This article has been cited by other articles in PMC. Data columns are as follows. Gene: a given predicted protein-coding or ncRNA-coding gene in the C. All further data columns are pertinent to that particular gene. Larvae: the expression level for a given gene in whole larvae, generated from a pooled set of all larval RNA-seq reads, measured in TPM.
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Search Menu Abstract The nematophagous fungus Arthrobotrys oligospora is a potential biological agent against parasitic gastrointestinal nematodes. Its subtilisin-like serine proteases play an important role in nematode cuticle breach. Our results show that the reXAoz1 had a molecular mass of 50 kDa after 3 days of 1. The reXAoz1 had the highest hydrolytic activity at pH 6. Moreover, the purified reXAoz1 displayed a highly toxic and biological activity to immobilize C.
Arthrobotrys oligospora , nematicidal activity , Pichia pastoris , recombinant expression , serine protease XAoz1 Introduction A chemotherapeutic approach has been used as the primary method for controlling parasitic gastrointestinal nematode disease Stear et al. Previous methods for treatment include anthelmintics but these are largely ineffective due to increase resistance to these drugs da Cruz et al.
Therefore, alternatives to the therapeutic chemical strategies are required Stear et al. Nematophagous fungi can enter the host through synergistic interactions of mechanical forces and hydrolytic enzymes that include protease, chitinase, and collagenase e.
Tosi et al. Increasing evidence has shown that extracellular subtilisin-like serine protease may be a key virulent factor involved in the penetration, immobilization, and digestion process Huang et al. These proteases have undergone selective pressure through the co-evolution of trapping structures and proteolytic enzymes Li et al. Among nematophagous fungi, the filamentous fungus Arthrobotrys oligospora is the most common fungus, with remarkable morphological adaptation, possessing the ability to form adhesive trapping nets once in contact with nematodes Huang et al.
Furthermore, A. In this study, the virulence gene of the serine protease XAoz1 of A. This subtilisin-like protease will be heterologously expressed in a methylotrophic Pichia pastoris. The cidal effects of the active recombinant XAoz1 reXAoz1 against the free-living nematode Caenorhabditis elegans and the animal-parasitic nematode Haemonchus contortus will be assessed.
The transformants were grown and selected on MD plates. Wild-type bacterivorous C. Infective larvae L3 of the animal-parasitic nematode H. The purified native XAoz1 obtained by the method of Zhao et al. Based on the serine protease gene of A. The purified amplicons were cloned into the vector pMDT. Generation of recombinant P.
One milliliter of ice-cold sorbitol solution 1 M was immediately added after electroporation. The transformants were patched on YPD plates containing different levels of G 0.
Expression of recombinant protease XAoz1 in P. Effects of different pHs on enzyme activity The pH profiles of the recombinant protease were investigated with the Britton—Robinson universal buffer system at different pH values ranging from 2 to 12 under standard assay conditions with casein as substrate.
Activity estimated as a percentage of the maximum. The experiment was performed in triplicate. Protein content was determined as described by Zhao et al. The purified protease and crude extract were pooled and desalted by ultrafiltration, lyophilized and their nematicidal bioassays investigated in vitro. The nematodes of either C. All solutions used were filter-sterilized via 0. All assays were performed in triplicate.
Effects of reXAoz1 on nematodes were detected at different time intervals under a light microscope Leica Microsystems, Germany. The originpro 8 v8. Results The adhesive traps of fungal cultures after nematode-induced incubation were harvested for RNA extraction.
This product consists of amino acids with a calculated molecular mass of Alignment results and phylogenetic analysis showed that XAoz1 shared a high degree of similarity with other serine proteases from closely related species of nematophagous fungi, especially Aoz1 and P II from A.
A single positive transformant, P. Lanes 1, 2, and 3 were the PCR products. M was the DNA marker, following lane M is the same. Lane 1, empty vector; lane 2, recombinant vector; lane 3, product after enzyme digestion. Lanes 1 and 2 were the PCR products.
The crude culture supernatant of P. Western blot analysis further revealed that the protein band reacted with anti-XAoz1 antibody raised against the native XAoz1 of A. Expression of reXAoz1 in P. High hydrolytic activity of reXAoz1 was observed between pH 6. This suggested that reXAoz1 is mostly active in an alkaline environment. Moreover, protease activity was most stable between pH 6. A sharp decline in the enzyme activity was seen at pH below 6. At the indicated times, a sample of the culture was removed and assayed for XAoz1 activity.
The results are the mean values of three independent assays relative to untransformed P.
Please note that this copy of the genome is not maintained by the author and is therefore not automatically updated. Nematode-trapping fungi are a heterogeneous group of organisms broadly distributed in terrestrial and aquatic ecosystems. These fungi are capable of developing specific trapping devices such as adhesive networks, adhesive knobs, and constricting rings to capture nematodes and then extract nutrients from their nematode prey. Most nematode-trapping fungi can live as both saprophytes and parasites. They play important roles in maintaining nematode population density in diverse natural environments.