An N-acetyl-beta-Dglucosaminidase gene, cr-nag1, from the biocontrol agent Clonostachys

Abstract

Clonostachys rosea is a widely distributed fungus that often acts as a parasite on other soil<br><br>fungi. This fungus has also been reported as a potential parasite against nematodes and<br><br>insects. The antagonistic activity is thought to be correlated with the secretion of cell<br><br>wall-degrading enzymes, including chitinases. In this work, we identified and characterized<br><br>an N-acetyl-b-D-glucosaminidase-encoding gene, cr-nag1, belonging to glycosyl hydrolase<br><br>family 20, from the C. rosea strain IK726 using a degenerated primer strategy designed<br><br>from conserved motifs. The complete gene, including its promoter region, was obtained by<br><br>genomic walking. Southern analysis showed that cr-nag1 is present as a single copy gene in<br><br>C. rosea. Phylogenetically, cr-nag1 showed the highest similarity to N-acetyl-b-D-glucosaminidase<br><br>genes from other mycoparasitic fungi. Enzymatic assays and RT-PCR showed that<br><br>the NAGase activity of C. rosea is specifically repressed in medium containing a high glucose<br><br>content and is expressed in media containing chitin or Fusarium culmorum cell walls<br><br>as sole carbon sources. Macroscopic and microscopic observations indicated that the mycelial<br><br>growth of F. culmorum and Pythium ultimum were inhibited during interactions with C.<br><br>rosea. High expression of cr-nag1 was found in interactions between C. rosea and F. culmorum,<br><br>whereas the expression of cr-nag1 in interactions between C. rosea and P. ultimum<br><br>was similar to the control. This indicates that although C. rosea secretes chitin-hydrolysing<br><br>agents in order to target the cell wall of F. culmorum, it seems to use another strategy for<br><br>controlling the development of the oomycete P. ultimum.