Integrative systems biology of wheat susceptibility to Fusarium graminearum uncovers a conserved gene regulatory network and identifies master regulators targeted by fungal core effectors.

BACKGROUND: Plant diseases are driven by an intricate set of defense mechanisms counterbalanced by the expression of host susceptibility factors promoted through the action of pathogen effectors. In spite of their central role in the establishment of the pathology, the primary components of plant susceptibility are still poorly understood and challenging to trace especially in plant-fungal interactions such as in Fusarium head blight (FHB) of bread wheat. Designing a system-level transcriptomics approach, we leveraged the analysis of wheat responses from a susceptible cultivar facing Fusarium graminearum strains of different aggressiveness and examined their constancy in four other wheat cultivars also developing FHB. RESULTS: In this study, we describe unexpected differential expression of a conserved set of transcription factors and an original subset of master regulators were evidenced using a regulation network approach. The dual-integration with the expression data of pathogen effector genes combined with database mining, demonstrated robust connections with the plant molecular regulators and identified relevant candidate genes involved in plant susceptibility, mostly able to suppress plant defense mechanisms. Furthermore, taking advantage of wheat cultivars of contrasting susceptibility levels, a refined list of 142 conserved susceptibility gene candidates was proposed to be necessary host's determinants for the establishment of a compatible interaction. CONCLUSIONS: Our findings emphasized major FHB determinants potentially controlling a set of conserved responses associated with susceptibility in bread wheat. They provide new clues for improving FHB control in wheat and also could conceivably leverage further original researches dealing with a broader spectrum of plant pathogens.

Transcriptomic monitoring of Douglas-fir heartwood formation.

OBJECTIVES: Molecular cues linked to heartwood formation open new (complementary) perspectives to genetic breeding programs of Douglas-fir, a tree species largely cultivated in Europe for the natural durability and civil engineering properties of its wood. DATA DESCRIPTION: RNAs from a single genotype of Douglas-fir, extracted from three distinct wood zones (outer sapwood, inner sapwood and transition zone) at four vegetative seasons to generate an extensive RNA-seq dataset used to apprehend the in-wood dynamic and seasonality of heartwood formation in this hardwood model species. Previously published data collected on somatic embryos of the same genotype could be merged with the present dataset to upgrade grade the Douglas-fir reference transcriptome.

Assessment of Tunisian Trichoderma Isolates on Wheat Seed Germination, Seedling Growth and Fusarium Seedling Blight Suppression.

Beneficial microorganisms, including members of the Trichoderma genus, are known for their ability to promote plant growth and disease resistance, as well as being alternatives to synthetic inputs in agriculture. In this study, 111 Trichoderma strains were isolated from the rhizospheric soil of Florence Aurore, an ancient wheat variety that was cultivated in an organic farming system in Tunisia. A preliminary ITS analysis allowed us to cluster these 111 isolates into three main groups, T. harzianum (74 isolates), T. lixii (16 isolates) and T. sp. (21 isolates), represented by six different species. Their multi-locus analysis (tef1, translation elongation factor 1; rpb2, RNA polymerase B) identified three T. afroharzianum, one T. lixii, one T. atrobrunneum and one T. lentinulae species. These six new strains were selected to determine their suitability as plant growth promoters (PGP) and biocontrol agents (BCA) against Fusarium seedling blight disease (FSB) in wheat caused by Fusarium culmorum. All of the strains exhibited PGP abilities correlated to ammonia and indole-like compound production. In terms of biocontrol activity, all of the strains inhibited the development of F. culmorum in vitro, which is linked to the production of lytic enzymes, as well as diffusible and volatile organic compounds. An in planta assay was carried out on the seeds of a Tunisian modern wheat variety (Khiar) by coating them with Trichoderma. A significant increase in biomass was observed, which is associated with increased chlorophyll and nitrogen. An FSB bioprotective effect was confirmed for all strains (with Th01 being the most effective) by suppressing morbid symptoms in germinated seeds and seedlings, as well as by limiting F. culmorum aggressiveness on overall plant growth. Plant transcriptome analysis revealed that the isolates triggered several SA- and JA-dependent defense-encoding genes involved in F. culmorum resistance in the roots and leaves of three-week-old seedlings. This finding makes these strains very promising in promoting growth and controlling FSB disease in modern wheat varieties.

Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome.

Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen's effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets.

Genome-Wide Identification, Structure Characterization, and Expression Pattern Profiling of the Aquaporin Gene Family in Betula pendula.

Aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life. They play key roles in the flux of water and many solutes across the membranes. The AQP diversity, protein features, and biological functions of silver birch are still unknown. A genome analysis of Betula pendula identified 33 putative genes encoding full-length AQP sequences (BpeAQPs). They are grouped into five subfamilies, representing ten plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). The BpeAQP gene structure is conserved within each subfamily, with exon numbers ranging from one to five. The predictions of the aromatic/arginine selectivity filter (ar/R), Froger's positions, specificity-determining positions, and 2D and 3D biochemical properties indicate noticeable transport specificities to various non-aqueous substrates between members and/or subfamilies. Nevertheless, overall, the BpePIPs display mostly hydrophilic ar/R selective filter and lining-pore residues, whereas the BpeTIP, BpeNIP, BpeSIP, and BpeXIP subfamilies mostly contain hydrophobic permeation signatures. Transcriptional expression analyses indicate that 23 BpeAQP genes are transcribed, including five organ-related expressions. Surprisingly, no significant transcriptional expression is monitored in leaves in response to cold stress (6 °C), although interesting trends can be distinguished and will be discussed, notably in relation to the plasticity of this pioneer species, B. pendula. The current study presents the first detailed genome-wide analysis of the AQP gene family in a Betulaceae species, and our results lay a foundation for a better understanding of the specific functions of the BpeAQP genes in the responses of the silver birch trees to cold stress.

Transcriptome profiling in susceptible and tolerant rubber tree clones in response to cassiicolin Cas1, a necrotrophic effector from Corynespora cassiicola.

Corynespora cassiicola, a fungal plant pathogen with a large host range, causes important damages in rubber tree (Hevea brasiliensis), in Asia and Africa. A small secreted protein named cassiicolin was previously identified as a necrotrophic effector required for the virulence of C. cassiicola in specific rubber tree clones. The objective of this study was to decipher the cassiicolin-mediated molecular mechanisms involved in this compatible interaction. We comparatively analyzed the RNA-Seq transcriptomic profiles of leaves treated or not with the purified cassiicolin Cas1, in two rubber clones: PB260 (susceptible) and RRIM600 (tolerant). The reads were mapped against a synthetic transcriptome composed of all available transcriptomic references from the two clones. Genes differentially expressed in response to cassiicolin Cas1 were identified, in each clone, at two different time-points. After de novo annotation of the synthetic transcriptome, we analyzed GO enrichment of the differentially expressed genes in order to elucidate the main functional pathways impacted by cassiicolin. Cassiicolin induced qualitatively similar transcriptional modifications in both the susceptible and the tolerant clones, with a strong negative impact on photosynthesis, and the activation of defense responses via redox signaling, production of pathogenesis-related protein, or activation of the secondary metabolism. In the tolerant clone, transcriptional reprogramming occurred earlier but remained moderate. By contrast, the susceptible clone displayed a late but huge transcriptional burst, characterized by massive induction of phosphorylation events and all the features of a hypersensitive response. These results confirm that cassiicolin Cas1 is a necrotrophic effector triggering a hypersensitive response in susceptible rubber clones, in agreement with the necrotrophic-effector-triggered susceptibility model.

Fungal X-Intrinsic Protein Aquaporin from Trichoderma atroviride: Structural and Functional Considerations.

