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.

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.

Comparative Genomics of Eight Fusarium graminearum Strains with Contrasting Aggressiveness Reveals an Expanded Open Pangenome and Extended Effector Content Signatures.

Fusarium graminearum, the primary cause of Fusarium head blight (FHB) in small-grain cereals, demonstrates remarkably variable levels of aggressiveness in its host, producing different infection dynamics and contrasted symptom severity. While the secreted proteins, including effectors, are thought to be one of the essential components of aggressiveness, our knowledge of the intra-species genomic diversity of F. graminearum is still limited. In this work, we sequenced eight European F. graminearum strains of contrasting aggressiveness to characterize their respective genome structure, their gene content and to delineate their specificities. By combining the available sequences of 12 other F. graminearum strains, we outlined a reference pangenome that expands the repertoire of the known genes in the reference PH-1 genome by 32%, including nearly 21,000 non-redundant sequences and gathering a common base of 9250 conserved core-genes. More than 1000 genes with high non-synonymous mutation rates may be under diverse selection, especially regarding the trichothecene biosynthesis gene cluster. About 900 secreted protein clusters (SPCs) have been described. Mostly localized in the fast sub-genome of F. graminearum supposed to evolve rapidly to promote adaptation and rapid responses to the host's infection, these SPCs gather a range of putative proteinaceous effectors systematically found in the core secretome, with the chloroplast and the plant nucleus as the main predicted targets in the host cell. This work describes new knowledge on the intra-species diversity in F. graminearum and emphasizes putative determinants of aggressiveness, providing a wealth of new candidate genes potentially involved in the Fusarium head blight disease.

Proteomics-Based Data Integration of Wheat Cultivars Facing Fusarium graminearum Strains Revealed a Core-Responsive Pattern Controlling Fusarium Head Blight.

Fusarium head blight (FHB), mainly occurring upon Fusarium graminearum infection in a wide variety of small-grain cereals, is supposed to be controlled by a range of processes diverted by the fungal pathogen, the so-called susceptibility factors. As a mean to provide relevant information about the molecular events involved in FHB susceptibility in bread wheat, we studied an extensive proteome of more than 7,900 identified wheat proteins in three cultivars of contrasting susceptibilities during their interaction with three F. graminearum strains of different aggressiveness. No cultivar-specific proteins discriminated the three wheat genotypes, demonstrating the establishment of a core proteome regardless of unequivocal FHB susceptibility differences. Quantitative protein analysis revealed that most of the FHB-induced molecular adjustments were shared by wheat cultivars and occurred independently of the F. graminearum strain aggressiveness. Although subtle abundance changes evidenced genotype-dependent responses to FHB, cultivar distinction was found to be mainly due to basal abundance differences, especially regarding the chloroplast functions. Integrating these data with previous proteome mapping of the three F. graminearum strains facing the three same wheat cultivars, we demonstrated strong correlations between the wheat protein abundance changes and the adjustments of fungal proteins supposed to interfere with host molecular functions. Together, these results provide a resourceful dataset that expands our understanding of the specific molecular events taking place during the wheat-F. graminearum interaction.

Chromatin phosphoproteomics unravels a function for AT-hook motif nuclear localized protein AHL13 in PAMP-triggered immunity.

In many eukaryotic systems during immune responses, mitogen-activated protein kinases (MAPKs) link cytoplasmic signaling to chromatin events by targeting transcription factors, chromatin remodeling complexes, and the RNA polymerase machinery. So far, knowledge on these events is scarce in plants and no attempts have been made to focus on phosphorylation events of chromatin-associated proteins. Here we carried out chromatin phosphoproteomics upon elicitor-induced activation of Arabidopsis The events in WT were compared with those in mpk3, mpk4, and mpk6 mutant plants to decipher specific MAPK targets. Our study highlights distinct signaling networks involving MPK3, MPK4, and MPK6 in chromatin organization and modification, as well as in RNA transcription and processing. Among the chromatin targets, we characterized the AT-hook motif containing nuclear localized (AHL) DNA-binding protein AHL13 as a substrate of immune MAPKs. AHL13 knockout mutant plants are compromised in pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species production, expression of defense genes, and PAMP-triggered immunity. Transcriptome analysis revealed that AHL13 regulates key factors of jasmonic acid biosynthesis and signaling and affects immunity toward Pseudomonas syringae and Botrytis cinerea pathogens. Mutational analysis of the phosphorylation sites of AHL13 demonstrated that phosphorylation regulates AHL13 protein stability and thereby its immune functions.

Searching for FHB Resistances in Bread Wheat: Susceptibility at the Crossroad.

