Rice Science ›› 2021, Vol. 28 ›› Issue (6): 567-578.DOI: 10.1016/j.rsci.2021.03.001
• Research Paper • Previous Articles Next Articles
Tianqiao Song1,2, Xiong Zhang3, You Zhang1, Dong Liang1, Jiaoling Yan1, Junjie Yu1, Mina Yu1, Huijuan Cao1, Mingli Yong1, Xiayan Pan1, Zhongqiang Qi1, Yan Du1, Rongsheng Zhang1, Yongfeng Liu1,4()
Received:
2020-09-14
Accepted:
2021-03-01
Online:
2021-11-28
Published:
2021-11-28
Tianqiao Song, Xiong Zhang, You Zhang, Dong Liang, Jiaoling Yan, Junjie Yu, Mina Yu, Huijuan Cao, Mingli Yong, Xiayan Pan, Zhongqiang Qi, Yan Du, Rongsheng Zhang, Yongfeng Liu. Genome-Wide Identification of Zn2Cys6 Class Fungal-Specific Transcription Factors (ZnFTFs) and Functional Analysis of UvZnFTF1 in Ustilaginoidea virens[J]. Rice Science, 2021, 28(6): 567-578.
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Fig. 1. Distribution of ZnFTFs in fungal species.Phylogenetic relationships of fungal species are shown in the left. The maximum likelihood tree was constructed from a concatenated alignment of core eukaryotic genes identified by the Core Eukaryotic Genes Mapping Approach analysis. The number of ZnFTFs, total number of genes, and percentage of ZnFTFs out of total number of genes in each species are listed in the right.
Fig. S1. Phylogenetic analysis of ZnFTFs from diverse fungal species.The genes included in this tree are ZnFTFs from Debaryomyces hansenii (CAG/CAR), Kluyveromyces lactis (CAG/CAH), Sporisorium reilianum (CBQ), Coprinopsis cinerea (CC1G), Claviceps purpurea (CCE), Ceriporiopsis subvermispora (Cersu1), Uncinocarpus reesii (EEP), Trichoderma reesei (EGR), Agaricus bisporus (EKM), Ustilaginoidea virens (KDB), Ustilago maydis (KIS), Magnaporthe oryzae (MGG), Fusarium solani (Necha), Tilletia indica (OAI99796), and Tremella mesenterica (Treme1). The tree was constructed using the neighbor-joining algorithm. These genes can be classified into thirteen groups. Each group is labeled in different background colors and with different Roman numerals. The gene IDs are shown on the outer ring of the circle. Gene IDs from representative pathogenic Ascomycota, non-pathogenic Ascomycota, pathogenic Basidiomycota, and non-pathogenic Basidiomycota species are colored as follows: U. virens, red; T. reesei, blue; S. reilianum, pink; C. subvermispora green. Gene IDs from all other species are shown in black.
Fig. 2. Characterization of UvZnFTFs in U. virens.A, Venn diagram shows the overlap in proteins, containing the fungal specific transcription factor domain and Zn2-Cys6 binuclear cluster domain.B, Phylogenetic analyses of ZnFTFs in U. virens. The phylogenetic tree was constructed using Geneious R11 by the neighbor-joining method. Domain organization is indicated below.C, UvZnFTF1 is located within a putative secondary metabolite-related gene cluster that encodes proteins possibly involved in toxin biosynthesis.MFS, Major facilitator superfamily; NRPS, Non- ribosomal peptide synthetase.
Fig. 3. UvZnFTF1 is involved in vegetative growth, pigment biosynthesis and conidiation of U. virens.A, Relative transcript levels of UvZnFTF1 in the wild type strain P1 and different transgenic U. virens lines (GFP, ST-2 and ST-3). The transcript levels were measured by quantitative real-time PCR using the β-tubulin gene as an internal reference.B, Colony morphology of P1, GFP, ST-2 and ST-3 cultured on potato sucrose agar plates after 14 d of incubation in the dark at 28 ºC. The upper and lower panels represent the front and back of the plates, respectively.C, Colony diameters of P1, GFP, ST-2 and ST-3.D, Statistical analysis of conidium concentration.A U. virens strain expressing GFP was served as a transgenic negative control. Data in A, C and D are Mean ± SE (n = 3). **, P < 0.01 by the Dunnett's test.
Fig. 4. UvZnFTF1-silenced transformants showed reduced pathogenicity on rice panicles.A, Representative photographs of disease symptoms on rice panicles at 30 d post inoculation with U. virens transformants (ST-2 and ST-3) and the wild type strain P1.B, Quantification of the number of false smut balls per panicle. Data are Mean ± SE (n = 3). **, P < 0.01 by the Dunnett's test.
Fig. S2. UvZnFTF1 expression profile during infection.Rice panicles were inoculated with a suspension of U. virens mycelia and conidia and collected at the indicated times post inoculation (hpi, hours post inoculation; dpi, days post inoculation). Values are the means ± SEM of three independent biological replicates. Letters represent significant differences P<0.01 as measured by Duncan’s multiple range test.
Fig. S3. Gene Ontology annotations of differentially expressed genes in UvZnFTF1-silenced transformants vs. the wild type strain.DEGs are genes with a more than 4-fold change in expression in the UvZNFTF1-silenced transformants compared with the WT strain. The x-axis represents the GO terms. The red bars represent ‘Biological Process’ terms, green bars represent ‘Cellular Component’ terms, and blue bars are the ‘Molecular Function’ terms. The y-axis shows the number of genes for each enriched GO term.
