Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (24): 5220-5229.doi: 10.3864/j.issn.0578-1752.2021.24.005

• PLANT PROTECTION • Previous Articles     Next Articles

The Resistance Mechanism of Apple Transcription Factor MdWRKY40b to Powdery Mildew

SHA RenHe1(),LAN LiMing1,WANG SanHong1,*(),LUO ChangGuo2,*()   

  1. 1College of Horticulture, Nanjing Agricultural University, Nanjing 210095
    2Institute of Fruits Sciences, Guizhou Academy of Agricultural Sciences, Guiyang 550006
  • Received:2021-04-19 Accepted:2021-05-31 Online:2021-12-16 Published:2021-12-28
  • Contact: SanHong WANG,ChangGuo LUO E-mail:1091595079@qq.com;wsh3xg@njau.edu.cn;376258195@qq.com

Abstract:

【Objective】The research aims to explore the mechanism of apple resistance to powdery mildew regulated by transcription factor gene MdWRKY40b, and to provide a theoretical basis for apple powdery mildew resistance breeding. 【Method】The 552 bp specific fragment of MdWRKY40b was cloned using the cDNA obtained from the ‘Gala’ leaf tissue, the forward and reverse sequence of this gene specific fragment was constructed into the RNAi expression vector pB7GWIWG2(Ⅱ) by the Gateway technique, and then transgenic plants were obtained by an Agrobacterium-mediated method. Notably, the young leaves at the stem tips of tissue culture seedlings were chosen as the materials for Agrobacterium-mediated infection experiments. The expression levels of MdWRKY40b, superoxide dismutase gene (SOD), catalase gene (CAT), peroxidase gene (POD), and β-1,3-glucanase in transgenic plants were analyzed by RT-qPCR technology. Furthermore, using one-year-old plants as materials, the resistance of transgenic plants to powdery mildew was analyzed through the inoculation test of the powdery mildew pathogen (Podosphaera leucotricha). The leaves of transgenic and control plants at 0, 5, 10, 15, and 20 d after inoculation with P. leucotricha were used as materials, and the accumulation of superoxide anion and hydrogen peroxide in plant materials during P. leucotricha infection was analyzed by staining with nitrotetrazolium chloride solution (NBT) and diaminobenzidine (DAB). Furthermore, the activities of SOD, POD, CAT, and β-1,3-glucanase in plants were determined during P. leucotricha infection. 【Result】Three MdWRKY40b gene silencing lines were identified by PCR detection. The RT-qPCR results showed that the gene silencing efficiency of RNAi-1, RNAi-2, and RNAi-3 was 95.2%, 92.2%, and 79.8%, respectively. The expression level of SOD, CAT, and β-1,3-glucanase in transgenic plants was significantly up-regulated compared with the wild-type. Under the condition of P. leucotricha infection, compared with wild-type plants, the area of powdery disease spots in the leaves of transgenic plants decreased significantly, the accumulation of superoxide anion and hydrogen peroxide was reduced significantly, and the activities of antioxidant enzymes such as SOD, CAT, and POD regulating superoxide anion metabolism and β-1,3-glucanase related to disease resistance were significantly enhanced. 【Conclusion】The silencing of MdWRKY40b enhances the resistance of apple plants to powdery mildew. It is speculated that the up-regulated expression level of SOD, CAT, and β-1,3-glucanase in transgenic plants improves the basic resistance of apple plants to powdery mildew, and increases the scavenging ability of plants to superoxide anions. It can maintain a low concentration of superoxide anion content in response to P. leucotricha infection, so as to reduce the damage of high concentrations of superoxide anion to plants.

Key words: apple, powdery mildew, MdWRKY40b, transgene, reactive oxygen species (ROS), antioxidant enzyme

Table 1

Primers used for cloning of MdWRKY40b and expression vector construction"

引物名称
Primer name
引物序列
Primer sequence
MdWRKY40b F: 5′-CACCAGCGAAAGGCAGACAGTGAAG-3′
R: 5′-CGTCGGAGCTTGAGTTTCTGG-3′
LOCUS1 F: 5′-GTGATTTTTGCGGACTCTAGCAT-3′
R: 5′-CAGGTCAGCTTGACACTGAACAT-3′
LOCUS2 F: 5′-CCATGAGGTTGCTTCTGATCC-3′
R: 5′-CAACATTACAATTTACTATTCTAGT-3′
Bar
F: 5′-CCGTTCTTCCGAATAGCA-3′
R: 5′-TCATACATGAGAATTAAGGGAG-3′

Table 2

Primers used for RT-qPCR"

引物名称
Primer name
引物序列
Primer sequence
Ubiquitin F: 5′-AAATCCAGGACAAGGAGGGC-3′
R: 5′-CACCACGGAGACGCAACAC-3′
MdWRKY40b
F: 5′-TGCTCAACTCCAAGGCAATG-3′
R: 5′-TTCACTGTCTGCCTTTCGCT-3′
CAT
F: 5′-GCAGCCAGTTGGACGTTTG-3′
R: 5′-CTGCAGATAGTTCGGGCCAA-3′
SOD
F: 5′-GTTCGATCCATCAAACGCCG-3′
R: 5′-ATGAAGTCCAGGCTTGAGGC-3′
POD
F: 5′-TGAAGAAACTGCTGGGGGTC-3′
R: 5′-AACGTCCAAATTCGCATTGAT-3′
β-1,3-glucanase F: 5′-GGGGTGAGGCTATTGGTGAG-3′
R: 5′-TCTGCCCCATCCCTTATTGC-3′

Fig. 1

Cloning and expression vector construction of MdWRKY40b"

Fig. 2

The identification and phenotype of transgenic plants"

Fig. 3

Expression analysis of antioxidant enzyme genes in transgenic lines"

Fig. 4

The phenotype observation after inoculated with P. leucotricha at 25 d"

Fig. 5

The observation of the leaf lesion area inoculated with P. leucotricha at 25 d"

Fig. 6

The leaf O2- accumulation after P. leucotricha inoculation"

Fig. 7

The leaf hydrogen peroxide accumulation after P. leucotricha inoculation"

Fig. 8

The quantitative analysis of leaf resistant enzyme activity after P. leucotricha inoculation"

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