Enzymes Of Secondary Metabolism Research Articles

Transcriptome analysis provides insights into preservation mechanism of postharvest Muscat Hamburg grapes treated with SO2

Sulfur dioxide (SO2) is the most frequently used preservative for table grapes, yet the preservation mechanism remains unclear. To ascertain the specific genes and pathways involved in SO2 preservation, RNA-seq technology was employed to characterize the transcriptome profile of grapes during postharvest storage. A total of 22,288 genes were identified, of which 377 genes were differentially expressed (≥ 2-fold change) between SO2 and control groups, mainly enriched in secondary metabolism, plant-pathogen interactions, plant hormone signaling, etc. Numerous genes encoding pathogenesis-related (PR) proteins exhibited higher expression levels in SO2 group, while the activities of disease-resistant enzymes, such as β-1,3-glucanase (PR2) and chitinase (PR3) significantly increased by 28.9 % and 29.3 % (P < 0.05), indicating SO2-induced plant resistance. Exposure to SO2 also enhanced the expression of genes encoding essential enzymes of secondary metabolism, and significantly increased both the activities of critical enzymes for the biosynthesis of secondary metabolites, such as phenylalanine ammonia-lyase (PAL) and 4-coumarate-CoA ligase (4CL), and the contents of secondary metabolites such as total phenol, flavonoid, anthocyanin, and lignin, demonstrating an enhanced chemical and physical barriers in SO2-fumigated grapes. Meanwhile, the genes associated with ethylene signaling and cell wall degradation were down-regulated by SO2 preservative, and some ethylene-responsive elements were identified in the promoter regions of cell wall hydrolase genes, suggesting that SO2-inhibited ethylene signaling might contribute to maintaining cell wall integrity. Altogether, gene expression patterns and cellular physiological processes were altered in grapes exposed to SO2, which helped maintain fruit quality and prolong postharvest life by regulating the defense responses and fruit ageing.

Grapevine woody tissues accumulate stilbenoids following bud burst

Main conclusionAfter bud burst, a transcriptional reprogramming of the shikimate and phenylpropanoid pathways occurs in grapevine canes resulting in the accumulation of stilbenoids like resveratrol and viniferin.Stilbenoids are phenylpropanoid compounds with important biological properties and biotechnological applications that are synthesized in grapevine in response to different stresses. Although they are found in woody tissues, such as canes and buds, their biosynthesis and accumulation have been essentially described in berries. We have previously shown that transcripts encoding secondary metabolism enzymes accumulate in grapevine canes following the transition from dormancy (E-L 1) to bud burst (E-L 4) suggesting that secondary metabolites may accumulate in grapevine canes during this transition. In the present study, using UPLC-MS we demonstrate the accumulation of important metabolites such as ferulic acid and the stilbenoids E-resveratrol, E-piceatannol and E-ε-viniferin. Stilbenoids accumulation correlated with the increased expression of several stilbene synthase genes and of VviMYB14, encoding a transcription factor that regulates stilbene biosynthesis. In addition, a general stimulation of the plastidial shikimate pathway was observed. Taken together, results show that important secondary metabolites accumulate in the woody canes during bud burst. These findings may aid biotechnological approaches aimed at extracting biologically active phenolic compounds, including stilbenoids, from grapevine woody tissues.

Sublethal effects of chlorantraniliprole on biological characteristics, detoxifying enzyme activity and gene expression profile in the Allium mongolicum Regel leaf beetle Galeruca daurica (Coleoptera: Chrysomelidae)

