Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (21): 4238-4247.doi: 10.3864/j.issn.0578-1752.2024.21.006

• PLANT PROTECTION • Previous Articles     Next Articles

Functional Analysis of SlSnRK1.2 in Regulating Tomato Resistance to Grey Mould

LI Jie(), LIANG ZhiLin(), SUN Yan, TAN GenJia(), HUAI BaoYu()   

  1. College of Plant Protection, Anhui Agricultural University/Anhui Province Key Laboratory of Crop Integrated Pest Management, Hefei 230036
  • Received:2024-06-27 Accepted:2024-08-06 Online:2024-11-01 Published:2024-11-10
  • Contact: TAN GenJia, HUAI BaoYu

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Abstract:

【Background】Grey mould, caused by Botrytis cinerea, poses serious threats to tomato production. Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) from plant is involved in the regulation of responses to biotic and abiotic stresses. However, whether tomato SnRK1 is involved in tomato resistance to grey mould remains unclear. 【Objective】In this study, SlSnRK1.2, which was up-regulated in the process of B. cinerea infecting tomato, was used as the research object to clone and analyze its function of regulating grey mould resistance, so as to provide theoretical basis and gene resources for the prevention and control of tomato grey mould. 【Method】The expression patterns of SlSnRK1.2 during the infection stage of B. cinerea and in different tomato tissues were examined through real-time fluorescence quantitative PCR (qRT-PCR); Subcellular localization of SlSnRK1.2 was analyzed using Agrobacterium-mediated transient transformation system; SlSnRK1.2 silencing plants were constructed by tobacco rattle virus (TRV)-mediated gene silencing (VIGS) technology, and the role of SlSnRK1.2 in the interaction between tomato and B. cinerea was preliminarily analyzed. The overexpression plants of SlSnRK1.2 were created by Agrobacterium-mediated tomato genetic transformation system, and the role of SlSnRK1.2 in regulating tomato resistance to grey mould was further clarified. NbSnRK1.2, a homologous gene of SlSnRK1.2, was silenced in N. benthamiana using TRV-mediated gene silencing technology to determine the function of NbSnRK1.2 during the interaction between N. benthamiana and B. cinerea. 【Result】Micro-Tom was used as the wild-type (WT) background, qRT-PCR technology was used to clarify that the transcriptional expression of SlSnRK1.2 was significantly induced by B. cinerea infection. Subcellular localization analysis revealed that SlSnRK1.2 was localized in the cytoplasm and nucleus. qRT-PCR analysis showed that SlSnRK1.2 was expressed in roots, stems, young leaves, mature leaves, flower buds, and flowers of tomato, with the highest relative expression level in the stems. Transient silencing of SlSnRK1.2 attenuated tomato resistance to grey mould, while overexpression of SlSnRK1.2 enhanced tomato resistance to grey mould. On this basis, transient silencing of NbSnRK1.2, a SlSnRK1.2 homologous gene, attenuated tobacco resistance to grey mould. 【Conclusion】SlSnRK1.2 positively regulates tomato resistance to grey mould and can be used as a genetic resource for molecular breeding of tomato resistance to grey mould.

Key words: grey mould of tomato (Solanum lycopersicum), Botrytis cinerea, SlSnRK1.2, disease resistance, virus induced gene silencing (VIGS)

Table 1

Primers used in this study"

引物Primer 序列Sequence (5′-3′) 用途Application
SlSnRK1.2-RT-F GGAAACATTATGCGAGAT 实时荧光定量PCR
qRT-PCR
SlSnRK1.2-RT-R AGGTGACTTGGAAGGGTGTAG
SlActin-RT-F TCTTTCCAATCATTGTGGTGCCTCC
SlActin-RT-R GAGCCTCCAATCCAGACAC
BcActin-RT-F TCCAAGCGTGGTATTCTTACCC
BcActin-RT-R TGGTGCTACACGAAGTTCGTTG
SlSnRK1.2-VIGS-F TAAGGTTACCGAATTCATGAGTTCCAGAGGTGGTGG 基因沉默
VIGS
SlSnRK1.2-VIGS-R GCTCGGTACCGGATCCCCCAGACTTGACATACTCCA
SlSnRK1.2-GFP-F GGGACTCTAGAGGATCCATGAGTTCCAGAGGTGGTGG 亚细胞定位
Subcellular localization
SlSnRK1.2-GFP-R GCTCGAATTCGGATCCTCTTGTGGCCCCTCTAGCTGCC
OE-SlSnRK1.2-F ACACGGGGGACTTTGCAACATGAGTTCCAGAGGTGGTG 番茄过表达载体构建
Overexpression vector construction of tomato
OE-SlSnRK1.2-R CCTCGCCCTTCACGATACATTGTGGCCCCTCTAGCTGC
NbSnRK1.2-VIGS-F TAAGGTTACCGAATTCCCATGGATCCTTTGGTAAAG 基因沉默
VIGS
NbSnRK1.2-VIGS-R GCTCGGTACCGGATCCATCTGCTGGAAAAAATGGCG
NbActin-F ACCAGATTAATGAGCCCAAGAG 实时荧光定量PCR
qRT-PCR
NbActin-R CCAACAGGGACAGTACCAATAC
NbSnRK1.2-RT-F TTACAGCCACAAACAGCTGTG
NbSnRK1.2-RT-R TCTTTCCAATCATTGTGGTGCCTCC

Fig. 1

Relative expression level of SlSnRK1.2 during B. cinerea infection in tomato leaves"

Fig. 2

Subcellular localization (A) and tissue-specific expression analysis (B) of SlSnRK1.2 The statistical analyses were performed using Student’s t-test, different lowercases on the bars indicated significant differences among treatments (P<0.05)"

Fig. 3

Silencing of SlSnRK1.2 reduced the resistance of tomato against grey mould"

Fig. 4

Overexpression of SlSnRK1.2 enhanced the resistance of tomato against grey mould"

Fig. 5

Silencing of NbSnRK1.2 reduced the resistance of tobacco against grey mould"

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