Repression of autocrine pheromone signaling leads to fusaric acid over-production

Abstract Fusaric acid (FA), a picolinic acid derivative, is a natural substance produced by a wide variety of fungal plant pathogens belonging to the Fusarium genus. As a metabolite, fusaric acid exerts several biological activities including metal chelation, electrolyte leakage, repression of ATP synthesis, and direct toxicity on plants, animals and bacteria. Prior studies on the structure of fusaric acid revealed a co-crystal dimeric adduct between FA and 9,10-dehydrofusaric acid. During an ongoing search for signaling genes differentially regulating FA production in the fungal pathogen Fusarium oxysporum (Fo), we found that mutants lacking pheromone expression have an increased production of FA compared to the wild type strain. Noteworthy, crystallographic analysis of FA extracted from Fo culture supernatants showed that crystals are formed by a dimeric form of two FA molecules (1:1 molar stoichiometry). Overall, our results suggest that pheromone signaling in Fo is required to regulate the synthesis of fusaric acid. Graphical Abstract


Introduction
Fusaric acid (Fa), a picolinic acid derivative initially isolated from Fusarium heterosporium (Yabuta et al. 1937), is a natural product also synthesized by a wide variety of other fungal plant pathogens belonging to the Fusarium genus.Firstly described as a phytotoxin (Gäumann 1957), several other biological activities were associated to Fa, including metal chelation, and direct toxicity on animals and bacteria (d'alton and Etherton 1984;marrè et al. 1993;porter et al. 1995;pavlovkin 1998;Bacon et al. 2006;Ruiz et al. 2015;Yin et al. 2015).
Fa functions as a metal-chelating agent owing to the presence of a carboxylic and pyridinic moieties being able to form conjugates with divalent ions including zinc, copper, manganese, or iron(iii) that are fundamental regulators of several cell biological processes (Ruiz et al. 2015).Fusaric acid is also able to penetrate cell membranes, due to the butyl side chain that enhances the molecule's lipophilicity (arumugam et al. 2021).as a result, Fa showed potential teratogen effects for animals consuming plants infected with Fusarium sp.(Yin et al. 2015).
Similar to most fungal secondary metabolites, Fa biosynthesis requires a complex biosynthetic gene cluster that includes at least twelve genes (FUB1 to FUB12) (Brown et al. 2015;Keller 2019).the expression of FUB genes is regulated by a variety of environmental conditions including pH, metal ion availability, and nitrogen sources (Brown et al. 2012;López-díaz et al. 2018;palmieri et al. 2023).moreover, nitrogen sources and pH values also influence the production of two fusaric acid-like compounds: fusarinolic acid and 9,10-dehydrofusaric acid (niehaus et al. 2014).
in Fusarium oxysporum (Fo), environmental regulation of Fa production is under control of the cell wall integrity (CWi) map kinase pathway, a signaling cascade regulating both responses to fungal pheromones and plant secreted chemical cues (ding et al. 2015;turrà et al. 2015;Vitale et al. 2019;palmieri et al. 2023).Here, in order to understand if Fo regulation of Fa production is dependent or independent on autocrine perception of pheromone peptides via the CWi pathway, we used a previously generated MFaΔMFαΔ mutant strain lacking both pheromone genes (Vitale et al. 2019).

Results and discussion
in this study, we have readily obtained Fa in a pure crystalline form, after extraction of culture supernatants from both wild type and MFaΔMFαΔ Fo strains.We have further characterized the compound by mass spectrometry and single crystal X-ray analyses.
the structure of fusaric acid is characterized by a dimeric adduct of two Fa molecules (1:1 molar stoichiometry) including one zwitterionic moiety of the compound, as shown in Figure 1. the structure reveals the presence of the same molecule in a neutral and zwitterionic form.the dimer is stabilized by a hydrogen bond between the -oH group of the neutral carboxylic atom group of a fusaric acid and the negatively charged -o atom group of zwitterion [n…o = 2.51(1)], as shown in Figure 1.interestingly, a database survey for Fa revealed a co-crystal structure of a similar dimer between Fa and 9,10-dehydrofusaric acid (Wang et al. 2005).the X-ray structure of the fusaric acid is shown in Figure S1. the compound crystallizes in centrosymmetric (monoclinic) space group p 2 1 /n with one dimer in the asymmetric unit.the dimer is stabilized by the intramolecular hydrogen bond of 2.650 Å between the -oH group and oxygen atom group of the ketone moiety.Crystal packing of fusaric acid shows anti parallel arrangement of the metabolite and exhibits pi -pi stacking interactions of the molecule core structure along the a-axis, in agreement with the inversion center of p 2 1 /n space group (Figure S1).
Fa presence was confirmed also by LC-mS and GC-mS analyses (Figure S2a and b).LC-mS mass spectrum (Figure S2a (Spitellek 1967;Capasso et al. 1996) GC-mS dataset for both Fo wild type and MFaΔMFαΔ mutant strains were subjected to univariate statistical analysis (Figure S3): Fa resulted more abundant (34%) in the MFaΔMFαΔ mutant compared to the wild type, with a Ln(FC) (natural logarithm of fold change) value of 0.44.
it has been previously reported that Fo mutants lacking the conserved CWi components Bck1, mkk2 and mpk1 produce significantly less Fa compared to the wild type strain.Similarly, production of Fa is largely reduced in knockout mutants of the upstream Ste2 pheromone-receptor which is required to sense both α-pheromone and additional host-derived stimuli through the CWi map kinase cascade (turrà et al. 2015; palmieri et al. 2023).as the MFaΔMFαΔ mutant is unable to produce both Fo pheromone peptides, we conclude that Fa biosynthesis in Fo is repressed by pheromone signaling while derepressed by alternative pheromone-independent signals via the CWi map kinase pathway.

Conclusions
this study focused on the isolation of fusaric acid from wild type and MFaΔMFαΔ mutant strains of Fo.We have structurally characterized Fa by X-ray single structure analysis and mass spectrometry.the X-ray structure has revealed a dimeric adduct made of two fusaric acids including a zwitterionic form.moreover, we found an accumulation of Fa in the MFaΔMFαΔ mutant compared to wild type strain.together with our previous results, this finding gives new insight into Fa biosynthesis, that we conclude is being regulated by pheromone signaling and the CWi map kinase pathway in Fo.