Isolation of mycoparasitic-related transcripts by SSH during interaction of the mycoparasite Stachybotrys elegans with its host Rhizoctonia solani.

Mycoparasitism by antagonistic fungi involves changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during mycoparasite-host interaction represents a powerful strategy to obtain insight into the molecular events underlying these changes. The aim of this study is to identify genes whose expression is upregulated when the mycoparasite Stachybotrys elegans is in direct confrontation with its host Rhizoctonia solani. Suppression subtractive hybridization (SSH) was used to create a subtracted cDNA library, and differential screening was applied to identify the over-expressed transcripts. We report the analysis of 2,166 clones, among which 47% were upregulated during mycoparasitism. Two hundred and sixty-one clones were sequenced that corresponded to 94 unique genes. Forty-four of these were identified as novel genes, while the remainder showed similarity to a broad diversity of genes with putative functions related to toxin production, pathogenicity, and metabolism. As a result of mycoparasitism, 15 genes belonged to R. solani among which 9 genes were assigned putative functions. Quantitative RT-PCR was used to examine the upregulation of 12 genes during the course of mycoparasitism. Seven genes showed significant upregulation at least at one-time point during interaction of the mycoparasite with its host. This study describes a first step toward knowledge of S. elegans genome. The results present the useful application of EST analysis on S. elegans and provide preliminary indication of gene expression putatively involved in mycoparasitism.

Molecular cloning, characterization, and expression of a cDNA encoding an endochitinase gene from the mycoparasite Stachybotrys elegans.

Stachybotrys elegans is a mycoparasite of the soilborne plant pathogenic fungus Rhizoctonia solani. The mycoparasitic activity of S. elegans is correlated with the production of cell wall degrading enzymes such as chitinases. This report details the cloning by RACE-PCR and characterization of a full-length cDNA clone, sechi44, that appears to encode an extracellular endochitinase. An analysis of the sechi44 sequence indicates that this gene contains a 1269-bp ORF and encodes a 423-aa polypeptide. The SECHI44 protein has a calculated molecular weight of 44.1kDa and pI of 5.53. Since the SECHI44 protein also appears to encode a signal peptide, an extracellular location for the corresponding protein is predicted. Comparison of SECHI44 sequence with known sequences of fungal endochitinases revealed that SECHI44 is grouped with endochitinases from other mycoparasites. Real-time quantitative RT-PCR analysis showed an elevated level of expression of sechi44 (21-fold) in chitin-rich (induced) as compared to no-carbon (non-induced) culture conditions. In dual culture, the temporal expression of sechi44 increased after 2 days of contact with R. solani, reaching a 10-fold increase after 9 days, followed by a decrease to basic expression level at 12 days. Interestingly, inhibition of sechi44 expression was observed when S. elegans hyphae were in close proximity with R. solani hyphae.