转录组和代谢组联合分析揭示稻曲病菌的致病因子

doi:10.16819/j.1001-7216.2024.230603

摘要/Abstract

摘要：

【目的】 由稻曲病菌引起的稻曲病是一种世界性水稻真菌病害，该病严重威胁水稻的产量和品质。探明稻曲病菌的致病分子机制，可为制定稻曲病防治策略和抗病分子育种开辟新思路。【方法】 用稻曲病菌接种水稻幼穗，采用转录组和代谢组测序技术对接种后9 d显症的样本（S）和未接种的PXD25菌丝（CK）进行代谢组和转录组测序，以稻曲病菌菌株UV-8b基因组作为参考基因组进行对比，利用FPKM法计算基因表达量，以|log₂fold change| ≥ 1且P≤0.05为条件筛选差异表达基因（DEG）；以P≤0.05 且 VIP≥1 为标准筛选差异代谢物（DAM）。【结果】 转录组测序分析表明，S vs. CK 有6708个DEG，通过 GO 富集和 KEGG 代谢途经分析，将DEG划分为 GO 功能下的3708个词条、110条代谢途径；DEG中有91个转录因子，分属23个转录因子家族，包括bZIP、C6、C2H2等；分析显著富集代谢途径发现，线粒体自噬、次生代谢和氨基酸代谢等途径显著富集，基因表达量都发生显著变化，其中次生代谢途径中的差异表达基因都显著上调，推测这些基因在稻曲病菌致病中发挥重要作用。非靶代谢组共鉴定出392个差异代谢物（DAM），DAM分析发现丙氨酸、酪氨酸、组氨酸、蛋氨酸、半胱氨酸以及脂肪酸类物质（亚油酸、棕榈酸、月桂酸、肉豆蔻酸）等都显著积累。转录组和代谢组联合分析发现苯丙氨酸、酪氨酸、半胱氨酸和蛋氨酸等氨基酸代谢途径与淀粉和蔗糖等与糖代谢相关途径显著富集，表明这些代谢物和基因可能与稻曲病菌的致病性密切相关。【结论】 线粒体自噬、次生代谢和氨基酸代谢等代谢途径中DEG在稻曲病菌致病中发挥重要作用，丙氨酸、酪氨酸、组氨酸、蛋氨酸、半胱氨酸以及脂肪酸类物质等代谢物与稻曲病菌的致病性密切相关。

关键词: 稻曲病菌, 转录组, 代谢组, 差异表达基因, 差异代谢物

Abstract:

【Objective】 Rice false smut, caused by Ustilaginoidea virens, is a global fungal disease that severely impacts rice yield and quality. Understanding the molecular mechanisms behind U. virens pathogenicity could inspire novel control strategies and molecular breeding for disease resistance. 【Method】 Transcriptome and metabolome analyses were conducted on the initial symptomatic panicle nine days after inoculation (S) and uninoculated PXD25 mycelium (CK). The genome of strain UV-8b served as the reference for sequence alignment. Gene expression levels were quantified using fragments per kilobase per million fragments (FPKM), and differentially expressed genes (DEGs) were identified using |LOG₂ fold change|≥1 and q-value≤0.05 criteria. Differentially accumulated metabolites (DAM) were identified with P≤0.05 and VIP≥1 thresholds. 【Result】 In the S vs. CK comparison, 6078 DEGs were identified. GO enrichment analyses revealed 3708 terms, and KEGG pathway analysis identified 110 metabolic pathways. Ninety-one transcription factors (TFs) belonging to 23 TF families, such as bZIP and C2H2, were among the DEGs. Significant enrichment of mitophagy, secondary metabolism, and amino acid metabolism pathways was observed, with upregulation of DEGs in secondary metabolic pathways suggesting their crucial role in U. virens pathogenesis. Additionally, 392 DAMs were identified, including alanine, tyrosine, histidine, methionine, cysteine, and fatty acids (linoleic acid, palmitic acid, lauric acid, myristic acid), indicating their association with U. virens pathogenicity. Combined transcriptome and metabolome analysis highlighted enrichment in amino acid metabolic pathways (e.g., phenylalanine, tyrosine, cysteine, and methionine) and carbohydrate-related pathways (e.g., starch and sucrose). 【Conclusion】 DEGs involved in mitophagy, secondary metabolism, and amino acid metabolism, along with DAMs like alanine, tyrosine, histidine, methionine, cysteine, and fatty acids, are closely linked to U. virens pathogenicity.

