PaTrx1 and PaTrx3, two cytosolic thioredoxins of the filamentous ascomycete Podospora anserina involved in sexual development and cell degeneration

Abstract

In various organisms, thioredoxins are known to be involved in the reduction of protein disulfide bonds and in protecting the cell from oxidative stress. Genes encoding thioredoxins were found by searching the complete genome sequence of the filamentous ascomycete Podospora anserina. Among them, PaTrx1, PaTrx2, and PaTrx3 are predicted to be canonical cytosolic proteins without additional domains. Targeted disruption of PaTrx1, PaTrx2, and PaTrx3 shows that PaTrx1 is the major thioredoxin involved in sulfur metabolism. Deletions have no effect on peroxide resistance; however, data show that either PaTrx1 or PaTrx3 is necessary for sexual reproduction and for the development of the crippled growth cell degeneration (CG), processes that also required the PaMpk1 mitogen-activated protein kinase (MAPK) pathway. Since PaTrx1 PaTrx3 mutants show not an enhancement but rather an impairment in CG, it seems unlikely that PaTrx1 and PaTrx3 thioredoxins participate in the inhibition of this MAPK pathway. Altogether, these results underscore a role for thioredoxins in fungal development.

FIG. 1.

Phylogenetic tree of the proteins indicated produced by using the neighbor-joining method with 1,000 replication for bootstrap. Analysis was performed with the thioredoxin domain underlined on Fig. S2 in the supplemental material. Similar trees were obtained with the maximum-parsimony and minimum evolution algorithms. The GenBank accession numbers for PaTrx1 is AY077728 and that for PaTrx2 is AY077731.

FIG. 2.

Mycelium morphology of strains with the thioredoxin genes deleted on M2 and on M2 supplemented with methionine. WT, wild type; Δ1, ΔPaTrx1, Δ2: ΔPaTrx2; Δ3, ΔPaTrx3.

FIG. 3.

Lack of CG in the ΔPaTrx1 ΔPaTrx3 and ΔPaTrx1 ΔPaTrx2 ΔPaTrx3 strains. On the left is depicted the experimental setup. A culture is grown for 7 days on M2 medium supplemented with methionine, and explants taken at various distances from the growing edge are replicated onto M2 medium supplemented with yeast extract. The C element responsible for CG is induced in the stationary phase and triggers CG in explants taken from the part of the thallus that is in the stationary phase. In contrast, explants taken from the growing edge yield mycelia in normal growth (NG) conditions. CG is visible in wild type grown on M2 plus yeast extract as a flat and spindly growth with pigment accumulation (8, 37). In the ΔPaTrx1 ΔPaTrx3 mutant, growth impairment is much reduced and visible as a slightly flatter mycelium. In the ΔPaTrx1 ΔPaTrx2 ΔPaTrx3 mutant no discernible CG was observed. Note that the mycelium from this strain grows more slowly, produces fewer aerial hyphae, and accumulates more pigment on M2 supplemented with yeast extract than normal-growing wild-type strains.

FIG. 4.

Effect of thioredoxin deletions during sexual reproduction. (Top row) Fertility defect of the ΔPaTrx1 ΔPaTrx3 and ΔPaTrx1 ΔPaTrx2 ΔPaTrx3 strains. mat+/mat− heterokaryotic mycelia of the indicated genotypes were incubated at 27°C for 1 week, at which time the perithecia are fully mature and starting to eject ascospores in wild-type strains. Incubation times of more than 3 weeks did not permit further maturation of perithecia in the ΔPaTrx1 ΔPaTrx3 and ΔPaTrx1 ΔPaTrx2 ΔPaTrx3 mutants. (Middle row) The fertility defect of the indicated mat+/mat− heterokaryons was not corrected when Δmat nuclei were added in the heterokaryons. (Bottom row) The perithecial envelope of the thioredoxin mutants/193 heterokaryon derived mostly or exclusively from the 193 parent, showing that expression of PaTrx1 or PaTrx3 was required in the perithecial envelope.

FIG. 5.

Lack of fertility on M2 medium and M2 medium supplemented with methionine of strains lacking PaTrx1/PaTrx3 and overexpressing PaTrx2 (surT2 Δ1Δ3). mat+/mat− heterokaryotic strains of the indicated genotypes were incubated for 10 days on M2 medium or M2 medium supplemented with 20 μg of methionine/ml, at which time the wild type produces fully mature perithecia, whereas strains with PaTrx1 and PaTrx3 deleted do not produce mature fructifications even if PaTrx2 is overexpressed. A longer incubation time does not result in perithecium maturation in these mutants.