The major intrinsic protein (MIP) superfamily is a key part of the fungal transmembrane transport network. It facilitates the transport of water and low molecular weight solutes across biomembranes. The fungal uncharacterized X-Intrinsic Protein (XIP) subfamily includes the full protein diversity of MIP. Their biological functions still remain fully hypothetical. The aim of this study is still to deepen the diversity and the structure of the XIP subfamily in light of the MIP counterparts-the aquaporins (AQPs) and aquaglyceroporins (AQGPs)-and to describe for the first time their function in the development, biomass accumulation, and mycoparasitic aptitudes of the fungal bioagent Trichoderma atroviride. The fungus-XIP clade, with one member (TriatXIP), is one of the three clades of MIPs that make up the diversity of T. atroviride MIPs, along with the AQPs (three members) and the AQGPs (three members). TriatXIP resembles those of strict aquaporins, predicting water diffusion and possibly other small polar solutes due to particularly wider ar/R constriction with a Lysine substitution at the LE2 position. The XIP loss of function in ∆TriatXIP mutants slightly delays biomass accumulation but does not impact mycoparasitic activities. ∆TriatMIP forms colonies similar to wild type; however, the hyphae are slightly thinner and colonies produce rare chlamydospores in PDA and specific media, most of which are relatively small and exhibit abnormal morphologies. To better understand the molecular causes of these deviant phenotypes, a wide-metabolic survey of the ∆TriatXIPs demonstrates that the delayed growth kinetic, correlated to a decrease in respiration rate, is caused by perturbations in the pentose phosphate pathway. Furthermore, the null expression of the XIP gene strongly impacts the expression of four expressed MIP-encoding genes of T. atroviride, a plausible compensating effect which safeguards the physiological integrity and life cycle of the fungus. This paper offers an overview of the fungal XIP family in the biocontrol agent T. atroviride which will be useful for further functional analysis of this particular MIP subfamily in vegetative growth and the environmental stress response in fungi. Ultimately, these findings have implications for the ecophysiology of Trichoderma spp. in natural, agronomic, and industrial systems.

Early transcriptional response to gravistimulation in poplar without phototropic confounding factors.

In response to gravistimulation under anisotropic light, tree stems showing an active cambium produce reaction wood that redirects the axis of the trees. Several studies have described transcriptomic or proteomic models of reaction wood relative to the opposite wood. However, the mechanisms leading to the formation of reaction wood are difficult to decipher because so many environmental factors can induce various signalling pathways leading to this developmental reprogramming. Using an innovative isotropic device where the phototropic response does not interfere with gravistimulation we characterized the early molecular responses occurring in the stem of poplar after gravistimulation in an isotropic environment, and without deformation of the stem. After 30 min tilting at 35° under anisotropic light, we collected the upper and lower xylems from the inclined stems. Controls were collected from vertical stems. We used a microarray approach to identify differentially expressed transcripts. High-throughput real-time PCR allowed a kinetic experiment at 0, 30, 120 and 180 min after tilting at 35°, with candidate genes. We identified 668 differentially expressed transcripts, from which we selected 153 candidates for additional Fluidigm qPCR assessment. Five candidate co-expression gene clusters have been identified after the kinetic monitoring of the expression of candidate genes. Gene ontology analyses indicate that molecular reprogramming of processes such as 'wood cell expansion', 'cell wall reorganization' and 'programmed cell death' occur as early as 30 min after gravistimulation. Of note is that the change in the expression of different genes involves a fine regulation of gibberellin and brassinosteroid pathways as well as flavonoid and phosphoinositide pathways. Our experimental set-up allowed the identification of genes regulated in early gravitropic response without the bias introduced by phototropic and stem bending responses.

Voltage-gating of aquaporins, a putative conserved safety mechanism during ionic stresses.

