The Identification, Characterization, and Functional Analysis of the Sugar Transporter Gene Family of the Rice False Smut Pathogen, Villosiclava virens

Abstract

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H₂O₂ treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity.

Keywords: STP; Ustilaginoidea virens; Villosiclava virens; expression pattern; false smut; pathogenicity; sugar transporter.

Figure 1

Gene structure of 35 VvSTPs. Scales of the exons are represented by blue boxes. Gray lines connecting two exons represent the introns. Left panel shows phylogenetic tree built with all 35 VvSTPs through maximum-likelihood (ML) method.

Figure 2

Distribution of conserved motifs of all 35 VvSTPs identified by MEME with the amino acid sequences. Conserved motifs are length-proportional and presented by boxes of different colors among the none-conserved sequences represented by gray lines.

Figure 3

Phylogenetic relation of VvSTPs with three other fungal STP proteins. Labels of STP proteins from Villosiclava virens, Saccharomyces cerevisiae, Neurospora crassa, and Ustilago maydis are shown in different colors, red, purple, brown, and blue, respectively. Branch of different clades is also shown in different colors.

Figure 4

Expression pattern of VvSTPs on different sole carbon sources analyzed through qRT-PCR. The expression of each VvSTP gene in NM medium without sugar was regarded as a reference and the α-tubulin gene was used as an internal control to normalize the data, and relative expression level was calculated through 2^−ΔΔCt method. SDs, shown by error bar, were obtained from three biological replicates.

Figure 5

Expression response of VvSTPs to H₂O₂ through analysis of relative expression with qRT-PCR after treatment of 0.5 and 1 h. The expression of each VvSTP gene at 0 h was regarded as a reference and the α-tubulin gene was used as an internal control to normalize the data, and relative expression level was calculated through 2^−ΔΔCt method. SDs shown by error bar were obtained from three biological replicates.

Figure 6

Expression pattern of VvSTPs after inoculation of a resistant variety and a susceptible variety (IR28 and WX98) with Villosiclava virens. The heatmap was constructed based on the expression level of each RPKM value of VvSTP genes from RNA-Seq data. Colors from blue to red in boxes indicate expression levels from highest to lowest, respectively. DPI, days post inoculation.

Figure 7

Phenotype assays of VvSTP gene deletion mutants. (A) Mycelium growth; (B) conidiation; (C) pathogenicity. Error bars represent the standard deviation, and asterisks represent a significant difference (p < 0.05).