香蕉枯萎病是由尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense, Foc）引起的香蕉维管束病害,给全世界香蕉产业带来了严重的危害[1]。该病菌1号小种（Foc 1）曾导致香蕉主栽品种‘大蜜哈’的毁灭,直到抗1号小种的Cavendish香蕉品种应用,世界香蕉生产才得以峰回路转。但是,近半个世纪来,侵染Cavendish香蕉的4号小种（Foc 4）出现迅速蔓延,使香蕉产业重新面临严重的困境[2]。因此,研究Foc的致病机理及其2个小种的致病性分化机制,将为香蕉枯萎病防治技术研发提供新的途径。
毒素是香蕉枯萎病菌的主要致病因子之一[3,4,5]。Foc分泌细胞壁降解酶突破寄主细胞壁,随后在寄主根部维管束组织内大量增殖,并沿着维管束向根茎和假茎部分蔓延,堵塞木质部导管,阻碍水分和营养的运输,同时产生真菌毒素（如镰刀菌酸和脱氢镰刀菌酸等）造成维管束坏死和叶片黄化,最终导致植株枯萎死亡[6]。
大部分真菌毒素属于次生代谢产物（secondary metabolites, SMs）,如白僵菌素（beauvericin, BEA）和镰刀菌酸（fusaric acid, FA）[7,8]。真菌毒素能够破坏寄主细胞膜,影响寄主正常的代谢过程,导致寄主生理失调和细胞死亡,在死体营养型真菌的致病过程中起着决定性作用[9]。毒素的生物合成是由一系列基因簇（secondary metabolite gene clusters, SMC）控制,包括非核糖体多肽合成酶（nonribosomal peptide synthetase, NRPS）和聚酮合成酶（polyketide synthase, PKS）基因,其他酶类（如氢化酶、氧化酶和转运蛋白等）基因以及相关调控基因[10]。例如,白僵菌素主要是由BEA合成酶（NRPS）催化合成,并且镰刀菌属真菌BEA生物合成还需要一系列不同功能的SMC参与催化代谢[11];镰刀菌酸的生物合成主要由FA基因簇中的聚酮合成酶Fub 1控制,敲除Fub1能够使F. oxysporum f. sp. lycopersici不产生FA,并且显著降低其对番茄的致病力[12]。
本研究前期通过RNA-seq测序分析,成功获得了3种碳源（寄主细胞壁、果胶和葡萄糖）条件下Foc 1和Foc 4的转录组数据,并发现在寄主细胞壁多糖条件下能有效模拟Foc侵染寄主的基因表达情况[13]。本研究将利用antiSMASH数据库对该转录组数据进行次生代谢物合成（secondary metabolism biosynthesis, SMB）基因的预测和表达分析,挖掘Foc致病相关的SMB基因,分析SMB基因在2个小种致病过程中的表达差异,为Foc致病基因的筛选和致病性分化的分子机理研究提供参考。
1 材料与方法
1.1 材料
本研究所用3种碳源条件下Foc 1和Foc 4转录组数据由华南农业大学植物病理生理研究室提供。该转录组测序所用菌株为香蕉枯萎病菌1号生理小种菌株C2和4号生理小种菌株DZ1。3种碳源分别为寄主细胞壁多糖的主要成分：寄主细胞壁（粉蕉细胞壁用于Foc 1菌株的培养,以FCW表示;巴西蕉细胞壁用于Foc 4菌株的培养,以BCW表示）、果胶（以P表示）和葡萄糖（以G表示）。该转录组raw reads序列已提交至NCBI,登录号为SRA486974。
1.2 方法
1.2.1 次生代谢物合成基因的预测 本研究利用antiSMASH数据库对转录组23 147个unigene序列进行SMB基因预测,比对参数为默认值。
1.2.2 次生代谢物合成基因的致病相关性分析 本研究利用病原物与寄主互作数据库PHI-base（http://www.phi-base.org/）对发掘的SMB基因的致病功能进行预测,比对阈值（e-value）为1e-5。
1.2.3 不同寄主细胞壁多糖条件下次生代谢物合成基因的表达分析 对转录组数据进行参考序列比对,基因表达水平进行FPKM（fragments per kilobase of exon model per million reads mapped）换算,随后将基因差异表达数据进行标准化处理,最后进行基因差异表达水平DEGseq分析,差异表达基因（differentially expressed gene, DEG）的筛选阈值为q-value<0.005且|log2 (Fold Change)| >1。
差异基因维恩图的制作使用Draw Venn Diagrum软件（http://bioinformatics.psb.ugent.be/ webtools/Venn/）;差异基因表达水平热图制作使用OmicShare Tools Heat map软件（https://www.omicshare.com/ tools）。
2 结果与分析
2.1 次生代谢物合成基因的预测
本研究以不同的寄主细胞壁多糖条件下Foc 1和Foc 4的转录组数据进行SMB基因分析,通过antiSMASH数据库的注释,共预测到486个unigene。其中,83个unigene被预测为SMC的主要合成基因,并分类到32个SMC中（表1）。32个SMC包括3个T1pks基因簇,1个T3pks基因簇,9个Nrps基因簇,1个Nrps-T1pks基因簇,10个Terpene基因簇,3个Indole基因簇和5个其他基因簇。根据上述SMB基因分析推测,3种碳源条件下Foc主要次生代谢物类型为I型聚酮、III型聚酮、T1pks-nrps聚酮、萜类和吲哚类。
表1 3种碳源条件下Foc转录组预测的次生代谢物合成基因簇Tab. 1 The precited secondary metabolites synthesis gene clusters in Foc transcriptome |
| 基因簇 Cluster | 类型 Type | 主要合成基因 Backbone gene | smCOG注释 smCOG annotation | 序列特征 Signature pHMM hits | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Cluster 1 | Nrps | comp18389_c0 | SMCOG1034(cytochrome P450) | |||||||||
| comp26123_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | Condensation; AMP-binding; PP-binding | ||||||||||
