Molecular Characterization of Two Totiviruses from the Commensal Yeast Geotrichum candidum

Abstract

Mycoviruses can infect many of the major taxa of fungi including yeasts. Mycoviruses in the yeast fungus Geotrichum candidum are not well studied with only three G. candidum-associated viral species characterized to date, all of which belong to the Totiviridae genus Totivirus. In this study, we report the molecular characteristics of another two totiviruses co-infecting isolate Gc6 of G. candidum. The two totiviruses were tentatively named Geotrichum candidum totivirus 2 isolate Gc6 (GcTV2-Gc6) and Geotrichum candidum totivirus 4 isolate Gc6 (GcTV4-Gc6). Both viruses have the typical genome organization of totiviruses comprising two ORFs encoding capsid protein (CP) and RNA-dependent RNA polymerase (RdRp) at the N and C termini, respectively. The genomes of GcTV2-Gc6 and GcTV4-Gc6 are 4592 and 4530 bp long, respectively. Both viruses contain the-frameshifting elements and their proteins could be expressed as a single fusion protein. GcTV2-Gc6 is closely related to a totivirus isolated from the same host whereas GcTV4-Gc6 is related to insect-associated totiviruses. The phylogenetic analysis indicated that GcTV2-Gc6 and GcTV4-Gc6 belong to two different sister clades, I-A and I-B, respectively. It is interesting that all viruses identified from G. candidum belong to the genus Totivirus; however, this might be due to the lack of research reporting the characterization of mycoviruses from this fungal host. It is possible that the RNA interference (RNAi) mechanism cannot actively suppress totivirus accumulation in G. candidum Gc6.

Keywords: Geotrichum; RNA interference; Totivirus; dsRNA; high-throughput sequencing; mycoviruses; siRNA; usRNA.

Figure 1

(a) Colony morphology of Geotrichum candidum isolate Gc6 grown on PDA media. (b) Agarose gel electrophoresis of dsRNAs purified from G. candidum Gc6. Purified dsRNA fraction was treated with DNase and RNase in a high salt buffer before electrophoretic separation. M: 1 kb plus DNA marker (Invitrogen). (c) Schematic representation of the genome organization of G. candidum Gc6 totiviruses. Coding regions are indicated by colored boxes whereas untranslated regions are indicated by short lines at both termini. The potential −1 frameshifting site (−1FS) is indicated by black arrows and its three components (slippery heptamer, spacer, and pseudoknot) are shown as an inset in each totiviral genome schematic representation. EFE refers to estimated free energy values for the H-type pseudoknots.

Figure 2

(a) Partial multiple amino acid (aa) sequence alignment of the CP sequences of totiviruses including those of G. candidum. The conserved histidine residue required for cap snatching is indicated by a red arrow. (b) aa sequence alignments showing the conserved motifs (I–VIII) of RNA-dependent RNA polymerase (RdRp) sequences of G. candidum totiviruses and other members of the genus Totivirus.

Figure 3

Maximum likelihood phylogenetic tree based on multiple alignments of RNA-dependent RNA polymerase amino acid (aa) sequences of Geotrichum candidum totivirus 2 (GcTV2-Gc6), GcTV4-Gc6, and other members of the genus Totivirus. The phylogenetic tree was constructed using MEGA-X software and LG + G + I as the best evolutionary model with 1000 bootstrap replicates.

Figure 4

Maximum likelihood phylogenetic tree based on multiple alignments of capsid protein (CP) amino acid (aa) sequences of Geotrichum candidum totivirus 2 (GcTV2-Gc6), GcTV4-Gc6, and other members of the genus Totivirus. The phylogenetic tree was constructed using MEGA-X software and rtREV + G + F as the best evolutionary model with 1000 bootstrap replicates.

Figure 5

Length distribution in log scale of small RNAs (sRNAs) mapped against Geotrichum candidum totivirus 2 (GcTV2-Gc6) and GcTV4-Gc6.