Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (3): 619-628.doi: 10.3864/j.issn.0578-1752.2021.03.015

• HORTICULTURE • Previous Articles     Next Articles

CmWRKY15-1 Regulates Resistance of Chrysanthemum White Rust Through Salicylic Acid Signaling Pathway

BI MengMeng1(),LIU Di1,GAO Ge1,ZHU PengFang1,2,MAO HongYu1,2()   

  1. 1College of Forestry, Shenyang Agruicultural University, Shenyang 100866
    2Key Laboratory of Forest Tree Genetics, Breeding and Cultivation of Liaoning Province, Shenyang 100866
  • Received:2020-05-04 Accepted:2020-07-13 Online:2021-02-01 Published:2021-02-01
  • Contact: HongYu MAO E-mail:1262665213@qq.com;maohongyu@syau.edu.cn

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Abstract:

【Objective】Chrysanthemum White Rust is one of the most important diseases of chrysanthemum, which seriously affects its ornamental quality. This study would provide theoretical reference for the molecular mechanism of Chrysanthemum White Rust through the preliminary analysis of the function of CmWRKY15-1.【Method】In this study, the overexpression and interference vector was constructed based on CmWRKY15-1 gene sequence, which were transformed into resistant cultivar Huangying by Agrobacterium mediated method. The function of CmWRKY15-1 gene in response to Chrysanthemum White Rust in salicylic acid signal pathway was explored by phenotype observation, disease index statistics, changes of endogenous SA content in transgenic plants, expression analysis of key genes of SA synthesis, pathogenesis-related genes and defense enzyme genes. 【Result】The OE-9 and the RNAi-4 strains were obtained after transformation. The disease index of RNAi-4 strain was 53.67 in phenotype identification and disease index investigation, and the resistant response was susceptible, but the phenotype of wild type plants had no change. Meanwhile, the content of endogenous Salicylic Acid (SA) in OE-9 lines increased, compared with WT after inoculation, while in RNAi-4 lines decreased. The overexpression and silencing of CmWRKY15-1 significantly changed SA content, which had a positive regulatory effect on SA accumulation. The expression of SA synthesis genes in OE-9, RNAi-4 and WT showed that the expression of ICS1 in OE-9 upregulated first and then down-regulated, and the highest expression was 3.8 times as much as that in WT. Whereas, the expression level of ICS1 in RNAi-4 decreased after inoculation and remained at a lower level. The PAL expression indicated an increasing trend, and the highest expression was 2.6 times as much as that of the control. PAL expression in RNAi-4 was down-regulated, basically consistent with the change of SA content, which further confirmed that CmWRKY15-1 was a novel regulator mediated by SA signal pathway in respond to Chrysanthemum White Rust. In addition, the analysis of the expression of disease resistance related genes in response to SA signal showed that the expression of these genes peaked at 40 h in OE-9. However, the PR5 was relatively delayed and peaked at 48 h, and their expression in RNAi-4 was significantly lower than that in WT and OE-9 except PR5. The overexpression of CmWRKY15-1 increased the transcription level of PR gene in SA signaling pathway. On the contrary, the silencing of CmWRKY15-1 reduced the expression of these genes.【Conclusion】Therefore, CmWRKY15-1 had a positive regulatory effect on resistance of Chrysanthemum White Rust, and it might respond to Chrysanthemum White Rust through the regulation of SA signal pathway.

Key words: chrysanthemum, CmWRKY15-1 gene, Chrysanthemum White Rust, SA, functional verification

Table 1

Primers used for cloning"

引物Primer 序列Sequence（5′-3′）
Npt Ⅱ F GGCTATGACTGGGCACAACA
Npt Ⅱ R GATACCGTAAAGCACGAGGAA
Actin-F TCCGTTGCCCTGAGGTTCT
Actin-R GATTTCCTTGCTCATCCTGTCA
CmWRKY15-1-F TGGTGGCTGCATCACAT
CmWRKY15-1-R GGAAGAATCAGTGCTAATACATTAA

Table 2

Primers used for cloning"

引物 Primer 序列 Sequence（5′-3′）
ICS1-F TCCCTACTGAAGAGGCACGG
ICS1-R CCAACAGCGGGTTCACTCTC
PAL-F ATGGCACCGAAGCAAGTCACAC
PAL-R GATACCCGAGTAACCCTGGAGGAG
NPR1-F TGTCGAGAAGGATGGAAAGCC
NPR1-R GGAGGCACCCATCATCAACA
PR1-F CTCAACCAAAAGGAATAGTCGG
PR1-R CCCTGCCAGTTTACGCTGTA
PR2-F GGCAATGGTGGTGTTGGAAC
PR2-R CTTCCTCCGTCAGCAGAAGG
PR5-F CCAATGGAGTTTAGCCCCGT
PR5-R GTCCACAACTACCACGCTCA

Fig. 1

PCR identification of potential transgenic plants a: PCR of pBI121-CmWRKY15-1; 1-18: Potential transgenic plants of pBI121-CmWRKY15-1. b: PCR of RNAi-CmWRKY15-1; 1-19: Potential transgenic plants of RNAi-CmWRKY15-1. M: DNA marker; W: Negative control; P: Positive control. The same as below"

Fig. 2

Semi quantitative detection of CmWRKY15-1 gene expression level"

Fig. 3

Relative expression level of CmWRKY15-1 in transgenic lines and WT Different small letters show significant difference (P<0.05). The same as below"

Fig. 4

Phenotypic differences of plant lines RNAi-4 after inoculated R1 and R2: The third leaves after inoculation in RNAi-4 (leaf surface and back); W1 and W2: The third leaves after inoculation in WT (leaf surface and back)"

Fig. 5

SA standard curve of plants WT: Wild-type plants; OE-9: Overexpression plants; RNAi-4: Silence plants. Different small letters show significant difference (P<0.05). The same as below"

Fig. 6

Relative expression levels of ICS1 and PAL"

Fig. 7

Relative expression levels of NPR1, PR1, PR2 and PR5"

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