Fusarium head blight (FHB), primarily caused by Fusarium graminearum, is one of the most devastating fungal wheat diseases. During the past decades, many efforts have been deployed to dissect FHB resistance, investigating both the wheat responses to infection and, more recently, the fungal determinants of pathogenicity. Although no total resistance has been identified so far, they demonstrated that some plant functions and the expression of specific genes are needed to promote FHB. Associated with the increasing list of F. graminearum effectors able to divert plant molecular processes, this fact strongly argues for a functional link between susceptibility-related factors and the fate of this disease in wheat. In this review, we gather more recent data concerning the involvement of plant and fungal genes and the functions and mechanisms in the development of FHB susceptibility, and we discuss the possibility to use them to diversify the current sources of FHB resistance.

Time-resolved dissection of the molecular crosstalk driving Fusarium head blight in wheat provides new insights into host susceptibility determinism.

Fungal plant diseases are controlled by a complex molecular dialogue that involves pathogen effectors able to manipulate plant susceptibility factors at the earliest stages of the interaction. By probing the wheat-Fusarium graminearum pathosystem, we profiled the coregulations of the fungal and plant proteins shaping the molecular responses of a 96-hr-long infection's dynamics. Although no symptoms were yet detectable, fungal biomass swiftly increased along with an extremely diverse set of secreted proteins and candidate effectors supposed to target key plant organelles. Some showed to be early accumulated during the interaction or already present in spores, otherwise stored in germinating spores and detectable in an in vitro F. graminearum exudate. Wheat responses were swiftly set up and were evidenced before any visible symptom. Significant wheat protein abundance changes co-occurred along with the accumulation of putative secreted fungal proteins and predicted effectors. Regulated wheat proteins were closely connected to basal cellular processes occurring during spikelet ontogeny, and particular coregulation patterns were evidenced between chloroplast proteins and fungal proteins harbouring a predicted chloroplast transit peptide. The described plant and fungal coordinated responses provide a resourceful set of data and expand our understanding of the wheat-F. graminearum interaction.

Unbalanced Roles of Fungal Aggressiveness and Host Cultivars in the Establishment of the Fusarium Head Blight in Bread Wheat.

Fusarium head blight (FHB), caused mainly by Fusarium graminearum, is the foremost destructive disease of cereals worldwide. Effector-like molecules produced by F. graminearum play key roles in the infection process and are assumed to be one of the essential components of the pathogen's aggressiveness. However, their nature and role in the disease are still largely misunderstood. As a mean to provide relevant information about the molecular determinism of F. graminearum aggressiveness, we surveyed three F. graminearum strains on three wheat cultivars contrasted by their susceptibility to FHB. F. graminearum strains revealed large differences in aggressiveness which were mostly unchanged when facing hosts of contrasted susceptibility, suggesting that their behavior rely on intrinsic determinants. Surveying the fungal mass progress and the mycotoxin production rate in the spikes did not evidence any simple relationship with aggressiveness differences, while clues were found through a qualitative and quantitative characterization of the three strain proteomes established in planta especially with regards to early synthesized putative effectors. Independently of the wheat cultivar, the three F. graminearum strains produced systematically the same protein set during the infection but substantial differences in their abundance enabled the categorization of fungal aggressiveness. Overall, our findings show that the contrasts in F. graminearum aggressiveness were not based on the existence of strain-specific molecules but rather on the ability of the strain to ensure their sufficient accumulation. Protein abundance variance was mostly driven by the strain genetics and part was also influenced by the host cultivar but strain by cultivar interactions were marginally detected, depicting that strain-specific protein accumulations did not depend on the host cultivar. All these data provide new knowledge on fungal aggressiveness determinants and provide a resourceful repertoire of candidate effector proteins to guide further research.

Genome Sequence of Fusarium graminearum Strain MDC_Fg1, Isolated from Bread Wheat Grown in France.

Fusarium graminearum is a major fungal pathogen that induces Fusarium head blight (FHB), a devastating disease of small-grain cereals worldwide. This announcement provides the whole-genome sequence of a highly virulent and toxin-producing French isolate, MDC_Fg1.

Quantitative Phosphoproteomic Analysis Reveals Shared and Specific Targets of Arabidopsis Mitogen-Activated Protein Kinases (MAPKs) MPK3, MPK4, and MPK6.

In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4, and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. Although some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4, and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. One hundred fifty-two peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar nonfermenting (SnRK) protein kinases.

Phosphoproteomic Analysis of Isolated Mitochondria in Yeast.

