In Vitro Antifungal Activity of Dihydrochelerythrine and Proteomic Analysis in Ustilaginoidea virens

doi:10.1016/j.rsci.2023.03.009

摘要/Abstract

. [J]. Rice Science, 2023, 30(3): 257-266.

图/表 8

Table 1. Growth inhibitory effect of dihydrochelerythrine (DHCHE) on five species of rice pathogenic fungi. %

Pathogen	DHCHE concentration (mg/L)
Pathogen	5.0	7.5	10.0
Ustilaginoidea virens	56.3 ± 1.2 a	68.8 ± 1.5 b	75.0 ± 1.0 c
Aspergillus ruber	6.3 ± 1.3 a	20.3 ± 2.2 b	30.8 ± 1.7 c
Magnaporthe oryzae	31.7 ± 1.5 a	44.3 ± 2.0 b	59.5 ± 1.0 c
Nigrospora oryzae	68.3 ± 2.3 a	81.3 ± 1.1 b	84.2 ± 1.2 c
Cochliobolus miyabeanus	36.5 ± 1.0 a	50.0 ± 1.3 b	66.2 ± 1.7 c

Fig. 1. Inhibition of mycelium growth of Ustilaginoidea virens by dihydrochelerythrine (DHCHE) and other isoquinoline alkaloids. 1, DHCHE; 2, Chelerythrine chloride; 3, Dihydrosanguinarine; 4, Sanguinarine hydrochloride; 5, 6-methoxydihydrosanguinarine; 6, Validamycin (control).The drug media were used at the concentration of 7.5 mg/L. Values are Mean ± SD (n = 3). *, P < 0.05; **, P < 0.01.

Fig. 2. Transmission electron micrographs of Ustilaginoidea virens spores. A and B, Control group. C and D, Group treated with 15.0 mg/L dihydrochelerythrine. CW, Cell wall; L, Lipid globule; N, Nucleus; NO, Nucleolus; IO, Incomplete organelle; MNM, Malformed nuclear membrane. Scale bars are 1 μm in A and C, and 500 nm in B and D.

Fig. 3. Effects of dihydrochelerythrine (DHCHE) on reactive oxygen species accumulation. A, Control group; B, 5.0 mg/L DHCHE treatment; C, 7.5 mg/L DHCHE treatment; D, 15.0 mg/L DHCHE treatment. P1 and P2 are groups divided according to the intensity of fluorescence signal, and FITC is fluorescence intensity.

Fig. 4. Effects of dihydrochelerythrine (DHCHE) on mitochondrial membrane potential of Ustilaginoidea virens. A, Positive control; B, 5.0 mg/L DHCHE treatment; C, 7.5 mg/L DHCHE treatment; D, 15.0 mg/L DHCHE treatment. Phylum P2 represents cells with high mitochondrial membrane potential (normal cells), and phylum P3 represents a population with low mitochondrial membrane potential (damaged or apoptotic cells). FITC is fluorescence intensity. With the increase of drug concentration, the proportion of cells in P3 phylum increased significantly, that is, the proportion of cell damage increased.

Fig. 5. Gene Ontology (GO) enrichment analysis of differentially expressed proteins in control and dihydrochelerythrine treatment groups (top 20 terms). A?C, Top 20 GO terms in biological process (A), cellular component (B) and molecular function (C).The numbers after the columns and in the parentheses refer to the number of proteins enriched in the corresponding functional level of the GO concentration and the corresponding P values, respectively.

Fig. 6. Enrichment items of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway between control and dihydrochelerythrine treatment groups. A, Differentially expressed proteins (DEPs) in the significantly enriched KEGG pathway. The ordinate on the left shows the specific metabolic pathway enriched. The ordinate on the right shows the abbreviation of the first and second classifications of the metabolic pathway enriched. Green nodes indicate species-specific. Nodes with red boxes indicate up-regulation, while nodes with blue boxes indicate down-regulation. The metabolism is mainly enriched in two different pathways: (1) Metabolism includes one metabolism, namely energy metabolism, and the specific pathway is oxidative phosphorylation; (2) Genetic information processing includes two metabolic pathways, which are folding, sorting and degradation as well as replication and repair. The specific pathway enriched in folding, sorting and degradation is ubiquitin mediated proteolysis, and the replication and repair pathway is concentrated in mismatch repair.B, Pie chart of DEPs. The KEGG enrichment results were sorted according to the P value, and the enriched protein amount in the top 20 items with the smallest P value was counted. Among them, oxidative phosphorylation and mismatch repair accounted for 27%, ubiquitin mediated proteolysis accounted for 28%, and regulation of mitophagy-yeast accounted for 18%.

Fig. 7. Average fold change ratios in expression of proteins in Ustilaginoidea virens cells after dihydrochelerythrine (DHCHE) treatment. A, Protein expression in DHCHE-treated U. virens cells by tandem mass tag (TMT)-based quantitative proteomics assay. B, Protein expression in DHCHE-treated U. virens cells by parallel reaction monitoring (PRM) assay. U. virens in the treatment groups was cultured in potato sucrose medium at 28 oC for 10 d, followed by incubation with 7.5 mg/L of DHCHE for 24 h. No DHCHE was added to the control (CK) groups. Values are Mean ± SD (n = 3). **, P < 0.01.

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