| Cluster 2 | T3pks | comp11818_c0 | SMCOG1023(enoy1-CoA hydratase) | |||||||||
| comp14386_c0 | SMCOG1294(Nitrilase/cyanide hydratase and apolipoprotein) | |||||||||||
| comp18161_c0 | SMCOG1024(chalcone and stilbene synthase domain protein) | Chal_sti_synt_C | ||||||||||
| Cluster 3 | T1pks | comp19458_c0 | SMCOG1050(monooxygenase FAD-binding | |||||||||
| comp22370_c0 | - | PP-binding; PKS_AT; itr_KS | ||||||||||
| Cluster 4 | Nrps-T1pks | comp15124_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||
| comp19424_c0 | SMCOG1168(O-succinylhomoserine sulfhydrylase) | |||||||||||
| comp22100_c0 | SMCOG1021(malonyl CoA-acyl carrier protein transacylase) | condensation; AMP-binding; PP-binding; PKS_AT;itr_KS; adh_short; NAD_binding_4 | ||||||||||
| comp22100_c0 | SMCOG1022(Beta-ketoacyl synthase) | itr_KS | ||||||||||
| comp9422_c0 | SMCOG1001(short-chain detydrogenase / reductase SDR) | |||||||||||
| comp12044_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||||
| comp1756_c0 | SMCOG1081(cysteine synthase) | |||||||||||
| Cluster 5 | Indole | comp18974_c0 | SMCOG1105(amiddase) | |||||||||
| comp16167_c0 | — | dmat | ||||||||||
| comp9570_c1 | SMCOG1103(FAD dependent oxidoreductase) | |||||||||||
| comp18661_c0 | SMCOG1010(NAD-dependent epimerase / dehydratase) | |||||||||||
| Cluster 6 | Nrps | comp1045_c0 | SMCOG1017(aldehyde dehydrogenase) | |||||||||
| comp22375_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | Condensation; AMP-binding; PP-binding | ||||||||||
| comp13778_c0 | SMCOG1028(crotonyl-CoA reductase / alcohol dehydrogenase) | |||||||||||
| Cluster 7 | Terpene | comp11592_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||
| Cluster 8 | Other | comp20521_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding; PP-binding; adh_short; NAD_binding_4 | ||||||||
| comp12071_c0 | SMCOG1072(dehydrogenase) | |||||||||||
| Cluster 9 | Terpene | comp16795_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||
| comp14836_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||||
| comp21246_c0 | SMCOG1036(alpha/beta hydrolase fold protein) | |||||||||||
| comp22098_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding; PP-binding; NAD_binding_4 | ||||||||||
| 基因簇 Cluster | 类型 Type | 主要合成基因 Backbone gene | smCOG注释 smCOG annotation | 序列特征 Signature pHMM hits | ||||||||
| Cluster 10 | Terpene | comp20117_c0 | SMCOG1039(aldo / keto reductase family oxidoreductase) | |||||||||
| comp20666_c0 | SMCOG1182(Polyprenyl synthetase) | fung_ggpps | ||||||||||
| Cluster 11 | Nrps | comp1167546_c0 | SMCOG1127(condensation domain-containing protein) | Condensation | ||||||||
| comp19826_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding | ||||||||||
| Cluster 12 | Other | comp4239_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | |||||||||
