γ-氨基丁酸对暹罗炭疽菌的转录调控及胁迫耐受性的影响

doi:10.13560/j.cnki.biotech.bull.1985.2026-0437

摘要/Abstract

摘要：

目的探究暹罗炭疽菌（Colletotrichum siamense）侵染橡胶树过程中宿主γ-氨基丁酸（GABA）的动态变化规律，解析外源GABA对该病原菌碳氮代谢、抗氧化防御及细胞壁完整性的转录调控机制，为阐明橡胶树炭疽病菌的致病机理提供理论依据。方法通过比色法测定暹罗炭疽菌侵染后不同时间点（0-96 hpi）橡胶树叶片GABA含量变化；分别以GABA和NaNO₃为氮源分析菌株生长情况，并进行比较转录组测序，结合荧光定量PCR验证关键差异基因；通过在含H₂O₂、NaCl、山梨醇、刚果红和DTT等胁迫因子的察氏培养基（以硝酸钠/GABA为氮源）培养，分析GABA对病原菌逆境耐受性和细胞壁完整性的影响。结果暹罗炭疽菌侵染诱导宿主GABA含量自48 hpi后显著升高，至96 hpi达到峰值（1.9 mg/g），约为对照的2.4倍。该菌可利用GABA为碳氮源维持生长；转录组测序和荧光定量PCR分析结果均表明，以GABA为氮源培养下GABA代谢支路核心酶基因（GABA-T和SSADH）及下游三羧酸循环关键酶基因（SDH-2）均显著上调表达，而内源GABA合成关键酶基因（GAD）则被显著抑制；同时，抗氧化系统相关基因（CSase、GR、GST、SOD、PRX1）及磷酸戊糖途径关键酶基因（G6PD）均显著上调。表型分析表明，GABA不仅显著增强了病原菌对非生物胁迫的耐受性，且在细胞壁合成相关基因普遍下调的情况下，提高了菌株对刚果红和DTT的耐受性。结论暹罗炭疽菌侵染能诱导橡胶树叶片GABA大量积累，而该菌可以外源GABA为碳氮源经GABA代谢支路回补三羧酸循环，同时激活抗氧化防御系统并维持细胞壁完整性。

关键词: 暹罗炭疽菌, 橡胶树, γ-氨基丁酸, GABA代谢支路, 抗氧化防御, 细胞壁完整性

Abstract:

Objective This study aimed to investigate the dynamic changes of host γ‑aminobutyric acid (GABA) during Colletotrichum siamense infection in rubber trees, and to elucidate the transcriptional regulatory mechanisms by exogenous GABA modulates carbon/nitrogen metabolism, antioxidant defense, and cell wall integrity in this pathogen. The findings are expected to provide a theoretical basis for elucidating the pathogenic mechanisms of C. siamense in rubber trees. Method GABA content variations were measured in rubber tree leaves from 0 to 96 hours post-inoculation (hpi) with C. siamense infection. Comparative transcriptome sequencing (RNA-seq) was performed on mycelia cultured with GABA versus sodium nitrate as nitrogen sources, followed by quantitative real-time PCR (RT-qPCR) to verify expression of key genes. Phenotypic plate assays using Czapek-Dox with NaNO₃ or GABA as sole nitrogen source, supplemented with stress factors such as hydrogen peroxide, sodium chloride, sorbitol, Congo red, and dithiothreitol (DTT), to assess the effects of exogenous GABA on stress tolerance and cell wall integrity of the pathogen. Result C. siamense infection induced a significant increase in host GABA content starting from 48 hpi, reaching a peak of 1.9 mg/g at 96 hpi, approximately 2.4-fold that of the control. The fungus effectively utilized exogenous GABA as a dual carbon and nitrogen source for growth maintenance. Transcriptome sequencing and RT-qPCR analyses revealed significant up-regulation in GABA shunt core enzyme genes (GABA-T and SSADH) and the downstream tricarboxylic acid (TCA) cycle key enzyme gene (SDH-2). Conversely, the endogenous GABA synthesis key enzyme gene (GAD) exhibited significant down-regulation. Furthermore, antioxidant system genes (CSase, GR, GST, SOD, and PRX1) and the pentose phosphate pathway key enzyme gene (G6PD) showed significant up-regulation. Phenotype assays demonstrated that exogenous GABA supplementation profoundly enhanced pathogen abiotic stress tolerance of the pathogen. Notably, exogenous GABA increased strain tolerance to Congo red and DTT, despite the widespread down‑regulation of cell wall synthesis genes. Conclusion C. siamense infection induces substantial GABA accumulation in rubber tree leaves. The pathogen efficiently assimilates exogenous host GABA and replenishes the TCA cycle via the GABA shunt, simultaneously activating the pathogen's antioxidant defense system and maintaining cell wall integrity.