AbstractAllium mongolicum Regel leaf beetle, Galeruca daurica (Coleoptera: Chrysomelidae) is rampantly harmful in Inner Mongolia grassland. However, the current management strategies for this pest still heavily rely on chemical control using traditional insecticides or those with novel action. In the study, we conducted an indoor bioassay to evaluate the sublethal effects of chlorantraniliprole on the biological characteristics. Additionally, we assessed the activity of detoxification enzymes, specifically the primary enzymes carboxylesterase (CarE) and cytochrome P450, and insect's secondary metabolic enzymes, namely glutathione S‐transferase (GST) and uridine diphosphate glycosyltransferase (UGT), as well as its gene expression profile. The developmental period of the 3rd instar larvae of G. daurica was significantly prolonged after treatment with chlorantraniliprole, which negatively affected the hatching, pupation and diapause rates as well as their body weight. The larvae showed different dynamics of the enzyme activities within 24 h of chlorantraniliprole treatment. Different sublethal concentrations of chlorantraniliprole showed an inducing effect on the activities, while no significant difference in activity was observed for P450. Expression profiling of detoxifying enzyme genes screened by transcriptome data has revealed that 6 CarE, 2 GST, 7 UGT and 31 P450 genes were up‐regulated. In conclusion, chlorantraniliprole's sublethal effect on 3rd instar G. daurica larvae resulted in a deceleration of their growth and developmental processes and significantly increased the activity levels of CarE, GST and UGT. The detoxifying proteins encoded by up‐regulation genes may involve in the detoxification metabolism of chlorantraniliprole. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in this key grassland insect pest.

Multiplexed Assessment of Promiscuous Non-Canonical Amino Acid Synthase Activity in a Pyridoxal Phosphate-Dependent Protein Family.

Pyridoxal phosphate (PLP)-dependent enzymes afford access to a variety of non-canonical amino acids (ncAAs), which are premier buildings blocks for the construction of complex bioactive molecules. The vinylglycine ketimine (VGK) subfamily of PLP-dependent enzymes plays a critical role in sulfur metabolism and is home to a growing set of secondary metabolic enzymes that synthesize γ-substituted ncAAs. Identification of VGK enzymes for biocatalysis faces a distinct challenge because the subfamily contains both desirable synthases as well as lyases that break down ncAAs. Some enzymes have both activities, which may contribute to pervasive mis-annotation. To navigate this complex functional landscape, we used a substrate multiplexed screening approach to rapidly measure the substrate promiscuity of 40 homologs in the VGK subfamily. We found that enzymes involved in transsulfuration are less likely to have promiscuous activities and often possess undesirable lyase activity. Enzymes from direct sulfuration and secondary metabolism generally had a high degree of substrate promiscuity. From this cohort, we identified an exemplary γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). This enzyme is thermostable and has high expression (~400 mg protein per L culture), enabling preparative scale synthesis of thioether containing ncAAs. When assayed with l-allylglycine, CahyGS catalyzes a stereoselective γ-addition reaction to afford access to a unique set of γ-methyl branched ncAAs. We determined high-resolution crystal structures of this enzyme that define an open-close transition associated with ligand binding and set the stage for future engineering within this enzyme subfamily.

Phosphorus and Serendipita indica synergism augments arsenic stress tolerance in rice by regulating secondary metabolism related enzymatic activity and root metabolic patterns

The multifarious problems created by arsenic (As), for collective environment and human health, serve a cogent case for searching integrative agricultural approaches to attain food security. Rice (Oryza sativa L.) acts as a sponge for heavy metal(loid)s accretion, specifically As, due to anaerobic flooded growth conditions facilitating its uptake. Acclaimed for their positive impact on plant growth, development and phosphorus (P) nutrition, ‘mycorrhizas’ are able to promote stress tolerance. Albeit, the metabolic alterations underlying Serendipita indica (S. indica; S.i) symbiosis-mediated amelioration of As stress along with nutritional management of P are still understudied. By using biochemical, RT-qPCR and LC-MS/MS based untargeted metabolomics approach, rice roots of ZZY-1 and GD-6 colonized by S. indica, which were later treated with As (10 µM) and P (50 µM), were compared with non-colonized roots under the same treatments with a set of control plants. The responses of secondary metabolism related enzymes, especially polyphenol oxidase (PPO) activities in the foliage of ZZY-1 and GD-6 were enhanced 8.5 and 12-fold, respectively, compared to their respective control counterparts. The current study identified 360 cationic and 287 anionic metabolites in rice roots, and the commonly enriched pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was biosynthesis of phenylalanine, tyrosine and tryptophan, which validated the results of biochemical and gene expression analyses associated with secondary metabolic enzymes. Particularly under As+S.i+P comparison, both genotypes exhibited an upregulation of key detoxification and defense related metabolites, including fumaric acid, L-malic acid, choline, 3,4-dihydroxybenzoic acid, to name a few. The results of this study provided the novel insights into the promising role of exogenous P and S. indica in alleviating As stress.

