, 1773 gene-protein-reaction associations and 1938 metabolites involved in 2072 reactions. The optimization of the model for biomass synthesis maximization in three infection time points suggested a suppression of the photosynthetic capacity related to the decrease of metabolic flux in light reactions and carbon fixation reactions. In addition, a variation pattern in the flux of carboxylation to oxygenation reactions catalyzed by RuBisCO was also identified, likely to be associated to a defense response in the compatible interaction between P. infestans and S. tuberosum. CONCLUSIONS: In this work, we introduced simultaneously the first metabolic network of S. tuberosum and the first genome-scale metabolic model of the compatible interaction of a plant with P. infestans.

Variation in Genetic Diversity of Phytophthora infestans Populations in Mexico from the Center of Origin Outwards.

The Toluca valley, located in central Mexico, is thought to be the center of origin of the potato late blight pathogen Phytophthora infestans. We characterized over 500 individuals of P. infestans sampled from populations with a geographical distance of more than 400 km in six regions adjacent to the Toluca valley in three states including Michoacán, Mexico, and Tlaxcala. Our sampling occurred on a predominant east to west gradient and showed significant genetic differentiation. The most western sampling location found in Michoacán was most differentiated from the other populations. Populations from San Gerónimo, Juchitepec, and Tlaxcala clustered together and appeared to be in linkage equilibrium. This work provides a finer understanding of gradients of genetic diversity in populations of P. infestans at the center of origin.

Comparative analysis of constitutive proteome between resistant and susceptible tomato genotypes regarding to late blight.

Late blight is one of the most destructive diseases of the tomato, resulting in substantial economic losses. There is difficulty in controlling this disease, so the molecular characterization of tomato genotypes may help in the selection of higher resistance tomato plants against Phytophthora infestans, late blight's pathogen. The objective was to analyze the differences with regard to the constitutive proteome between the access Vegetable Germplasm Bank (BGH)-2127, resistant genotype, and Santa Clara-susceptible genotype to late blight. Proteomic analysis of leaf samples by two-dimensional electrophoresis (2-DE) followed by identification by mass spectrometry (MALDI TOF/TOF) was performed. Nineteen proteins were identified, which were then related to metabolism and energy, photosynthesis, transcription, stress, and defenses. Approximately 90% of these proteins were more abundant in Santa Clara, a susceptible cultivar. Acidic 26 kDa endochitinase and ribonuclease T2 proteins were more abundant in BGH-2127 access. The enzymatic activity confirmed a greater abundance of chitinase in the BGH-2127 access as compared to the cultivar Santa Clara. Gene expression analyses by real-time PCR demonstrated that the mRNA levels were not correlated with the respective protein levels. Abundance of the acidic 26 kDa endochitinase and ribonuclease T2 proteins in the constitutive proteomes of BGH-2127 may be associated with the answer to the resistance of this access.

Analysis of the potato calcium-dependent protein kinase family and characterization of StCDPK7, a member induced upon infection with Phytophthora infestans.

KEY MESSAGE: We describe the potato CDPK family and place StCDPK7 as a player in potato response to Phytophthora infestans infection, identifying phenylalanine ammonia lyase as its specific phosphorylation target in vitro. Calcium-dependent protein kinases (CDPKs) decode calcium (Ca2+) signals and activate different signaling pathways involved in hormone signaling, plant growth, development, and both abiotic and biotic stress responses. In this study, we describe the potato CDPK/CRK multigene family; bioinformatic analysis allowed us to identify 20 new CDPK isoforms, three CDPK-related kinases (CRKs), and a CDPK-like kinase. Phylogenetic analysis indicated that 26 StCDPKs can be classified into four groups, whose members are predicted to undergo different acylation patterns and exhibited diverse expression levels in different tissues and in response to various stimuli. With the aim of characterizing those members that are particularly involved in plant-pathogen interaction, we focused on StCDPK7. Tissue expression profile revealed that StCDPK7 transcript levels are high in swollen stolons, roots, and mini tubers. Moreover, its expression is induced upon Phytophthora infestans infection in systemic leaves. Transient expression assays showed that StCDPK7 displays a cytosolic/nuclear localization in spite of having a predicted chloroplast transit peptide. The recombinant protein, StCDPK7:6xHis, is an active Ca2+-dependent protein kinase that can phosphorylate phenylalanine ammonia lyase, an enzyme involved in plant defense response. The analysis of the potato CDPK family provides the first step towards the identification of CDPK isoforms involved in biotic stress. StCDPK7 emerges as a relevant player that could be manipulated to deploy disease resistance in potato crops.

Phytophthora infestans: New Tools (and Old Ones) Lead to New Understanding and Precision Management.

New tools have revealed that migrations of Phytophthora infestans have been a dominant feature of the population biology of this pathogen for the past 50 years, and maybe for the past 170 years. We now have accurate information on the composition of many P. infestans populations. However, migration followed by selection can lead and has led to dramatically rapid changes in populations over large regions. Except for the highlands of central Mexico, many populations of P. infestans have probably been in flux over the past several decades. There is some evidence that this pathogen has different characteristics in the field than it does in the lab, and early field phenotypic analyses of hypotheses concerning fitness and pathogenicity would be beneficial. The newly available capacity to acquire and process vast amounts of weather and weather forecast data in combination with advancements in molecular diagnostics enables much greater precision in late blight management to produce recommendations that are site, host, and pathogen specific.

