The Adaptation of Botrytis cinerea Extracellular Vesicles Proteome to Surrounding Conditions: Revealing New Tools for Its Infection Process.

Extracellular vesicles (EVs) are membranous particles released by different organisms. EVs carry several sets of macromolecules implicated in cell communication. EVs have become a relevant topic in the study of pathogenic fungi due to their relationship with fungal-host interactions. One of the essential research areas in this field is the characterization protein profile of EVs since plant fungal pathogens rely heavily on secreted proteins to invade their hosts. However, EVs of Botrytis cinerea are little known, which is one of the most devastating phytopathogenic fungi. The present study has two main objectives: the characterization of B. cinerea EVs proteome changes under two pathogenic conditions and the description of their potential role during the infective process. All the experimental procedure was conducted in B. cinerea growing in a minimal salt medium supplemented with glucose as a constitutive stage and deproteinized tomato cell walls (TCW) as a virulence inductor. The isolation of EVs was performed by differential centrifugation, filtration, ultrafiltration, and sucrose cushion ultracentrifugation. EVs fractions were visualised by TEM using negative staining. Proteomic analysis of EVs cargo was addressed by LC-MS/MS. The methodology used allowed the correct isolation of B. cinerea EVs and the identification of a high number of EV proteins, including potential EV markers. The isolated EVs displayed differences in morphology under both assayed conditions. GO analysis of EV proteins showed enrichment in cell wall metabolism and proteolysis under TCW. KEGG analysis also showed the difference in EVs function under both conditions, highlighting the presence of potential virulence/pathogenic factors implicated in cell wall metabolism, among others. This work describes the first evidence of EVs protein cargo adaptation in B. cinerea, which seems to play an essential role in its infection process, sharing crucial functions with the conventional secretion pathways.

LysM-mediated signaling in Marchantia polymorpha highlights the conservation of pattern-triggered immunity in land plants.

Pattern-recognition receptor (PRR)-triggered immunity (PTI) wards off a wide range of pathogenic microbes, playing a pivotal role in angiosperms. The model liverwort Marchantia polymorpha triggers defense-related gene expression upon sensing components of bacterial and fungal extracts, suggesting the existence of PTI in this plant model. However, the molecular components of the putative PTI in M. polymorpha and the significance of PTI in bryophytes have not yet been described. We here show that M. polymorpha has four lysin motif (LysM)-domain-containing receptor homologs, two of which, LysM-receptor-like kinase (LYK) MpLYK1 and LYK-related (LYR) MpLYR, are responsible for sensing chitin and peptidoglycan fragments, triggering a series of characteristic immune responses. Comprehensive phosphoproteomic analysis of M. polymorpha in response to chitin treatment identified regulatory proteins that potentially shape LysM-mediated PTI. The identified proteins included homologs of well-described PTI components in angiosperms as well as proteins whose roles in PTI are not yet determined, including the blue-light receptor phototropin MpPHOT. We revealed that MpPHOT is required for negative feedback of defense-related gene expression during PTI. Taken together, this study outlines the basic framework of LysM-mediated PTI in M. polymorpha and highlights conserved elements and new aspects of pattern-triggered immunity in land plants.

Grafting systems for plant cadmium research: Insights for basic plant physiology and applied mitigation.

Cadmium (Cd) is a highly toxic and carcinogenic pollutant that poses a threat to human and animal health by affecting several major organ systems. Urbanization and human activities have led to significant increases in Cd concentration in the environment, including in agroecosystems. To protect against the harmful effects of Cd, efforts are being made to promote safe crop production and to clean up Cd-contaminated agricultural lands and water, reducing Cd exposure through the consumption of contaminated agricultural products. There is a need for management strategies that can improve plant Cd tolerance and reduce Cd accumulation in crop plant tissues, all of which involve understanding the impacts of Cd on plant physiology and metabolism. Grafting, a longstanding plant propagation technique, has been shown to be a useful approach for studying the effects of Cd on plants, including insights into the signaling between organs and organ-specific modulation of plant performance under this form of environmental stress. Grafting can be applied to the large majority of abiotic and biotic stressors. In this review, we aim to highlight the current state of knowledge on the use of grafting to gain insights into Cd-induced effects as well as its potential applicability in safe crop production and phytoremediation. In particular, we emphasize the utility of heterograft systems for assessment of Cd accumulation, biochemical and molecular responses, and tolerance in crop and other plant species under Cd exposure, as well as potential intergenerational effects. We outline our perspectives and future directions for research in this area and the potential practical applicability of plant grafting, with attention to the most obvious gaps in knowledge. We aim at inspiring researchers to explore the potential of grafting for modulating Cd tolerance and accumulation and for understanding the mechanisms of Cd-induced responses in plants for both agricultural safety and phytoremediation purposes.

