Fungal endophytes express chitinolytic enzymes in the infected plants

Neotyphodium endophyte-grass associations are naturally occurring mutualistic symbioses. Endophyte-infection generally confers insect resistance, and in some cases disease resistance, to the infected plant. Because of these benefits, endophytes are often incorporated into turfgrass cultivars of several species. The factors that are involved in the establishment of these mutualistic interactions and the mechanisms underlying the endophyte-enhanced traits are the targets of continued research interest. Apoplastic secreted proteins, both plant and fungal, are likely to be important components of the mutualistic interaction since they are located at the interface of the two species. I have therefore begun investigating some of the proteins secreted in culture and apoplastic proteins isolated from infected plants. A chitinase was identified by peptide sequencing of an abundant extracellular 52 kDa protein expressed in culture. Activity gel analysis using the substrates 4-MU-(GlcNAc) ₂ and 4-MU-(GlcNAc)₃ indicated the 52 kDa protein had endochitinase activity. A full-length cDNA of the chitinase, Nchi52, had an open-reading frame of 1377 bp. The deduced amino acid sequence of the clone had 37–40% identity to other fungal chitinases, including those of several mycoparasitic and entomopathogenic fungi. The native enzyme was purified from the culture filtrate by binding to colloidal chitin and filtering through a hydrophobic interaction column. The mature enzyme was a 421 as protein starting with GIHKGKLDG determined by N-terminal sequencing. RNA gel blot analysis revealed Nchi52 is highly expressed in infected Poa ampla leaf sheaths. The peptide sequences of NCHI52 were identified as a major component of the 52 kDa apoplastic protein band isolated from infected leaf sheaths. An N-acetyl-glucosaminidase was partially purified from the culture filtrate. The full sequence of cDNA, Nnag60, had an open-reading frame of 1920 bp. Sequence alignment showed a high homology with N-acetyl-glucosaminidases/N-acetyl-hexosaminidases from other fungal species. RNA gel blot analysis revealed Nnag60 is expressed in infected P. ampla leaf sheaths. Activity gel analysis using the substrate 4-MU-GlcNAc revealed an N-acetyl-glucosaminidase activity was associated with endophyte infection in both plant crude proteins and apoplastic proteins. Homologous transcripts of Nchi52 and Nnag60 were also detected in endophyte-infected perennial ryegrass, tall fescue and Chewings fescue. (Abstract shortened by UMI.)...