Aquaporins are transmembrane water channels found in almost every living organism. Numerous studies have brought a good understanding of both water transport through their pores and the regulations taking place at the molecular level, but subtleties remain to be clarified. Recently, a voltage-related gating mechanism involving the conserved arginine of the channel's main constriction was captured for human aquaporins through molecular dynamics studies. With a similar approach, we show that this voltage-gating could be conserved among this family and that the underlying mechanism could explain part of plant AQPs diversity when contextualized to high ionic concentrations provoked by drought. Finally, we identified residues as adaptive traits which constitute good targets for drought resistance plant breeding research.

Genome Wild Analysis and Molecular Understanding of the Aquaporin Diversity in Olive Trees (Olea Europaea L.).

Cellular aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life, playing important roles in the uptake of water and many solutes across the membranes. In olive trees, AQP diversity, protein features and their biological functions are still largely unknown. This study focuses on the structure and functional and evolution diversity of AQP subfamilies in two olive trees, the wild species Olea europaea var. sylvestris (OeuAQPs) and the domesticated species Olea europaea cv. Picual (OleurAQPs), and describes their involvement in different physiological processes of early plantlet development and in biotic and abiotic stress tolerance in the domesticated species. A scan of genomes from the wild and domesticated olive species revealed the presence of 52 and 79 genes encoding full-length AQP sequences, respectively. Cross-genera phylogenetic analysis with orthologous clustered OleaAQPs into five established subfamilies: PIP, TIP, NIP, SIP, and XIP. Subsequently, gene structures, protein motifs, substrate specificities and cellular localizations of the full length OleaAQPs were predicted. Functional prediction based on the NPA motif, ar/R selectivity filter, Froger's and specificity-determining positions suggested differences in substrate specificities of Olea AQPs. Expression analysis of the OleurAQP genes indicates that some genes are tissue-specific, whereas few others show differential expressions at different developmental stages and in response to various biotic and abiotic stresses. The current study presents the first detailed genome-wide analysis of the AQP gene family in olive trees and it provides valuable information for further functional analysis to infer the role of AQP in the adaptation of olive trees in diverse environmental conditions in order to help the genetic improvement of domesticated olive trees.

Cytological, Biochemical and Molecular Events of the Embryogenic State in Douglas-fir (Pseudotsuga menziesii [Mirb.]).

Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.

Repetitive somatic embryogenesis induced cytological and proteomic changes in embryogenic lines of Pseudotsuga menziesii [Mirb.].

BACKGROUND: To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2ry) and tertiary (3ry) lines from cotyledonary somatic embryos (SEs) of two Douglas-fir genotypes: SD4 and TD17. The 2ry lines exhibited significantly higher embryogenic potential (SE yields) than the 1ry lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g- 1 f.w.). Moreover, we observed similar differences in yield between 2ry and 3ry lines of SD4 (2400 vs 3921 g- 1 f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2ry vs 1ry and 2ry vs 3ry lines at histo-cytological (using LC-MS/MS) and proteomic levels. RESULTS: Repetitive somatic embryogenesis dramatically improved the proliferating lines' cellular organization (genotype SD4's most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2ry and (even more) 3ry lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2ry vs 1ry and 3ry vs 2ry lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2ry relative to 1ry lines, but up-regulated in 3ry relative to 2ry lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same "Plant development" and "Defense response" biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3ry lines. CONCLUSIONS: This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines' embryogenic status.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

MIP diversity from Trichoderma: Structural considerations and transcriptional modulation during mycoparasitic association with Fusarium solani olive trees.