Mitochondria play a central role in cellular energy metabolism and cell death. Deregulation of mitochondrial functions is associated with several human pathologies (neurodegenerative diseases, neuromuscular diseases, type II diabetes, obesity, cancer). The steadily increasing number of identified mitochondrial phosphoproteins, kinases, and phosphatases in recent years suggests that reversible protein phosphorylation plays an important part in the control of mitochondrial processes. In addition, many mitochondrial phosphoproteins probably still remain to be identified, considering that 30% of proteins are expected to be phosphorylated in eukaryotes. In this chapter, we describe two procedures for the analysis of the mitochondrial phosphoproteome. The first one is a qualitative method that combines blue native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D-BN/SDS-PAGE) and specific phosphoprotein staining. The second one is a quantitative approach that associates mitochondrial peptide labeling, phosphopeptide enrichment, and mass spectrometry.

X!TandemPipeline: A Tool to Manage Sequence Redundancy for Protein Inference and Phosphosite Identification.

X!TandemPipeline is a software designed to perform protein inference and to manage redundancy in the results of phosphosite identification by database search. It provides the minimal list of proteins or phosphosites that are present in a set of samples using grouping algorithms based on the principle of parsimony. Regarding proteins, a two-level classification is performed, where groups gather proteins sharing at least one peptide and subgroups gather proteins that are not distinguishable according to the identified peptides. Regarding phosphosites, an innovative approach based on the concept of phosphoisland is used to gather overlapping phosphopeptides. The graphical interface of X!TandemPipeline allows the users to launch X!tandem identification, to inspect spectra and to manually validate their assignment to peptides, to launch the grouping program, and to visualize elementary data as well as grouping and redundancy information. Identification results obtained from other search engines can also be processed. X!TandemPipeline results can be exported as ready-to-use tabulated files or as XML files that can be directly used by the PROTICdb database or by the MassChroQ quantification software. X!TandemPipeline runs fast, is easy to use, and can process hundreds of samples simultaneously. It is freely available under the GNU General Public License v3.0 at http://pappso.inra.fr/bioinfo/xtandempipeline/ .

Transcriptome dynamics of a susceptible wheat upon Fusarium head blight reveals that molecular responses to Fusarium graminearum infection fit over the grain development processes.

In many plant/pathogen interactions, host susceptibility factors are key determinants of disease development promoting pathogen growth and spreading in plant tissues. In the Fusarium head blight (FHB) disease, the molecular basis of wheat susceptibility is still poorly understood while it could provide new insights into the understanding of the wheat/Fusarium graminearum (Fg) interaction and guide future breeding programs to produce cultivars with sustainable resistance. To identify the wheat grain candidate genes, a genome-wide gene expression profiling was performed in the French susceptible wheat cultivar, Recital. Gene-specific two-way ANOVA of about 40 K transcripts at five grain developmental stages identified 1309 differentially expressed genes. Out of these, 536 were impacted by the Fg effect alone. Most of these Fg-responsive genes belonged to biological and molecular functions related to biotic and abiotic stresses indicating the activation of common stress pathways during susceptibility response of wheat grain to FHB. This analysis revealed also 773 other genes displaying either specific Fg-responsive profiles along with grain development stages or synergistic adjustments with the grain development effect. These genes were involved in various molecular pathways including primary metabolism, cell death, and gene expression reprogramming. An increasingly complex host response was revealed, as was the impact of both Fg infection and grain ontogeny on the transcription of wheat genes. This analysis provides a wealth of candidate genes and pathways involved in susceptibility responses to FHB and depicts new clues to the understanding of the susceptibility determinism in plant/pathogen interactions.

Cross-talk in host-parasite associations: What do past and recent proteomics approaches tell us?

A cross-talk in host-parasite associations begins when a host encounters a parasite. For many host-parasite relationships, this cross-talk has been taking place for hundreds of millions of years. The co-evolution of hosts and parasites, the familiar 'arms race' results in fascinating adaptations. Over the years, host-parasite interactions have been studied extensively from both the host and parasitic point of view. Proteomics studies have led to new insights into host-parasite cross-talk and suggest that the molecular strategies used by parasites attacking animals and plants share many similarities. Likewise, animals and plants use several common molecular tactics to counter parasite attacks. Based on proteomics surveys undertaken since the post-genomic era, a synthesis is presented on the molecular strategies used by intra- and extracellular parasites to invade and create the needed habitat for growth inside the host, as well as strategies used by hosts to counter these parasite attacks. Pitfalls in deciphering host-parasite cross-talk are also discussed. To conclude, helpful advice is given with regard to new directions that are needed to discover the generic and specific molecular strategies used by the host against parasite invasion as well as by the parasite to invade, survive, and grow inside their hosts, and to finally discover parasitic molecular signatures associated with their development.

A proteomics survey on wheat susceptibility to Fusarium head blight during grain development.

