The oomycete Pythium oligandrum expresses putative effectors during mycoparasitism of Phytophthora infestans and is amenable to transformation

Abstract

The oomycete Pythium oligandrum is a mycoparasitic biocontrol agent that is able to antagonise several plant pathogens, and can promote plant growth. In order to test the potential usefulness of P. oligandrum as a biocontrol agent against late blight disease caused by the oomycete Phytophthora infestans, we investigated the interaction between P. oligandrum and Ph. infestans using the green fluorescent protein (GFP) as a reporter gene. A CaCl(2) and polyethylene-glycol-based DNA transformation protocol was developed for P. oligandrum and transformants constitutively expressing GFP were produced. Up to 56 % of P. oligandrum transformants showed both antibiotic resistance and fluorescence. Mycoparasitic interactions, including coiling of P. oligandrum hyphae around Ph. infestans hyphae, were observed with fluorescent microscopy. To gain further insights into the nature of P. oligandrum mycoparasitism, we sequenced 2376 clones from cDNA libraries of P. oligandrum mycelium grown in vitro, or on heat-killed Ph. infestans mycelium as the sole nutrient source. 1219 consensus sequences were obtained including transcripts encoding glucanases, proteases, protease inhibitors, putative effectors and elicitors, which may play a role in mycoparasitism. This represents the first published expressed sequence tag (EST) resource for P. oligandrum and provides a platform for further molecular studies and comparative analysis in the Pythiales.

Pythium oligandrum: an example of opportunistic success

“…Most of our knowledge about the relationships established between non-pathogenic Pythium species and their prey derives from a number of cytological and molecular studies that have been conducted to gain a deeper insight into the mechanisms underlying the antagonistic process of P. oligandrum El-Katatny et al, 2006;Horner et al, 2012). A considerable body of work indicates that direct mycoparasitism is a key component of the antagonistic process Picard et al, 2000a), even though antibiosis and competition for nutrients in the rhizosphere may also account for the observed antagonistic effect against certain preys.…”

“…Whether similar mechanisms occur at the onset of contact between P. oligandrum and its prey has not yet been elucidated. However, the recent discovery that transcripts encoding cellulases, glucanases, proteases, protease inhibitors, putative effectors and elicitors are produced by P. oligandrum grown under biocontrol conditions suggest that P. oligandrum operates similarly in the earliest stages of the mycoparasitic process (Horner et al, 2012). Obviously, further work is needed to functionally characterize the potential protease-encoding transcripts identified by Horner et al (2012) before concluding that P. oligandrum proteases operate as extracellular sensing mechanisms.…”

“…Positive correlation between surface-associated components and recognition events in fungal-fungal and oomycete-fungal interactions have often been reported (Benhamou & Chet, 1997) and are considered to be key determinants in the outcome of any mycoparasitic interaction. In their investigation using the green fluorescent protein (GFP) Pythium oligandrum: an example of opportunistic success reporter gene, Horner et al (2012) identified a group of very similar transcripts predicted to encode tyrosine-and glycine-rich proteins in P. oligandrum. Such proteins are characteristic of cell wall and extracellular matrix proteins and may well collaborate to allow the recognition and attachment of P. oligandrum to its prey.…”

“…This crucial observation suggests that cellulase synthesis by P. oligandrum is a mechanism induced in response to a signal produced by the pathogen itself. The importance of these hydrolytic enzymes in the mycoparasitic activity of P. oligandrum is also reflected by the presence of transcripts putatively involved in cell wall degradation (Horner et al, 2012). Indeed, a number of sequences from cDNA libraries of P. oligandrum have been annotated as having a key role in the degradation of carbohydrates.…”

“…A more comprehensive analysis performed on P. oligandrum by the use of the expressed sequence tag (EST) sequencing has recently provided the first overview on the molecules putatively involved in the P. oligandrum-prey biochemical cross-talk (Horner et al, 2012). Although of major interest, the data obtained remain speculative and are quite difficult to interpret in the absence of entire genome sequence information.…”