番茄糖转运蛋白SlSTP2在防御细菌性叶斑病中的功能

doi:10.3864/j.issn.0578-1752.2022.16.007

摘要/Abstract

摘要：

【背景】近年来,我国番茄生产中面临的不良气候环境导致露地和设施栽培中番茄病害日益严重。由丁香假单胞菌番茄致病变种（Pseudomonas syringae pv. tomato,Pst）所引发的番茄细菌性叶斑病频发,严重影响了番茄的产量、品质。糖是植物体内重要的信号分子与营养物质,在植物遭受病原菌侵染时,糖既可以作为信号参与调控植物抗病性,也可以作为主要碳源为免疫反应提供能量。STP（sugar transport protein）家族是一类糖转运蛋白家族,在植物生长发育过程与病原防御中起着重要作用。【目的】明确番茄中STP家族是否参与调控植株对细菌性叶斑病的抗性。【方法】以番茄CR品种（Solanum lycopersicum cv. Condine Red）为材料,通过接种Pst DC3000,明确STP基因家族对Pst DC3000的响应。构建SlSTP2的突变材料与过表达材料,并进行鉴定、繁种。在野生型和SlSTP2基因突变体、过表达植株上接种Pst DC3000,观察比较叶片发病情况、台盼蓝染色显示的死细胞数量,检测叶片菌落数（colony-forming units,CFU）和光系统II实际光化学效率,明确SlSTP2在植株防御细菌性叶斑病中的作用。为探究SlSTP2防御Pst DC3000的内在分子机制,通过双分子荧光互补（bimolecular fluorescent complimentary,BiFC）试验筛选互作蛋白,构建编码该蛋白的基因突变与过表达材料,并接种Pst DC3000明确其在植株防御细菌性叶斑病中的作用。【结果】在番茄植株上接种Pst DC3000后,SlSTP1和SlSTP2表达上调,选取上调最显著的SlSTP2作为后续研究对象,构建其突变材料与过表达材料。在番茄野生型和Slstp2突变植株、OE:SlSTP2过表达植株上接种Pst DC3000,相比野生型植株,Slstp2植株细菌性叶斑病的发病情况更严重,叶片CFU与台盼蓝染色显示出的死细胞数均更多,光系统II实际光化学效率下降,OE:SlSTP2植株则相反。通过BiFC试验发现,SlSTP2与G蛋白β亚基SlAGB1互作。接种Pst DC3000后,Slagb1突变体发病较野生型重,呈现与Slstp2突变体一致的发病表型;OE:SlAGB1则与OE:SlSTP2植株同样表现出更强的抗性。【结论】SlSTP2受Pst DC3000诱导,并正调控番茄对细菌性叶斑病的抗性,且SlSTP2与正调控因子SlAGB1互作,推测SlSTP2调控番茄细菌性叶斑病抗性与SlAGB1有关。

关键词: 番茄, 糖转运蛋白, SlSTP2, 细菌性叶斑病, 抗病性

Abstract:

