Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (16): 3110-3223.doi: 10.3864/j.issn.0578-1752.2023.16.006

• PLANT PROTECTION • Previous Articles     Next Articles

Mechanism of StLAC2 and StLAC6 Differentially Affecting Setosphaeria turcica Based on Non-Targeted Metabonomics Analysis

ZOU JinPeng1,2(), YUE HaoFeng2, LI HaiXiao1,2, LIU Zheng3, LIU Ning1,2(), CAO ZhiYan1,2(), DONG JinGao1,2   

  1. 1 College of Plant Protection, Hebei Agricultural University, Baoding 071001, Hebei
    2 Key Laboratory of Hebei Province for Plant Physiology and Molecular Pathology/State Key Laboratory of North China Crop Improvement and Regulation, Baoding 071001, Hebei
    3 Academy of Educational Sciences of Baoding, Baoding 071066, Hebei
  • Received:2023-05-11 Accepted:2023-06-06 Online:2023-08-16 Published:2023-08-18

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Abstract:

【Background】As a polyphenol oxidase, laccase plays an important role in fungal growth, development and secondary metabolism. A plurality of laccase genes are encoded in the genome of Setosphaeria turcica, among which StLAC2 and StLAC6 have differential effects on the growth, development, and pathogenicity of S. turcica.【Objective】To clarify the differential mechanisms of StLAC2 and StLAC6 on S. turcica and explore new targets for developing new fungicides and disease control strategies by mining differential metabolites.【Method】StLAC6 was connected with pHZ100-GFP plasmid by seamless cloning, and the complementary expression vector of StLAC6 was constructed. Using PEG-mediated protoplast transformation method, the constructed vector was transferred into the protoplast of StLAC6 gene knockout mutant, and the positive transformants were identified by PCR, RT-qPCR and GFP fluorescence verification, and the StLAC6 revertant strain was successfully constructed. The effects of knocking out and reverting StLAC2 and StLAC6 on melanin synthesis and oxidation resistance in and out of S. turcica were analyzed. Taking wild-type (WT), StLAC2 and StLAC6 gene knockout mutants as experimental materials, the differential metabolites were analyzed by non-targeted metabonomics, and the mechanism of the differential action of StLAC2 and StLAC6 was analyzed by KEGG.【Result】StLAC2 and StLAC6 have differential effects on melanin synthesis in mycelium and secreted into culture medium, and StLAC2 also affects antioxidant activity of S. turcica. Metabolomic analysis found that compared with the WT strain of S. turcica, there were more differential metabolites in the mycelium or secreted into the culture medium after knocking out StLAC2, and KEGG analysis showed that the differential metabolites were mainly lipids, especially phospholipids. Meanwhile, the absence of the StLAC2 caused down-regulation of various flavonoids and polyphenols. The contents of intermediates of the 1, 8-dihydroxynaphthalene melanin biosynthesis pathway, scytalone and vermelone, significantly increased in ΔStLAC2 and decreased in ΔStLAC6.【Conclusion】The StLAC2 participates in melanin polymerization, the StLAC6 negatively regulates melanin biosynthesis in S. turcica, and the differential effects of StLAC2 and StLAC6 affect lipid metabolism and intermediates of the melanin biosynthesis pathway in S. turcica. The absence of StLAC2 caused down-regulation of various flavonoids and polyphenols, leading to decreased antioxidant activity.

Key words: Setosphaeria turcica, laccase, metabonomics, differential metabolite, melanin

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
StLAC6-F CCACCGCGGTGGCGGCCGCTCTAGAATGGTCTTTTCCATCTCAAG
StLAC6-R CGCCCTTGCTCACCCTATCGAATTCCCGACGCAGTCCAGAGTCGC
GFP-F GCGGCCGCCATGAGTAAAGGAGAAGAACT
GFP-R GAATTCTGTATAGTTCATCCATGCCA
RT-StLAC6-F TTACCGACGCAGTCCAGAGTCGC
RT-StLAC6-R ATGGTCTTTTCCATCTCAAGGGTAG
β-tubulin-F GCGTTTCCCTGGTCAGCTTA
β-tubulin-R GGGAAGGGCACCATGTTG

Fig. 1

PCR, RT-qPCR, and fluorescence verification of the transformant"

Fig. 2

Colony morphology of different strains"

Fig. 3

The melanin content of WT, ΔStLAC2 and ΔStLAC6"

Fig. 4

Determination of antioxidant activity of WT and mutant strains"

Fig. 5

Sample principal component analysis"

Fig. 6

Volcanic diagram of differential metabolites of each strain"

Fig. 7

Venn diagram of metabolite group"

Fig. 8

KEGG pathway enrichment of the differential metabolites"

Fig. 9

KEGG compound classification of differential metabolites affected by StLAC2 and StLAC6"

Table 2

Main lipid differential metabolites"

分类
Classification		 化合物
Compound		 ΔStLAC2 vs WT ΔStLAC6 vs WT
FC P value FC P value
二酰甘油
Diacylglycerol		 CDP-DG (16:0/18:1(11Z)) 0.9652 1.59E-05 1.6565 6.37E-05
CDP-DG (16:0/18:2(9Z,12Z)) 0.9751 3.32E-05 1.8412 8.23E-05
CDP-DG (18:1(11Z)/18:1(11Z)) 0.8606 4.48E-03 1.9461 4.49E-02
鞘磷脂
Sphingolipid		 C17 sphingosine-1-phosphocholine 1.5874 1.64E-05 1.0236 8.45E-02
Sphingosine 1-phosphate 1.5328 3.53E-06 0.9084 9.29E-06
磷脂酰胆碱
Phosphatidyl cholines
(PC and LysoPC)		 PC (17:1/0:0) 2.6534 0.000814 1.0452 4.66E-03
PC (18:0/0:0) 1.9638 3.42E-03 1.0620 0.000339
PC (16:1(9Z)/20:4(8Z,11Z,14Z,17Z)) 1.1043 5.33E-06 0.2862 8.27E-05
PC (14:0/22:5(7Z,10Z,13Z,16Z,19Z)) 0.9632 1.52E-05 0.3511 2.23E-05
磷脂酰甘油
Phosphatidyl glycerol (PG)		 PG (16:0/0:0)[U] 2.4214 4.96E-05 1.1026 1.45E-03
PG (18:0/0:0)[U] 1.5639 3.61E-02 1.0628 0.000153
PG (18:1(9Z)/0:0) 1.8132 0.000566 1.1901 1.45E-03

Fig. 10

Box diagram of flavonoids metabolites"

Fig. 11

Box diagram of intermediate metabolites of melanin synthesis"

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