| Cluster 13 | Terpene | comp17133_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||
| comp17791_c0 | SMCOG1109(8-amino-7-oxononanoate synthase) | |||||||||||
| Cluster 14 | Nrps | comp13338_c0 | SMCOG1017(aldehyde dehydrogenase) | |||||||||
| comp20790_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | Condensation; AMP-binding | ||||||||||
| comp13794_c0 | SMCOG1001(short-chain detydrogenase / reductase SDR) | |||||||||||
| Cluster 15 | Nrps | comp21540_c0 | SMCOG1127(condensation domain-containing protein) | Condensation; AMP-binding; PP-binding | ||||||||
| comp4617_c1 | SMCOG1038(phenylalanine-specific permease) | |||||||||||
| comp21825_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||||
| Cluster 16 | Nrps | comp22368_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | Condensation; AMP-binding; PP-binding | ||||||||
| comp21749_c0 | SMCOG1109(8-amino-7-oxononanoate synthase) | |||||||||||
| Cluster 17 | Terpene | comp980601_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||
| Cluster 18 | Nrps | comp20665_c0 | SMCOG1001(short-chain detydrogenase / reductase SDR) | |||||||||
| comp20785_c0 | SMCOG1127(condensation domain-containing protein) | Condensation; AMP-binding | ||||||||||
| comp6376_c0 | SMCOG1180(Decarboxylase,pyridoxal-dependent) | |||||||||||
| comp16435_c0 | SMCOG1013(aminotransferase class-Ⅲ) | |||||||||||
| comp19140_c0 | SMCOG1055(Pyruvate oxidase / decarboxylase) | |||||||||||
| comp1976779_c0 | SMCOG1053(beta-lactamase) | |||||||||||
| comp17350_c0 | SMCOG1089(methyltransferase) | |||||||||||
| Cluster 19 | Terpene | comp18903_c0 | SMCOG1028(crotonyl-CoA reductase / alcohol dehydrogenase) | |||||||||
| comp8549_c0 | — | phytoene_synt | ||||||||||
| comp22259_c0 | SMCOG1105(amiddase) | |||||||||||
| Cluster 20 | T1pks | comp8972_c0 | SMCOG1034(cytochrome P450) | |||||||||
| comp21052_c0 | SMCOG1022(Beta-ketoacyl synthase) | PP-binding; PKS_AT; itr_KS; adh_short | ||||||||||
| Cluster 21 | Nrps | comp14287_c0 | SMCOG1017(aldehyde dehydrogenase) | |||||||||
| comp20689_c0 | SMCOG1138(FAD linked oxidase domain protein) | |||||||||||
| comp22376_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | Condensation; AMP-binding; PP-binding | ||||||||||
| comp2100_c0 | SMCOG1040(alcohol dehydrogenase) | |||||||||||
| comp17240_c0 | SMCOG1081(cysteine synthase) | |||||||||||
| Cluster 22 | Indole | comp22755_c0 | — | dmat | ||||||||
| Cluster 23 | Nrps | comp18186_c0 | SMCOG1138(FAD linked oxidase domain protein) | |||||||||
| comp19075_c0 | — | Condensation | ||||||||||
| comp1549937_c0 | SMCOG1001(short-chain detydrogenase / reductase SDR) | |||||||||||
| comp5072_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding | ||||||||||
| Cluster 24 | Terpene | comp53030_c0 | SMCOG1040(alcohol dehydrogenase) | |||||||||