New Insights into rice pyrimidine catabolic enzymes.

Rice is a primary global food source, and its production is affected by abiotic stress, caused by climate change and other factors. Recently, the pyrimidine reductive catabolic pathway, catalyzed by dihydropyrimidine dehydrogenase (DHPD), dihydropyrimidinase (DHP) and β-ureidopropionase (β-UP), has emerged as a potential participant in the abiotic stress response of rice. The rice enzymes were produced as recombinant proteins, and two were kinetically characterized. Rice dihydroorotate dehydrogenase (DHODH), an enzyme of pyrimidine biosynthesis often confused with DHPD, was also characterized. Salt-sensitive and salt-resistant rice seedlings were subjected to salt stress (24 h) and metabolites in leaves were determined by mass spectrometry. The OsDHPD sequence was homologous to the C-terminal half of mammalian DHPD, conserving FMN and uracil binding sites, but lacked sites for Fe/S clusters, FAD, and NADPH. OsDHPD, truncated to eliminate the chloroplast targeting peptide, was soluble, but inactive. Database searches for polypeptides homologous to the N-terminal half of mammalian DHPD, that could act as co-reductants, were unsuccessful. OsDHODH exhibited kinetic parameters similar to those of other plant DHODHs. OsDHP, truncated to remove a signal sequence, exhibited a kcat/Km = 3.6 x 103 s-1M-1. Osb-UP exhibited a kcat/Km = 1.8 x 104 s-1M-1. Short-term salt exposure caused insignificant differences in the levels of the ureide intermediates dihydrouracil and ureidopropionate in leaves of salt-sensitive and salt-resistant plants. Allantoin, a ureide metabolite of purine catabolism, was found to be significantly higher in the resistant cultivar compared to one of the sensitive cultivars. OsDHP, the first plant enzyme to be characterized, showed low kinetic efficiency, but its activity may have been affected by truncation. Osb-UP exhibited kinetic parameters in the range of enzymes of secondary metabolism. Levels of two pathway metabolites were similar in sensitive and resistant cultivars and appeared to be unaffected by short-term salt exposure."

Research progress on application of multi-enzyme-catalyzed cascade reactions in enzymatic synthesis of natural products

As a biocatalyst, enzyme has the advantages of high catalytic efficiency, strong reaction selectivity, specific target products, mild reaction conditions, and environmental friendliness, and serves as an important tool for the synthesis of complex organic molecules. With the continuous development of gene sequencing technology, molecular biology, genetic manipulation, and other technologies, the diversity of enzymes increases steadily and the reactions that can be catalyzed are also gradually diversified. In the process of enzyme-catalyzed synthesis, the majority of common enzymatic reactions can be achieved by single enzyme catalysis, while many complex reactions often require the participation of two or more enzymes. Therefore, the combination of multiple enzymes together to construct the multi-enzyme cascade reactions has become a research hotspot in the field of biochemistry. Nowadays, the biosynthetic pathways of more natural products with complex structures have been clarified, and secondary metabolic enzymes with novel catalytic activities have been identified, discovered, and combined in enzymatic synthesis of natural/unnatural molecules with diverse structures. This study summarized a series of examples of multi-enzyme-catalyzed cascades and highlighted the application of cascade catalysis methods in the synthesis of carbohydrates, nucleosides, flavonoids, terpenes, alkaloids, and chiral molecules. Furthermore, the existing problems and solutions of multi-enzyme-catalyzed cascade method were discussed, and the future development direction was prospected.

