Fungi belonging to the genus Trichoderma are among the most active and ecologically successful microbes found in natural environments, as they are able to use a variety of substrates and affect the growth of other microbes and virtually any plant species. We isolated and characterized a novel type II hydrophobin secreted by the biocontrol strain MK1 of Trichoderma longibrachiatum. The corresponding gene (Hytlo1) has a multiple role in the Trichoderma-plant-pathogen three-way interaction, while the purified protein displayed a direct antifungal as well as a MAMP and a plant growth promotion (PGP) activity. Leaf infiltration with the hydrophobin systemically increased resistance to pathogens and activated defence-related responses involving ROS, SOD, oxylipins, phytoalexins and PR-proteins formation or activity. The hydrophobin was found to enhance development of a variety of plants when applied at very low doses. It particularly stimulated root formation and growth, as demonstrated also by transient expression of the encoding gene in tobacco and tomato. Targeted knock-out of Hytlo1 significantly reduced both antagonistic and PGP effect of the WT strain. We conclude that this protein represents a clear example of a molecular factor developed by Trichoderma to establish a mutually beneficial interaction with the colonized plant.
Fusarium verticillioides is one of the most important fungal pathogens causing ear and stalk rot in maize, even if frequently asymptomatic, producing a harmful series of compounds named fumonisins. Plant and fungal oxylipins play a crucial role in determining the outcome of the interaction between the pathogen and its host. Moreover, oxylipins result as signals able to modulate the secondary metabolism in fungi. In keeping with this, a novel, quantitative LC-MS/MS method was designed to quantify up to 17 different oxylipins produced by F. verticillioides and maize kernels. By applying this method, we were able to quantify oxylipin production in vitro - F. verticillioides grown into Czapek-Dox/yeast extract medium amended with 0.2% w/v of cracked maize - and in vivo, i.e. during its growth on detached mature maize ears. This study pinpoints the role of oxylipins in a plant pathogen such as F. verticillioides and sets up a novel tool aimed at understanding the role oxylipins play in mycotoxigenic pathogens during their interactions with respective hosts.
In some filamentous fungi, the pathways related to the oxidative stress and oxylipins production are involved both in the process of host-recognition and in the pathogenic phase. In fact, recent studies have shown that the production of oxylipins in filamentous fungi, yeasts and chromists is also related to the development of the organism itself and to mechanisms of communication with the host at the cellular level. The oxylipins, also produced by the host during defense reactions, are able to induce sporulation and to regulate the biosynthesis of mycotoxins in several pathogenic fungi. In A. flavus, the oxylipins play a crucial role as signals for regulating the biosynthesis of aflatoxins, the conidiogenesis and the formation of sclerotia. To investigate the involvement of an oxylipins based cross-talk into Z. mays and A. flavus interaction, we analyzed the oxylipins profile of the wild type strain and of three mutants of A. flavus that are deleted at the Aflox1 gene level also during maize kernel invasion. A lipidomic approach has been addressed through the use of LC-ToF-MS, followed by a statistical analysis of the principal components (PCA). The results showed the existence of a difference between the oxylipins profile generated by the WT and the mutants onto challenged maize. In relation to this, aflatoxin synthesis which is largely hampered in vitro, is intriguingly restored. These results highlight the important role of maize oxylipin in driving secondary metabolism in A. flavus.
Aspergillus flavus is a cosmopolitan fungus able to respond to external stimuli and to shift both its trophic behaviour and the production of secondary metabolites, including that of the carcinogen aflatoxin (AF). To better understand the adaptability of this fungus, we examined genetic and phenotypic responses within the fungus when grown under four conditions that mimic different ecological niches ranging from saprophytic growth to parasitism. Global transcription changes were observed in both primary and secondary metabolism in response to these conditions, particularly in secondary metabolism where transcription of nearly half of the predicted secondary metabolite clusters changed in response to the trophic states of the fungus. The greatest transcriptional change was found between saprophytic and parasitic growth, which resulted in expression changes in over 800 genes in A. flavus. The fungus also responded to growth conditions, putatively by adaptive changes in conidia, resulting in differences in their ability to utilize carbon sources. We also examined tolerance of A. flavus to oxidative stress and found that growth and secondary metabolism were altered in a superoxide dismutase (sod) mutant and an alkyl-hydroperoxide reductase (ahp) mutant of A. flavus. Data presented in this study show a multifaceted response of A. flavus to its environment and suggest that oxidative stress and secondary metabolism are important in the ecology of this fungus, notably in its interaction with host plant and in relation to changes in its lifestyle (i.e. saprobic to pathogenic).
In filamentous fungi, peroxisomes are crucial for the primary metabolism and play a pivotal role in the formation of some secondary metabolites. Further, peroxisomes are important site for fatty acids ?-oxidation, the formation of reactive oxygen species and for their scavenging through a complex of antioxidant activities. Oxidative stress is involved in different metabolic events in all organisms and it occurs during oxidative processes within the cell, including peroxisomal ?-oxidation of fatty acids. In Aspergillus flavus, an unbalance towards an hyper-oxidant status into the cell is a prerequisite for the onset of aflatoxin biosynthesis. In our preliminary results, the use of bezafibrate, inducer of both peroxisomal ?-oxidation and peroxisome proliferation in mammals, significantly enhanced the expression of pex11 and foxA and stimulated aflatoxin synthesis in A. flavus. This suggests the existence of a correlation among peroxisome proliferation, fatty acids ?-oxidation and aflatoxin biosynthesis. To investigate this correlation, A. flavus was transformed with a vector containing P33, a gene from Cymbidium ringspot virus able to induce peroxisome proliferation, under the control of the promoter of the Cu,Zn-sod gene of A. flavus. This transcriptional control closely relates the onset of the antioxidant response to ROS increase, with the proliferation of peroxisomes in A. flavus. The AfP33 transformant strain show an up-regulation of lipid metabolism and an higher content of both intracellular ROS and some oxylipins. The combined presence of a higher amount of substrates (fatty acids-derived), an hyper-oxidant cell environment and of hormone-like signals (oxylipins) enhances the synthesis of aflatoxins in the AfP33 strain. The results obtained demonstrated a close link between peroxisome metabolism and aflatoxin synthesis.
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