Purification and Identification of the Nematicidal Activity of S1 Family Trypsin-Like Serine Protease (PRA1) from Trichoderma longibrachiatum T6 Through Prokaryotic Expression and Biological Function Assays.

Background/objectives: Heterodera avenae is a highly significant plant-parasitic nematode, causing severe economic losses to global crop production each year. Trichoderma species have been found to parasitize nematodes and control them by producing enzymes that degrade eggshells. The T. longibrachiatum T6 (T6) strain has been demonstrated the parasitic and lethal effects on H. avenae cysts and eggs, associated with the increased serine protease activity and trypsin-like serine protease gene (PRA1) expression.

Methods: Our present study aimed to purify the recombinant PRA1 protease through a prokaryotic expression system and identify its nematicidal activity.

Results: The recombinant PRA1 protease was identified as S1 family trypsin-like serine protease, with a molecular weight of 43.16 kDa. The purified soluble protease exhibited the optimal activity at 35 °C and pH 8.0, and also demonstrating higher hydrolytic ability toward casein and skimmed milk. Meanwhile, the Ca²⁺ and Mg²⁺ enhanced its activity, while the inhibitor PMSF significantly reduced it. The contents of H. avenae eggs leaked out after treatment with the recombinant PRA1 protease, with egg hatching inhibition and relative hatching inhibition rates at 70.60% and 66.58%, respectively. In contrast, there was no sign of content dissolution, and embryos developed normally in the control group.

Conclusions: Our present study revealed that the PRA1 protease of T6 strain has a lethal effect on H. avenae eggs, which providing a theoretical basis for developing biocontrol agents to control nematodes.