Genomic analysis of the basal lineage fungus Rhizopus oryzae reveals a whole-genome duplication

Abstract

Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called "zygomycetes," R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.

Figure 1. Relationship of major phyla within the fungal kingdom.

Phylogeny is shown as a dendrogram using H. sapiens (Metazoa) as the out-group. B. dendrobatidis (phylum Chytridiomycota) is a unicellular organism with flagellated spores. The terrestrial multicellular fungi include the monophyletic Dikaryomycota (Ascomycota and Basidiomycota) and the more basal fungal lineages, including R. oryzae. In contrast to the Dikaryomycota fungi that form hyphae divided by septa (white arrows), the hyphae of R. oryzae are multinucleate but not divided into separate cells (coenocytic).

Figure 2. R. oryzae genomic structure showing duplicated regions retained after WGD and distribution of LTR transposable elements.

The length of the light blue background for each linkage group is defined by the optical map. For each chromosome, row a represents the genomic scaffolds positioned on the optical linkage groups. The red oval indicates linkage to telomeric repeat arrays. Row b displays the 256 duplicated regions capturing 648 gene pairs and spanning 12% of the genome. The shaded backgrounds around some duplicated regions illustrate the duplicated blocks by merging duplicated regions that are within 200 kb after discounting the transposon sequences. These extended duplicated blocks contain the same amount of the duplicates but span 23% of the genome. A pair of corresponding duplicated regions between linkage 2 and linkage 9 are shown in the zoomed images. The numbers in the gene boxes are gene IDs. Row c corresponds to the distribution of the LTR retroelements.

Figure 3. Estimation of duplication dates using P. blakesleeanus as an outgroup.

(A) An unrooted tree diagram for the duplicated gene pairs in R. oryzae and their homologous gene in P. blakesleeanus. Midpoint rooting is used to calculate of the relative age of each duplication (R) in relation to the root. The branch lengths as substitutions per site for the unrooted tree topology were calculated using the WAG evolutionary model employing a maximum likelihood-based package, PhyML . The distance between two duplicated genes in R. oryzae is t ₁+t ₂, and the distances between the duplicates and their orthologous gene in P. blaskesleeanus are t+t ₃+t ₁ and t+t ₃+t ₂, respectively. (B) The distribution of the relative duplication time for each duplicated region in comparison to the root (R). R is normalized within each duplicated region by averaging the divergences of all the duplicated gene pairs within the region. If the divergence time between R. oryzae and P. blakesleeanus is defined as t using midpoint rooting, approximately 78% of all these regions were estimated to be duplicated within one standard deviation (0.115) of the mean (0.386t).

Figure 4. RT–PCR of R. oryzae chitin synthases (CHSs).

Presence of a transcript was detected from mycelia grown with four different growth phases: 1L, 1-day-old liquid culture; 1S, 1-day-old agar plate; 2S, 2-day-old agar plate; and 3S, 3-day-old agar plate. Gene pairs retained after WGD as detected in the duplicated regions are shown in blue.