Characteristics and adaptability of Flavobacterium panici BSSL-CR3 in tidal flat revealed by comparative genomic and enzymatic analysis

Tidal flat microbes play an important ecological role by removing organic pollutants and providing an energy source. However, bacteria isolated from tidal flatsand their genomes have been scarcely reported, making it difficult to elucidate which genes and pathways are potentially involved in the above roles. In this study, strain BSSL-CR3, the third reported species among the tidal flat Flavobacterium was analyzed using whole-genome sequencing to investigate its adaptability and functionality in tidal flats. BSSL-CR3 is comprised of a circular chromosome of 5,972,859bp with a GC content of 33.84%. Genome annotation and API ZYM results showed that BSSL-CR3 has a variety of secondary metabolic gene clusters and enzyme activities including α-galactosidase. BSSL-CR3 had more proteins with a low isoelectric point (pI) than terrestrial Flavobacterium strains, and several genes related to osmotic regulation were found in thegenomic island(GI). Comparative genomic analysis with other tidal flat bacteria also revealed that BSSL-CR3 had the largest number of genes encoding Carbohydrate Active EnZymes (CAZymes) which are related to algae degradation. This study will provide insight into the adaptability of BSSL-CR3 to the tidal flats and contribute to facilitating future comparative analysis of bacteria in tidal flats.

Identification and characterization of UDP-glycosyltransferase genes and the potential role in response to insecticides exposure in Bactrocera dorsalis.

The oriental fruit fly, Bactrocera dorsalis (Hendel) is a worldwide pest damaging a wide range of hosts. Due to the long-term indiscriminate use of insecticides, B. dorsalis has developed serious resistance to several insecticides. UDP-glycosyltransferases (UGTs) are secondary metabolic enzymes involved in biotransformation and play an important role in the metabolism of plant secondary metabolites and synthetic insecticides in insects. Thus, we suspect that UGTs in B. dorsalis play an important role in insecticide tolerance. In this study, 31 UGT genes were identified in the genome of B. dorsalis, belonging to 13 subfamilies. Real-time quantitative polymerase chain reaction (RT-qPCR) results revealed that 12 UGT genes were highly expressed in the antennae, midgut, Malpighian tubule and fat body. The mRNA expressions of 17 UGT genes were up-regulated upon exposure to λ-cyhalothrin, imidacloprid, abamectin and chlorpyrifos. Knockdown of the selected five UGT genes (BdUGT301D2, BdUGT35F2, BdUGT36K2, BdUGT49D2, BdUGT50B5) by RNA interference increased the mortality of B. dorsalis from 9.29% to 27.22% upon exposure to four insecticides. The abundance of UGTs in B. dorsalis is similar to other insect species, and 12 out of 31 UGTs were specifically expressed in metabolic tissues, suggesting a key role in detoxification. Down-regulation of five selected UGT genes increased the susceptibility of B. dorsalis to various insecticides, indicating that UGTs may play an important role in tolerance of B. dorsalis to multiple insecticides. © 2022 Society of Chemical Industry.

A Secondary Metabolic Enzyme Functioned as an Evolutionary Seed of a Primary Metabolic Enzyme.

