Water-Soluble Trichogin GA IV-Derived Peptaibols Protect Tomato Plants From Botrytis cinerea Infection With Limited Impact on Plant Defenses.

Peptaibols are non-ribosomal linear peptides naturally produced by a wide variety of fungi and represent the largest group of peptaibiotic molecules produced by Trichoderma species. Trichogin GA IV is an 11-residue lipopeptaibol naturally produced by Trichoderma longibrachiatum. Peptaibols possess the ability to form pores in lipid membranes or perturb their surface, and have been studied as antibiotics or anticancer drugs in human medicine, or as antimicrobial molecules against plant pathogens. When applied to plants, peptaibols may also elicit defense responses. A major drawback to the exploitation and application of peptaibols in agriculture is their poor water solubility. In a previous study, we designed water-soluble Lys-containing Trichogin GA IV analogs, which were able to inhibit the growth of several fungal plant pathogens in vitro. In the present study, we shed light on the mechanism underpinning their efficacy on plants, focusing on six Trichogin GA IV analogs. Our results highlighted peptide hydrophilicity, rather than helix stability, as the major determinant of their activity against B. cinerea infection in tomato leaves. The peptides showed preventive but not curative efficacy against infection, and lack of translaminar activity, with results reproducible on two tomato cultivars, Marmande and Micro-Tom. Reactive oxygen species (ROS) detection analysis in tomato and Arabidopsis, and expression of defense genes in tomato, highlighted a transient and limited impact of the peptides on the plant defense system. The treatment did not result in significant modulation of defense genes or defense priming. The antimicrobial effect thus emerges as the only mechanism behind the plant protection ability exerted by water-soluble Trichogin GA IV analogs, and limited effects on the plant metabolism are expected to occur.

Cold Stress, Freezing Adaptation, Varietal Susceptibility of Olea europaea L.: A Review.

Olive (Olea europaea L.) is an evergreen xerophytic tree characterizing vegetative landscape and historical-cultural identity of the Mediterranean Basin. More than 2600 cultivars constitute the rich genetic patrimony of the species cultivated in approximately 60 countries. As a subtropical species, the olive tree is quite sensitive to low temperatures, and air temperature is the most critical environmental factor limiting olive tree growth and production. In this present review, we explored the detrimental effects caused of low temperatures on olive cultivars, and analyzed the most frequently experimental procedures used to evaluate cold stress. Then, current findings freezing stress physiology and gene are summarized in olive tree, with an emphasis on adaptive mechanisms for cold tolerance. This review might clear the way for new research on adaptive mechanisms for cold acclimation and for improvement of olive growing management.

Moderate Salinity Stress Affects Expression of Main Sugar Metabolism and Transport Genes and Soluble Carbohydrate Content in Ripe Fig Fruits (Ficus carica L. cv. Dottato).

Fig trees (Ficus carica L.) are commonly grown in the Mediterranean area, where salinity is an increasing problem in coastal areas. Young, fruiting plants of cv. Dottato were subjected to moderate salt stress (100 mM NaCl added to irrigation water) for 48 days before fruit sampling. To clarify the effect of salinity stress, we investigated changes in the transcription of the main sugar metabolism-related genes involved in the synthesis, accumulation and transport of soluble carbohydrates in ripe fruits by quantitative real-time PCR as well as the content of soluble sugars by quantitative 1H nuclear magnetic resonance spectroscopy. A general increase in the transcript levels of genes involved in the transport of soluble carbohydrates was observed. Alkaline-neutral and Acid Invertases transcripts, related to the synthesis of glucose and fructose, were up-regulated in ripe fruits of NaCl-stressed plants without a change in the content of D-glucose and D-fructose. The increases in sucrose and D-sorbitol contents were likely the result of the up-regulation of the transcription of Sucrose-Synthase- and Sorbitol-Dehydrogenase-encoding genes.

Straw Competition and Wheat Root Endophytism of Trichoderma gamsii T6085 as Useful Traits in the Biological Control of Fusarium Head Blight.