Proximity-Dependent In Vivo Biotin Labeling for Interactome Mapping in Marchantia polymorpha.

Weak or transient protein-protein interactions (PPIs) are involved in a manifold of cellular processes in all living organisms, including plants. However, many of these interactions may remain undiscovered by co-immunoprecipitation (Co-IP) approaches due to their low binding affinities or transitory nature. Enzyme-mediated proximity-dependent in vivo biotin labeling can be a powerful strategy to efficiently capture weak and transient PPIs and has been successfully applied in different model angiosperm species. Here, we provide an optimized and robust protocol for biotin ligase-mediated proximity labeling for interactome mapping in the model liverwort Marchantia polymorpha.

OXIDATIVE SIGNAL-INDUCIBLE1 induces immunity by coordinating N-hydroxypipecolic acid, salicylic acid, and camalexin synthesis.

Expression of OXIDATIVE SIGNAL-INDUCIBLE1 (OXI1) is induced by a number of stress conditions and regulates the interaction of plants with pathogenic and beneficial microbes. In this work, we generated Arabidopsis OXI1 knockout and genomic OXI1 overexpression lines and show by transcriptome, proteome, and metabolome analysis that OXI1 triggers ALD1, SARD4, and FMO1 expressions to promote the biosynthesis of pipecolic acid (Pip) and N-hydroxypipecolic acid (NHP). OXI1 contributes to enhanced immunity by induced SA biosynthesis via CBP60g-induced expression of SID2 and camalexin accumulation via WRKY33-targeted transcription of PAD3. OXI1 regulates genes involved in reactive oxygen species (ROS) generation such as RbohD and RbohF. OXI1 knock out plants show enhanced expression of nuclear and chloroplast genes of photosynthesis and enhanced growth under ambient conditions, while OXI1 overexpressing plants accumulate NHP, SA, camalexin, and ROS and show a gain-of-function (GOF) cell death phenotype and enhanced pathogen resistance. The OXI1 GOF phenotypes are completely suppressed when compromising N-hydroxypipecolic acid (NHP) synthesis in the fmo1 or ald1 background, showing that OXI1 regulation of immunity is mediated via the NHP pathway. Overall, these results show that OXI1 plays a key role in basal and effector-triggered plant immunity by regulating defense and programmed cell death via biosynthesis of salicylic acid, N-hydroxypipecolic acid, and camalexin.

miniTurbo-based interactomics of two plasma membrane-localized SNARE proteins in Marchantia polymorpha.

Marchantia polymorpha is a model liverwort and its overall low genetic redundancy is advantageous for dissecting complex pathways. Proximity-dependent in vivo biotin-labelling methods have emerged as powerful interactomics tools in recent years. However, interactomics studies applying proximity labelling are currently limited to angiosperm species in plants. Here, we established and evaluated a miniTurbo-based interactomics method in M. polymorpha using MpSYP12A and MpSYP13B, two plasma membrane-localized SNARE proteins, as baits. We show that our method yields a manifold of potential interactors of MpSYP12A and MpSYP13B compared to a coimmunoprecipitation approach. Our method could capture specific candidates for each SNARE. We conclude that a miniTurbo-based method is a feasible tool for interactomics in M. polymorpha and potentially applicable to other model bryophytes. Our interactome dataset on MpSYP12A and MpSYP13B will be a useful resource to elucidate the evolution of SNARE functions.

Cavity surface residues of PAD4 and SAG101 contribute to EDS1 dimer signaling specificity in plant immunity.

Arabidopsis pathogen effector-triggered immunity (ETI) is controlled by a family of three lipase-like proteins (EDS1, PAD4, and SAG101) and two subfamilies of HET-S/LOB-B (HeLo)-domain "helper" nucleotide-binding/leucine-rich repeats (ADR1s and NRG1s). EDS1-PAD4 dimers cooperate with ADR1s, and EDS1-SAG101 dimers with NRG1s, in two separate defense-promoting modules. EDS1-PAD4-ADR1 and EDS1-SAG101-NRG1 complexes were detected in immune-activated leaf extracts but the molecular determinants for specific complex formation and function remain unknown. EDS1 signaling is mediated by a C-terminal EP domain (EPD) surface surrounding a cavity formed by the heterodimer. Here we investigated whether the EPDs of PAD4 and SAG101 contribute to EDS1 dimer functions. Using a structure-guided approach, we undertook a comprehensive mutational analysis of Arabidopsis PAD4. We identify two conserved residues (Arg314 and Lys380) lining the PAD4 EPD cavity that are essential for EDS1-PAD4-mediated pathogen resistance, but are dispensable for the PAD4-mediated restriction of green peach aphid infestation. Positionally equivalent Met304 and Arg373 at the SAG101 EPD cavity are required for EDS1-SAG101 promotion of ETI-related cell death. In a PAD4 and SAG101 interactome analysis of ETI-activated tissues, PAD4R314A and SAG101M304R EPD variants maintain interaction with EDS1 but lose association, respectively, with helper nucleotide-binding/leucine-rich repeats ADR1-L1 and NRG1.1, and other immune-related proteins. Our data reveal a fundamental contribution of similar but non-identical PAD4 and SAG101 EPD surfaces to specific EDS1 dimer protein interactions and pathogen immunity.

