Effect of Amino Acids on Fusarium oxysporum Growth and Pathogenicity Regulated by TORC1- Tap42 Gene and Related Interaction Protein Analysis

Abstract

Free amino acids (AAs) formed in fermented meat products are important nitrogen sources for the survival and metabolism of contaminating fungi. These AAs are mainly regulated by the TORC1-Tap42 signaling pathway. Fusarium spp., a common contaminant of fermented products, is a potential threat to food safety. Therefore, there is an urgent need to clarify the effect of different AAs on Fusarium spp. growth and metabolism. This study investigated the effect of 18 AAs on Fusarium oxysporum (Fo17) growth, sporulation, T-2 toxin (T-2) synthesis and Tri5 expression through Tap42 gene regulation. Co-immunoprecipitation and Q Exactive LC-MS/MS methods were used to detect the interacting protein of Tap42 during specific AA treatment. Tap42 positively regulated L-His, L-Ile and L-Tyr absorption for Fo17 colony growth. Acidic (L-Asp, L-Glu) and sulfur-containing (L-Cys, L-Met) AAs significantly inhibited the Fo17 growth which was not regulated by Tap42. The L-Ile and L-Pro addition significantly activated the sporulation of ΔFoTap42. L-His and L-Ser inhibited the sporulation of ΔFoTap42. In T-2 synthesis, ΔFoTap42 was increased in GYM medium, but was markedly inhibited in L-Asp and L-Glu addition groups. Dose-response experiments showed that 10-70 mg/mL of neutral AA (L-Thr) and alkaline AA (L-His) significantly increased the T-2 production and Tri5 expression of Fo17, but Tri5 expression was not activated in ΔFoTap42. Inhibition of T-2 synthesis and Tri5 expression were observed in Fo17 following the addition of 30-70 mg/mL L-Asp. KEGG enrichment pathway analysis demonstrated that interacting proteins of Tap42 were from glycerophospholipid metabolism, pentose phosphate pathway, glyoxylate and dicarboxylate metabolism, glycolysis and gluconeogenesis, and were related to the MAPK and Hippo signaling pathways. This study enhanced our understanding of AA regulation in fermented foods and its effect on Fusarium growth and metabolism, and provided insight into potential ways to control fungal contamination in high-protein fermented foods.

Keywords: Fusarium oxysporum; KEGG; T-2 toxin; Tap42; amino acids.

Figure 1

Colony morphology of wild-type F. oxysporum Fo17, ΔFoTap42 and ΔFoTap42-C in dextrose agar medium, supplemented with 18 amino acids cultured for 7 d at 28 °C (n = 3). Control was cultured in the PDA medium.

Figure 2

Changed patterns of sporation (A) and T-2 synthesis (B) by F. oxysporum Fo17, ΔFoTap42 and ΔFoTap42-C exposed to 18 amino acids as nitrogen sources (n = 3). Control was cultured in CDA and GYM media.

Figure 3

Effects of L-Threonine (L-Thr) (A), L-Histidine (L-His) (B) and L-Aspartic acid (L-Asp) (C) on T-2 toxin production and Tri5 expression of different strains (n = 3), cultured at 28 °C for 14 d. The control was cultured in GYM medium. (a)–(c): T-2 toxin production of wild-type Fo17, ΔFoTap42 and ΔFoTap42-C; (d)–(f): Tri5 expression of wild-type Fo17, ΔFoTap42 and ΔFoTap42-C. Different letters “a–d” in the same plot indicate significant differences (p < 0.05).

Figure 4

(A) PCR amplification analysis of a plasmid digested by the XbaI-XhoI enzyme. A1: plasmid; A2: XbaI-XhoI digestion plasmid; M: DNA marker. (B) Prokaryotic expression of pET28a(+)-Tap42 fusion protein. B1: protein not induced by IPTG, B2-B4: clone 1–3 protein induced by IPTG. (C) Purification of pET28a(+)-Tap42 fusion protein. M: marker; C1: unpurified protein; C2: washed protein; C3: eluted protein; M: molecular protein marker. (D) Co-immunoprecipitation products of Tap42 protein identified by SDS-PAGE. D1: GYM control group, D2: L-Histidine (L-His)-treated group, D3: L-Threonine (L-Thr)-treated group; D4: L-Aspartic acid (L-Asp)-treated group, M: molecular protein marker.

Figure 5

The common and specific numbers of potential Tap42-interacting proteins in F. oxysporum following L-threonine (L-Thr), L-Histidine (L-His) and L-Aspartic acid (L-Asp) treatments (n = 3).

Figure 6

The KEGG enrichment analysis of the signaling pathway of Tap42, potentially interacting with proteins of F. oxysporum Fo17 cultured with L−threonine (L−Thr), L−Histidine (L−His) and L−Aspartic acid (L−Asp) (n = 3).