The antibiotic alaremycin has a structure that resembles that of 5-aminolevulinic acid (ALA), a universal precursor of porphyrins, and inhibits porphyrin biosynthesis. Genome sequencing of the alaremycin-producing bacterial strain and enzymatic analysis revealed that the first step of alaremcyin biosynthesis is catalysed by the enzyme, AlmA, which exhibits a high degree of similarity to 5-aminolevulinate synthase (ALAS) expressed by animals, protozoa, fungi, and α-proteobacteria. Site-directed mutagenesis of AlmA revealed that the substitution of two amino acids residues around the substrate binding pocket transformed its substrate specificity from that of alaremycin precursor synthesis to ALA synthesis. To estimate the evolutionary trajectory of AlmA and ALAS, we performed an ancestral sequence reconstitution analysis based on a phylogenetic tree of AlmA and ALAS. The reconstructed common ancestral enzyme of AlmA and ALAS exhibited alaremycin precursor synthetic activity, rather than ALA synthetic activity. These results suggest that ALAS evolved from an AlmA-like enzyme. We propose a new evolutionary hypothesis in which a non-essential secondary metabolic enzyme acts as an ‘evolutionary seed’ to generate an essential primary metabolic enzyme.

BSH-TRAP: Bile salt hydrolase tagging and retrieval with activity-based probes.

Bile acids are important molecules that participate in digestion and regulate many host physiological processes, including metabolism and inflammation. Primary bile acids are biosynthesized from cholesterol in the liver, where they are conjugated to glycine and taurine before secretion into the intestines. A small fraction of these molecules remain in the gut, where they are modified by a microbial enzyme, bile salt hydrolase (BSH), which deconjugates the glycine and taurine groups. This deconjugation precedes all subsequent biotransformation in the intestines, including regioselective dehydroxylation and epimerization reactions, to produce numerous secondary bile acids. Thus, BSH is considered the gatekeeper enzyme of secondary bile acid metabolism, and, as a result, it controls the overall bile acid composition in the host. Despite the critical role that BSH plays in bile acid metabolism, there exist few tools to probe its activity in complex biological mixtures. In this chapter, we describe a chemoproteomic approach termed BSH-TRAP (bile salt hydrolase tagging and retrieval with activity-based probes) that enables visualization and identification of BSH activity in bacteria. Here, we describe application of BSH-TRAP to cultured bacterial strains and the gut microbes derived from mice. We envision that BSH-TRAP could be used to profile changes in BSH activity and identify novel BSH enzymes in complex biological samples, such as the gut microbiome.

Physiological and antioxidant responses of Euryale ferox salisb seedlings to microcystins

Lake Taihu is the third largest freshwater lake located in eastern China. In recent years, it has experienced extensive cyanobacterial (Microcystis spp.) blooms that produce toxic microcystins (MCs), which may have acute and chronic hepatotoxic effects in animals and humans. Although the impact of MCs on both terrestrial and aquatic plants is well documented, the effects and underlying mechanisms of the harmful toxin MC-LR on Euryale ferox Salisb seedlings have rarely been reported. Thus, herein, the antioxidant response mechanisms and the biosynthesis of secondary metabolites during the exposure of E. ferox Salisb seedlings to varying MC-LR concentrations (0.05, 0.2, 1, and 5 μg/L) were thoroughly investigated after exposure periods (7, 14, 21 d). Our study revealed that the seedling growth was inhibited with increasing MC-LR exposure concentration that significantly induced at 1 μg/L and reached a maximum level at 5 μg/L, whereas the activity of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) in the seedling cells increased gradually with increasing MC-LR concentration and longer exposure time. The maximum malondialdehyde (MDA) content was 4.3-fold higher than that of the control group under an MC-LR concentration of 5.0 μg/L after 7 days of exposure treatment. The study of the seedling detoxification mechanism revealed that the content of total glutathione (tGSH) and reduced glutathione (GSH), as well as the activities of GSH sparse transferase (GST) and glutathione reductase (GR), increased to varying degrees and reached a maximum level at 1 μg/L. Therefore, the exposure to MC-LR can promote the accumulation of secondary metabolites and increase the activities of secondary metabolic enzymes in the seedlings. Further investigation of these antioxidative mechanisms will provide additional information for the identification and development of bio-indicators to evaluate the environmental impact of MCs on aquatic ecosystems.

Glutathione S-transferases GhGSTF1 and GhGSTF2 involved in the anthocyanin accumulation in Gossypium hirsutum L.