Trichoderma gamsii T6085 has been investigated for many years as a beneficial isolate for use in the biocontrol of Fusarium head blight (FHB) of wheat caused primarily by Fusarium graminearum. Previous work focused on application of T6085 to wheat spikes at anthesis, whereas application to soil before or at sowing has received limited attention. In the present study, the competitive ability of T6085 on plant residues against F. graminearum was investigated. Results showed a significant reduction of wheat straw colonization by the pathogen and of the development of perithecia, not only when T6085 was applied alone but also in the presence of a F. oxysporum isolate (7121), well known as a natural competitor on wheat plant residues. T6085 was able to endophytically colonize wheat roots, resulting in internal colonization of the radical cortex area, without reaching the vascular system, as confirmed by confocal microscopy. This intimate interaction with the plant resulted in a significant increase of the expression of the plant defense-related genes PAL1 and PR1. Taken together, competitive ability, endophytic behavior, and host resistance induction represent three important traits that can be of great use in the application of T6085 against FHB not only on spikes at anthesis but potentially also in soil before or at sowing.

Ray flower initiation in the Helianthus radula inflorescence is influenced by a functional allele of the HrCYC2c gene.

The radiate pseudanthium, with actinomorphic disk flowers surrounded by showy marginal zygomorphic ray flowers, is the most common inflorescence in the Helianthus genus. In Helianthus radula, ray flower primordia are normally absent at the dorsal domain of the inner phyllaries (discoid heads) while the occurrence of radiate inflorescences is uncommon. In Helianthus spp., flower symmetry and inflorescence architecture are mainly controlled by CYCLOIDEA (CYC)-like genes but the putative role of these genes in the development of discoid inflorescences has not been investigate. Three CYC genes of H. radula with a role in ray flower identity (HrCYC2c, HrCYC2d, and HrCYC2e) were isolated. The phylogenetic analysis placed these genes within the CYC2 subclade. We identified two different alleles for the HrCYC2c gene. A mutant allele, designed HrCYC2c-m, shows a thymine to adenine transversion, which generates a TGA stop codon after a translation of 14 amino acids. We established homozygous dominant (HrCYC2c/HrCYC2c) and recessive (HrCYC2c-m/HrCYC2c-m) plants for this nonsense mutation. Inflorescences of both HrCYC2c/HrCYC2c and HrCYC2c/HrCYC2c-m plants initiated ray flowers, despite at low frequency. By contrast, plants homozygous for the mutant allele (HrCYC2c-m/HrCYC2c-m) failed at all to develop ray flowers. The results support, for the first time, a role of the HrCYC2c gene on the initiation of ray flower primordia. However, also in the two dominant phenotypes, discoid heads are the prevalent architecture suggesting that this gene is required but not sufficient to initiate ray flowers in pseudanthia. Other unknown major genes are most likely required in the shift from discoid to radiate inflorescence.

Combined Comparative Genomics and Gene Expression Analyses Provide Insights into the Terpene Synthases Inventory in Trichoderma.

Trichoderma is a fungal genus comprising species used as biocontrol agents in crop plant protection and with high value for industry. The beneficial effects of these species are supported by the secondary metabolites they produce. Terpenoid compounds are key players in the interaction of Trichoderma spp. with the environment and with their fungal and plant hosts; however, most of the terpene synthase (TS) genes involved in their biosynthesis have yet not been characterized. Here, we combined comparative genomics of TSs of 21 strains belonging to 17 Trichoderma spp., and gene expression studies on TSs using T. gamsii T6085 as a model. An overview of the diversity within the TS-gene family and the regulation of TS genes is provided. We identified 15 groups of TSs, and the presence of clade-specific enzymes revealed a variety of terpenoid chemotypes evolved to cover different ecological demands. We propose that functional differentiation of gene family members is the driver for the high number of TS genes found in the genomes of Trichoderma. Expression studies provide a picture in which different TS genes are regulated in many ways, which is a strong indication of different biological functions.

