A Metabolome Analysis and the Immunity of Phlomis purpurea against Phytophthora cinnamomi.

Phlomis purpurea grows spontaneously in the southern Iberian Peninsula, namely in cork oak (Quercus suber) forests. In a previous transcriptome analysis, we reported on its immunity against Phytophthora cinnamomi. However, little is known about the involvement of secondary metabolites in the P. purpurea defense response. It is known, though, that root exudates are toxic to this pathogen. To understand the involvement of secondary metabolites in the defense of P. purpurea, a metabolome analysis was performed using the leaves and roots of plants challenged with the pathogen for over 72 h. The putatively identified compounds were constitutively produced. Alkaloids, fatty acids, flavonoids, glucosinolates, polyketides, prenol lipids, phenylpropanoids, sterols, and terpenoids were differentially produced in these leaves and roots along the experiment timescale. It must be emphasized that the constitutive production of taurine in leaves and its increase soon after challenging suggests its role in P. purpurea immunity against the stress imposed by the oomycete. The rapid increase in secondary metabolite production by this plant species accounts for a concerted action of multiple compounds and genes on the innate protection of Phlomis purpurea against Phytophthora cinnamomi. The combination of the metabolome with the transcriptome data previously disclosed confirms the mentioned innate immunity of this plant against a devastating pathogen. It suggests its potential as an antagonist in phytopathogens' biological control. Its application in green forestry/agriculture is therefore possible.

Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi.

The soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.

Multiple new cryptic pathogenic Phytophthora species from Fagaceae forests in Austria, Italy and Portugal.

During surveys of Phytophthora diversity in natural and semi-natural Fagaceae forests in Austria, Italy and Portugal, four new cryptic species were isolated from rhizosphere soil samples. Multigene phylogeny based on nuclear ITS, ß-tubulin and HSP90 and mitochondrial cox1 and NADH1 gene sequences demonstrated that two species, P. tyrrhenica and P. vulcanica spp. nov., belong to phylogenetic Clade 7a, while the other two species, P. castanetorum and P. tubulina spp. nov., clustered together with P. quercina forming a new clade, named here as Clade 12. All four new species are homothallic and have low optimum and maximum temperatures for growth and very slow growth rates at their respective optimum temperature. They differed from each other and from related species by a unique combination of morphological characters, cardinal temperatures, and growth rates. Pathogenicity of all Phytophthora species to the root system of their respective host species was demonstrated in soil infestation trials.

De novo assembly of Phlomis purpurea after challenging with Phytophthora cinnamomi.

BACKGROUND: Phlomis plants are a source of biological active substances with potential applications in the control of phytopathogens. Phlomis purpurea (Lamiaceae) is autochthonous of southern Iberian Peninsula and Morocco and was found to be resistant to Phytophthora cinnamomi. Phlomis purpurea has revealed antagonistic effect in the rhizosphere of Quercus suber and Q. ilex against P. cinnamomi. Phlomis purpurea roots produce bioactive compounds exhibiting antitumor and anti-Phytophthora activities with potential to protect susceptible plants. Although these important capacities of P. purpurea have been demonstrated, there is no transcriptomic or genomic information available in public databases that could bring insights on the genes underlying this anti-oomycete activity. RESULTS: Using Illumina technology we obtained a de novo assembly of P. purpurea transcriptome and differential transcript abundance to identify putative defence related genes in challenged versus non-challenged plants. A total of 1,272,600,000 reads from 18 cDNA libraries were merged and assembled into 215,739 transcript contigs. BLASTX alignment to Nr NCBI database identified 124,386 unique annotated transcripts (57.7%) with significant hits. Functional annotation identified 83,550 out of 124,386 unique transcripts, which were mapped to 141 pathways. 39% of unigenes were assigned GO terms. Their functions cover biological processes, cellular component and molecular functions. Genes associated with response to stimuli, cellular and primary metabolic processes, catalytic and transporter functions were among those identified. Differential transcript abundance analysis using DESeq revealed significant differences among libraries depending on post-challenge times. Comparative cyto-histological studies of P. purpurea roots challenged with P. cinnamomi zoospores and controls revealed specific morphological features (exodermal strips and epi-cuticular layer), that may provide a constitutive efficient barrier against pathogen penetration. Genes involved in cutin biosynthesis and in exodermal Casparian strips formation were up-regulated. CONCLUSIONS: The de novo assembly of transcriptome using short reads for a non-model plant, P. purpurea, revealed many unique transcripts useful for further gene expression, biological function, genomics and functional genomics studies. The data presented suggest a combination of a constitutive resistance and an increased transcriptional response from P. purpurea when challenged with the pathogen. This knowledge opens new perspectives for the understanding of defence responses underlying pathogenic oomycete/plant interaction upon challenge with P. cinnamomi.

