Carbon Catabolite Repressor UvCreA is Required for Development and Pathogenicity in Ustilaginoidea virens

doi:10.1016/j.rsci.2023.11.008

Abstract

Abstract:

The rice false smut disease, caused by Ustilaginoidea virens, has emerged as a significant global threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in the efficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritional conditions. Although significant progress has been made in understanding carbon catabolite repression in fungi such as Aspergillus nidulans and Magnaporthe oryzae, its role in U. virens remains unclear. To address this knowledge gap, we identified UvCreA, a pivotal component of carbon catabolite repression, in U. virens. Our investigation revealed that UvCreA localized to the nucleus. Deletion of UvCreA resulted in decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that the knockout of UvCreA led to the up-regulation of 514 genes and down-regulation of 640 genes. Moreover, UvCreA was found to be involved in the transcriptional regulation of pathogenic genes and genes associated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA is important in fungal development, virulence, and the utilization of carbon sources through transcriptional regulation, thus making it a critical element of carbon catabolite repression.

Key words: Ustilaginoidea virens, virulence, carbon catabolite repression, Oryza sativa, rice false smut

Xie Shuwei, Shi Huanbin, Wen Hui, Liu Zhiquan, Qiu Jiehua, Jiang Nan, Kou Yanjun. Carbon Catabolite Repressor UvCreA is Required for Development and Pathogenicity in Ustilaginoidea virens[J]. Rice Science, 2024, 31(2): 203-214.

Figures/Tables 8

Fig. 1. Identification and targeted knockout of UvCreA in Ustilaginoidea virens. A, Functional domain analysis of CreA homologs from Magnaporthe oryzae (XP_003714427.1), Aspergillus nidulans (XP_663799.1), Beauveria bassiana (KAH8718674.1), U. virens (XP_042997845.1), Fusarium graminearum (XP_011327987.1), and Alternaria citri (BAF74640.1). B, Map of UvCreA knockout strategy and sites for restriction enzyme NcoI. HYG, Hygromycin resistance gene cassette.C, Southern blot analysis of UvCreA knockout mutants. The genomic DNA of the indicated strains was digested by NcoI and subjectedcom to Southern blotting with a probe located downstream of the UvCreA coding region. In the correct UvCreA knockout mutants, the 3.0 kb band in wild type (WT) was shifted to 6.8 kb. D, Expression level of UvCreA was determined by qRT-PCR assay. The UvCreA expression was detected in the WT and complementation (ΔUvcreA-C) strains but not in the ΔUvcreA-5 and ΔUvcreA-6 mutants. Data are Mean ± SD (n = 3). Asterisks (***) represent statistically significant differences at P ˂ 0.001.

Fig. 2. Expression pattern and subcellular location of UvCreA. A, Relative expression levels of UvCreA gene during infection stages in Ustilaginoidea virens. With the β-tubulin gene as an internal control, the expression level of UvCreA at the vegetative growth stage was used as a criterion. Data are Mean ± SD (n = 3).B and C, UvCreA-mCherry co-localized with Hoechst-stained nuclei on hyphae (B) and conidia (C). Hoechst is used for staining nuclei in cells. Scale bars, 10 μm.

Fig. 3. Deletion of UvCreA resulted in decreased vegetative growth and increased spore production in Ustilaginoidea virens. A, Mycelium growths of wild type (WT), ΔUvcreA-5, ΔUvcreA-6, and complementation (ΔUvcreA-C) strains were measured after 14 d of cultivating on potato sucrose agar medium at 28 ºC. B, Colony diameters of WT, ΔUvcreA-5, ΔUvcreA-6, and ΔUvcreA-C strains. C and D, Deletion of UvCreA increased spore production in liquid potato sucrose medium for 7 d. Scale bar, 2.5 μm.Data are Mena ± SD (n = 3). Asterisks (***) represent statistically significant differences at P ˂ 0.001.

Fig. 4. Disruption of UvCreA gene resulted in reduced pathogenicity. A, Infection assays of wild type (WT), ΔUvcreA-5, ΔUvcreA-6, and complementation (ΔUvcreA-C) strains. At the booting stage, the panicles of Wanxian 98 were injected with spores and mycelia from the WT, ΔUvcreA-5, ΔUvcreA-6, and ΔUvcreA-C strains. After 3 weeks, the symptoms were determined.B, ΔUvcreA-5 and ΔUvcreA-6 strains exhibited a lower number of rice false smut balls compared with the WT and ΔUvcreA-C strains. Data are Mena ± SD (n = 4). Asterisks indicate statistically significant differences at P ˂ 0.01 (**) or P ˂ 0.001 (***).

Fig. 5. RNA-seq analysis revealed effects of UvCreA deletion on transcription in Ustilaginoidea virens. A, Differentially expressed genes (DEGs) between ΔUvcreA and the wild type are depicted on a volcano plot. The x-axis represents the fold changes (FC) of up-regulated or down-regulated DEGs, and the y-axis indicates the levels of statistical significance. The P-value signifies the degree of change in gene expression. Blue, gray, and red dots correspond to down-regulated genes, genes with no significant change, and up-regulated genes, respectively.B, The enriched Gene Ontology (GO) classifications for DEGs were depicted in a doughnut chart. These classifications were categorized into three broader processes: biological process, molecular function, and cellular component.C, Encyclopedia of Genes and Genomes (KEGG) enrichment of the top 30 in the comparison of ΔUvcreA and wild type in U. virens.

Fig. 6. UvCreA regulates transcription of glucose-repressed genes. A‒D, Relative transcription levels of xylanolytic gene (Uv8b_00917) (A), d-galactose utilization gene (Uv8b_05392) (B), maltose utilization gene (Uv8b_04729) (C), and galacturonic acid utilization gene (Uv8b_02619) (D) were up-regulated in the ΔUvcreA mutant compared with the wild type (WT) strain. Total mRNA was extracted from the mycelia of both the WT and ΔUvcreA mutant strains cultured in potato sucrose media after 7 d, with the β-tubulin gene serving as an internal control.

Fig. 7. UvCreA is required for utilization of various carbon sources. A, Strains grew on potato agar medium supplemented by various carbon sources at 28 ºC for 14 d.B, Growth rates of different strains were assessed at 14 d post-inoculation. Data are Mean ± SD (n = 3). Asterisks represent statistically significant differences at P ˂ 0.01 (**) and P ˂ 0.05 (*).

Fig. 8. UvCreA regulates transcription of genes involved in mitogen-activated protein kinase (MAPK) signaling pathway and pathogenesis. A‒C, Relative transcription levels of three identified pathogenic genes, including UvEc1 (A), UvPro1 (B), and UvScre1 (C), were down-regulated in the ΔUvcreA compared with the wild type (WT). Total mRNA was extracted from the mycelia of both the WT and ΔUvcreA mutant strains cultured in potato sucrose media after 7 d, with the β-tubulin gene serving as an internal control. Data are Mean ± SD (n = 3). D, The heatmap demonstrates the expression patterns of genes associated with the MAPK signaling pathway in the compared group, relative to the WT group.E, The heatmap illustrates the dynamic expression of pathogenic genes in the ΔUvcreA mutant in comparison with the WT strain. The colored cells in the images of D and E represent the detected expression levels of genes via RNA-seq.

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