The mycotoxigenic fungal species Fusarium graminearum is able to attack several important cereal crops, such as wheat and barley. By causing Fusarium Head Blight (FHB) disease, F. graminearum induces yield and quality losses and poses a public health concern due to in planta mycotoxin production. The molecular and physiological plant responses to FHB, and the cellular biochemical pathways used by F. graminearum to complete its infectious process remain still unknown. In this study, a proteomics approach, combining 2D-gel approach and mass spectrometry, has been used to determine the specific protein patterns associated with the development of the fungal infection during grain growth on susceptible wheat. Our results reveal that F. graminearum infection does not deeply alter the grain proteome and does not significantly disturb the first steps of grain ontogeny but impacts molecular changes during the grain filling stage (impact on starch synthesis and storage proteins). The differentially regulated proteins identified were mainly involved in stress and defence mechanisms, primary metabolism, and main cellular processes such as signalling and transport. Our survey suggests that F. graminearum could take advantage of putative susceptibility factors closely related to grain development processes and thus provide new insights into key molecular events controlling the susceptible response to FHB in wheat grains.

Proteomic and phosphoproteomic analyses of chromatin-associated proteins from Arabidopsis thaliana.

The nucleus is the organelle where basically all DNA-related processes take place in eukaryotes, such as replication, transcription, and splicing as well as epigenetic regulation. The identification and description of the nuclear proteins is one of the requisites toward a comprehensive understanding of the biological functions accomplished in the nucleus. Many of the regulatory mechanisms of protein functions rely on their PTMs among which phosphorylation is probably one of the most important properties affecting enzymatic activity, interaction with other molecules, localization, or stability. So far, the nuclear and subnuclear proteome and phosphoproteome of the model plant Arabidopsis thaliana have been the subject of very few studies. In this work, we developed a purification protocol of Arabidopsis chromatin-associated proteins and performed proteomic and phosphoproteomic analyses identifying a total of 879 proteins of which 198 were phosphoproteins that were mainly involved in chromatin remodeling, transcriptional regulation, and RNA processing. From 230 precisely localized phosphorylation sites (phosphosites), 52 correspond to hitherto unidentified sites. This protocol and data thereby obtained should be a valuable resource for many domains of plant research.

Quantitative variations of the mitochondrial proteome and phosphoproteome during fermentative and respiratory growth in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a facultative aerobe able to adapt its metabolism according to the carbon substrate. The mechanisms of these adaptations involve at least partly the mitochondria but are not yet well understood. To address the possible role of protein phosphorylation event in their regulation, it is necessary in a first instance to determine precisely the phosphorylation sites that show changes depending on the carbon source. In this aim we performed an overall quantitative proteomic and phosphoproteomic study of isolated mitochondria extracted from yeast grown on fermentative (glucose or galactose) and respiratory (lactate) media. Label free quantitative analysis of protein accumulation revealed significant variation of 176 mitochondrial proteins including 108 proteins less accumulated in glucose medium than in lactate and galactose media. We also showed that the responses to galactose and glucose are not similar. Stable isotope dimethyl labeling allowed the quantitative comparison of phosphorylation levels between the different growth conditions. This study enlarges significantly the map of yeast mitochondrial phosphosites as 670 phosphorylation sites were identified, of which 214 were new and quantified. Above all, we showed that 90 phosphosites displayed a significant variation according to the medium and that variation of phosphorylation level is site-dependent. BIOLOGICAL SIGNIFICANCE: This proteomic and phosphoproteomic study is the first extensive study providing quantitative data on phosphosites responses to different carbon substrates independent of the variations of protein quantities in the yeast S. cerevisiae mitochondria. The significant changes observed in the level of phosphorylation according to the carbon substrate open the way to the study of the regulation of mitochondrial proteins by phosphorylation in fermentative and respiratory media. In addition, the identification of a large number of new phosphorylation sites show that the characterization of mitochondrial phosphoproteome is not yet completed.

Identification of novel PAMP-triggered phosphorylation and dephosphorylation events in Arabidopsis thaliana by quantitative phosphoproteomic analysis.

Signaling cascades rely strongly on protein kinase-mediated substrate phosphorylation. Currently a major challenge in signal transduction research is to obtain high confidence substrate phosphorylation sites and assign them to specific kinases. In response to bacterial flagellin, a pathogen-associated molecular pattern (PAMP), we searched for rapidly phosphorylated proteins in Arabidopsis thaliana by combining multistage activation (MSA) and electron transfer dissociation (ETD) fragmentation modes, which generate complementary spectra and identify phosphopeptide sites with increased reliability. Of a total of 825 phosphopeptides, we identified 58 to be differentially phosphorylated. These peptides harbor kinase motifs of mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs), as well as yet unknown protein kinases. Importantly, 12 of the phosphopeptides show reduced phosphorylation upon flagellin treatment. Since protein abundance levels did not change, these results indicate that flagellin induces not only various protein kinases but also protein phosphatases, even though a scenario of inhibited kinase activity may also be possible.