Dynamic light- and acetate-dependent regulation of the proteome and lysine acetylome of Chlamydomonas.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate) or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a false discovery rate of <1%. As a major result of this study, our data show that dynamic changes in the abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are dependent on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may be of great value for genetic engineering of metabolic pathways in Chlamydomonas.

The EDS1-PAD4-ADR1 node mediates Arabidopsis pattern-triggered immunity.

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.

Comparative phosphoproteomic analysis of tomato genotypes with contrasting cadmium tolerance.

KEY MESSAGE: A first insight into the effects of cadmium exposure on the phosphoproteome of tomato plants by performing a comparative analysis of tomato genotypes with contrasting cadmium tolerance.

Pathogen effector recognition-dependent association of NRG1 with EDS1 and SAG101 in TNL receptor immunity.

Plants utilise intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors to detect pathogen effectors and activate local and systemic defence. NRG1 and ADR1 "helper" NLRs (RNLs) cooperate with enhanced disease susceptibility 1 (EDS1), senescence-associated gene 101 (SAG101) and phytoalexin-deficient 4 (PAD4) lipase-like proteins to mediate signalling from TIR domain NLR receptors (TNLs). The mechanism of RNL/EDS1 family protein cooperation is not understood. Here, we present genetic and molecular evidence for exclusive EDS1/SAG101/NRG1 and EDS1/PAD4/ADR1 co-functions in TNL immunity. Using immunoprecipitation and mass spectrometry, we show effector recognition-dependent interaction of NRG1 with EDS1 and SAG101, but not PAD4. An EDS1-SAG101 complex interacts with NRG1, and EDS1-PAD4 with ADR1, in an immune-activated state. NRG1 requires an intact nucleotide-binding P-loop motif, and EDS1 a functional EP domain and its partner SAG101, for induced association and immunity. Thus, two distinct modules (NRG1/EDS1/SAG101 and ADR1/EDS1/PAD4) mediate TNL receptor defence signalling.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.

Proteomic profiling of Botrytis cinerea conidial germination.

Botrytis cinerea is one of the most relevant plant pathogenic fungi. The first step during its infection process is the germination of the conidia. Here, we report on the first proteome analysis during the germination of B. cinerea conidia, where 204 spots showed significant differences in their accumulation between ungerminated and germinated conidia by two-dimensional polyacrylamide gel electrophoresis and qPCR. The identified proteins were grouped by gene ontology revealing that the infective tools are mainly preformed inside the ungerminated conidia allowing a quick fungal development at the early stages of conidial germination. From 118 identified spots, several virulence factors have been identified while proteins, such as mannitol-1-phosphate dehydrogenase, 6,7-dimethyl-8-ribityllumazine synthase or uracil phosphoribosyltransferase, have been disclosed as a new potential virulence factors in botrytis whose role in pathogenicity needs to be studied to gain new insights about the role of these proteins as therapeutic targets and virulence factors.

Broad-range glycosidase activity profiling.

Plants produce hundreds of glycosidases. Despite their importance in cell wall (re)modeling, protein and lipid modification, and metabolite conversion, very little is known of this large class of glycolytic enzymes, partly because of their post-translational regulation and their elusive substrates. Here, we applied activity-based glycosidase profiling using cell-permeable small molecular probes that react covalently with the active site nucleophile of retaining glycosidases in an activity-dependent manner. Using mass spectrometry we detected the active state of dozens of myrosinases, glucosidases, xylosidases, and galactosidases representing seven different retaining glycosidase families. The method is simple and applicable for different organs and different plant species, in living cells and in subproteomes. We display the active state of previously uncharacterized glycosidases, one of which was encoded by a previously declared pseudogene. Interestingly, glycosidase activity profiling also revealed the active state of a diverse range of putative xylosidases, galactosidases, glucanases, and heparanase in the cell wall of Nicotiana benthamiana. Our data illustrate that this powerful approach displays a new and important layer of functional proteomic information on the active state of glycosidases.

Staurosporine and extracellular matrix proteins mediate the conversion of small cell lung carcinoma cells into a neuron-like phenotype.