Genotypic Characterization of Torymus sinensis (Hymenoptera: Torymidae) After Its Introduction in Tuscany (Italy) for the Biological Control of Dryocosmus kuriphilus (Hymenoptera: Cynipidae).

Torymus sinensis Kamijo (Hymenoptera: Torymidae) is an alien parasitoid that is used in many areas of the world for biological control the Asian chestnut gall wasp, Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae). In Italy, this parasitoid was imported from Japan in 2003 and subsequently multiplied and released throughout the country. In this study, a phylogenetic investigation was carried out on insects from three different sites in northern Tuscany (Italy). Moreover, the possible hybridization between T. sinensis and some native Torymus species was evaluated. The conserved region 18S rRNA gene and the hypervariable ITS2 (Internal Transcribed Spacer 2) region of the ribosomal cistrone were selected as molecular markers. Sequencing the amplified products, after cloning, ruled out any hybridization between T. sinensis and the native Torymus species, and also confirmed the presence of two haplotypes for the Tuscan population of T. sinensis both for the region of the 18S rRNA gene as well as for the ITS2 region. These results confirm that the environmental impact of the alien parasitoid T. sinensis in the study site is acceptable, although an extensive and repeated monitoring would be desirable.

Discerning between Two Tuscany (Italy) Ancient Apple cultivars, 'Rotella' and 'Casciana', through Polyphenolic Fingerprint and Molecular Markers.

Ancient apple cultivars usually have higher nutraceutical value than commercial ones, but in most cases their variability in pomological traits does not allow us to discriminate among them. Fruit of two Tuscany ancient apple cultivars, 'Casciana' and 'Rotella', picked from eight different orchards (four for each cultivar) were analyzed for their pomological traits, organoleptic qualities, polyphenolic profile and antiradical activity. The effectiveness of a polyphenol-based cluster analysis was compared to molecular markers (internal transcribed spacers, ITS1 and ITS2) to unequivocally discern the two apples. 'Casciana' and 'Rotella' fruit had a higher nutraceutical value than some commercial cultivars, in terms of phenolic abundance, profile and total antiradical activity. Although pedo-climatic conditions of different orchards influenced the phenolic profile of both apples, the polyphenolic discriminant analysis clearly separated the two cultivars, principally due to higher amounts of procyanidin B2, procyanidin B3 and p-coumaroylquinic acid in 'Casciana' than in 'Rotella' fruit. These three polyphenols can be used proficiently as biochemical markers for distinguishing the two apples when pomological traits cannot. Conversely, ITS1 and ITS2 polymorphism did not allow us to distinguish 'Casciana' from 'Rotella' fruit. Overall, the use of polyphenolic fingerprint might represent a valid tool to ensure the traceability of products with a high economic value.

UV-B exposure reduces the activity of several cell wall-dismantling enzymes and affects the expression of their biosynthetic genes in peach fruit (Prunus persica L., cv. Fairtime, melting phenotype).

Softening processes after ripening are a major factor contributing to the perishability of fleshy fruit and, together with mechanical damage, represent the onset of physiological decay. Softening involves multiple co-ordinated events leading to modifications of the cell wall architecture. Several studies described that UV-B radiation positively affects both the nutraceutical and aesthetical qualities of fruit. However, very few studies investigated the effect of UV-B irradiation on the activity of cell wall-related enzymes. This research aimed at studying how different UV-B treatments (10 min and 60 min) affect the activity of cell wall-modifying enzymes (pectin methylesterase, polygalacturonase and ß-galactosidase) together with the expression of some of their isoforms up to 36 h after UV-B treatment of peach (cv. Fairtime, melting phenotype) fruit. Results revealed that UV-B radiation did not affect the soluble solid content and the titratable acidity, two important parameters influencing consumers' choice and taste. In contrast, UV-B was effective at reducing the loss of firmness 24 h after the 60 min irradiation. Generally, a lower activity of the hydrolytic enzymes compared to untreated fruit was observed, regardless of the UV-B dose. However, gene expression did not reflect the corresponding enzymatic activity. Based on these results, UV-B irradiation might be a successful tool in reducing the loss of firmness of peach fruit during post-harvest, thus improving their quality and shelf-life.