Major intrinsic proteins (MIP) are characterized by a transmembrane pore-type architecture that facilitates transport across biomembranes of water and a variety of low molecular weight solutes. They are found in all parts of life, with remarkable protein diversity. Very little is known about MIP from fungi. And yet, it can legitimately be stated that MIP are pivotal molecular components in the privileged relationships fungi enjoy with plants or soil fauna in various environments. To date, MIP have never been studied in a mycoparasitism situation. In this study, the diversity, expression and functional prediction of MIP from the genus Trichoderma were investigated. Trichoderma spp. genomes have at least seven aquaporin genes. Based on a phylogenetic analysis of the translated sequences, members were assigned to the AQP, AQGP and XIP subfamilies. In in vitro and in planta assays with T. harzianum strain Ths97, expression analyses showed that four genes were constitutively expressed. In a mycoparasitic context with Fusarium solani, the causative agent of fusarium dieback on olive tree roots, these genes were up-regulated. This response is of particular interest in analyzing the MIP promoter cis-regulatory motifs, most of which are involved in various carbon and nitrogen metabolisms. Structural analyses provide new insights into the possible role of structural checkpoints by which these members transport water, H2O2, glycerol and, more generally, linear polyols across the membranes. Taken together, these results provide the first evidence that MIP may play a key role in Trichoderma mycoparasitism lifestyle.

Integrated drought responses of black poplar: how important is phenotypic plasticity?

Climate change is expected to increase drought frequency and intensity which will threaten plant growth and survival. In such fluctuating environments, perennial plants respond with hydraulic and biomass adjustments, resulting in either tolerant or avoidant strategies. Plants' response to stress relies on their phenotypic plasticity. The goal of this study was to explore physiology of young Populus nigra in the context of a time-limited and progressive water deficit in regard to their growth and stress response strategies. Fourteen French 1-year-old black poplar genotypes, geographically contrasted, were subjected to withholding water during 8 days until severe water stress. Water fluxes (i.e. leaf water potentials and stomatal conductance) were analyzed together with growth (i.e. radial and longitudinal branch growth, leaf senescence and leaf production). Phenotypic plasticity was calculated for each trait and response strategies to drought were deciphered for each genotype. Black poplar genotypes permanently were dealing with a continuum of adjusted water fluxes and growth between two extreme strategies, tolerance and avoidance. Branch growth, leaf number and leaf hydraulic potential traits had contrasted plasticities, allowing genotype characterization. The most tolerant genotype to water deficit, which maintained growth, had the lowest global phenotypic plasticity. Conversely, the most sensitive and avoidant genotype ceased growth until the season's end, had the highest plasticity level. All the remaining black poplar genotypes were close to avoidance with average levels of traits plasticity. These results underpinned the role of plasticity in black poplar response to drought and calls for its wider use into research on plants' responses to stress.

Genetic Determinism of Sensitivity to Corynespora cassiicola Exudates in Rubber Tree (Hevea brasiliensis).

An indirect phenotyping method was developed in order to estimate the susceptibility of rubber tree clonal varieties to Corynespora Leaf Fall (CLF) disease caused by the ascomycete Corynespora cassiicola. This method consists in quantifying the impact of fungal exudates on detached leaves by measuring the induced electrolyte leakage (EL%). The tested exudates were either crude culture filtrates from diverse C. cassiicola isolates or the purified cassiicolin (Cas1), a small secreted effector protein produced by the aggressive isolate CCP. The test was found to be quantitative, with the EL% response proportional to toxin concentration. For eight clones tested with two aggressive isolates, the EL% response to the filtrates positively correlated to the response induced by conidial inoculation. The toxicity test applied to 18 clones using 13 toxinic treatments evidenced an important variability among clones and treatments, with a significant additional clone x treatment interaction effect. A genetic linkage map was built using 306 microsatellite markers, from the F1 population of the PB260 x RRIM600 family. Phenotyping of the population for sensitivity to the purified Cas1 effector and to culture filtrates from seven C. cassiicola isolates revealed a polygenic determinism, with six QTL detected on five chromosomes and percentages of explained phenotypic variance varying from 11 to 17%. Two common QTL were identified for the CCP filtrate and the purified cassiicolin, suggesting that Cas1 may be the main effector of CCP filtrate toxicity. The CCP filtrate clearly contrasted with all other filtrates. The toxicity test based on Electrolyte Leakage Measurement offers the opportunity to assess the sensitivity of rubber genotypes to C. cassiicola exudates or purified effectors for genetic investigations and early selection, without risk of spreading the fungus in plantations. However, the power of this test for predicting field susceptibility of rubber clones to CLF will have to be further investigated.

