Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (16): 3144-3154.doi: 10.3864/j.issn.0578-1752.2022.16.007

• PLANT PROTECTION • Previous Articles     Next Articles

Function of Sugar Transport Protein SlSTP2 in Tomato Defense Against Bacterial Leaf Spot

LI YiMei1(),WANG Jiao1(),WANG Ping1,SHI Kai1,2()   

  1. 1College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058
    2Hainan Institute of Zhejiang University, Sanya 572025, Hainan
  • Received:2022-02-22 Accepted:2022-03-19 Online:2022-08-16 Published:2022-08-11
  • Contact: Kai SHI E-mail:liyimei@zju.edu.cn;11616046@zju.edu.cn;kaishi@zju.edu.cn

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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

Table 1

Primers used in this study"

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
OE-SlSTP2 Solyc09g075820 AGGCGCGCCATGGCCGGTGGAGGATTTAC ACGCGTCGACCAACCGAGAAGTGGGATCAT OE载体构建
OE vector construction
OE-SlAGB1 Solyc01g109560 AGGCGCGCCATGTCAGTTGCGGAGCTGAAAG GGGGTACCGATCACACTTCTGTGTCCTC
SlSTP2 Solyc09g075820 TATCAACTCTTTCATTACAT CAGAAACTCCAACATCATAAC CRISPR/Cas9植株鉴定CRISPR/Cas9 plant identification
SlAGB1 Solyc01g109560 GCTAATAGGGTGTCCCACACG CGTATAGTTAGTGCATCCCAAG
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

Fig. 1

Phylogenetic tree of tomato and A. thaliana STP family"

Fig. 2

SlSTP1 and SlSTP2 in tomato STP gene family were induced by Pst DC3000"

Fig. 3

SlSTP2 contains 12 transmembrane domains and is located on the plasma membrane Subcellular localization of SlSTP2, Scale bar=25 μm"

Fig. 4

SlSTP2 positively regulates the defense against bacterial leaf spot in tomato"

Fig. 5

Bimolecular fluorescent complimentary experiment shows that SlSTP2 interacts with SlAGB1 SlSTP2-cYFP interacts with SlAGB1-nYFP. Scale bar=25 μm"

Fig. 6

SlAGB1 positively regulates the defense against bacterial leaf spot in tomato"

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