芒果胶孢炭疽病菌应答菌丝机械损伤产生无性孢子的分子机制

doi:10.11983/CBB20026

植物学报 ›› 2020, Vol. 55 ›› Issue (5): 551-563.DOI: 10.11983/CBB20026

芒果胶孢炭疽病菌应答菌丝机械损伤产生无性孢子的分子机制

王丽妍¹, 卢梦瑶¹, 童悦¹, 徐祥斌¹, 张正科¹, 孟兰环¹, 史学群¹^,^*(), 宋海超²^,^*()

¹海南大学食品科学与工程学院, 海口 570228
²海南大学生命科学与药学院, 海口 570228

收稿日期:2020-02-22 接受日期:2020-05-08 出版日期:2020-09-01 发布日期:2020-09-03
通讯作者: 史学群,宋海超
作者简介:songhaichao@sohu.com
*E-mail: shixuequn@163.com;
基金资助:
海南省科技厅创新团队(2019CXTD399)

Molecular Mechanism of the Generation of Asexual Spores of the Mango Fungal Pathogen (Colletotrichum gloeosporioides) Induced by Mechanical Injuries

Liyan Wang¹, Mengyao Lu¹, Yue Tong¹, Xiangbin Xu¹, Zhengke Zhang¹, Lanhuan Meng¹, Xuequn Shi¹^,^*(), Haichao Song²^,^*()

¹College of Food Science and Engineering, Hainan University, Haikou 570228, China
²School of Life and Pharmaceutical Sciences, Hainan University, Haikou 570228, China

Received:2020-02-22 Accepted:2020-05-08 Online:2020-09-01 Published:2020-09-03
Contact: Xuequn Shi,Haichao Song

摘要/Abstract

摘要：

胶孢炭疽菌(Colletotrichum gloeosporioides)是引发芒果(Mangifera indica)炭疽病的主要病原体。室内平板培养胶孢炭疽菌不产生或产生很少分生孢子的情况时有发生, 但菌丝在机械损伤后24-48小时会产生大量分生孢子。胶孢炭疽菌应答机械损伤诱导产孢的核心基因及关键代谢通路尚未见报道。基于转录组测序(RNA-seq)技术检测了芒果胶孢炭疽菌菌丝在机械损伤处理后2小时内5个时间点的基因表达变化, 对差异表达基因进行GO富集和KEGG代谢通路富集分析, 并对菌丝响应胁迫的基因动态表达数据进行分析。基于常微分方程ODE模型结合变量选择技术, 构建了动态基因调控网络。结果表明, 有417个差异表达基因参与应答胶孢炭疽菌菌丝机械损伤, 分属12个聚类模块, 有4条通路存在显著富集, 分别是丙酮酸代谢、硫代谢、黄曲霉素合成途径和二萜合成途径。结合功能注释筛选出12个应答菌丝损伤胁迫的核心基因。研究结果为后续深入开展芒果胶孢炭疽菌产孢和致病机理研究奠定了重要基础。

关键词: 芒果, 胶孢炭疽菌, 损伤胁迫, RNA-seq, 动态表达

Abstract:

Colletotrichum gloeosporioides is a prevalent pathogen that causes anthracnose in mango (Mangifera indica). Mycelium of C. gloeosporioides will accumulate a large number of conidia in 24-48 hours after mechanical injuries. However, it often accumulates none or few conidia during indoor culture, and the gene regulatory networks of the response to injury for a short-time (ST), or the key metabolic pathways involved in the response has not been explored. In this study, RNA-seq was carried out on RNA samples obtained at 5 time points within 2 hours after mechanical injuries. The differentially expressed genes were enriched by GO enrichment and KEGG metabolic pathway. The expression dynamics of mycelia in response to ST injury stress was analyzed. Based on a nonlinear ordinary differential equation model coupled with variable selection techniques, inter-module networks were constructed. The results showed that 417 differentially expressed genes were obtained, which belong to 12 clustered modules. KEGG enrichment analysis of differentially expressed genes was enriched in the process of pyruvate metabolism, sulfur metabolism, aflatoxin biosynthesis, diterpenoid biosynthesis. Combined with functional annotation, 12 core genes were identified that significantly correlated with ST injury-induced expression. These results provide valuable references for further research on asexual development and pathogenicity in C. gloeosporioides.

Key words: mango, Colletotrichum gloeosporioides, injury stress, RNA sequencing, expression dynamics

王丽妍, 卢梦瑶, 童悦, 徐祥斌, 张正科, 孟兰环, 史学群, 宋海超. 芒果胶孢炭疽病菌应答菌丝机械损伤产生无性孢子的分子机制. 植物学报, 2020, 55(5): 551-563.

Liyan Wang, Mengyao Lu, Yue Tong, Xiangbin Xu, Zhengke Zhang, Lanhuan Meng, Xuequn Shi, Haichao Song. Molecular Mechanism of the Generation of Asexual Spores of the Mango Fungal Pathogen (Colletotrichum gloeosporioides) Induced by Mechanical Injuries. Chinese Bulletin of Botany, 2020, 55(5): 551-563.

