Discovery of Phytophthora infestans Genes Expressed in Planta through Mining of cDNA Libraries

BACKGROUND: Phytophthora infestans (Mont.) de Bary causes late blight of potato and tomato, and has a broad host range within the Solanaceae family. Most studies of the Phytophthora--Solanum pathosystem have focused on gene expression in the host and have not analyzed pathogen gene expression in planta. METHODOLOGY/PRINCIPAL FINDINGS: We describe in detail an in silico approach to mine ESTs from inoculated host plants deposited in a database in order to identify particular pathogen sequences associated with disease. We identified candidate effector genes through mining of 22,795 ESTs corresponding to P. infestans cDNA libraries in compatible and incompatible interactions with hosts from the Solanaceae family. CONCLUSIONS/SIGNIFICANCE: We annotated genes of P. infestans expressed in planta associated with late blight using different approaches and assigned putative functions to 373 out of the 501 sequences found in the P. infestans genome draft, including putative secreted proteins, domains associated with pathogenicity and poorly characterized proteins ideal for further experimental studies. Our study provides a methodology for analyzing cDNA libraries and provides an understanding of the plant--oomycete pathosystems that is independent of the host, condition, or type of sample by identifying genes of the pathogen expressed in planta

Stress influences the dynamics of hippocampal structural remodeling associated with fear memory extinction

Fear extinction is defined as a decline in fear-conditioned responses following non-reinforced exposure to a fear conditioned stimulus, therefore the conditioned stimulus gains new predictive properties. Patients with anxiety related disorders (e.g.: PTSD) subjected to extinction-like exposure treatments often experience a relapse of symptoms. Stress is a risk factor for those psychiatric disorders and a critical modulator of fear learning that turns the memory resistant to the extinction process. Dendritic spines are the anatomical sites where neuronal activity reshapes brain networks during learning and memory processes. Thus, we planned to characterize the dynamics of synaptic remodeling before and after contextual fear extinction in the dorsal hippocampus (DH), and how this process is affected by a previous stress experience. Animals with or without previous stress were contextually fear conditioned and one day later trained in an extinction paradigm. Rats were sacrificed one day after conditioning (pre-extinction) or one day after extinction for spine density analysis in the DH. We confirmed that stress exposure induced a deficit in extinction learning. Further, a higher density of dendritic spines, particularly mature ones, was observed in the DH of non-stressed conditioned animals at pre-extinction. Interestingly, after extinction, the spine levels returned to the control values. Conversely, stressed animals did not show such spines boost (pre-extinction) or any other change (post-extinction). In contrast, such standard dynamics of dendritic changes as well as the behavioral extinction was recovered when stressed animals received an intra-basolateral amygdala infusion of midazolam prior to stress. Altogether, these findings suggest that stress hinders the normal dynamic of dendritic remodeling after fear extinction and this could be part of the neurobiological substrate that makes those memories resistant to be extinguished.Fil: Bender, Crhistian Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Giachero, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Comas Mutis, Ramiro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Molina, Víctor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; ArgentinaFil: Calfa, Gaston Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Farmacología Experimental de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Farmacología Experimental de Córdoba; Argentin

