Ammonia and Nematode Ascaroside Are Synergistic in Trap Formation in Arthrobotrys oligospora

Abstract

Nematode-trapping (NT) fungi are natural predators of the soil living nematodes. Diverse external signals mediate the generation of predatory devices of NT fungi. Among these, broad ascarosides and nitrogenous ammonia are highly efficient inducers for trap structure initiation. However, the overlay effect of ammonia and ascaroside on the trap morphogenesis remains unclear. This study demonstrated that the combination of nitrogenous substances with nematode-derived ascarosides led to higher trap production compared to the single inducing cues; notably, ammonia and Ascr#18 had the most synergistic effect on the trap in A. oligospora. Further, the deletion of ammonia transceptor Amt43 blocked trap formation against ammonia addition in A. oligospora but not for the ascaroside Ascr#18 induction. Moreover, ammonia addition could promote plasma endocytosis in the process of trap formation. In contrast, ascaroside addition would facilitate the stability of intracellular organization away from endocytosis. Therefore, there is a synergistic effect on trap induction from different nitrogenous and ascaroside signals.

Keywords: Acsr#18; Amt43; Arthrobotrys oligospora; ammonia; synergy; trap formation.

Figure 1

Measurement of trap-inducing activity by nitrogenous and ascaroside signals. (A) Comparison of adhesive traps produced by the overlay effect of Ascr#18 and Am (Bar = 100 μm). (B) The mix of Ascr#18 and Am significantly promoted the formation of traps. (C) Comparison of traps produced by the overlay effect of Ascr#18 and the different nitrogen signals; ** p < 0.01, *** p < 0.001.

Figure 2

Domain analyses of ammonia transport proteins. (A) Comparison of gene functional domains between Saccharomyces cerevisiae Mep2 and Arthrobotrys oligospora AmtBs (AoAmt6, AoAmt43, AoAmt80). The numerical scale shows the length of amino acid chains; it was presented by NCBI and TBtools software ((version 1.09861, CJ chen, Guangdong Province, China). (B) The phylogenetic tree of Amt in fungi (Arthrobotrys oligospora, Drechslerella stenobrocha, Dactylellina haptotyla, Aspergillus niger, Aspergillus nidulans, Saccharomyces cerevisiae); the tree was constructed using Mega 7 software (version 7.0, Mega Limited, Auckland, New Zealand). (C) The 11 helices of Amt43 and the ammonia transport process in fungi, referenced from “Hypothetical transport mechanisms of Mep/Amt proteins” [41].

Figure 3

The dynamic response of A. oligospora by the addition of Ascr#18 or/and Am. (A) Schematic diagram of Amt43::GFP fluorescent labeling. (B) Green fluorescence shows that Amt43 is mainly localized on the cell membrane of the hyphal of the NT fungus A. oligospora (bar = 10 μm). (C) Amt43::GFP dynamics responding to Ascr#18 or/and Am (bar = 10 μm), v is for vacuole. (D) Dynamics response of endocytosis by Ascr#18 or/and Am (bar = 10 μm).

Figure 4

The growth and trap-inducing activity in strains of the WT and ΔAoAmt43. (A) Measuring the growth and spore number of ΔAoAmt43 (PDA bar = 1 cm, CMY bar = 100 μm). (B) Comparison of the spore numbers of WT, ΔAoAmt43, and ΔAoAmt43::Amt43. (C) The trap-inducing activity of ΔAoAmt43 under the different nitrogen signals. Am, 25% ammonia diluted 10³ times; NH₄Cl, 37.4 mM; urea, 33.3 mM; Glu (glutamine), 3.4 mM. (D) The trap-inducing activity of ΔAoAmt43 under ammonia or/and Ascr#18 (Am, 25% ammonia diluted 10³ times; Ascr#18, 10 nM); unpaired t-test, parametric test, ** p < 0.01, *** p < 0.001.

Figure 5

The model of ammonia and Ascr#18 on trap formation in A. oligospora. Entry of exogenous ammonia into A. oligospora mycelial cells via ammonia transport proteins in the cell membrane, and synergism with exogenous ascarosides in the cell, promotes the formation of adhesive nets of traps by A. oligospora, which in turn traps and kills nematodes more rapidly and controls nematode populations.