【Background】In recent years, tomato diseases occur frequently in open field and protected cultivation due to the suboptimal environmental conditions. Tomato bacterial leaf spot caused by Pseudomonas syringae pv. tomato (Pst) is a common bacterial disease, which severely affects the yield and quality of tomato. Sugar is an important signaling molecule as well as nutrient substance in plants. When plants are subjected to pathogen attack, sugar not only functions as a signal to regulate the plant defense, but also provides energy for immune responses as a main carbon source. Sugar transport protein (STP) family is responsible for the sugar transport, and plays an important role in plant growth, development and defense. 【Objective】The objective of this study is to clarify whether STP family is involved in the regulation of plant defense against bacterial leaf spot. 【Method】CR (Solanum lycopersicum cv. Condine Red) was used as the wild-type (WT) background in this study. The responses of STP gene family to Pst DC3000 were determined by Pst DC3000 inoculation. The SlSTP2, which was up-regulated most significantly after Pst DC3000 inoculation, was selected for further investigation. The Slstp2 homozygous mutant and overexpression materials were generated. To explore the role of SlSTP2 in plant defense against bacterial leaf spot, WT and Slstp2 mutants, OE:SlSTP2 plants were inoculated with Pst DC3000, then the disease symptoms were assessed by disease-associated cell death and bacterial growth as well as the photochemical efficiency. In order to explore the potential mechanism of SlSTP2 in the disease resistance, the interacting proteins were screened by bimolecular fluorescence complementary (BiFC) assay. The mutant and overexpression materials of candidate interacting protein were generated and inoculated with Pst DC3000 to investigate its role in plant defense against bacterial leaf spot. 【Result】After inoculation with Pst DC3000 on tomato plants, the expressions of SlSTP1 and SlSTP2 were up-regulated. SlSTP2 was up-regulated most significantly, so it was selected for further investigation and its mutant and overexpression materials were generated. WT and Slstp2 mutants, OE:SlSTP2 plants were subjected to Pst DC3000 inoculation. Compared with WT plants, Slstp2 mutants showed significantly increased susceptibility, as evidenced by more severe disease symptoms, increased disease-associated cell death, an enhanced bacterial population and a decreased photochemical efficiency in the leaves. On the contrary, OE:SlSTP2 plants showed enhanced defense against Pst DC3000 compared with WT plants. It was further found that SlSTP2 interacted with the G protein β subunit SlAGB1 using BiFC assay. Similar to the Slstp2 mutants, the Slagb1 mutants also showed significantly increased susceptibility to Pst DC3000 compared with WT plants, and OE:SlAGB1 plants showed enhanced defense against Pst DC3000 as OE:SlSTP2 plants. 【Conclusion】SlSTP2 is significantly induced by Pst DC3000 inoculation, and positively regulates the defense against bacterial leaf spot in tomato. SlSTP2 interacts with SlAGB1, which also plays a positive role in defense against bacterial leaf spot, suggesting that SlSTP2 associated with SlAGB1 regulate tomato resistance to bacterial leaf spot.

Key words: tomato (Solanum lycopersicum), sugar transport protein (STP), SlSTP2, bacterial leaf spot, disease resistance

李依镁,王娇,王萍,师恺. 番茄糖转运蛋白SlSTP2在防御细菌性叶斑病中的功能[J]. 中国农业科学, 2022, 55(16): 3144-3154.

LI YiMei,WANG Jiao,WANG Ping,SHI Kai. Function of Sugar Transport Protein SlSTP2 in Tomato Defense Against Bacterial Leaf Spot[J]. Scientia Agricultura Sinica, 2022, 55(16): 3144-3154.

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https://www.chinaagrisci.com/CN/Y2022/V55/I16/3144

图/表 7

表1

本试验所用引物"