Effects of chlorogenic acid, epicatechin gallate, and quercetin on mucin expression and secretion in the Caco-2/HT29-MTX cell model.

Mucins are a family of large glycoproteins that represent the major structural components of the mucus and are encoded by 20 different mucin genes. Mucin expression can be modulated by different stimuli. In this study, we analyzed four mucins (MUC2, MUC3, MUC13, and MUC17) in coculture of Caco-2/HT29-MTX cells to demonstrate the variation in gene expression in the presence of antioxidant compounds like chlorogenic acid, epicatechin gallate, and quercetin (apple, tea, and coffee polyphenols, respectively). coculture of Caco-2/HT29-MTX cells was treated with polyphenols, and the expression of four mucins was determined by reverse-transcriptase PCR. In addition, the secretion levels of MUC2 were established by enzyme-linked immunoassay (ELISA) analysis. The results showed that each polyphenol compound induces different expression patterns of the mucin genes. Statistically significant up-regulation of MUC17 was observed following incubation with epicatechin gallate and quercetin. ELISA results did not prove any significant differences in protein levels of MUC2 after treatment by the polyphenol compounds. The polyphenols considered in this study may influence mucin secretion and act on diverse salivary substrates to change the barrier properties of mucins for mucus secretion in different ways.

Is Exploitation Competition Involved in a Multitrophic Strategy for the Biocontrol of Fusarium Head Blight?

Trichoderma gamsii T6085 was used in combination with a Fusarium oxysporum isolate (7121) in order to evaluate, in a multitrophic approach, their competitive ability against F. graminearum, one of the main causal agents of Fusarium head blight (FHB) on wheat. The two antagonists and the pathogen were coinoculated on two different natural substrates, wheat and rice kernels. Both T6085 and 7121, alone and coinoculated, significantly reduced the substrate colonization and mycotoxin production by the pathogen. The two antagonists did not affect each other. Using a metabolic approach (Biolog), we investigated whether exploitation competition could explain this antagonistic activity. The aim was to define whether the three fungi coexist or if one isolate nutritionally dominates another. Results obtained from Biolog suggest that no exploitative competition occurs between the antagonists and the pathogen during the colonization of the natural substrates. Interference competition was then preliminarily evaluated to justify the reduction in the pathogen's growth and to better explain mechanisms. A significant reduction of F. graminearum growth was observed when the pathogen grew in the cultural filtrates of T. gamsii T6085, both alone and cocultured with F. oxysporum 7121, thus suggesting the involvement of secondary metabolites. As far as we know, this is the first time that an ecological study has been performed to explain how and which kind of competition could be involved in a multitrophic biocontrol of FHB.

How an ancient, salt-tolerant fruit crop, Ficus carica L., copes with salinity: a transcriptome analysis.

Although Ficus carica L. (fig) is one of the most resistant fruit tree species to salinity, no comprehensive studies are currently available on its molecular responses to salinity. Here we report a transcriptome analysis of F. carica cv. Dottato exposed to 100 mM sodium chloride for 7 weeks, where RNA-seq analysis was performed on leaf samples at 24 and 48 days after the beginning of salinization; a genome-derived fig transcriptome was used as a reference. At day 24, 224 transcripts were significantly up-regulated and 585 were down-regulated, while at day 48, 409 genes were activated and 285 genes were repressed. Relatively small transcriptome changes were observed after 24 days of salt treatment, showing that fig plants initially tolerate salt stress. However, after an early down-regulation of some cell functions, major transcriptome changes were observed after 48 days of salinity. Seven weeks of 100 mM NaCl dramatically changed the repertoire of expressed genes, leading to activation or reactivation of many cell functions. We also identified salt-regulated genes, some of which had not been previously reported to be involved in plant salinity responses. These genes could be potential targets for the selection of favourable genotypes, through breeding or biotechnology, to improve salt tolerance in fig or other crops.

