番茄SlβCA3在防御丁香假单胞菌番茄致病变种中的功能

doi:10.3864/j.issn.0578-1752.2022.14.005

摘要/Abstract

摘要：

【背景】在全球气候变化的背景下,大气CO₂浓度的升高会影响植物病害的发生,进而影响农业生产。β型碳酸酐酶（β- carbonic anhydrase,βCA）是植物CO₂感应和浓缩系统中的重要组成元件,参与拟南芥和烟草的植物免疫过程,但在番茄（Solanum lycopersicum）等园艺作物中的研究较少。【目的】通过探究番茄SlβCA3在抵御植物病害中的作用及机制,为番茄生产中的抗性调控提供科学依据。【方法】以拟南芥AtβCA氨基酸系列为参考序列,在番茄Sol genomics network 数据库中鉴定到4个SlβCA。进一步以野生型（wild-type,WT）番茄‘Ailsa Craig’（AC）为材料接种丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato DC3000,Pst DC3000）,利用qRT-PCR技术测定叶片中SlβCA的表达量,筛选出受Pst DC3000诱导表达的基因SlβCA3。在此基础上,以AC为背景,利用农杆菌介导法进行番茄遗传转化,构建SlβCA3稳定过表达植株（OE-SlβCA3）。通过观察OE-SlβCA3植株接种Pst DC3000后的抗性表型,明确SlβCA3在番茄抵御Pst DC3000过程中的作用。为了研究SlβCA3调控植物抗病性的内在机制,比较WT和OE-SlβCA3植株接种Pst DC3000与对照条件下转录组的变化,并利用KEGG数据库对差异基因进行功能分析,推测糖代谢与SlβCA3介导的免疫反应有关。最后,通过测定WT和OE-SlβCA3植株糖代谢及其信号途径相关基因表达量以及葡萄糖、果糖和蔗糖含量,对转录组结果进行验证及分析。【结果】OE-SlβCA3植株对Pst DC3000的抗性增强,接种Pst DC3000后,叶片中的细菌生长量、病斑数以及死细胞积累量明显减少。转录组测序结果显示,正常条件下,OE-SlβCA3植株转录谱没有发生明显变化;接种Pst DC3000后,在WT和OE-SlβCA3植株中检测到2 100个Pst DC3000诱导基因,其中有63.3%的基因在OE-SlβCA3植株中表达量更高。KEGG分析结果显示,依赖于SlβCA3过表达的Pst DC3000诱导基因富集在糖代谢相关路径中,包括淀粉和蔗糖代谢,内质网中的蛋白质加工（糖基化）,氨基糖和核苷酸糖代谢,真核生物中的核糖体生物合成以及光合作用等路径。糖代谢与糖信号密不可分,qRT-PCR及糖含量测定结果显示,接种Pst DC3000后,OE-SlβCA3植株叶片中糖代谢及其信号传导途径相关基因表达量与葡萄糖、果糖和蔗糖的含量较WT更高。【结论】番茄SlβCA3的过表达增强了植株对Pst DC3000的抗性,该过程可能与糖代谢及其信号通路在植物免疫中的作用有关。

关键词: 番茄, SlβCA, 丁香假单胞菌番茄致病变种, 抗病性, 糖代谢, 糖信号

Abstract:

