Marion Brodhagen

Signalling pathways connecting mycotoxin production and sporulation

SUMMARY Mycotoxin contamination of food and feed presents a serious food safety issue on a global scale, causing tremendous yield and economic losses. These toxins, produced largely by members of the genera Aspergillus and Fusarium, represent a subset of the impressive array of secondary metabolites produced by filamentous fungi. Some secondary metabolites are associated temporally and functionally with sporulation. In Aspergillus and Fusarium, sporulation and mycotoxin production are both regulated by G protein signalling pathways. G protein signalling pathways commonly regulate fungal development, stress response and expression of virulence traits. In addition, fungal development is influenced by external factors. Among these are lipids, and in particular, oxylipin signals, which may be derived from either the fungus or infected seeds. Regardless of origin, oxylipins have the potential to elicit profound changes in both sporulation and mycotoxin production in the fungus. Signal transduction via G protein signalling pathways represents one mechanism by which oxylipin signals might elicit these changes. Therefore, in this review we integrate discussion of oxylipin signals and of G protein signalling cascades as regulators of fungal development.

Inactivation of the Lipoxygenase ZmLOX3 Increases Susceptibility of Maize to Aspergillus spp.

Plant and fungal lipoxygenases (LOX) catalyze the oxidation of polyunsaturated fatty acids, creating fatty-acid hydroperoxides (oxylipins). Fungal oxylipins are required for normal fungal development and secondary metabolism, and plant host-derived oxylipins interfere with these processes in fungi, presumably by signal mimicry. The maize LOX gene ZmLOX3 has been implicated previously in seed-Aspergillus interactions, so we tested the interactions of a mutant maize line (lox3-4, in which ZmLOX3 is disrupted) with the mycotoxigenic seed-infecting fungi Aspergillus flavus and Aspergillus nidulans. The lox3-4 mutant was more susceptible than wild-type maize to both Aspergillus species. All strains of A. flavus and A. nidulans produced more conidia and aflatoxin (or the precursor sterigmatocystin) on lox3-4 kernels than on wild-type kernels, in vitro and under field conditions. Although oxylipins did not differ detectably between A. flavus-infected kernels of the lox3-4 and wild-type (WT) maize, oxylipin precursors (free fatty acids) and a downstream metabolite (jasmonic acid) accumulated to greater levels in lox3-4 than in WT kernels. The increased resistance of the lox3-4 mutant to other fungal pathogens (Fusarium, Colletotrichum, Cochliobolus, and Exserohilum spp.) is in sharp contrast to results described herein for Aspergillus spp., suggesting that outcomes of LOX-governed host-pathogen interactions are pathogen-specific.

Reciprocal oxylipin-mediated cross-talk in the Aspergillus-seed pathosystem

In Aspergilli, mycotoxin production and sporulation are governed, in part, by endogenous oxylipins (oxygenated, polyunsaturated fatty acids and metabolites derived therefrom). In Aspergillus nidulans, oxylipins are synthesized by the dioxygenase enzymes PpoA, PpoB and PpoC. Structurally similar oxylipins are synthesized in seeds via the action of lipoxygenase (LOX) enzymes. Previous reports have shown that exogenous application of seed oxylipins to Aspergillus cultures alters sporulation and mycotoxin production. Herein, we explored whether a plant oxylipin biosynthetic gene (ZmLOX3) could substitute functionally for A. nidulans ppo genes. We engineered ZmLOX3 into wild‐type A. nidulans, and into a ΔppoAC strain that was reduced in production of oxylipins, conidia and the mycotoxin sterigmatocystin. ZmLOX3 expression increased production of conidia and sterigmatocystin in both backgrounds. We additionally explored whether A. nidulans oxylipins affect seed LOX gene expression during Aspergillus colonization. We observed that peanut seed pnlox2–3 expression was decreased when infected by A. nidulansΔppo mutants compared with infection by wild type. This result provides genetic evidence that fungal oxylipins are involved in plant LOX gene expression changes, leading to possible alterations in the fungal/host interaction. This report provides the first genetic evidence for reciprocal oxylipin cross‐talk in the Aspergillus–seed pathosystem.