Key words: Colletotrichum siamense, rubber tree, γ-aminobutyric acid, GABA shunt, antioxidant defense, cell wall integrity

何其光, 冼雪梅, 刘辉, 袁坤, 杨洁, 王玉婷, 王真辉, 杨洪. γ-氨基丁酸对暹罗炭疽菌的转录调控及胁迫耐受性的影响[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0437.

HE Qi-guang, XIAN Xue-mei, LIU Hui, YUAN Kun, YANG Jie, WANG Yu-ting, WANG Zhen-hui, YANG Hong. Effects of γ-aminobutyric Acid on Transcriptional Regulation and Stress Tolerance in Colletotrichum siamense[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0437.

图/表 7

表1 本研究所用RT-qPCR引物信息

Table 1 RT-qPCR primers used in this study

引物名称 Primer name	上游引物序列 Upstream primer sequence （5′-3′）	下游引物序列 Downstream primer sequence （5′-3′）
qCsActin	GATGTGGATATCAGGAAGG	CATACTGCTTGGAGCAAGA
qCsGAD-1	GGTTTGCCTGTGGTCTCGTTC	CGGTAGTGGGTAGTTGGGAATG
qCsGAD-2	GGCTGGGTTGTTCCGGCTT	GTGGTTGTGCGACGACTTGGT
qCsGABAT-1	GAGCACCTCATCAAGACCTACCA	CTCGTGAGCCCAGAACTTGC
qCsGABAT-2	GGCAGTCTGTCTACCACCCG	CCGCCCTCGCTCTGAATAG
qCsSAADH-1	CCAAGGGTTCTCAGCCGATAC	CGAAGTCGGAAAGGTTGGAAG
qCsSAADH-2	CGTGCCCTGAATTTGATGAGA	CCTGGATGGTATCGCCGTAT
qCS-1	TGACCAAGGAGGAGGCTTACAT	TCGGTGGGAGCACCAGAGT
qCS-2	GTCCAACGAGCTGAACAACATTATC	GTCGGCCTGGTATCTGAGCAA
qCsSDH-1	CTTCGTTTGTGCGACCTGCT	TTCTGGACGGTAGGCTTGAGG
qCsSDH-2	GATGGAGCAGACGTGGTTCGG	GGTGGCGGATGCCGTTGA
qCsMDH	CGTCAACTCCACCGTTCCTATTT	AAGGCGTGACTTGGCTGAAAA
qGDH-1	CCCCTACAAGGGAGGTCTGA	CCTTGGGGTTGAAGTCGGAA
qGDH-2	GCAGCGCAAGAACAAAGACA	CCGGTGAAGAACGACTTCCA
qCsCSase-1	GGAATACCTGAACCCAACCG	GAGTGCAAACGAAGGCCAGT
qCsCSase-2	GGCCCTTCTTCTGGGTTCAA	TCGTTCGGGATTACGCTTTC
qCsCSase-3	TGGTGTTGACCTCTACCGATACGA	TCCTCGGGCTGACGCAAGT
qCsTRX-1	ATGGTCGTCATCCACGCTTC	TCTCGTACATGGGCTTGATTTG
qCsTRX-2	CTTTGGTCCCGTCCGTGTCT	TGTTCGTGTAGAGGTGGATGGTG
qCsTRX-3	ACGAGTGCTTGTGCTTGCGATAC	GAGGACACGCCTTGCGACATT
qCsG6PD	CCCCATTTCTTTCGCCTCTGA	TTGAATTGGATGCGGATCTCG
qCsGR	GTCTGGCGGAGCGTCTGTT	GCGTAGTACATGGCGGTAAAGT
qCsGST-1	CATTTCGCCAACGGCTCCC	TCTCGCCCGCCAACCACTT
qCsGST-2	GGAGGTCTTCCATCGCAACAA	TGGCATCTCGTCCACTCTTCG
qCsPRX	CATCATTGCCGACAAGGAGC	AGGAGAGGATGGTGCGGATG
qCsSOD-1	TACGATGCGTTGGAACCTCAC	CGTTGTATGTTTCGATAGCCTTGT
qCsSOD-2	GCTACTCTGCCCGATATGCC	GCCTCCTGGACGGTCTCAA
qCskatG	ATCTCATGGGCGGACCTGTAC	CCAGTAGACGGACTCATCGGAC