The glutathione S-transferases (GSTs) are important enzymes of secondary metabolism in plants. In this study, two putative GSTs, GhGSTF1 and GhGSTF2, were identified as anthocyanin-related GSTs by the transcriptome data of the leaves of Gossypium hirsutum L. TM-1 and T586. The quantitative real-time PCR showed that GhGSTF1 and GhGSTF2 were highly expressed in red leaves and stems of Gossypium hirsutum L. T586. Orthologous genes of GhGSTF2 in two Gossypium barbadense L. 3-79 and Xinhai21 contain bases deletion in N-terminal (GbGSTF2a) and C-terminal (GbGSTF2b) respectively. Among which, GhGSTF1 and GhGSTF2 can restore pigmentation in hypocotyls of Arabidopsis thaliana mutant tt19-7 while GbGSTF2a and GbGSTF2b cannot. Furthermore, in vitro assays showed the recombinant GhGSTF1 and GhGSTF2 had Glutathione S-transferase activities. Fluorescence quenching assays showed that Cya could obviously quench the fluorescence of GhGSTF1, GhGSTF2, GbGSTF2a and GbGSTF2b to lower levels as compared to C3G. Moreover, the transient dual-luciferase assays showed that the promoters of GhGSTF1 and GhGSTF2 could be activated by GhPAP1D at different levels. GUS staining assays showed that their promoters have different activities to light. This study indicated that GhGSTF1 and GhGSTF2 play important roles in anthocyanin accumulation and the regulatory mechanism of anthocyanin accumulation in allotetraploid Gossypium are complicated.

Isolation, Characterization and Targeted Metabolic Evaluation of Endophytic Fungi Harbored in 14 Seed-Derived Hypericum Species.

Medicinal plants of the genus Hypericum are rich sources of bioactive naphthodianthrones, which are unique in the plant kingdom, but quite common in fungal endophytes. Cultivable endophytic fungi were isolated from 14 different Hypericum spp. originating from seeds grown under in vitro conditions and further acclimated to outdoor conditions. Among 37 fungal isolates yielded from the aerial and underground plant organs, 25 were identified at the species level by the fungal barcode marker internal transcribed spacer rDNA and protein-coding gene region of tef1α. Ten of them were isolated from Hypericum spp. for the first time. The axenic cultures of the isolated endophytes were screened for the production of extracellular enzymes, as well as bioactive naphthodianthrones and their putative precursors by Bornträger's test and HPLC-HRMS. Traces of naphthodianthrones and their intermediates, emodin, emodin anthrone, skyrin, or pseudohypericin, were detected in the fungal mycelia of Acremonium sclerotigenum and Plectosphaerella cucumerina isolated from Hypericum perforatum and Hypericum maculatum, respectively. Traces of emodin, hypericin, and pseudohypericin were released in the broth by Scedosporium apiospermum, P. cucumerina, and Fusarium oxysporum during submerged fermentation. These endophytes were isolated from several hypericin-producing Hypericum spp. Taken together, our results reveal the biosynthetic potential of cultivable endophytic fungi harbored in Hypericum plants as well as evidence of the existence of remarkable plant-endophyte relationships in selected non-native ecological niches. A possible role of the extracellular enzymes in plant secondary metabolism is discussed.

Quaternary Structure of the Tryptophan Synthase α-Subunit Homolog BX1 from Zea mays.

BX1 from Zea mays (zmBX1) is an enzyme of plant secondary metabolism that generates indole for the synthesis of plant defensins. It is a homologue of the tryptophan synthase α-subunit, TrpA. Whereas TrpA itself is a monomer in solution, zmBX1 is dimeric, confirmed in our work by native MS. Using cross-linking and mutagenesis, we identified the physiological dimerization interface of zmBX1. We found that homodimerization has only minor effects on catalysis and stability. A comparison of the zmBX1-zmBX1 homodimer and zmTrpA-zmTrpB heterodimer interfaces suggest that homodimerization in zmBX1 might, at an early point in evolution, have served as a mechanism to exclude the interaction with the tryptophan synthase β-subunit (zmTrpB), marking its transition from primary to secondary metabolism.

