Comprehensive genome-wide analysis reveals different classes of enigmatic old yellow enzyme in fungi

Abstract

In this study, we systematically identify Old Yellow Enzymes (OYEs) from a diverse range of economically important fungi representing different ecology and lifestyle. Using active site residues and sequence alignments, we present a classification for these proteins into three distinct classes including a novel class (Class III) and assign names to sequences. Our in-depth phylogenetic analysis suggests a complex history of lineage-specific expansion and contraction for the OYE gene family in fungi. Comparative analyses reveal remarkable diversity in the number and classes of OYE among fungi. Quantitative real-time PCR (qRT-PCR) of Ascochyta rabiei OYEs indicates differential expression of OYE genes during oxidative stress and plant infection. This study shows relationship of OYE with fungal ecology and lifestyle, and provides a foundation for future functional analysis and characterization of OYE gene family.

Figure 1. The distribution of OYE homologues in 60 fungi.

Blue bars showed the numbers of fungi that have the members of specific OYE family.

Figure 2. Comparative analysis of fungal OYEs.

The numbers of OYEs in each species are represented as horizontal bars. Different colors of the bar represent different classes of OYE.

Figure 3. Conserved motifs in ArOYEs.

(A) Conserved core active site and YGGS motif arrangement among the three OYE Classes, and (B) OYE Class specific conserved motifs. The result shows the top five hits that were obtained through MEME analysis of each OYE Class. Numbers on the right shows the E-value of each motif. Some of these motifs correlate with the motifs identified by Oberdorfer et al..

Figure 4. Comparison of OYEs among fungi, (A) among unicellular fungi (black), dimorphic fungi (red) and multi-cellular fungi (blue), (B) among animal pathogenic fungi (black), plant pathogenic fungi (red) and saprophytic fungi (blue) and (C) among biotrophic (black), hemibiotrophic (red), and necrotrophic (blue) fungi.

Figure 5. Evolutionary relationship of 424 deduced OYE proteins from 60 fungal species based on Bayesian inference analysis of the structure-based amino acid sequence alignment.

The multiple sequence alignment of full length OYE proteins was carried out using multiple sequence and structure alignment program PROMALS3D. The numbers at the nodes indicates the Bayesian posterior probabilities. The OYE proteins were classified into three distinct classes, designated as Class I, II, and III. Different colour was assigned to each class.

Figure 6. Lineage specific expansion and contraction of OYE gene family.

Evolutionary relationships were inferred by the Bayesian Markov Chain Monte Carlo (MCMC) for (A) Aspergillus sp., (B) Trichoderma sp. and (C) Penicillium sp. For each node, Bayesian posterior probabilities supporting the phylogeny are indicated. The phylogeny shows clustering based mainly on orthology instead of paralogy, indicating that the expansion of OYEs occurred prior to speciation. In all the three species analysed, several groups display additional instances of gene duplications and losses supporting for birth-and-death model of evolution.

Figure 7. Expression profiles of six ArOYE genes during oxidative stress and in planta, assayed by qRT-PCR.

Bar diagrams representing the expression pattern of 6 genes are shown as the fold-change compared to the control. (A) Expression was analyzed at 0.25, 0.5, 1, 3 and 6 h after treatment with menadione. (B) Expression pattern of 6 genes from A. rabiei-infected chickpea samples are shown as fold-change compared to control. Expression was analyzed at 12, 24, 72 and 144 hours post inoculation (hpi). Error bars represent ±SE for three biological replicates. ArEF1α gene was used as an internal reference gene.