引物名称 Primer name	基因编号 Accession number	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)	用途 Amplification
CRISPR-SlSTP2	Solyc09g075820	GATTGTAACGGAGGCACGCATTTCG	AAACCGAAATGCGTGCCTCCGTTAC	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
CRISPR-SlAGB1	Solyc01g109560	GATTGTTGAGCTCGACTAGCAACT	AAACAGTTGCTAGTCGAGCTCAAC	CRISPR/Cas9载体构建 CRISPR/Cas9 vector construction
OE-SlSTP2	Solyc09g075820	AGGCGCGCCATGGCCGGTGGAGGATTTAC	ACGCGTCGACCAACCGAGAAGTGGGATCAT	OE载体构建 OE vector construction
OE-SlAGB1	Solyc01g109560	AGGCGCGCCATGTCAGTTGCGGAGCTGAAAG	GGGGTACCGATCACACTTCTGTGTCCTC	OE载体构建 OE vector construction
SlSTP2	Solyc09g075820	TATCAACTCTTTCATTACAT	CAGAAACTCCAACATCATAAC	CRISPR/Cas9植株鉴定CRISPR/Cas9 plant identification
SlAGB1	Solyc01g109560	GCTAATAGGGTGTCCCACACG	CGTATAGTTAGTGCATCCCAAG	CRISPR/Cas9植株鉴定CRISPR/Cas9 plant identification
RT-SlSTP1	Solyc02g079220	GGGGTGTGACATCAATGGAC	ATGCCACCAGAGAAGACACA	qRT-PCR
RT-SlSTP2	Solyc09g075820	ACCGGAGCAGTCAACGTTCT	ACCATGGCTAAGGTTGTCTGAATGA
RT-SlSTP3	Solyc07g006970	GGCTGGTTTAGTTGCATCTCTGG	GGAACTGCCTGGTTGCCAAA
RT-SlSTP4	Solyc04g074070	GGTGCATTCAGCACAGGCTT	TGTCACGGCTGGGACTATGG
RT-SlSTP5	Solyc01g010530	AGCAATCGGGGCAATTTTGT	GTACACACCAACACCACCAC
RT-SlSTP6	Solyc08g080300	GGAACAAGTGGCGATCCAGGAA	GGGACTAACCACCCTAGTGGAC
RT-SlSTP7	Solyc03g005140	GGCTCTTGTCGCGAGCTTT	CCGAAGCCAACACCAACACC
RT-SlSTP8	Solyc00g009030	TGCAATGGGAGGGTTGATCT	GGTCACTTTTCTGCCGTGTT
RT-SlSTP9	Solyc01g008240	GTGACGGAGCTGCGTTGATG	TGATGGAACCAACAGCAATCTGAC
RT-SlSTP10	Solyc03g006650	CTTGCCCGAGACGAAGAATG	GCTCTCCCCTTTGCCATTTC
RT-SlSTP11	Solyc06g054270	CAACGGGTGGAGGGTGTCA	TTGCCCTGTTCGTCTTTGCC
RT-SlSTP12	Solyc05g018230	AGTTGATGCTGAGTTTGCGG	GAGTTCATGCCGGTAAGCTG
RT-SlSTP13	Solyc03g005150	TTTGCTTCAAAGGCAGCCACT	CCGAATCCAATACCAACGCCAA
RT-SlSTP14	Solyc03g078600	ATGGCAGCAATGGGAGGGTT	AAGGGTGAGCAACTGGCTGT
RT-SlSTP15	Solyc03g093400	GGAGGAGCTAACTTCCTCGCT	ACGGTACAGCCTGATTGGCA
RT-SlSTP16	Solyc03g093410	CGTGATCTCCTTCTACGCTCCA	GCCTACCACACCTGTCACCA
RT-SlSTP17	Solyc03g094170	CCAGACCATAGGCCTAGGTG	ACATAGCCCTTCGTCTGACC
RT-SlSTP18	Solyc12g008320	TTTGGCTCTGGTGCTGCTCT	AGCTACCGCTACCATGACACA
RT-SlAGB1	Solyc01g109560	TGGGTATGCAAAGACGCAAG	CTGGCTTGTCAGAGCATTCC
SlSTP2-GFP	Solyc09g075820	CCGCTCGAGATGGCCGGTGGAGGATTTAC	TCCCCCGGGCAACCGAGAAGTGGGATCAT	亚细胞定位 Subcellular localization
SlSTP2-PacI-F SlSTP2-AscI-R	Solyc09g075820	CCCTTAATTAACATGGCCGGTGGAGGATTTAC	AAAGGCGCGCCCCAACCGAGAAGTGGGATCAT	双分子荧光互补载体构建 BiFC vector construction
SlAGB1-PacI-F SlAGB1-AscI-R	Solyc01g109560	CCCTTAATTAACATGTCAGTTGCGGAGCTGAAAG	AAAGGCGCGCCCGATCACACTTCTGTGTCCTC	双分子荧光互补载体构建 BiFC vector construction

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