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https://www.chinbullbotany.com/CN/Y2020/V55/I5/551

图/表 8

表1 实时荧光定量PCR所用引物

Table 1 Primers for real-time quantitative PCR

Transcript	Upstream primer sequence (5°?3°)	Downstream primer sequence (5°?3°)
ELA27200	CCTCAAACTTCGCAGATG	GGCTGTCGTGTAGAACTG
ELA31518	ACTGCGAATCACATCATCTCA	TAAGAGGTCGGCGTCAGA
ELA26856	CAGCAGATGGTCAAGAGG	GAGGAGACGAAGGAAGGA
ELA23533	TCAGCCTTGTTGGACCTTAG	CGTAGCATCGGACCTTGT
ELA34567	GAGCAGGTATCAGAAGACA	CCAGTTCGTAAGCCAGAT
Actin	ACGCTTCTCATCTCCAAGATCCGT	AGAGAGCCTCGTTGTCAATGCAGA

图1 不同处理样品基因表达数据和差异表达基因GO富集结果 (A) 不同样本基因表达数据的分布; (B) 不同样本间的相关性; (C) 差异表达基因的GO富集分析。MF: 分子功能; BP: 生物过程; CC: 细胞组分

Figure 1 Gene expression data and GO enrichment for differentially expressed genes under different treatments (A) Gene expression in different samples; (B) Correlation between different samples; (C) GO enrichment for differentially expressed genes. MF: Molecular function; BP: Biological process; CC: Cell components

表2 差异表达基因KEGG富集结果

Table 2 KEGG enrichment result for differentially expressed genes

Description	Gene ratio	Bg ratio	P-value	Gene ID	Count
Aflatoxin biosynthesis	6/113	46/2826	0.009071	CGGC5_14023/CGGC5_13120/CGGC5_13638/ CGGC5_13456/CGGC5_10453/CGGC5_9572	6
Pyruvate metabolism	7/113	72/2826	0.023327	CGGC5_14045/CGGC5_7569/CGGC5_3411/ CGGC5_10319/CGGC5_11829/CGGC5_3455/ CGGC5_5764	7
Sulfur metabolism	3/113	19/2826	0.037811	CGGC5_5794/CGGC5_12200/CGGC5_15291	3
Diterpenoid biosynthesis	3/113	21/2826	0.048998	CGGC5_1921/CGGC5_15293/CGGC5_11451	3

图2 损伤胁迫条件下差异表达基因聚类模式

Figure 2 Expression profiles for each cluster of differentially expressed genes

图3 差异表达基因聚类模块的富集分析 (A) GO富集分析; (B) KEGG富集分析。图中横坐标表示不同聚类, 纵坐标表示富集通路的名称。不同颜色的点代表不同P-value值, 点的大小表示该通路下差异表达基因的多少。P-value取值范围为0-0.15, 数值越接近0, 表示富集越显著。

Figure 3 Enrichment analysis for each gene cluster of differentially expressed genes (A) GO enrichment analysis for each cluster; (B) KEGG enrichment analysis for each cluster. The x-axis represents the different cluster and the y-axis is the name of enrichment pathway. Different P-value are represented by dots with different colors, while the number of differentially expressed genes in each pathway are displayed for the size of the dots. P-value ranges from 0-0.15. The closer the value to 0, the more significant the enrichment is.

表3 差异表达基因12个聚类模块中的12个核心基因

Table 3 Twelve core genes identified in 12 cluster modules of differentially expressed genes

Module	Gene numbers	Transcript	Gene description
1	1	ELA23757	Pep1
1	6	ELA25282	Cytochrome p450
1	16	ELA23240	Nad-dependent epimerase dehydratase
2	20	EFCGT00000013177	5.8S_rRNA
2	25	ELA35632	Polyketide synthase
2	44	ELA25574	Integral membrane protein
2	49	ELA24193	Short chain dehydrogenase reductase family
2	57	ELA35820	Ankyrin repeat-containing protein
2	189	ELA25228	Ankyrin repeat protein
2	26	ELA33919	Laccase
6	76	ELA24504	Major facilitator superfamily transporter
11	232	ELA37190	Catalase

图4 差异表达基因聚类模块内基因间调控网络重构建不同颜色对应不同的聚类模块。方框中的数字代表核心模块中相互作用的基因编号, 12个核心基因包含其中。

Figure 4 Inter-module regulatory network reconstruction of differentially expressed genes Different colors correspond to different clustered modules. The numbers in the boxes represent the interacting gene numbers in core modules. The 12 core genes are included.

图5 5个核心基因的动态表达模式及数据相关性不同小写字母表示各处理间差异显著(P<0.05)。

Figure 5 The dynamic expression patterns and data correlation of five core genes Different lowercase letters indicate significant differences among treatments (P<0.05).

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芒果胶孢炭疽病菌应答菌丝机械损伤产生无性孢子的分子机制

Molecular Mechanism of the Generation of Asexual Spores of the Mango Fungal Pathogen (Colletotrichum gloeosporioides) Induced by Mechanical Injuries

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