Signalling molecules responsive to ozone-induced oxidative stress in Salvia officinalis.

Tropospheric ozone (O3) is the most important gaseous pollutant and induces a mass of negative impacts on vegetation at functional and genic levels. The aim of the present study was to investigate the role of reactive oxygen species and signalling molecules in sage plants exposed to O3 (200 ppb, 5 h). Ozone exposure induced only a transient oxidative burst, as confirmed by the rapid peak of anion superoxide during the first hours of exposure (+16% compared to controls). The spontaneous reaction of O3 with membrane fatty acids stimulates peroxidative processes, as demonstrated by the rise of thiobarbituric acid reactive substances concentration starting after 1 h of exposure (+25%). The formation of lipid-based signalling molecules (e.g. jasmonic acid) may be regarded as a sort of O3-perception. The concomitant accumulation of salicylic acid suggests that sage responds early to O3 by inducing cellular antioxidants mechanisms in order to minimize O3-oxidative burst. The transient increase of abscisic acid (+25% at the end of the treatment) twinned with the maximal ethylene emission (about two-fold higher than controls) could be interpreted as a first attempt by plants to regulate the signalling responses induced by O3. In order to investigate the involvement of transcription factors in managing oxidative protection, BLASTX analysis against the Salvia miltiorrhiza sequence genome was carried out using Arabidopsis thaliana WRKY sequences as queries. Six gene sequences were identified for sage WRKYs and their relative gene expression analyses were characterized. WRKY4, WRKY5, WRKY11 and WRKY46 were up-regulated by O3 at 2 and 5 h of exposure and they showed similarity with AtWRKY48, AtWRKY22 and AtWRKY53 in A. thaliana. These results suggest that WRKYs could play a pivotal role in the signalling mechanisms during the responses of plants to O3.

Transcriptome changes induced by arbuscular mycorrhizal fungi in sunflower (Helianthus annuus L.) roots.

Arbuscular mycorrhizal (AM) fungi are essential elements of soil fertility, plant nutrition and productivity, facilitating soil mineral nutrient uptake. Helianthus annuus is a non-model, widely cultivated species. Here we used an RNA-seq approach for evaluating gene expression variation at early and late stages of mycorrhizal establishment in sunflower roots colonized by the arbuscular fungus Rhizoglomus irregulare. mRNA was isolated from roots of plantlets at 4 and 16 days after inoculation with the fungus. cDNA libraries were built and sequenced with Illumina technology. Differential expression analysis was performed between control and inoculated plants. Overall 726 differentially expressed genes (DEGs) between inoculated and control plants were retrieved. The number of up-regulated DEGs greatly exceeded the number of down-regulated DEGs and this difference increased in later stages of colonization. Several DEGs were specifically involved in known mycorrhizal processes, such as membrane transport, cell wall shaping, and other. We also found previously unidentified mycorrhizal-induced transcripts. The most important DEGs were carefully described in order to hypothesize their roles in AM symbiosis. Our data add a valuable contribution for deciphering biological processes related to beneficial fungi and plant symbiosis, adding an Asteraceae, non-model species for future comparative functional genomics studies.

Cultivar-specific transcriptome prediction and annotation in Ficus carica L.

The availability of transcriptomic data sequence is a key step for functional genomics studies. Recently, a repertoire of predicted genes of a Japanese cultivar of fig (Ficus carica L.) was released. Because of the great phenotypic variability that can be found in this species, we decided to study another fig genotype, the Italian cv. Dottato, in order to perform comparative studies between the two cultivars and extend the pan genome of this species. We isolated, sequenced and assembled fig genomic DNA from young fruits of cv. Dottato. Then, putative gene sequences were predicted and annotated. Finally, a comparison was performed between cvs. Dottato and Horaishi predicted transcriptomes. Our data provide a resource (available at the Sequence Read Archive database under SRP109082) to be used for functional genomics of fig, in order to fill the gap of knowledge still existing in this species concerning plant development, defense and adaptation to the environment.