Small cell lung carcinomas (SCLCs) represent highly aggressive tumors with an overall five-year survival rate in the range of 5 to 10%. Here, we show that four out of five SCLC cell lines reversibly develop a neuron-like phenotype on extracellular matrix constituents such as fibronectin, laminin or thrombospondin upon staurosporine treatment in an RGD/integrin-mediated manner. Neurite-like processes extend rapidly with an average speed of 10 µm per hour. Depending on the cell line, staurosporine treatment affects either cell cycle arrest in G2/M phase or induction of polyploidy. Neuron-like conversion, although not accompanied by alterations in the expression pattern of a panel of neuroendocrine genes, leads to changes in protein expression as determined by two-dimensional gel electrophoresis. It is likely that SCLC cells already harbour the complete molecular repertoire to convert into a neuron-like phenotype. More extensive studies are needed to evaluate whether the conversion potential of SCLC cells is suitable for therapeutic interventions.

Large-scale proteomic analysis of the grapevine leaf apoplastic fluid reveals mainly stress-related proteins and cell wall modifying enzymes.

BACKGROUND: The extracellular space or apoplast forms a path through the whole plant and acts as an interface with the environment. The apoplast is composed of plant cell wall and space within which apoplastic fluid provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Large-scale proteomic analysis reveals the protein content of the grapevine leaf apoplastic fluid and the free interactive proteome map considerably facilitates the study of the grapevine proteome. RESULTS: To obtain a snapshot of the grapevine apoplastic fluid proteome, a vacuum-infiltration-centrifugation method was optimized to collect the apoplastic fluid from non-challenged grapevine leaves. Soluble apoplastic protein patterns were then compared to whole leaf soluble protein profiles by 2D-PAGE analyses. Subsequent MALDI-TOF/TOF mass spectrometry of tryptically digested protein spots was used to identify proteins. This large-scale proteomic analysis established a well-defined proteomic map of whole leaf and leaf apoplastic soluble proteins, with 223 and 177 analyzed spots, respectively. All data arising from proteomic, MS and MS/MS analyses were deposited in the public database world-2DPAGE. Prediction tools revealed a high proportion of (i) classical secreted proteins but also of non-classical secreted proteins namely Leaderless Secreted Proteins (LSPs) in the apoplastic protein content and (ii) proteins potentially involved in stress reactions and/or in cell wall metabolism. CONCLUSIONS: This approach provides free online interactive reference maps annotating a large number of soluble proteins of the whole leaf and the apoplastic fluid of grapevine leaf. To our knowledge, this is the first detailed proteome study of grapevine apoplastic fluid providing a comprehensive overview of the most abundant proteins present in the apoplast of grapevine leaf that could be further characterized in order to elucidate their physiological function.

Proteomic analysis of conidia germination in Colletotrichum acutatum.

Colletotrichum acutatum is an important phytopathogenic fungus causing anthracnose in commercially important fruit crops, such as strawberry. The conidia produced by the fungus are survival structures which play a key role in host infection and fungal propagation. Despite its relevance to the fungal life cycle, conidial biology has not been extensively investigated. Here, we provide the first proteomic description of the conidial germination in C. acutatum by comparing the proteomic profiles of ungerminated and germinated conidia. Using two-dimensional electrophoresis combined with MALDI-TOF/TOF mass spectrometry, we have identified 365 proteins in 354 spots, which represent 245 unique proteins, including some proteins with key functions in pathogenesis. All these proteins have been classified according to their molecular function and their involvement in biological processes, including cellular energy production, oxidative metabolism, stress, fatty acid synthesis, protein synthesis, and folding. This report constitutes the first comprehensive study of protein expression during the early stage of the C. acutatum conidial germination. It advances our understanding of the molecular mechanisms involved in the conidial germination process, and provides a useful basis for the further characterization of proteins involved in fungal biology and fungus life cycles.

Identification of proteins associated with malting quality in a subset of wild barley introgression lines.

Malted barley is an important ingredient used in the brewing and distilling industry worldwide. In this study, we used a proteomics approach to investigate the biochemical function of previously identified quantitative trait loci (QTLs) on barley chromosomes 1H and 4H that influence malting quality. Using a subset of barley introgression lines containing wild barley (Hordeum vulgare ssp. spontaneum) alleles at these QTLs, we validated that wild barley alleles at the chromosome 1H QTL reduced overall malting quality, whereas wild barley alleles at the chromosome 4H QTL improved the malting quality parameters α-amylase activity, VZ45, and Kolbach index compared to the control genotype Scarlett. 2DE was used to detect changes in protein expression during the first 72 h of micromalting associated with these QTLs. In total, 16 protein spots showed a significant change in expression between the introgression lines and Scarlett, of which 14 were successfully identified with MS. Notably, the wild barley alleles in the line containing the chromosome 4H QTL showed a sixfold increased expression of a limit dextrinase inhibitor. The possible role of the identified proteins in malting quality is discussed. The knowledge gained will assist ongoing research toward cloning the genes underlying these important QTL.