【Background】With global climate change, the increase of atmospheric CO₂ concentration is predicted to exert an influence on plant diseases, which seriously affects agricultural production. Plant β-carbonic anhydrases (βCAs) are important components in plant CO₂ sensing and concentration systems and are involved in the immunity of Arabidopsis and tobacco. However, little is known about the functions of βCAs in the regulation of disease resistance in tomato (Solanum lycopersicum). 【Objective】The objective of this study is to explore the role and mechanism of tomato SlβCA3 in disease resistance, so as to provide scientific basis for resistance regulation of tomato in agricultural production. 【Method】Based on the similarity to the amino acid sequences of AtβCAs, four SlβCAs were identified in the Sol genomics network database. Wild-type (WT) tomato ‘Ailsa Craig’ (AC) was used to inoculate Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in the study. Then qRT-PCR was used to determine the transcript abundance of SlβCAs in leaves to screen the Pst DC3000-induced gene SlβCA3. Furthermore, SlβCA3 stable over-expression lines (OE-SlβCA3) were generated by Agrobacterium tumefaciens-mediated genetic transformation technology as the background of AC. OE-SlβCA3 plants were inoculated with Pst DC3000 to investigate the role of SlβCA3 in disease defense. For exploring the intrinsic mechanism of SlβCA3 regulating plant disease resistance, the transcriptome changes of WT and OE-SlβCA3 plants between inoculation with Pst DC3000 and control conditions were compared, and KEGG (Kyoto encyclopedia of genes and genomes) database was used to analyze functions of the differentially expressed genes. It is speculated that sugar metabolism pathways are involved in SlβCA3-mediated plant immunity. To verify and further analyze the conclusion, the expression of genes related to the sugar metabolism and signaling, as well as the contents of glucose, fructose and sucrose in WT and OE-SlβCA3 plants were determined. 【Result】OE-SlβCA3 plants enhanced the resistance to Pst DC3000, and showed less disease-associated cell death and a lower number of bacteria compared to the WT controls. RNA-Seq results showed that OE-SlβCA3 did not greatly change the overall transcript profile in the absence of the pathogen. In total, 2 100 Pst DC3000-induced transcripts were differentially changed in abundance. Of these, 63.3% were more abundant following Pst DC3000 inoculation in the OE-SlβCA3 plants. KEGG analysis showed that Pst DC3000-induced genes, which are dependent on SlβCA3-overexpression, were enriched in the pathways related to sugar metabolism, including starch and sucrose metabolism, protein processing in the endoplasmic reticulum (glycosylation), amino sugar and nucleotide sugar metabolism, ribosomal biosynthesis in eukaryotes and photosynthesis. Sugar metabolism is closely related to sugar signaling. Further studies found that the expression of genes related to sugar metabolism and signal transduction pathways, as well as the contents of glucose, fructose and sucrose, were higher in the leaves of OE-SlβCA3 plants than those of WT after inoculation with Pst DC3000. 【Conclusion】Overexpression of SlβCA3 in tomato enhances the resistance of plants to Pst DC3000, which may be related to the role of sugar metabolism and signaling in plant immunity.

Key words: tomato (Solanum lycopersicum), SlβCA, Pst DC3000, disease resistance, sugar metabolism, sugar signaling

方瀚墨,胡璋健,马巧梅,丁淑婷,王萍,王安然,师恺. 番茄SlβCA3在防御丁香假单胞菌番茄致病变种中的功能[J]. 中国农业科学, 2022, 55(14): 2740-2751.

FANG HanMo,HU ZhangJian,MA QiaoMei,DING ShuTing,WANG Ping,WANG AnRan,SHI Kai. Function of SlβCA3 in Plant Defense Against Pseudomonas syringae pv. tomato DC3000[J]. Scientia Agricultura Sinica, 2022, 55(14): 2740-2751.

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基因ID Accession number	基因Gene	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
Solyc02g086820	SlβCA1	CAGCGAGAAAGCAGAACTTG	TTTCATGTGCTCAACAGGGT
Solyc05g005490	SlβCA2	CGAGTTTGCCCATCACACAT	TGCATATTCGACTGCTGCAC
Solyc02g067750	SlβCA3	AAATTGGGTTACCTGCCAAG	TGGATAGGTCAGCAAGTTGG
Solyc09g010970	SlβCA4	CTTGCAGACGAACAATCACC	CTCCTGGTTGAAATCCCAGT
Solyc10g083290	CWIN	GAATCACAGTTGCACAGGCT	GCGCATAAAGATCAGCCCAA
Solyc06g073760	BGLU	AAGCCCACCTCATGCTAACT	CCGCTTCACAGCATCATCAA
Solyc07g006500	TPS	CTGGTACCTGCAGACACTGA	AGAAGCTCTTTAGCCTGCCA
Solyc04g076810	SnRK	CACAGGCGGGGAACTTTTTG	ATGTTGACTCCTTCGCCAGG
Solyc01g100460	bZIP	TTCCAACAGGGAATCTGCGA	CTGCTCACTTCCCCTGTCAA
Solyc03g078400	SlACTIN	TGGTCGGAATGGGACAGAAG	CTCAGTCAGGAGAACAGGGT