Metabolite profiling during graft union formation reveals the reprogramming of primary metabolism and the induction of stilbene synthesis at the graft interface in grapevine

BackgroundGrafting with rootstocks is essential for the culture of many perennial fruit crops and is increasing being used in the production of annual fruits and vegetables. Our previous work based on microarrays showed that transcripts encoding enzymes of both primary and secondary metabolism were differentially expressed during graft union formation in both homo-grafts (a genotype grafted with itself) and hetero-grafts (two different genotypes grafted together). The aim of this study was to profile primary and secondary metabolites, and quantify the activity of phenylalanine ammonia lyase (PAL) and neutral invertase (NI) in the scion and rootstock tissues and the graft interface of homo and hetero-grafts of grapevine 1 month after grafting. Table-top grafting was done on over-wintering stems (canes) of grapevine and the graft interface tissues (containing some woody stem tissues and callus) were compared to the surrounding rootstock and scion tissues. The objective was to identify compounds involved in graft union formation and hetero-grafting responses.ResultsA total of 54 compounds from primary and secondary metabolism (19 amino acids, five primary and 30 secondary compounds metabolites) and the activity of two enzymes were measured. The graft interface was associated with an increase in the accumulation of the branched-chain amino acids, basic amino acids, certain stilbene compounds and higher PAL and NI activity in comparison to the surrounding woody stem tissues. Some amino acids and stilbenes were identified as being accumulated differently between the graft interfaces of the scion/rootstock combinations in a manner which was unrelated to their concentrations in the surrounding woody stem tissues.ConclusionsThis study revealed the modification of primary metabolism to support callus cell formation and the stimulation of stilbene synthesis at the graft interface, and how these processes are modified by hetero-grafting. Knowledge of the metabolites and/or enzymes required for successful graft union formation offer us the potential to identify markers that could be used by nurseries and researchers for selection and breeding purposes.

Comparative quantitative proteomics of osmotic signal transduction mutants in Botrytis cinerea explain mutant phenotypes and highlight interaction with cAMP and Ca2+ signalling pathways

Signal transduction (ST) is essential for rapid adaptive responses to changing environmental conditions. It acts through rapid post-translational modifications of signalling proteins and downstream effectors that regulate the activity and/or subcellular localisation of target proteins, or the expression of downstream genes. We have performed a quantitative, comparative proteomics study of ST mutants in the phytopathogenic fungus Botrytis cinerea during axenic growth under non-stressed conditions to decipher the roles of two kinases of the hyper-osmolarity pathway in B. cinerea physiology. We studied the mutants of the sensor histidine kinase Bos1 and of the MAP kinase Sak1. Label-free shotgun proteomics detected 2425 proteins, 628 differentially abundant between mutants and wild-type, 270 common to both mutants, indicating independent and shared regulatory functions for both kinases. Gene ontology analysis showed significant changes in functional categories that may explain in vitro growth and virulence defects of both mutants (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress). The proteome data also highlight a new link between Sak1 MAPK, cAMP and Ca2+ signalling. This study reveals the potential of proteomic analyses of signal transduction mutants to decipher their biological functions. TEXT-VULGARISATION: The fungus Botrytis cinerea is responsible for grey mold disease of hundreds of plant species. During infection, the fungus has to face important changes of its environment. Adaptation to these changing environmental conditions involves proteins of such called signal transduction pathways that regulate the production, activity or localisation of cellular components, mainly proteins. While the components of such signal transduction pathways are well known, their role globally understood, the precise impact on protein production remains unknown. In this study we have analysed and compared the global protein content of two Botrytis cinerea signal transduction mutants - both avirulent - to the pathogenic parental strain. The data of 628 differential proteins between mutants and wild-type, showed significant changes in proteins related to plant infection (secondary metabolism enzymes, lytic enzymes, proteins linked to osmotic, oxidative and cell wall stress) that may explain the virulence defects of both mutants. Moreover, we observed intracellular accumulation of secreted proteins in one of the mutants suggesting a potential secretion defect.

