Global characterization of the Quercus suber ectomycorrhizal transcriptome using 454 pyrosequencing

In temperate forests, trees live in symbiosis with fungi and depend on them for their survival. Species from the Fagaceae family, including oaks, establish a symbiotic relationship in their roots with basidiomycetous fungi, called ectomycorrhizas (ECM). ECM symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with well developed ECM root tips are more tolerant to environmental stresses, such as drought, and iotic stresses such as root pathogens. There is a general agreement that ECM fungi increase plant survival and productivity. Cork oak is well adapted to water scarcity typical of the Mediterranean summer, due to a root system that can reach several metres in depth, and to the abundance of roots at the soil surface associated with ECM. The development of ECM symbiosis is characterized by the successive development of three structural components: a mantle of fungal tissue that encloses the root, the intraradical network of hyphae (Hartig net), where plant and fungus exchange metabolites, and the extraradical mycelium, which extends into the soil and is responsible for nutrient and water uptake. All these processes are highly regulated and are accompanied by alterations on gene expression in both partners. The current project is integrated in the effort for covering the transcriptome of Quercus suber and is focused on the identification of ESTs involved in the development of ECM symbiosis in cork oak. Since only 236 ESTs from Quercus suber are currently known, the use of genomics approaches for gene discovery or functional studies is far from being achieved for this plant species.This work was supported by the FCT project SOBREIRO/0034/200

Organogenic nodule development in hop (Humulus lupulus L.): Transcript and metabolic responses

Plant science

Oak protein profile alterations upon root colonization by an ectomycorrhizal fungus

An increased knowledge on the real impacts of ectomycorrhizal symbiosis in forest 32 species is needed to optimize forest sustainable productivity and thus to improve forests 33 services and their capacity to act as carbon sinks. In this study we investigated the 34 response of an oak species to ectomycorrhizae formation using a proteomics approach 35 complemented by biochemical analysis of carbohydrates levels. Comparative proteome 36 analysis between mycorrhizal and non-mycorrhizal cork oak plants revealed no 37 differences at the foliar level. However, the protein profile of 34 unique oak proteins 38 was altered in the roots. Consistent with the results of the biochemical analysis, the 39 proteome analysis of the mycorrhizal roots suggests a decreasing utilization of sucrose 40 for the metabolic activity of mycorrhizal roots which is consistent with an increased 41 allocation of carbohydrates from the plant to the fungus in order to sustain the 42 symbiosis. In addition, a promotion of protein unfolding mechanisms, attenuation of 43 defense reactions, increased nutrient mobilization from the plant-fungus interface (N 44 and P), as well as cytoskeleton rearrangements and induction of plant cell wall 45 loosening for fungal root accommodation in colonized roots, are also suggested by the 46 results. The suggested improvement in root capacity to take up nutrients accompanied 47 by an increase of root biomass without apparent changes in aboveground biomass 48 strongly re-enforce the potential of mycorrizal inoculation to improve cork oak forest 49 resistance capacity to cope with coming climate change

Characterization of the transcriptional signatures associated with resistance and susceptibility to Hemileia vastatrix in the Kawisari coffee hybrid

Coffee leaf rust (CLR), a disease caused by the biotrophic fungus Hemileia vastatrix (Hv), is the main threat to the worldwide production of Arabica coffee. The gradual breakdown of resistance in coffee varieties in the last years has highlighted the need for novel sources of resistance to CLR. This work aimed to unveil the cellular and molecular resistance profile of the Kawisari hybrid (C. arabica x C. liberica), a genotype used as a resistance donor in Arabica breeding programs in India. This coffee genotype was inoculated with two Hv races that triggered either resistance or susceptibility. Progress of infection was monitored using light microscopy. Simultaneously, we conducted a time-course RNA-seq characterization of the transcriptional responses. The microscopic studies showed that the post-haustorial resistance of Kawisari was associated with the hypersensitive response, accumulation of phenolic-like compounds and haustorium encasement with callose. The transcriptomic analysis suggest the downregulation of host primary metabolism genes at the early onset of infection, followed later by activation of genes functionally associated with multiple plant defense responses, including salicylic acid and jasmonate hormonal signaling. Resistance was also accompanied by the differential regulation of genes associated with phenylpropanoid metabolism and lignin biosynthesis. Our results, further validated by qPCR, provide important new insight into the molecular mechanisms underpinning resistance against CLR in this coffee genotype.Foundation for Science and Technology (FCT) and FEDER funds through PORNorte under the project CoffeeRES PTDC/ASPPLA/ 29779/2017 and by FCT UNIT LEAF (UID/AGR/04129/2020).info:eu-repo/semantics/publishedVersio