Structure, anti-Phytophthora and anti-tumor activities of a nortriterpenoid from the rhizome of Phlomis purpurea (Lamiaceae).

To investigate bioactive compounds potentially involved in the biotic interactions exhibited by Phlomis purpurea (Lamiaceae) in rhizospheres infested with Phytophthora cinnamomi, the plant rhizome was chemically analysed. The nortriterpenoid (17S)-2α,3α,11α,23,24-pentahydroxy-19(18 â†’ 17)-abeo-28-norolean-12-en-18-one, was isolated and its structure was elucidated by comprehensive spectroscopic analysis, chiefly using 2D NMR experiments, and X-ray analysis. It was shown to be exuded by roots and to exhibit anti-Phytophthora and antitumor activities.

Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection.

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-ß-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-ß-glucanase (QsGlu). Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), ß-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work. Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Cloning, characterization and in vitro and in planta expression of a glucanase inhibitor protein (GIP) of Phytophthora cinnamomi.

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.

Association of polymorphism of the ß(1, 4)-galactosyltransferase-I gene with milk production traits in Holsteins.

The ß(1,4)-galactosyltransferase-I gene (ß4galt1) encodes the catalytic part of the enzyme lactose synthase, responsible of lactose synthesis in the mammary gland. The complete coding region of the gene was screened for the presence of allelic variation among a sample of 1,200 Iranian Holstein cows, using PCR-SSCP technique followed by sequencing. Nine polymorphic nucleotide sites were identified-one in exons I and VI, two in exons II and III, and three in exon V. Altogether 18 different genotypes were assigned. Statistical analysis showed that the genotypes of Β4GALT1 significantly affect milk, lactose, protein and total solid productions in both the first and second lactation (P < 0.001). Variance component analysis considering restricted maximum likelihood showed that the major factor making differences in milk, lactose, protein and total solid productions among the studied cow is the ß4galt1 genotype. We concluded that the ß4galt1 gene is potentially associated with milk production traits in dairy cows and should be considered for further studies on genetics of the milk production traits.

Molecular genetic analysis of a cattle population to reconstitute the extinct Algarvia breed.

BACKGROUND: Decisions to initiate conservation programmes need to account for extant variability, diversity loss and cultural and economic aspects. Molecular markers were used to investigate if putative Algarvia animals could be identified for use as progenitors in a breeding programme to recover this nearly extinct breed. METHODS: 46 individuals phenotypically representative of Algarvia cattle were genotyped for 27 microsatellite loci and compared with 11 Portuguese autochthonous and three imported breeds. Genetic distances and factorial correspondence analyses (FCA) were performed to investigate the relationship among Algarvia and related breeds. Assignment tests were done to identify representative individuals of the breed. Y chromosome and mtDNA analyses were used to further characterize Algarvia animals. Gene- and allelic-based conservation analyses were used to determine breed contributions to overall genetic diversity. RESULTS: Genetic distance and FCA results confirmed the close relationship between Algarvia and southern Portuguese breeds. Assignment tests without breed information classified 17 Algarvia animals in this cluster with a high probability (q > 0.95). With breed information, 30 cows and three bulls were identified (q > 0.95) that could be used to reconstitute the Algarvia breed. Molecular and morphological results were concordant. These animals showed intermediate levels of genetic diversity (MNA = 6.0 +/- 1.6, Rt = 5.7 +/- 1.4, Ho = 0.63 +/- 0.19 and He = 0.69 +/- 0.10) relative to other Portuguese breeds. Evidence of inbreeding was also detected (Fis = 0.083, P < 0.001). The four Algarvia bulls had Y-haplotypes H6Y2 and H11Y2, common in Portuguese cattle. The mtDNA composition showed prevalence of T3 matrilines and presence of the African-derived T1a haplogroup. This analysis confirmed the genetic proximity of Algarvia and Garvonesa breeds (Fst = 0.028, P > 0.05). Algarvia cattle provide an intermediate contribution (CB = 6.18, CW = -0.06 and D1 = 0.50) to the overall gene diversity of Portuguese cattle. Algarvia and seven other autochthonous breeds made no contribution to the overall allelic diversity. CONCLUSIONS: Molecular analyses complemented previous morphological findings to identify 33 animals that can be considered remnants of the Algarvia breed. Results of genetic diversity and conservation analyses provide objective information to establish a management program to reconstitute the Algarvia breed.

Associations among milk production traits and glycosylated haemoglobin in dairy cattle; importance of lactose synthesis potential.