Generation of a Stand-Alone Tryptophan Synthase α-Subunit by Mimicking an Evolutionary Blueprint.

The αββα tryptophan synthase (TS), which is part of primary metabolism, is a paradigm for allosteric communication in multienzyme complexes. In particular, the intrinsically low catalytic activity of the α-subunit TrpA is stimulated several hundredfold through the interaction with the β-subunit TrpB1. The BX1 protein from Zea mays (zmBX1), which is part of secondary metabolism, catalyzes the same reaction as that of its homologue TrpA, but with high activity in the absence of an interaction partner. The intrinsic activity of TrpA can be significantly increased through the exchange of several active-site loop residues, which mimic the corresponding loop in zmBX1. The subsequent identification of activating amino acids in the generated "stand-alone" TrpA contributes to an understanding of allostery in TS. Moreover, findings suggest an evolutionary trajectory that describes the transition from a primary metabolic enzyme regulated by an interaction partner to a self-reliant, stand-alone, secondary metabolic enzyme.

The pathogenic mechanisms of Tilletia horrida as revealed by comparative and functional genomics

Tilletia horrida is a soil-borne, mononucleate basidiomycete fungus with a biotrophic lifestyle that causes rice kernel smut, a disease that is distributed throughout hybrid rice growing areas worldwide. Here we report on the high-quality genome sequence of T. horrida; it is composed of 23.2 Mb that encode 7,729 predicted genes and 6,973 genes supported by RNA-seq. The genome contains few repetitive elements that account for 8.45% of the total. Evolutionarily, T. horrida lies close to the Ustilago fungi, suggesting grass species as potential hosts, but co-linearity was not observed between T. horrida and the barley smut Ustilago hordei. Genes and functions relevant to pathogenicity were presumed. T. horrida possesses a smaller set of carbohydrate-active enzymes and secondary metabolites, which probably reflect the specific characteristics of its infection and biotrophic lifestyle. Genes that encode secreted proteins and enzymes of secondary metabolism, and genes that are represented in the pathogen-host interaction gene database genes, are highly expressed during early infection; this is consistent with their potential roles in pathogenicity. Furthermore, among the 131 candidate pathogen effectors identified according to their expression patterns and functionality, we validated two that trigger leaf cell death in Nicotiana benthamiana. In summary, we have revealed new molecular mechanisms involved in the evolution, biotrophy, and pathogenesis of T. horrida.

Transcriptome Profiling Reveals Distinct Gene Activations in Barley Responding to Scald and Spot blotch

Scald (Rhynchosporium secalis; Rs) and spot blotch (Cochliobolus sativus; Cs) are important diseases of barley (Hordeum vulgare L.) worldwide. Similar mechanisms and gene transcripts are assumed to be involved in the barley defense response since both these pathogens are necrotrophic fungi. In the current study, the transcriptome in leaves of the same barley genotype WI2291 inoculated with Rs and Cs was compared at different times postinoculation. Comparison of data for barley Rs- and Cs- inoculated plants with mock-inoculated plants revealed gene expression changes that included basal defense transcripts and transcripts specific to the establishment of a necrotrophic interaction with associated fungi. During barley–pathogen interaction pathway, WI2291 activated a higher number of genes and pathways in response to Rs infection than in response to Cs invasion. However, families of genes encoding pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded to cover Rs and Cs at an early stage following inoculation. Our results demonstrate differences in the pathways and activated genes of barely cv. WI291challenged by Rs and Cs, and that expression patterns of the same defense-associated genes were altered in adaptation to different pathogens. Our work provides new insights into the underlying mechanisms related to regulation of different pathways in response to fungal infection.