Functional analyses of fungal genomes are expanding our view of the metabolic pathways involved in the production of secondary metabolites. These genomes contains a significant number of genes encoding key biosynthetic enzymes such as polyketides synthases (PKS), non-ribosomal peptide synthases (NRPS) and their hybrids (PKS-NRPS), as well as terpene synthases (TS). Magnaporthe grisea has the highest number of such key enzymes (22 PKS, 8 NRPS, 10 PKS-NRPS, and 5 TS) among fungal plant pathogens, suggesting that this fungal species produce a large number of secondary metabolites. In particular, it has 10 hybrid PKS-NRPS that likely produce polyketides containing a single an amino-acid. Three of them (ACE1, SYN2 and SYN8) have the same expression pattern that is specific of early stages of infection (appressorium-mediated penetration), suggesting that the corresponding metabolites are delivered to the first infected cells. M. grisea mutants deleted for ACE1 or SYN2 by targeted gene replacement are as pathogenic as wild type Guy11 isolate on susceptible rice cultivars. Such a negative result could result from a functional redundancy between these pathways. However, ACE1 null mutants become specifically pathogenic on resistant rice cultivars carrying the Pi33 resistance gene compared to wild type Guy11 isolate that is unable to infect such rice cultivars. Introduction of a Guy11 wild type ACE1 allele in Pi33 virulent M. grisea isolates restore their avirulence on Pi33 resistant rice cultivars, showing that ACE1 behaves as a classical avirulence gene (AVR). ACE1 differs from other fungal AVR genes (proteins secreted into host tissues during infection) as it likely controls the production of a secondary metabolite specifically recognized by resistant rice cultivars. Arguments toward this hypothesis involve the fact that the protein Ace1 is only detected in the cytoplasm of appressoria and is not translocated into infectious hyphae inside epidermal cells. Furthermore, Ace1-ks0, an ACE1 allele obtained by site-directed mutagenesis of a single amino acid essential for the enzymatic activity of Ace1, is unable to confer avirulence. According to this hypothesis, resistant rice plants carrying Pi33 are able to recognize its fungal pathogen M. grisea through the perception of one fungal secondary metabolite produced during infection. The map based cloning of the Pi33 rice gene was initiated and this gene maps at a locus rich in classical NBS-LRR resistance genes. Further work is ongoing to identify which gene is Pi33. In order to characterize the secondary metabolite produced by ACE1, this gene was expressed in a heterologous fungal host such as Aspergillus oryzae under the control of an inducible promoter. The removal of the three introns of ACE1 allowed the expression of the enzyme in A. oryzae. Characterization of the novel metabolite produced by Ace1 is in progress.
Full conference title:
10th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 10th (2010)