Glucose is the major precursor of lactose synthesis in the mammary gland. Lactose the major carbohydrate and osmolyte of milk, controls milk volume and its concentration. Glycosylated haemoglobin (HbG) is a retrospective measure of mean blood glucose level and it is largely unaffected by recent physiological conditions and environmental events. The purposes of this study were to determine the correlations between lactose traits and other milk production traits in dairy cattle and to investigate whether HbG level can be correlated with milk and lactose production traits. Here, HbG percentage, milk and lactose production traits including milk yield, lactose, protein, SNF, total solid and fat percentages and yields were measured in 485 second calved Iranian Holstein cattle. Statistically significant negative correlations were established between HbG and milk yield (r = -0.88), lactose yield (r = -0.83), SNF yield (r = -0.81), protein yield (r = -0.79) and total solid yield (r = -0.74). Positive correlations were established between lactose yield and milk (r = 0.96), protein (r = 0.81), SNF (r = 0.92) and total solid (r = 0.79) yields. The negative correlation between HbG and milk and total lactose production is probably related to the higher glucose demands in the lactating mammary gland of more productive cows. The positive correlation between lactose yield and milk, protein, SNF and total solid yield indicates that the level of lactose synthesis influences milk production traits in ways other than merely via its osmolytic action.

Identification and characterization of four splicing variants of ovine POU1F1 gene.

Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly (p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.

Effects of genetic polymorphisms at the growth hormone gene on milk yield in Serra da Estrela sheep.

The five exons and the 5' and 3'-untranslated regions (5'-UTR and 3'-UTR) of the oGH gene were screened for mutations using PCR-single strand conformation polymorphism (PCR-SSCP) procedures in 523 Serra da Estrela ewes and were found to be highly polymorphic. The region extending across and between the GH2-N and GH2-Z copies was sequenced allowing the design of primers for the specific PCR amplification of each copy. These were cloned and sequenced in 20 animals representative of all SSCP patterns. The corresponding genotypes were established for each copy following nucleotide sequencing of SSCP alleles. Twenty-four polymorphic sites were found at the GH2-N (or GH1) and fourteen at the GH2-Z copies. Eight amino acid substitutions were predicted at the GH2-N and six at the GH2-Z copies. Milk yield adjusted to 150 lactation days was analysed for the genotype of each oGH gene copy taken separately or together (associated genotypes) by restricted maximum likelihood (REML) through a univariate best linear unbiased prediction (BLUP) animal model with repeated measures. Significant associations between genotypes and milk yield were observed. Within GH2-N genotypes there was a milk yield differential of 21.4+/-0.2 l/150 d between the most (N7) and the least (N5) productive ones. Within GH2-Z genotypes there was a differential of 21.6+/-0.2 l/150 d between the most (Z8) and the least (Z1) productive ones. The effect of associated GH2-N and GH2-Z genotypes revealed a differential of 39.6+/-0.3 l/150 d between the most (N1+Z7) and the least (N3+Z2) productive associated genotypes. The results show that GH2-N and GH2-Z genotypes significantly affect milk yield in Serra da Estrela ewes. Moreover, the apparent joint effect of GH2-N and GH2-Z genotype could improve milk yield in 25% as compared with the mean milk production of the analysed population.

Ovis aries POU1F1 gene: cloning, characterization and polymorphism analysis.

POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed 'Churra da Terra Quente'. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.

Crystal structures of the free and sterol-bound forms of beta-cinnamomin.

The crystal structure of the elicitin beta-cinnamomin (beta-CIN) was determined in complex with ergosterol at 1.1 A resolution. beta-CIN/ergosterol complex crystallized in the monoclinic space group P2(1), with unit cell parameters of a = 31.0, b = 62.8, c = 50.0 A and beta = 93.4 degrees and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 A structure of beta-CIN (P4(3)2(1)2 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of beta-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to beta-CIN provided an explanation for the apparent inability of beta-CIN to bind this ligand, as observed experimentally.

Structure of beta-cinnamomin, a protein toxic to some plant species.

Phytophthora and Pythium species are among the most aggressive plant pathogens, as they invade many economically important crops and forest trees. They secrete large amounts of 10 kDa proteins called elicitins that can act as elicitors of plant defence mechanisms. These proteins may also induce a hypersensitive response (HR) including plant cell necrosis, with different levels of toxicity depending on their pI. Recent studies showed that elicitins function as sterol carrier proteins. The crystallographic structure of the highly necrotic recombinant beta-cinnamomin (beta-CIN) from Phytophthora cinnamomi has been determined at 1.8 A resolution using the molecular-replacement method. beta-CIN has the same overall structure as beta-cryptogein (beta-CRY), an elicitin secreted by Phytophthora cryptogea, although it shows a different surface electrostatic potential distribution. The protein was expressed in Pichia pastoris and crystallized in the triclinic space group with two monomers in the asymmetric unit. The interface formed by these two monomers resembles that from beta-CRY dimer, although with fewer interactions.