Rhizophagus intraradices or its associated bacteria affect gene expression of key enzymes involved in the rosmarinic acid biosynthetic pathway of basil.

In recent years, arbuscular mycorrhizal fungi (AMF) have been reported to enhance plant biosynthesis of secondary metabolites with health-promoting activities, such as polyphenols, carotenoids, vitamins, anthocyanins, flavonoids and lycopene. In addition, plant growth-promoting (PGP) bacteria were shown to modulate the concentration of nutraceutical compounds in different plant species. This study investigated for the first time whether genes encoding key enzymes of the biochemical pathways leading to the production of rosmarinic acid (RA), a bioactive compound showing antioxidant, antibacterial, antiviral and anti-inflammatory properties, were differentially expressed in Ocimum basilicum (sweet basil) inoculated with AMF or selected PGP bacteria, by using quantitative real-time reverse transcription PCR. O. basilicum plants were inoculated with either the AMF species Rhizophagus intraradices or a combination of two PGP bacteria isolated from its sporosphere, Sinorhizobium meliloti TSA41 and Streptomyces sp. W43N. Present data show that the selected PGP bacteria were able to trigger the overexpression of tyrosine amino-transferase (TAT), hydroxyphenylpyruvate reductase (HPPR) and p-coumaroyl shikimate 3'-hydroxylase isoform 1 (CS3'H iso1) genes, 5.7-fold, 2-fold and 2.4-fold, respectively, in O. basilicum leaves. By contrast, inoculation with R. intraradices triggered TAT upregulation and HPPR and CS3'H iso1 downregulation. Our data suggest that inoculation with the two selected strains of PGP bacteria utilised here could represent a suitable biotechnological tool to be implemented for the production of O. basilicum plants with increased levels of key enzymes for the biosynthesis of RA, a compound showing important functional properties as related to human health.

Cerato-platanin induces resistance in Arabidopsis leaves through stomatal perception, overexpression of salicylic acid- and ethylene-signalling genes and camalexin biosynthesis.

Microbe-associated molecular patterns (MAMPs) lead to the activation of the first line of plant defence. Few fungal molecules are universally qualified as MAMPs, and proteins belonging to the cerato-platanin protein (CPP) family seem to possess these features. Cerato-platanin (CP) is the name-giving protein of the CPP family and is produced by Ceratocystis platani, the causal agent of the canker stain disease of plane trees (Platanus spp.). On plane tree leaves, the biological activity of CP has been widely studied. Once applied on the leaf surface, CP acts as an elicitor of defence responses. The molecular mechanism by which CP elicits leaves is still unknown, and the protective effect of CP against virulent pathogens has not been clearly demonstrated. In the present study, we tried to address these questions in the model plant Arabidopsis thaliana. Our results suggest that stomata rapidly sense CP since they responded to the treatment with ROS signalling and stomatal closure, and that CP triggers salicylic acid (SA)- and ethylene (ET)-signalling pathways, but not the jasmonic acid (JA)-signalling pathway, as revealed by the expression pattern of 20 marker genes. Among these, EDS1, PAD4, NPR1, GRX480, WRKY70, ACS6, ERF1a/b, COI1, MYC2, PDF1.2a and the pathogenesis-related (PR) genes 1-5. CP rapidly induced MAPK phosphorylation and induced the biosynthesis of camalexin within 12 hours following treatment. The induction of localised resistance was shown by a reduced susceptibility of the leaves to the infection with Botrytis cinerea and Pseudomonas syringae pv. tomato. These results contribute to elucidate the key steps of the signalling process underlying the resistance induction in plants by CP and point out the central role played by the stomata in this process.

Cerato-platanin shows expansin-like activity on cellulosic materials.