Oak root response to ectomycorrhizal symbiosis establishment: RNA-Seq derived transcript identification and expression profiling

Ectomycorrhizal symbiosis is essential for the life and health of trees in temperate and boreal forests where it plays a major role in nutrient cycling and in functioning of the forest ecosystem. Trees with ectomycorrhizal root tips are more tolerant to environmental stresses, such as drought, and biotic stresses such as root pathogens. Detailed information on these molecular processes is essential for the understanding of symbiotic tissue development in order to optimize the benefits of this natural phenomenon. Next generation sequencing tools allow the analysis of non model ectomycorrhizal plant-fungal interactions that can contribute to find the "symbiosis toolkits" and better define the role of each partner in the mutualistic interaction. By using 454 pyrosequencing we compared ectomycorrhizal cork oak roots with non-symbiotic roots. From the two cDNA libraries sequenced, over 2 million reads were obtained that generated 19,552 cork oak root unique transcripts. A total of 2238 transcripts were found to be differentially expressed when ECM roots were compared with non-symbiotic roots. Identification of up- and down-regulated gens in ectomycorrhizal roots lead to a number of insights into the molecular mechanisms governing this important symbiosis. In cork oak roots, ectomycorrhizal colonization resulted in extensive cell wall remodelling, activation of the secretory pathway, alterations in flavonoid biosynthesis, and expression of genes involved in the recognition of fungal effectors. In addition, we identified genes with putative roles in symbiotic processes such as nutrient exchange with the fungal partner, lateral root formation or root hair decay. These findings provide a global overview of the transcriptome of an ectomycorrhizal host root, and constitute a foundation for future studies on the molecular events controlling this important symbiosis.This work was funded by the Portuguese Foundation for Science and Technology (www.fct.pt) in the frame of the project Cork Oak EST Consortium SOBREIRO/0034/2009. Post-doc grant to MS was supported by the Portuguese Foundation for Science and Technology (SFRH/BPD/25661/2005). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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.Peer Reviewe

A possible approach for gel-based proteomic studies in recalcitrant woody plants

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry

New advances on ectomycorrhizal establishment : learning from transcriptome and metabolome analysis

