Role of Low-Affinity Calcium System Member Fig1 Homologous Proteins in Conidiation and Trap-Formation of Nematode-trapping Fungus Arthrobotrys oligospora

Abstract

Arthrobotrys oligospora is a typical nematode-trapping fungus capturing free-living nematodes by adhesive networks. Component of the low-affinity calcium uptake system (LACS) has been documented to involve in growth and sexual development of filamentous fungi. Bioassay showed incapacity of trap formation in A. oligospora on Water Agar plate containing 1 mM ethylene glycol tetraacetic acid (EGTA) due to Ca²⁺ absorbing block. The functions of homologous proteins (AoFIG_1 and AoFIG_2) of LACS were examined on conidiation and trap formation of A. oligospora. Compared with wild type, ΔAoFIG_1 (AOL_s00007g566) resulted in 90% of trap reduction, while ΔAoFIG_2 (AOL_s00004g576) reduced vegetative growth rate up to 44% and had no trap and conidia formed. The results suggest that LACS transmembrane protein fig1 homologs play vital roles in the trap-formation and is involved in conidiation and mycelium growth of A. oligospora. Our findings expand fig1 role to include development of complex trap device and conidiation.

Figure 1

Trap-formation of A. oligospora wild type strain on Water Agar (WA) medium and WA medium containing EGTA. (A) Trap-formation of A. oligospora on WA medium containing different EGTA concentration. (B) Dosage response curve of trap numbers and different EGTA concentration. (C) Expression pattern of AoFIG_1 and AoFIG_2 during trap-formation. Bar 100× = 50 μm; Bar 40× = 200 μm.

Figure 2

Phylogenetic analysis and protein structures prediction. (A) Phylogenetic relationships of the orthologs of AoFIG_1 and AoFIG_2. Unrooted tree was constructed using FastTree based on protein sequences. (B) Putative trans-membrane regions predicted in AoFIG_1 and AoFIG_2 by TMHMM.

Figure 3

Gene disruption strategy and verification by PCR and Southern blot. (A) Disruption strategy and verification of AoFIG_1 (B) Disruption strategy and verification of AoFIG_2. WT: the wild-type strain. In PCR verification, M1: DNA marker D2000; M2: DNA marker 1Kb (1 Kb and 2 Kb were marked on the right side respectively); H: hygromycin resistance gene partial sequence which was absent in WT and present in the mutants; G: gene ORF partial sequence which was absent in mutants and present in WT; U: PCR was used to ensure the correct recombination using primers UP and HYG500_R; D: PCR results using primers DOWN and HYG500_F; L: PCR results using primers DOWN and UP. The location of primers and expected length of PCR products were marked on the figure.

Figure 4

A. oligospora bio-assays. (A) The colony morphology of A. oligospora wild type strain (WT), ΔAoFIG_1 and ΔAoFIG_2 on PDA medium. (B) Vegetative growth rates of ΔAoFIG_1 and ΔAoFIG_2 on PDA medium; colony diameters were measured every 24-hour cultivation. (C) Capacity of conidiation on PDA medium after 3- and 6-day cultivation. (D) Capacity of trap-formation after induced by nematodes for 24 and 48 hours. ^aNot found. ^bp < 0.01, t-test.

Figure 5

Trap-formation of A. oligospora wild-type strain (WT), ΔAoFIG_1 and ΔAoFIG_2. Traps were induced by nematodes for 24 and 48 hours. The morphological characteristics of the traps were shown under both 40× and 100× magnification. a: adhesive networks; b: conidia; c: nematodes. Bar A, C, E, G, I, K = 200 μm; Bar B, D, F, H, J, L = 50 μm.

Figure 6

Relative growth rates of A. oligospora wild type strain (WT), disruption mutants of ΔAoFIG_1 and ΔAoFIG_2 to abiotic stresses. Growth rates were calculated from 24-hour to 96-hour cultivation; the growth rates on PDA at 28 °C were used as control. (A) Cold stress at 22 °C. (B) Cold stress at 15 °C. (C) PDA containing 200 μg/mL Congo red at 28 °C. (D) Osmotic stress: PDA containing 1 mol/L sorbitol and PDA containing 0.5 mol/L KCl at 28 °C.