Cerato-platanin (CP) is a non-catalytic protein with a double ψß-barrel fold located in the cell wall of the phytopathogenic fungus Ceratocystis platani. CP is released during growth and induces defence-related responses in plants. CP is also the first member of the "cerato-platanin family" (CPF) (Pfam PF07249). In the CPF, the molecular mechanism of action on plants and above all the biological role in fungal life are little-known aspects. However, an expansin-like function has recently been suggested concerning CP. Expansin-like proteins have the ability to act non-hydrolytically on cellulose. In the present work, the expansin-like activity of CP and Pop1, a CP family member, was investigated. Like expansins, CP and Pop1 were able to weaken filter paper in a concentration-dependent manner and without the production of reducing sugars. A metal-dependent polysaccharide monooxygenase-like activity was excluded. The optimum of activity was pH5.0, 38 °C. CP was also able to cause fragmentation of the crystalline cellulose Avicel and the breakage and defibration of cotton fibres. However, the interaction did not involve a stable bond with the substrates and CP did not significantly enhance the hydrolytic activity of cellulase. On the other hand, CP and Pop1 bound quickly to chitin. We consider CP as a novel one-domain expansin-like protein. We propose a structural role for CP in the fungal cell wall due to the ability to bind chitin, and hypothesize a functional role in the interaction of the fungus with the plant for the weakening activity shown on cellulose.

Modulation of gene expression in contextual fear conditioning in the rat.

In contextual fear conditioning (CFC) a single training leads to long-term memory of context-aversive electrical foot-shocks association. Mid-temporal regions of the brain of trained and naive rats were obtained 2 days after conditioning and screened by two-directional suppression subtractive hybridization. A pool of differentially expressed genes was identified and some of them were randomly selected and confirmed with qRT-PCR assay. These transcripts showed high homology for rat gene sequences coding for proteins involved in different cellular processes. The expression of the selected transcripts was also tested in rats which had freely explored the experimental apparatus (exploration) and in rats to which the same number of aversive shocks had been administered in the same apparatus, but temporally compressed so as to make the association between painful stimuli and the apparatus difficult (shock-only). Some genes resulted differentially expressed only in the rats subjected to CFC, others only in exploration or shock-only rats, whereas the gene coding for translocase of outer mitochondrial membrane 20 protein and nardilysin were differentially expressed in both CFC and exploration rats. For example, the expression of stathmin 1 whose transcripts resulted up regulated was also tested to evaluate the transduction and protein localization after conditioning.

Differential timing of defense-related responses induced by cerato-platanin and cerato-populin, two non-catalytic fungal elicitors.

The cerato-platanin (CP) family consists of fungal-secreted proteins involved in various stages of the host-fungus interaction and acting as phytotoxins and elicitors of defense responses. The founder member of this family is CP, a non-catalytic protein with a six-stranded double-ψß-barrel fold. Cerato-populin (Pop1) is an ortholog showing low sequence identity with CP. CP is secreted by Ceratocystis platani, the causal agent of the canker stain of plane. Pop1 is secreted by Ceratocystis populicola, a pathogen of poplar. CP and Pop1 have been suggested to act as PAMPs (pathogen-associated molecular patterns) because they induce phytoalexin synthesis, transcription of defense-related genes, restriction of conidia growth and cell death in various plants. Here, we treated plane leaves with CP or Pop1, and monitored defense responses to define the role of these elicitors in the plant interactions. Both CP and Pop1 were able to induce mitogen-activated protein kinases (MAPKs) phosphorylation, production of reactive oxygen species and nitric oxide, and overexpression of defense related genes. The characteristic DNA fragmentation and the cytological features indicate that CP and Pop1 induce cell death by a mechanism of programmed cell death. Therefore, CP and Pop1 can be considered as two novel, non-catalytic fungal PAMPs able to enhance primary defense. Of particular interest is the observation that CP showed faster activity compared to Pop1. The different timing in defense activation could potentially be due to the structural differences between CP and Pop1 (i.e. different hydrophobic index and different helix content) therefore constituting a starting point in unraveling their structure-function relationships.