Ectomycorrhiza formation encompasses changes in soil fungi and vascular plants roots. Host plants metabolites released to the rhizosphere trigger spore germination, hyphae growth towards the root inducing mycorrhiza formation. Plant and fungal genetic switches necessary for ECM development remain unidentified and little is known on the initial contact phase. The model system studied is the mycorrhizal association Castanea sativa / Pisolithus tinctorius and Quercus suber / Pisolithus tinctorius. The mycorrhiza formation followed two different methods. The association C. sativa / P. tinctorius mycorrhization was induced in liquid medium as previously described (Sebastiana et al. 2009), while that of Q. suber / P. tinctorius was induced as follows:- (1) Pure culture of P. tinctorius was obtained from a sporocarp collected at the base of a cork oak tree (Minas de Aljustrel, Alentejo), development of mycorrhizal inoculum for maximum biomass production in a carrier substrate (peatmoss and vermiculite)inoculation of Q. suber seedlings in the greenhouse by transferring the roots to inoculated potting substrate. 4 months after P. tinctorius inoculation, 100% of the plants were mycorrhized with an average mycorrhized root length of 46%. Metabolome profiling of C. sativa / P. tinctorius revealed metabolites crucial for mycorrhiza formation, at the plant level:- stress and defence related amino acids (proline, alanine and glycine), cell wall synthesis and degradation related metabolites; saccharides (glucose, fructose and sucrose); galacturonic acid (indicator of cell wall destruction); aromatic amino acids and signalling related metabolites (myo-inositol, polyamines, galactinol) and at the fungi level:- trehalose-fungi (marker of fungi presence); gluconic acid (promoter of ectomycorrhizae formation); glutamine (marker of mantle and Hartig net formation). Transcriptome profiling revealed, up regulation of genes encoding for stress and defence response proteins (cystatin, cystatin-like protein, defensin and a universal stress protein). Early contact with the fungal mycelium altered expression of genes putatively involved in cellular processes:- signal transduction and communication (receptor kinase-related protein), protein fate (papain-like cystein proteinase) and water transport facilitation (water channel MipK protein). The transcriptome of mycorrhizal cork oak association revealed up-regulation of ACX1 (Acyl-CoA Oxidase), CYP74 (Allene oxide synthase), Allene Oxide Synthase, OPR3 (12-Oxophytodienoate reductase 3), OPCL1 (4-Coumarate-CoA Ligase), Fatty Acid Desaturase, Ethylene Transcription Factor, Hydroxyacyl-CoA Dehydrogenase, Acyl-CoA Oxidase, Omega-3 Fatty Acid Desaturase, Catalase, and of Long Chain Acyl-CoA Synthetase 4. Taking together the metabolome and transcriptome analysis in Chestnut and cork-oak mycorrhizal associations there is a strong evidence of an important signaling pathway. Results on the over expression of genes related to Jasmonic acid and proline biosynthesis are discussed. Expression profiling in early C. sativa / P. tinctorius contact revealed changes in gene expression occurring few hours after contact, long before developing a functional mycorrhiza. Induction of genes involved on stress and defense suggests a quick reaction of host plant to the mycobiont presence, eliciting a defence programme similar to that described for pathogenic interactions. Repression of genes related to water stress accounts for a water stress relief resulting from the initial contact with ectomycorrhizal fungus

Reference Gene Selection and Validation for the Early Responses to Downy Mildew Infection in Susceptible and Resistant <i>Vitis vinifera</i> Cultivars

<div><p>The pivotal role of cultivated grapevine (<i>Vitis vinifera</i> L.) in many countries economy is compromised by its high susceptibility to <i>Plasmopara viticola</i>, the causal agent of downy mildew disease. Recent research has identified a set of genes related to resistance which may be used to track downy mildew infection. Quantification of the expression of these resistance genes requires normalizing qPCR data using reference genes with stable expression in the system studied. In this study, a set of eleven genes (<i>VATP16</i>, <i>60 S</i>, <i>UQCC</i>, <i>SMD3</i>, <i>EF1α</i>, <i>UBQ</i>, <i>SAND</i>, <i>GAPDH</i>, <i>ACT</i>, <i>PsaB</i>, <i>PTB2</i>) was evaluated to identify reference genes during the first hours of interaction (6, 12, 18 and 24 hpi) between two <i>V. vinifera</i> genotypes and <i>P. viticola</i>. Two analyses were used for the selection of reference genes: direct comparison of susceptible, Trincadeira, and resistant, Regent, <i>V. vinifera</i> cultivars at 0 h, 6, 12, 18 and 24 hours post inoculation with <i>P. viticola</i> (genotype effect); and comparison of each genotype with mock inoculated samples during inoculation time-course (biotic stress effect). Three statistical methods were used, GeNorm, NormFinder, and BestKeeper, allowing to identify <i>UBQ</i>, <i>EF1α</i> and <i>GAPDH</i> as the most stable genes for the genotype effect. For the biotic stress effect, <i>EF1α</i>, <i>SAND</i> and <i>SMD3</i> were the most constant for the susceptible cultivar Trincadeira and <i>EF1α</i>, <i>GAPDH</i>, <i>UBQ</i> for the resistant cultivar Regent. In addition, the expression of three defense-related transcripts, encoding for subtilisin-like protein, <i>CYP</i> and <i>PR10</i>, was analysed, for both datasets, during inoculation time-course. Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the first hours of interaction between different grapevine cultivars and <i>P. viticola</i>.</p></div