Fig. 5. Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations of differentially expressed genes (DEGs) in UvZnFTF1- silenced transformants vs. wild type strain.A and B, KEGG pathway annotations of DEGs. The number of genes related to different KEGG pathways was calculated. The KEGG pathways are listed according to their molecular interactions and reaction networks within ‘Metabolism’ (light green bars), ‘Genetic information processing’ (purple bars), ‘Environmental information processing’ (blue bars), ‘Cellular processes’ (red bars) and ‘Organismal systems’ (orange bar).C and D, Top 20 down-regulated DEGs (C) and up-regulated DEGs (D) enriched KEGG pathways. The color of the bubble represents the corrected q value. The size of the bubble indicates the number of DEGs. The rich factor is the proportion of DEGs in each pathway.
Gene ID | Biological function | Up-/down-regulated a |
---|---|---|
Genes involved in signal transduction | ||
KDB11856, KDB16287, KDB16312, KDB18181, KDB18666, KDB11669 | G protein-coupled receptor gene | Up |
KDB12196, KDB13191, KDB12444, KDB17214, KDB11081, KDB16465, KDB12620 | G protein-coupled receptor gene | Down |
KDB17719, KDB14771, KDB17609, KDB18180, KDB17527, KDB17948, KDB13363, KDB18665, KDB14539, KDB13362, KDB13535, KDB12267 | Transcription factor | Up |
KDB13663, KDB18728, KDB14016, KDB13664, KDB12105, KDB11763, KDB15876, KDB18718, KDB18880, KDB11956, KDB17862, KDB11405 | Transcription factor | Down |
KDB14812, KDB14811, KDB11045 | Zn2Cys6 transcription factor | Up |
KDB11606 | Zn2Cys6 transcription factor | Down |
KDB13647 | Transcription factor for Polarity Control 1 | Down |
KDB18369 | Histidine kinase | Up |
KDB16000, KDB13339, KDB11031 | Protein kinase | Up |
KDB13650, KDB18230 | Protein kinase | Down |
Genes involved in transport | ||
KDB18524 | Putative mitochondrial ornithine transporter | Up |
KDB16595, KDB15193 | BCAA transporter | Up |
KDB14098 | BCAA transporter | Down |
KDB13509, KDB13238 | Copper transporter | Up |
KDB11479, KDB11766, KDB15819 | Glucose uptake and transport | Up |
Genes encoding secreted proteins | ||
KDB15463, KDB11767 | Necrosis inducing protein | Up |
KDB14361, KDB17416 | Effector protein | Down |
KDB17641 | LysM effector protein | Down |
KDB14258, KDB15607 | Small secreted fungal protein | Down |
KDB12171 | Proteinaceous elicitor | Down |
Table 1 Representative PHI genes regulated by UvZnFTF1.
Gene ID | Biological function | Up-/down-regulated a |
---|---|---|
Genes involved in signal transduction | ||
KDB11856, KDB16287, KDB16312, KDB18181, KDB18666, KDB11669 | G protein-coupled receptor gene | Up |
KDB12196, KDB13191, KDB12444, KDB17214, KDB11081, KDB16465, KDB12620 | G protein-coupled receptor gene | Down |
KDB17719, KDB14771, KDB17609, KDB18180, KDB17527, KDB17948, KDB13363, KDB18665, KDB14539, KDB13362, KDB13535, KDB12267 | Transcription factor | Up |
KDB13663, KDB18728, KDB14016, KDB13664, KDB12105, KDB11763, KDB15876, KDB18718, KDB18880, KDB11956, KDB17862, KDB11405 | Transcription factor | Down |
KDB14812, KDB14811, KDB11045 | Zn2Cys6 transcription factor | Up |
KDB11606 | Zn2Cys6 transcription factor | Down |
KDB13647 | Transcription factor for Polarity Control 1 | Down |
KDB18369 | Histidine kinase | Up |
KDB16000, KDB13339, KDB11031 | Protein kinase | Up |
KDB13650, KDB18230 | Protein kinase | Down |
Genes involved in transport | ||
KDB18524 | Putative mitochondrial ornithine transporter | Up |
KDB16595, KDB15193 | BCAA transporter | Up |
KDB14098 | BCAA transporter | Down |
KDB13509, KDB13238 | Copper transporter | Up |
KDB11479, KDB11766, KDB15819 | Glucose uptake and transport | Up |
Genes encoding secreted proteins | ||
KDB15463, KDB11767 | Necrosis inducing protein | Up |
KDB14361, KDB17416 | Effector protein | Down |
KDB17641 | LysM effector protein | Down |
KDB14258, KDB15607 | Small secreted fungal protein | Down |
KDB12171 | Proteinaceous elicitor | Down |
Fig. S4. Quantitative real-time PCR analysis to validate differentially expressed genes.The relative transcript levels of KDB11856 (GPCR gene), KDB13650 (protein kinase), KDB11479 (Glucose uptake and transport), KDB14258 (Small secreted fungal protein), KDB15463 (Necrosis inducing protein), and KDB16287(GPCR gene) in silencing transformants ST-2 was normalized to those in WT line, using the Tubulin gene as an internal reference. Means and standard deviations obtained from three replicates are shown.
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