图1 暹罗炭疽菌侵染过程橡胶树叶片GABA含量

Fig. 1 Dynamics of GABA accumulation in rubber tree leaves during C. siamense infection

图2 暹罗炭疽菌对GABA的同化利用

Fig. 2 Assimilation and utilization of GABA by C. siamese

图3 暹罗炭疽菌响应GABA同化利用的转录组分析A：不同处理组间样本聚类主成分分析（PCA）图；B：差异基因分布火山图；C：差异表达基因层次聚类热图；D：差异表达基因GO注释与功能分类；E：差异表达基因KEGG富集分析

Fig. 3 Transcriptomic analysis of C. siamense in response to GABA assimilation and utilizationA: Principal component analysis (PCA) showing the clustering of samples among different treatments. B: Volcano plot of DEGs. C: Hierarchical clustering heatmap of the DEGs. D: Gene Ontology (GO) functional classification of the DEGs. E: KEGG pathway enrichment analysis of the DEGs

图4 暹罗炭疽菌同化外源GABA的代谢通路核心基因表达模式A：转录组中参与GABA代谢途径的基因表达模式；B：GABA代谢途径基因表达的RT-qPCR验证。* P<0.05；** P<0.01，下同

Fig. 4 Expression patterns of key genes in the exogenous GABA assimilation pathway of C. siamenseA: Expression profiles of GABA pathway‑related genes in the transcriptome; B: RT-qPCR validation of gene expression in the GABA metabolic pathway. * P<0.05; ** P<0.01, the same below

图5 GABA介导暹罗炭疽菌逆境胁迫耐受力A：转录组中ROS代谢相关基因表达模式；B：ROS代谢相关基因表达的RT-qPCR验证；C：以GABA为氮源逆境胁迫下暹罗炭疽菌生长情况

Fig. 5 GABA-mediated stress tolerance in C. siamenseA: Expression patterns of key genes associated with ROS metabolism in the transcriptome; B: RT-qPCR validation of the expression of ROS metabolism-related genes; C: growth of C. siamense under various stresses with GABA as the nitrogen source

图6 GABA介导暹罗炭疽菌细胞壁完整性A：利用不同氮源培养时暹罗炭疽菌的生长情况；B：基于RNA-seq数据的细胞壁生物合成关键基因表达模式；C：不同氮源条件下对刚果红（Congo red）和DTT的敏感性

Fig. 6 GABA-mediated cell wall integrity in C. siamenseA: Growth of C. siamense cultured with different nitrogen sources; B: expression patterns of key genes involved in cell wall biosynthesis based on RNA‑seq data; C: sensitivities to Congo red and DTT under different nitrogen conditions

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