Effects of point-mutated profilins on the growth of Neurosporacrassa and the biochemical mechanisms

Abstract:

[Objective]In order to further clarify the functions of profilin (PFN) in filamentous fungi, we explored the effects of point-mutated PFNs on the colony growth and actin polymerization of Neurospora crassa. [Methods] The PFN F78 mutants (F78A and F78D) and V113 mutants (V113E, V113R and V113W) of N.crassa were obtained by techniques of site-directed mutagenesis, homologous recombination, filtration of conidia and PCR. With plate method, race tube assay and microscopy, the phenotypic changes of colonies were observed, and together with polyproline affinity chromatography, fluorescence spectrophotometry and high-speed co-sedimentation, the effect of point mutation of PFN on the actin nucleation and polymerization was analyzed. [Results] Mutants F78A, F78D, V113E, V113R, and V113W all grew slowly compared with the control strain ku70^RIP (P<0.05). Particularly, the colony diameters of F78D and V113W were only 20%–75.7% and 12.7%–39.2% that of the control at 12–48 h. Race tube analysis demonstrated that mycelia of F78D and V113W presented slow growth in comparison with the control. However, both of them exhibited near-normal conidiation rhythms. In addition, the purified wild-type PFN could inhibit the spontaneous nucleation of actin in a concentration-dependent manner, while the inhibition of mutant proteins PFN (F78D) and PFN (V113W) on the actin nucleation was weakened. Wild-type PFN could suppress the polymerization of actin in the concentration range of 0–5 mmol/L, and the suppression was enhanced with the increase of the concentration, causing the monomer actin content in the supernatant to peak at about 82%. However, the inhibitory effect of 5 mmol/L PFN (F78D) and PFN (V113W) on actin polymerization was significantly weakened, as manifested by the decrease of actin monomer content by 12.0% (P<0.01) and 30.7% (P<0.01), respectively, from the control. [Conclusion]This study confirmed that PFN played an important role in N.crassa, and F78 and V113 were important active sites for regulating the polymerization and depolymerization of actin and the growth and development of N.crassa.