The Hevea brasiliensis XIP aquaporin subfamily: genomic, structural and functional characterizations with relevance to intensive latex harvesting.

X-Intrinsic Proteins (XIP) were recently identified in a narrow range of plants as a full clade within the aquaporins. These channels reportedly facilitate the transport of a wide range of hydrophobic solutes. The functional roles of XIP in planta remain poorly identified. In this study, we found three XIP genes (HbXIP1;1, HbXIP2;1 and HbXIP3;1) in the Hevea brasiliensis genome. Comprehensive bioinformatics, biochemical and structural analyses were used to acquire a better understanding of this AQP subfamily. Phylogenetic analysis revealed that HbXIPs clustered into two major groups, each distributed in a specific lineage of the order Malpighiales. Tissue-specific expression profiles showed that only HbXIP2;1 was expressed in all the vegetative tissues tested (leaves, stem, bark, xylem and latex), suggesting that HbXIP2;1 could take part in a wide range of cellular processes. This is particularly relevant to the rubber-producing laticiferous system, where this isoform was found to be up-regulated during tapping and ethylene treatments. Furthermore, the XIP transcriptional pattern is significantly correlated to latex production level. Structural comparison with SoPIP2;1 from Spinacia oleracea species provides new insights into the possible role of structural checkpoints by which HbXIP2;1 ensures glycerol transfer across the membrane. From these results, we discuss the physiological involvement of glycerol and HbXIP2;1 in water homeostasis and carbon stream of challenged laticifers. The characterization of HbXIP2;1 during rubber tree tapping lends new insights into molecular and physiological response processes of laticifer metabolism in the context of latex exploitation.

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii).

BACKGROUND AND AIMS: In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of in vitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions in vitro. Embryogenesis in larch is well understood both in situ and in vitro and thus provides a suitable system for addressing this question. METHODS: Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis. KEY RESULTS: Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg(-1) f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g(-1) d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap. CONCLUSIONS: In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

Cotyledonary somatic embryos of Pinus pinaster Ait. most closely resemble fresh, maturing cotyledonary zygotic embryos: biological, carbohydrate and proteomic analyses.

Cotyledonary somatic embryos (SEs) of maritime pine are routinely matured for 12 weeks before being germinated and converted to plantlets. Although regeneration success is highly dependent on SEs quality, the date of harvesting is currently determined mainly on the basis of morphological features. This empirical method does not provide any accurate information about embryo quality with respect to storage compounds (proteins, carbohydrates). We first analyzed SEs matured for 10, 12 and 14 weeks by carrying out biological (dry weight, water content) and biochemical measurements (total protein and carbohydrate contents). No difference could be found between collection dates, suggesting that harvesting SEs after 12 weeks is appropriate. Cotyledonary SEs were then compared to various stages, from fresh to fully desiccated, in the development of cotyledonary zygotic embryos (ZEs). We identified profiles that were similar using hierarchical ascendant cluster analysis (HCA). Fresh and dehydrated ZEs could be distinguished, and SEs clustered with fresh ZEs. Both types of embryo exhibited similar carbohydrate and protein contents and signatures. This high level of similarity (94.5 %) was further supported by proteome profiling. Highly expressed proteins included storage, stress-related, late embryogenesis abundant and energy metabolism proteins. By comparing overexpressed proteins in developing and cotyledonary SEs or ZEs, some (23 proteins) could be identified as candidate biomarkers for the late, cotyledonary stage. This is the first report of useful generic protein markers for monitoring embryo development in maritime pine. Our results also suggest that improvements of SEs quality may be achieved if the current maturation conditions are refined.

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome [two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mass spectrometry (MS) identification] of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography-MS), soluble sugars (high-pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA-mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin-like protein and ubiquitin-protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.