Dendritic spines and IGF-1

Fil: Champarini, Leandro Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Champarini, Leandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Herrera, Macarena Lorena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Herrera, Macarena Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Comas Mutis, Ramiro Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Comas Mutis, Ramiro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Espejo, Pablo Javier. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Espejo, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Molina, Victor Alejandro. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Molina, Victor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentna.Fil: Calfa, Gaston Diego. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Calfa, Gaston Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Hereñú, Claudia Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Hereñú, Claudia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Growth factors, such as insulin-like growth factor 1 (IGF-1), among others are known for their critical involvement in learning and memory processes. IGF-1 regulates cognitive functions, synapse density, neurotransmission, and adult neurogenesis and induces structural and synaptic plasticity-specific changes. Although IGF-1 has been suggested to participate in different memory processes, its role in memories associated with negative emotional experiences still remains to be elucidated. The principal aim of the present study was to test whether IGF-1 overexpression using adenoviral vectors in basolateral amygdala (BLA) influences both the expression and formation of contextual fear memory, as well as the hippocampal structural plasticity associated with such memory trace.We found that IGF-1 overexpression promotes the formation and expression of a specific contextual fear memory trace, and such effect persisted at least 7 days after recall. Moreover, the overexpression of this growth factor in BLA upregulates the activation of the ERK/MAPK pathway in this brain structure. In addition, intra-BLA IGF-1 overexpression causes dorsal hippocampus (DH) structural plasticity modifications promoting changes in the proportion of mature dendritic spines in the CA1 region, after a weak conditioning protocol. The present findings contribute to the knowledge underlying BLA-DH trace memory of fear and reveal important new insights into the neurobiology and neurochemistry of fear acquisition modulated by IGF-1 overexpression. The understanding of how IGF-1 modulates the formation of a fear contextual trace may pave the way for the development of novel therapeutic strategies focused on fear, anxiety, and trauma-related disorders.Fil: Champarini, Leandro Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Champarini, Leandro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Herrera, Macarena Lorena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Herrera, Macarena Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Comas Mutis, Ramiro Gabriel. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Comas Mutis, Ramiro Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Espejo, Pablo Javier. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Espejo, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Molina, Victor Alejandro. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Molina, Victor Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentna.Fil: Calfa, Gaston Diego. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; ArgentinaFil: Calfa, Gaston Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina.Fil: Hereñú, Claudia Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Hereñú, Claudia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina

Number of ESTs falling into different GC content ranges among a total of 403 ESTs that had a hit against the <i>P. infestans</i> genes.

<p>Number of ESTs falling into different GC content ranges among a total of 403 ESTs that had a hit against the <i>P. infestans</i> genes.</p

Differential abundance of predicted CDS found in the 501 selected sequences and the total predicted <i>P. infestans</i> CDS, based on KOG functional categories.

<p>The differential abundance (<i>y</i> axis) of predicted CDS to assignable categories (<i>x</i> axis) is shown. KOG categories are as follows (from: <a href="http://www.ncbi.nlm.nih.gov/COG/" target="_blank">http://www.ncbi.nlm.nih.gov/COG/</a>): J, Translation; A, RNA processing and modification; K, Transcription; L, Replication, recombination and repair; B, Chromatin structure and dynamics; D, Cell cycle control, cell division, chromosome partitioning; Y, Nuclear structure; V, Defense mechanisms; T, Signal transduction mechanisms; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; Z, Cytoskeleton; W, Extracellular structures; U, Intracellular trafficking, secretion, and vesicular transport; O, Posttranslational modification, protein turnover, chaperones; C, Energy production and conversion; G, Carbohydrate transport and metabolism; E, Amino acid transport and metabolism; F Nucleotide transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S Function unknown.</p

Main workflow of analysis followed in this study.

<p>Main workflow of analysis followed in this study.</p

GenBank accession numbers and descriptions of the sequences used for this study.

<p>GenBank accession numbers and descriptions of the sequences used for this study.</p

Different criteria used to separate the <i>P. infestans</i> sequences from host sequences produces results that differ notably among them.

<p>12,900 unitigs containing host and pathogen sequences were used to test different approaches to separate both types of sequences. The scheme represents the number of sequences obtained using GC content and a BLAST cut-off e-value combined with different ratio cut-offs: ((Alignment length * %ID)/Unitig length). (a) GC >52%; (b) e-value cut-off of 10⁻¹⁵ (c) e-value cut-off of 10^-15and ratio >50%; (d) e-value cut-off of 10⁻¹⁵ and ratio >60%; (e) e-value cut-off of 10⁻¹⁵ and ratio >70%; (f) e-value cut-off of 10⁻¹⁵ and ratio >80%; (g) e-value cut-off of 10⁻¹⁵ and ratio >90%.</p

Distribution of ESTs within contigs after clustering (using CAP3) the 22,795 sequences downloaded from GenBank.

<p>Distribution of ESTs within contigs after clustering (using CAP3) the 22,795 sequences downloaded from GenBank.</p

Summary of resulting candidate <i>Phytophthora infestans</i> sequences after separating 12,900 unitigs containing pathogen and host sequences using different selection criteria.

<p>The sequences identified as belonging to <i>P. infestans</i> but also having a hit against potato and tomato ESTs or any Solanaceae are shown to the right.</p>a<p>((Alignment length * %ID)/Unitig length).</p