| comp16429_c0 | — | phytoene_synt | ||||||||||
| Cluster 25 | Terpene | comp12002_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||
| Cluster 26 | Indole | comp23658_c0 | — | dmat | ||||||||
| 基因簇 Cluster | 类型 Type | 主要合成基因 Backbone gene | smCOG注释 smCOG annotation | 序列特征 Signature pHMM hits | ||||||||
| Cluster 27 | Other | comp21904_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding; PP-binding; NAD_binding_4 | ||||||||
| Cluster 28 | Terpene | comp16713_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||
| comp7130_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||||
| Cluster 29 | Terpene | comp20481_c0 | SMCOG1172(methionine aminopeptidase) | |||||||||
| comp18246_c0 | SMCOG1235(polysaccharide deacetylase) | |||||||||||
| comp18246_c0 | SMCOG1052(Terpene synthase / cyclase metal-binding domain protein) | Terpene_synth_C | ||||||||||
| Cluster 30 | T1pks | comp779042_c0 | SMCOG1138(FAD linked oxidase domain protein) | |||||||||
| comp15302_c0 | SMCOG1217(NADH:flavin oxidoreductase / NADH oxidase) | |||||||||||
| comp38245_c0 | SMCOG1022(Beta-ketoacyl synthase) | PP-binding; PKS_AT; itr_KS; adh_short | ||||||||||
| comp21043_c0 | SMCOG1038(phenylalanine-specific permease) | |||||||||||
| Cluster 31 | Other | comp22060_c0 | SMCOG1176(alkyl hydroperoxide reductase subunit) | |||||||||
| comp5848_c0 | SMCOG1034(cytochrome P450) | |||||||||||
| comp22364_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding; PP-binding | ||||||||||
| comp8303_c0 | SMCOG1089(methyltransferase) | |||||||||||
| Cluster 32 | Other | comp21755_c0 | SMCOG1002(AMP-dependent synthetase and ligase) | AMP-binding; PP-binding; adh_short; NAD_binding_4 | ||||||||
通过antiSMASH数据库的预测,3个SMC（Cluster 4、Cluster 6和Cluster 21）预测到合成的次生代谢物核心结构,同时还发现Cluster 6和Cluster 21虽同为Nrps基因簇,但合成的次生代谢物组成单位存在明显差异（图1）。
2.2 次生代谢物合成基因的致病相关性分析
通过PHI-base对486个SMB基因的致病功能进行分析预测（e-value≤10-5）,结果发现有37个基因可能与Foc和寄主的互作相关（即PHI基因）,其中13个SMB基因缺失后可能导致Foc致病性丧失或致病力减弱。结果还显示,83个SMC主要合成基因在PHI-base中并无致病相关功能注释。
2.3 3种碳源条件下次生代谢物合成基因的差异表达分析
为了解次生代谢物在2个小种致病性分化中的作用,本研究对3种碳源条件下Foc 1和Foc 4的SMB基因表达情况进行分析,总共获得6个比较组（FCW_Foc1 vs. G_Foc1、BCW_Foc4 vs. G_Foc4、BCW_Foc4 vs. FCW_Foc1、P_Foc4 vs. G_Foc4、P_Foc1 vs. G_Foc1和P_Foc4 vs. P_Foc1）的差异表达基因。结果表明,在3种碳源条件下Foc 1或Foc 4表达的SMB基因均存在明显差异（图2）。同时,相较于Foc 1,Foc 4在降解寄主细胞壁多糖时SMB基因的差异表达数量较多（表2）。而在同种碳源条件下,2个小种SMB基因的表达模式也存在明显差异（图2）,具体表现在：Foc 4特异表达的SMB基因数量相较于Foc 1更多（图2A、图2B、图2C和图2D）;在Foc 4中高表达的SMB基因在Foc 1中基本都低表达（图2E）。
