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Chemical warfare between the host and the pathogen plays a crucial role in plant-necrotrophic pathogen interactions, but examples of its involvement in quantitative disease resistance in plants are poorly documented. In the Daucus carota-Alternaria dauci pathosystem, the novel toxin aldaulactone has been identified as a key factor in both fungal pathogenicity and the carrot’s partial resistance to the pathogen. Bioinformatic analyses have pinpointed a secondary metabolism gene cluster that harbors two polyketide synthase genes, AdPKS7 and AdPKS8 , that are likely responsible for the biosynthesis of aldaulactone. Here, we present the functional validation of AdPKS7 and AdPKS8 as genes responsible for aldaulactone production in A. dauci . We generated A. dauci knock-out mutants for AdPKS7 and AdPKS8 by replacing essential domains with a hygromycin resistance gene, marking the first reported case of genetic manipulation in A. dauci . Following transformation, the mutants were analyzed for toxin production via HPLC-UV and assessed for pathogenicity in planta . Aldaulactone production was abolished in all PKS mutants, which also exhibited significantly reduced pathogenicity on H1-susceptible carrot leaves. These findings confirm the roles of AdPKS7 and AdPKS8 in aldaulactone biosynthesis and their contribution to fungal pathogenicity.

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds. Terpene cyclases catalyze the formation of diverse hydrocarbon scaffolds found in terpenoids. Here, the authors report the cryptic function of class I terpene cyclases as aromatic prenyltransferases and the universality of this cryptic feature is confirmed using enzymes from different sources.

Iron is one of the most abundant elements on earth and essential for life. However, Fe 3+ ions are rather insoluble and microorganisms such as fungi may use siderophores as strong chelators for uptake. In addition, free cytoplasmic iron is rather toxic and intracellular siderophores are used to control the toxicity. Siderophores are also important for iron storage. We studied two siderophore systems in the plant necrotrophic fungus Alternaria alternata and show that the non-ribosomal peptide synthase, Nps2, is required for the biosynthesis of intracellular ferricrocin, whereas Nps6 is needed for the formation of extracellular coprogen and coprogen B. Whereas nps2 was dispensable for growth on iron-depleted medium, nps6 was essential under those conditions. nps2 deletion caused an increase in spore formation and reduced pathogenicity on tomato. Our results suggest that A. alternata employs an external and an internal siderophore system to adapt to low iron conditions.

The fungal strain Alternaria alternata JS0515 was isolated from Vitex rotundifolia (beach vitex). Twelve secondary metabolites, including one new altenusin derivative (1), were isolated. The isolated metabolites included seven known altenusin derivatives (2–8), two isochromanones (9, 10), one perylenequinone (11), and one benzocycloalkanone (12). Their structures were determined via 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), and computational electronic circular dichroism (ECD) analysis. Compounds 3 and 11 increased pyruvate dehydrogenase (PDH) activity in AD-293 human embryonic kidney cells and significantly inhibited PDH phosphorylation. The IC50 values of 3 and 11 were 32.58 and 27.82 μM, respectively.

Locoweeds, including Oxytropis and Astragalus species, are globally recognized as plants containing swainsonine (SW), a neurotoxic alkaloid that induces neurological dysfunction and growth inhibition in livestock. SW is produced by endophytic fungi in plants; the pyrroline-5-carboxylate reductase (P5CR) gene is critical in the fungal SW biosynthetic pathway. In this study, a P5CR gene knockout mutant (ΔP5CR) was constructed from the endophytic fungus Alternaria oxytropis OW7.8 isolated from Oxytropis glabra. Compared to the wild-type strain (A. oxytropis OW7.8), the SW content in the ΔP5CR mycelia was significantly reduced, indicating that the P5CR gene plays a crucial role in promoting SW biosynthesis. Compared to the wild-type strain A. oxytropis OW7.8, the ΔP5CR mutant exhibited distinct morphological alterations in both colony and mycelial structures. The transcriptomic analysis of A. oxytropis OW7.8 and ΔP5CR revealed the downregulation of six genes associated with SW biosynthesis. Metabolomic profiling further demonstrated altered levels of six metabolites linked to SW synthesis. These findings provide foundational insights into the molecular mechanisms and metabolic pathways underlying SW biosynthesis in fungi. They hold significant value for future strategies to control SW in Oxytropis glabra and contribute positively to the protection and sustainable development of grassland ecosystems.

Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH) and alternariol-9-methyl ether (AME) are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS). In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.

Swainsonine (SW) is a toxic alkaloid biosynthesized by the endophytic fungus Alternaria oxytropis in Oxytropis glabra . The biosynthetic pathway of SW is poorly understood. Saccharopine reductase/dehydrogenase of fungus plays an important role in this pathway. The gene knocked out mutant M1 in A. oxytropis was constructed in our previous work. In this study, the transcriptome of wild-strain OW7.8 and M1 was firstly sequenced to understand the biosynthetic pathway and molecular mechanism of SW in A. oxytropis . A total of 45,634 Unigenes were annotated. 5 genes were up-regulated and 11,213 genes were down-regulated. 41 Unigenes possibly related to the biosynthesis of SW were identified by data analyzing. The biosynthesis pathway of SW in the fungus was speculated, including two branches of P6C and P2C. Delta1-piperidine-2-carboxylate reductase, lysine 6-dehydrogenase, and saccharopine oxidase/L-pipecolate oxidase were involved in P6C. 1-piperidine-2-carboxylate/1-pyrroline-2- carboxylate reductase [NAD(P)H] and delta1-piperidine-2-carboxylate reductase were involved in P2C. Saccharopine reductase was involved in both. In addition, 1-indolizidineone was considered to be the direct precursor in the synthesis of SW, and the hydroxymethylglutaryl-CoA lyase catalyzed the synthesis of SW. Here we analyzed details of the metabolic pathway of A. oxytropis SW, which is of great significance for the follow-up research.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α- l -rhamnosidase from Alternaria sp . L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2′-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α- l -rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Background There is an imperative necessity for alternative sources of energy able to reduce the world dependence of fossil oil. One of the most successful options is ethanol obtained mainly from sugarcane and corn fermentation. The foremost residue from sugarcane industry is the bagasse, a rich lignocellulosic raw material uses for the production of ethanol second generation (2G). New cellulolytic and hemicellulytic enzymes are needed, in order to optimize the degradation of bagasse and production of ethanol 2G. Results The ability to produce hemicellulases and related enzymes, suitable for lignocellulosic biomass deconstruction, was explored using 110 endophytic fungi and 9 fungi isolated from spoiled books in Brazil. Two initial selections were performed, one employing the esculin gel diffusion assay, and the other by culturing on agar plate media with beechwood xylan and liquor from the hydrothermal pretreatment of sugar cane bagasse. A total of 56 isolates were then grown at 29°C on steam-exploded delignified sugar cane bagasse (DEB) plus soybean bran (SB) (3:1), with measurement of the xylanase, pectinase, β-glucosidase, CMCase, and FPase activities. Twelve strains were selected, and their enzyme extracts were assessed using different substrates. Finally, the best six strains were grown under xylan and pectin, and several glycohydrolases activities were also assessed. These strains were identified morphologically and by sequencing the internal transcribed spacer (ITS) regions and the partial β-tubulin gene (BT2). The best six strains were identified as Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49. These strains produced glycohydrolases with different profiles, and production was highly influenced by the carbon sources in the media. Conclusions The selected endophytic fungi Aspergillus niger DR02, Trichoderma atroviride DR17 and DR19, Alternaria sp. DR45, Annulohypoxylon stigyum DR47 and Talaromyces wortmannii DR49 are excellent producers of hydrolytic enzymes to be used as part of blends to decompose sugarcane biomass at industrial level.

produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B (FB ) in fumonisin-producing than in nonproducing , and likewise between isolates of and differing in production of sphinganine-analog toxins. It has been reported that in , ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of and by generating a double mutant strain in a background. Nearly unchanged resistance to added FB was observed compared to the parental strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast gene, but not , increased fumonisin resistance. High-level resistance was conferred by , but not by . Likewise, strong resistance to FB was caused by overexpression of the presumed \"housekeeping\" ceramide synthases , , and , located outside the fumonisin cluster, indicating that possesses a redundant set of insensitive targets as a self-resistance mechanism.