Functional Diversification and Redundancy of Hydrolase Maturases in the plant-pathogenic fungus Botrytis cinerea

REVUELTA MV; ARJEN TEN HAVE

Punta Ballena

Simposio; Yeast Systems Biology; 2011

Background  Recent genome sequence projects have identified a stunning amount of Aspartic Proteases (APs) in ascomycete fungi, of which some have unprecedented characteristics. The amount of APs identified in ascomycete genomes, typically more than 10, suggests both functional diversification and redundancy. Based on the cellular component of the AP gene ontology, their putative involvement in different physiological processes was hypothesized. Yapsins are a class of GPI anchored APs. Yapsin 1 was  firstly identified in Saccharomyces cerevisiae where it is involved in pheromone processing. Yapsin gene deletion in Candida albicans (human pathogenic fungus) was shown to attenuate virulence and biochemical  studies have suggested they function as maturases of secreted hydrolases. As such they might modulate several secreted hydrolases and the functions they are involved in. Botrytis cinerea is a plant pathogen that  heavily depends on hydrolases and we hypothesize that its yapsins are indirectly involved in the virulence process. Biochemical studies of yapsins have suggested they have a narrow substrate specificity. We  started a phylogenetic and in silico structure-function analysis in order to properly classify ascomycete APs and to study the evolution of the various AP gene families. We focus on the functional diversification and  redundancy of B. cinerea yapsins and their putative functional analogues, in relation to both its virulence system and saprophytic survival.    Results  Data mining was performed on 88 complete fungal genomes, including asco- but also basidiomycetes and 19 complete genomes from non-fungal model organisms. The obtained phylogeny demonstrates the existence of at least eight subfamilies of which six contain fungal sequences only. This corroborates the hypothesis of functional diversification. The critical differences between three of the subfamilies were then  analysed using WebLogo and JMol. This allowed us to identify the key residues responsible for branch formation. It appears that the loop regions are fundamental for the AP diversification. Comparative 3D  analysis shows that the basis of this substrate specificity is likely to be determined by two acidic residues on a surface loop that moves towards the active site. These two acidic residues (D77 and E129, according to  pig pepsin numeration) are likely to be responsible for the recognition of the monobasic signal in substrates.    Conclusions  Eukaryotic APs form interesting subjects for structure-function prediction since they show high sequence variability and are subject to functional diversification since the phylogeny indicates specialization of sequences. The subfamilies found show clear differences among each other, which allows us to speculate regarding their function. The plant-pathogen interaction process is complex and should actually be  understood as the result of multiple interactions through time. Yapsins, believed to be involved in the maturation of various virulence factors but also other hydrolases forrm an interesting model to study from a  Systems Biology point of view.