图2 3种碳源诱导下香蕉枯萎病菌差异表达的次生代谢物合成基因维恩图和次生代谢物主要合成基因的表达水平热图A：寄主细胞壁条件下上调差异表达的次生代谢物合成基因;B：下调差异表达的次生代谢物合成基因;C：果胶条件下上调差异表达的次生代谢物合成基因;D：下调差异表达的次生代谢物合成基因。维恩图非重叠部分为2个小种特异表达的次生代谢物合成基因（红色数字）。E：3种碳源条件下香蕉枯萎病菌2个小种次生代谢物合成基因的差异表达热图以FPKM绘制。G：葡萄糖;P：果胶;FCW：粉蕉细胞壁;BCW：巴西蕉细胞壁。 Fig. 2 The number of DEGs from secondary metabolites synthesis genes, derived from comparisons induced by host cell wall or pectin and Expression profiles of backbone genes from secondary metabolites synthesis gene clusters HeatmapA: Up-regulated differentially expressed secondary metabolite synthesis genes under host cell wall conditions; B: Down-regulated differentially expressed secondary metabolite synthesis genes under host cell wall conditions; C: Up-regulated differentially expressed secondary metabolite synthesis genes under pectin condition; D: Down-regulated differentially expressed secondary metabolite synthesis genes under pectin condition. The non-overlapping parts of the Venn diagram were the secondary metabolite synthesis genes (red number) specifically expressed by two Foc races. E: The differential expression heat map of the major secondary metabolite synthesis genes in two Foc races under three carbon sources was drawn by FPKM. G: Glucose; P: Pectin; FCW: Cell wall of Fenjiao cultivar; BCW: Cell wall of Brazil cultivar. |
表2 3种碳源诱导下香蕉枯萎病菌差异表达的次生代谢物合成相关基因数量Tab. 2 The number of DEGs involved in secondary metabolites synthesis in each comparison |
| BCW_Foc4 vs. G_Foc4 | FCW_Foc1 vs. G_Foc1 | BCW_Foc4 vs. FCW_Foc1 | P_Foc4 vs. G_Foc4 | P_Foc1 vs. G_Foc1 | P_Foc4 vs. G_Foc1 | ||
|---|---|---|---|---|---|---|---|
| 上调基因数 | 32 | 23 | 33 | 11 | 8 | 34 | |
| 下调基因数 | 45 | 28 | 28 | 23 | 20 | 26 | |
| 总数 | 77 | 51 | 61 | 34 | 28 | 60 | |
Note: G. Glucose; P. Pectin; FCW. Cell wall of Fenjiao cultivar; BCW. Cell wall of Brazil cultivar. | |
注：G. 葡萄糖;P. 果胶;FCW. 粉蕉细胞壁;BCW. 巴西蕉细胞壁。 |
上述结果表明,这些在Foc 4中特异表达或高表达的SMB基因可能参与Foc 4对Cavendish香蕉的侵染过程。
3 讨论
antiSMASH数据库是预测微生物次生代谢物合成基因簇的综合性数据库,可实现基因组与基因组之间的相关天然产物合成基因簇的查询[14]。PHI-base数据库整合了大量病原真菌和卵菌在侵染寄主时具有互作功能的基因研究数据[15]。目前,这2种数据库已广泛利用于植物病原菌基因组和转录组的致病基因预测。通过antiSMASH和PHI-base数据库对引起多种植物土传病害的菜豆壳球孢（Macrophmina phaseolina）基因组分析发现,该病原菌相对于F. oxysporum和F. graminearum拥有更多的致病酶类和毒素摧毁寄主防御,其中包括75个主要SMB基因和537个致病基因[16]。在小麦赤霉病菌（F. graminearum）与非致病型镰刀菌（F. venenatum）的比较基因组研究中,利用antiSMASH、PHI-base等数据库揭示了F. graminearum中5个特有的致病相关SMC参与了该菌侵染小麦的病程,另外2个PHI转录因子敲除后使其对小麦的致病力显著降低[17,18]。
本研究利用antiSMASH和PHI-base数据库对3种碳源条件下Foc 1和Foc 4表达的次生代谢物合成基因进行了预测分析,发现了32个SMC,总共获得486个候选的SMB基因（包含83个主要合成基因）,但大部分SMB基因的功能未知。该结果与Foc 1和Foc 4基因组的SMC注释结果基本相符[5]。Foc基因组中存在4个合成二甲基丙烯基色氨酸（DAMT）的基因簇,但在本研究中未发现合成DAMT的基因簇,可能是在突破寄主细胞壁防御时,DAMT合成途径并不参与Foc与寄主互作过程。
本研究发现3种碳源条件下Foc 1和Foc 4表达的13个SMB基因可能与其致病性相关,但83个主要合成基因致病功能未获得预测信息。这可能是由于SMB基因的功能在植物病原真菌中研究还较少,利用同源比对分析,从PHI-base收录的致病基因中不能找到Foc主要SMB基因的同源基因。因此,进一步开展SMB基因功能验证,将有助于深入了解次生代谢物在Foc致病过程中的作用。
本研究发现在3种碳源条件下,Foc的SMB基因具有不同的表达模式,说明不同营养条件能够影响Foc次生代谢物的合成。已有研究证明,Foc在PDA培养条件（in vitro）和侵染巴西蕉（in vivo）时SMB基因的表达存在明显差异[5];而在水稻恶苗病菌（F. fujikuroi）中也发现了相似结果,该病菌次生代谢物的合成受不同氮源和植物信号的影响[19]。本研究还发现,相较于Foc 1,Foc 4在侵染寄主细胞壁时有更多的次生代谢物合成基因特异表达或表达量升高,说明Foc 4能够产生更多的次生代谢物来适应不同的寄主环境,并成功侵染大部分香蕉主栽品种,这可能与2个小种寄主专化性和致病性分化密切相关。本研究结果将为进一步明确次生代谢产物在Foc致病性分化的作用提供参考依据。