Key words: Ustilaginoidea virens, transciptome, metabolome, differentially expressed genes, differential accumulated metabolites (DAM)

伏荣桃, 陈诚, 王剑, 赵黎宇, 陈雪娟, 卢代华. 转录组和代谢组联合分析揭示稻曲病菌的致病因子[J]. 中国水稻科学, 2024, 38(4): 375-385.

FU Rongtao, CHEN Cheng, WANG Jian, ZHAO Liyu, CHEN Xuejuan, LU Daihua. Combined Transcriptome and Metabolome Analyses Reveals the Pathogenic Factors of Ustilaginoidea virens[J]. Chinese Journal OF Rice Science, 2024, 38(4): 375-385.

图/表 10

图1 接种后第9天稻曲病的症状 A: 接种症状；B: 对照。

Fig. 1. Symptoms of rice false smut on 9 days after inoculation. A, Symptoms; B, CK.

表1 样本测序数据质量控制数据统计

Table 1. Statistical quality analysis of transcriptome sequencing data

Sample	Raw reads	Raw bases(G)	Clean reads	Clean bases(G)	Error rate （%）	Q20 （%）	Q30 （%）	GC content （%）	Total mapped （%）
S1	47 859 198	7.21	47 509 820	7.17	0.02	97.99	94.33	57.34	45 977 524 (96.77%)
S2	48 693 622	7.33	48 321 368	7.30	0.03	97.71	93.64	57.25	46 762 014 (96.77%)
S3	48 928 150	7.37	48 570 976	7.33	0.03	97.77	93.79	57.28	46 907 398 (96.57%)
CK1	47 241 470	7.08	46 561 454	7.03	0.03	97.49	93.31	56.62	45 375 848 (97.45%)
CK2	46 356 766	6.95	45 750 132	6.91	0.03	97.76	93.96	56.55	44 648 578 (97.59%)
CK3	47 564 308	7.12	46 926 056	7.09	0.03	97.91	94.21	56.13	45 579 618 (97.13%)
Total	286 643 514	43.06	283 639 806	42.83

图2 稻曲病菌侵染水稻后的差异表达基因

Fig. 2. Differentially expressed genes in S vs. CK

图3 差异表达基因的GO和KEGG富集分析 A: GO中显著性富集前20条功能分类；B: KEGG前20条显著性富集代谢通路。

Fig. 3. GO and KEGG enrichment analysis of differentially expressed genes. A, The top 20 functional classes were significantly enriched in GO. B, The top 20 metabolic pathways of KEGG were significantly enriched.

表2 稻曲病菌侵染水稻后的转录因子变化情况

Table 2. Transcription factors change of U. virens after infection

转录因子 Transcription factor family	上调 Up-regulated	下周 Down-regulated	合计 Total
bZIP transcription factor	8	3	11
C6 transcription factor	7	16	23
MADS-box	1	0	1
C2H2	0	5	5
bHLH	1	2	3
homeobox	1	1	2
Zinc finger transcription factor	1	1	2
GATA	1	2	3
PHD	0	1	1
Fungal specific transcription factor	6	12	18
APSES	1	1	2
binuclear zinc transcription factor	1	1	2
transcription factor tos4	0	1	1
TFIII	3	3	6
hoxa13	1	0	1
SipA3	1	1	2
G6G8.4	0	1	1
RNA polymerase II	1	0	1
CCAAT	0	1	1
activating transcription factor 7a	1	1	2
atf21	1	0	1
Zn2Cys6	1	0	1
vib-1	1	0	1
Total	38	53	91

图4 重要代谢途径差异表达基因分析 A: 线粒体自噬途径；B: 次生代谢途径；C: 氨基酸代谢途径。

Fig. 4. Analysis of differentially expressed genes involved in important metabolic pathways A, Mitophagy pathway; B, Secondary metabolic pathway; C, Amino acid metabolic pathway.

图5 差异代谢物分析 A:差异代谢物火山图；B: KEGG前20条显著性富集代谢通路。

Fig. 5. Differential metabolite analysis A, Volcanic map of differential metabolites; B, The top 20 metabolic pathways of KEGG were significantly enriched.

图6 氨基酸代谢和脂肪酸代谢途径中的DAMs分析

Fig. 6. Analysis of DAMs in amino acid metabolism and fatty acid metabolism pathways

图7 差异表达基因和差异代谢物数据联合分析 A: 韦恩图；B:代谢通路分析。

Fig. 7. Integrated analysis of differentially expressed gene and differentially accumulated metabolite data A, Venn diagram; B, Metabolic pathway analysis.

图8 苯丙氨酸代谢中DEG和DAM的相关性网络分析

Fig. 8. Correlation network analysis of DEG and DAM in phenylalanine metabolism

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