Aspergillus Oxylipin Signaling and Quorum Sensing Pathways Depend on G Protein-Coupled Receptors

Oxylipins regulate Aspergillus development and mycotoxin production and are also involved in Aspergillus quorum sensing mechanisms. Despite extensive knowledge of how these oxylipins are synthesized and what processes they regulate, nothing is known about how these signals are detected and transmitted by the fungus. G protein-coupled receptors (GPCR) have been speculated to be involved as they are known oxylipin receptors in mammals, and many putative GPCRs have been identified in the Aspergilli. Here, we present evidence that oxylipins stimulate a burst in cAMP in A. nidulans, and that loss of an A. nidulans GPCR, gprD, prevents this cAMP accumulation. A. flavus undergoes an oxylipin-mediated developmental shift when grown at different densities, and this regulates spore, sclerotial and aflatoxin production. A. flavus encodes two putative GprD homologs, GprC and GprD, and we demonstrate here that they are required to transition to a high-density development state, as well as to respond to spent medium of a high-density culture. The finding of GPCRs that regulate production of survival structures (sclerotia), inoculum (spores) and aflatoxin holds promise for future development of anti-fungal therapeutics.

Biodegradable plastic agricultural mulches and key features of microbial degradation

The development of biodegradable plastic mulch films for use in agriculture has been ongoing for decades. These films consist of mixtures of polymers with various additives. As a result, their physical and chemical properties differ from those of the pure polymers often used for in vitro enzymatic and microbial degradation studies, raising questions about the biodegradation capability of mulch films. Currently, standards exist for the biodegradation of plastics in composting conditions but not in soil. Biodegradation in soil or compost depends on a complex synergy of biological and abiotic degradative processes. This review discusses the physicochemical and structural properties of biodegradable plastic mulches, examines their potential for on-site decomposition in light of site-to-site variance due to environmental and biological conditions, and considers the potential for long-term effects on agroecosystem sustainability and functionality.

Immunocytochemical localization of taxol in Taxus cuspidata

The supply of taxol, a valuable anticancer compound, depends completely on the extraction of taxol from Taxus brevifolia (Pacific yew) plants. Although taxol is found in virtually all parts of the plant, nothing is known about the localization of taxol within the cells or tissues of the plant. Portions of T. cuspidata stems were chemically fixed, dehydrated in ethanol, and then embedded in standard resins. In these tissues, immunolocalization using polyclonal antitaxol antiserum indicates that taxol is associated with vacuolar tannin inclusions in axial phloem parenchyma cells. Little or no label is bound over the cell walls of any cell type. However, chemical preparation causes diffusion of taxol, which results in an erroneous localization of taxol. In contrast, using cryotechniques and a water-soluble melamine resin (Nanoplast), taxol is localized almost exclusively in the cell walls of phloem, vascular cambium, and xylem. Our method yields insight into the problems associated with the intra- and extracellular localization of lipophilic plant secondary compounds. It also offers an alternative tissue-preparation protocol that could be useful for the localization of other plant metabolites.

Isolation of native soil microorganisms with potential for breaking down biodegradable plastic mulch films used in agriculture.

Fungi native to agricultural soils that colonized commercially available biodegradable mulch (BDM) films were isolated and assessed for potential to degrade plastics. Typically, when formulations of plastics are known and a source of the feedstock is available, powdered plastic can be suspended in agar-based media and degradation determined by visualization of clearing zones. However, this approach poorly mimics in situ degradation of BDMs. First, BDMs are not dispersed as small particles throughout the soil matrix. Secondly, BDMs are not sold commercially as pure polymers, but rather as films containing additives (e.g. fillers, plasticizers and dyes) that may affect microbial growth. The procedures described herein were used for isolates acquired from soil-buried mulch films. Fungal isolates acquired from excavated BDMs were tested individually for growth on pieces of new, disinfested BDMs laid atop defined medium containing no carbon source except agar. Isolates that grew on BDMs were further tested in liquid medium where BDMs were the sole added carbon source. After approximately ten weeks, fungal colonization and BDM degradation were assessed by scanning electron microscopy. Isolates were identified via analysis of ribosomal RNA gene sequences. This report describes methods for fungal isolation, but bacteria also were isolated using these methods by substituting media appropriate for bacteria. Our methodology should prove useful for studies investigating breakdown of intact plastic films or products for which plastic feedstocks are either unknown or not available. However our approach does not provide a quantitative method for comparing rates of BDM degradation.