Improving the Pathogenicity of a Nematode-Trapping Fungus by Genetic Engineering of a Subtilisin with Nematotoxic Activity

Abstract

Nematophagous fungi are soil-living fungi that are used as biological control agents of plant and animal parasitic nematodes. Their potential could be improved by genetic engineering, but the lack of information about the molecular background of the infection has precluded this development. In this paper we report that a subtilisin-like extracellular serine protease designated PII is an important pathogenicity factor in the common nematode-trapping fungus Arthrobotrys oligospora. The transcript of PII was not detected during the early stages of infection (adhesion and penetration), but high levels were expressed concurrent with the killing and colonization of the nematode. Disruption of the PII gene by homologous recombination had a limited effect on the pathogenicity of the fungus. However, mutants containing additional copies of the PII gene developed a higher number of infection structures and had an increased speed of capturing and killing nematodes compared to the wild type. The paralyzing activity of PII was verified by demonstrating that a heterologousproduced PII (in Aspergillus niger) had a nematotoxic activity when added to free-living nematodes. The toxic activity of PII was significantly higher than that of other commercially available serine proteases. This is the first report showing that genetic engineering can be used to improve the pathogenicity of a nematode-trapping fungus. In the future it should be possible to express recombinant subtilisins with nematicidal activity in other organisms that are present in the habitat of parasitic nematodes (e.g., host plant).

A rapid isolation method of extracellular proteins produced by Pseudomonad strains

“…Extracellular proteins might be playing an important role in the microbial environment likewise as in nitrogen storage and for microbial growth etc. Such enzymes may also contribute to checking infection of hosts by degrading the host's protective barriers (Ahman et al, 2002;Huang et al, 2004). Many previous studies proposed that the microbial proteases are produced virulence factors against the pathogens (Ahman et al 2002;Siddiqui et al 2005;Tian et al 2006).…”

“…Such enzymes may also contribute to checking infection of hosts by degrading the host's protective barriers (Ahman et al, 2002;Huang et al, 2004). Many previous studies proposed that the microbial proteases are produced virulence factors against the pathogens (Ahman et al 2002;Siddiqui et al 2005;Tian et al 2006). This finding further evaluated using different microbial samples and also identifying the various extracellular biomolecules for medicinal, pest resistance, stress resistance and many other perspectives.…”

“…Catalytic function of protease is to hydrolyze peptide bonds and break them into free amino acids. Extracellular serine protease from many fungi inhibits pathogen infection by degrading cuticle proteins (Clarkson and Charnley, 1996;Ahman et al, 2002;Meyer, 2003). Naturally they have been isolated from all the life forms especially from bacteria (Devi et al, 2008).…”

Crystal structure and mutagenesis analysis of chitinase CrChi1 from the nematophagous fungus Clonostachys rosea in complex with the inhibitor caffeine

“…As a result, increasing efforts have been paid to developing biological control agents. Nematophagous fungi have been proposed as biological agents to control harmful nematodes because of their unique ability to infect and kill the nematodes (Å hman et al, 2002;Gan et al, 2007a;Yang et al, 2007). In this study, we determined the crystal structure of chitinase CrChi1 from the nematophagous fungus C. rosea.…”

The Function of Snodprot in the Cerato-Platanin Family fromDactylellina cionopagain Nematophagous Fungi

“…[1][2][3][4] A multi-copy PII gene encoding an alkaline serine protease integrated into the genome of Arthrobotrys oligospora could improve the pathogenicity of the fungus. 5) By the use of chitinase, egg-parasitic fungi were found to destroy the chitin component on the surface of nematode eggs and to inhibit subsequent egg growth. 6,7) M. haptotylum produces adhesive knobs to infect nematodes.…”