Potassium phosphite primes defense responses in potato against Phytophthora infestans.

Although phosphite is widely used to protect plants from pathogenic oomycetes on a wide range of horticultural crops, the molecular mechanisms behind phosphite induced resistance are poorly understood. The aim of this work was to assess the effects of potassium phosphite (KPhi) on potato plant defense responses to infection with Phytophtora infestans (Pi). Pathogen development was severely restricted and there was also an important decrease in lesion size in infected KPhi-treated leaves. We demonstrated that KPhi primed hydrogen peroxide and superoxide anion production in potato leaves at 12 h post-inoculation with Pi. Moreover, the KPhi-treated leaves showed an increased and earlier callose deposition as compared with water-treated plants, beginning 48 h after inoculation. In contrast, callose deposition was not detected in water-treated leaves until 72 h after inoculation. In addition, we carried out RNA gel blot analysis of genes implicated in the responses mediated by salicylic (SA) and jasmonic acid (JA). To this end, we examined the temporal expression pattern of StNPR1 and StWRKY1, two transcription factors related to SA pathway, and StPR1 and StIPII, marker genes related to SA and JA pathways, respectively. The expression of StNPR1 and StWRKY1 was enhanced in response to KPhi treatment. In contrast, StIPII was down regulated in both KPhi- and water-treated leaves, until 48 h after infection with Pi, suggesting that the regulation of this gene could be independent of the KPhi treatment. Our results indicate that KPhi primes the plant for an earlier and more intense response to infection and that SA would mediate this response.

Comparison of transcript profiles in late blight-challenged Solanum cajamarquense and B3C1 potato clones.

Two Solanum genotypes, a wild relative of cultivated potato S. cajamarquense (Cjm) and an advanced tetraploid clone B3C1 (B3), were inoculated with two Phytophthora infestans isolates and leaves were sampled at 72 and 96 h after inoculation. Gene expression in the inoculated versus noninoculated samples was monitored using the Institute of Genomic Research (TIGR) 10K potato array and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The current experiment is study number 83 of the TIGR expression profiling service project, and all data are publicly available in the Solanaceae Gene Expression Database (SGED) at ftp://ftp.tigr.org/pub/data/s_tuberosum/SGED. Differentially regulated cDNA clones were selected separately for each isolate-time point interaction by significant analysis of microarray (SAM), and differentially regulated clones were classified into functional categories by MapMan. The results show that the genes activated in B3 and Cjm have largely the same biological functions and are commonly activated when plants respond to pathogen attack. The genes activated within biological function categories were considerably different between the genotypes studied, suggesting that the defence pathways activated in B3 and Cjm during the tested conditions may involve unique genes. However, as indicated by real-time RT-PCR, some of the genes thought to be genotype specific may be activated across genotypes at other time points during disease development.

Intraspecific functional diversity in hosts and its effect on disease risk across a climatic gradient.

The effects of host biodiversity on disease risk may vary greatly depending on host population structure and climatic conditions. Agricultural diseases such as potato late blight, caused by Phytophthora infestans, provide the opportunity to study the effects of intraspecific host diversity that is relatively well-defined in terms of disease resistance phenotypes and may have functional impacts on disease levels. When these systems are present across a climatic gradient, it is also possible to study how season length and conduciveness of the environment to disease may influence the effects of host diversity on disease risk. We developed a simple model of epidemic progress to evaluate the effects on disease risk of season length, environmental disease conduciveness, and host functional divergence for mixtures of a susceptible host and a host with some resistance. Differences in disease levels for the susceptible vs. resistant genotypes shifted over time, with the divergence in disease levels first increasing and then decreasing. Disease reductions from host diversity were greatest for high host divergence and combinations of environmental disease conduciveness and season length that led to moderate disease severity. We also compared the effects of host functional divergence on potato late-blight risk in Ecuador (long seasons), two sites in Peru (intermediate seasons) in El Niño and La Niña years, and the United States (short seasons). There was some evidence for greater disease risk reduction from host diversity where seasons were shorter, probably because of lower regional inoculum loads. There was strong evidence for greater disease reduction when host functional divergence was greater. These results indicate that consideration of season length, environmental conduciveness to disease, and host functional divergence can help to explain the variability in disease response to host diversity.

[Morphological and molecular characterization of the antagonistic interaction between the endophyte Diaporthe sp. isolated from frailejón (Espeletia sp.) and the plant pathogen Phytophthora infestans]. / Caracterización morfológica y molecular del antagonismo entre el endofito Diaporthe sp. aislado de frailejón (Espeletia sp.) y el fitopatógeno Phytophthora infestans.

Endophytic fungi produce a great variety of secondary metabolites both in vivo and in vitro. In this study, we characterized the ability of a sterile-mycelium endophytic fungus isolated from Espeletia sp. to control the growth of Phytophthora infestans in Petri dishes. Sequence from the ITS regions (internal transcribed spacer) of the endophyte showed 94% similarity to Diaporthe phaseolorum's. The antagonistic interaction between Diaporthe sp. and P. infestans was evaluated in three different culture media. Diaporthe sp. showed an antagonistic effect towards P. infestans, with some variation depending on which medium was used. In an attempt to identify possible genes involved in this antagonism, we detected a gene from the endophyte encoding an amylase, which was differentially expressed during this biotic interaction.