Anthocyanins from Malus spp. inhibit the activity of Gymnosporangium yamadae by downregulating the expression of WSC, RLM1, and PMA1

Abstract

Malus plants are frequently devastated by the apple rust caused by Gymnosporangium yamadae Miyabe. When rust occurs, most Malus spp. and cultivars produce yellow spots, which are more severe, whereas a few cultivars accumulate anthocyanins around rust spots, forming red spots that inhibit the expansion of the affected area and might confer rust resistance. Inoculation experiments showed that Malus spp. with red spots had a significantly lower rust severity. Compared with M. micromalus, M. 'Profusion', with red spots, accumulated more anthocyanins. Anthocyanins exhibited concentration-dependent antifungal activity against G. yamadae by inhibiting teliospores germination. Morphological observations and the leakage of teliospores intracellular contents evidenced that anthocyanins destroyed cell integrity. Transcriptome data of anthocyanins-treated teliospores showed that differentially expressed genes were enriched in cell wall and membrane metabolism-related pathways. Obvious cell atrophy in periodical cells and aeciospores was observed at the rust spots of M. 'Profusion'. Moreover, WSC, RLM1, and PMA1 in the cell wall and membrane metabolic pathways were progressively downregulated with increasing anthocyanins content, both in the in vitro treatment and in Malus spp. Our results suggest that anthocyanins play an anti-rust role by downregulating the expression of WSC, RLM1, and PMA1 to destroy the cell integrity of G. yamadae.

Keywords: Gymnosporangium yamadae; anthocyanins; cell membrane; cell wall; teliospores; transcriptome.

Figure 1

Rust infection severity in two Malus spp. (A) Malus ‘Profusion’. (B) Malus micromalus. RIT, rust-infected leaf tissue; UIT, uninfected leaf tissue. Scale bars: 2 cm.

Figure 2

Anthocyanins inhibit the germination of G. yamadae teliospores. (A) Teliospores treated with different concentrations of anthocyanins, BM: basidium. (B) Cumulative germination rate of teliospores. Each value is the mean of three replicates. The vertical bar indicates the standard error. a–e Statistically significant differences between control and treatments. (C) Cell breakage rate of teliospores. Each value is the mean of three replicates. The vertical bar indicates the standard error. a–e Statistically significant differences between control and treatments. (D) Morphological effects of anthocyanins on teliospores. Asterisks indicate broken spores after anthocyanins treatment. Scale bars: (A) 60 μm; (D) 25 μm.

Figure 3

The effects of anthocyanins on G. yamadae cell integrity. (A) The extracellular AKP activities. (B) Electrical conductivity. (C) nucleic acids content. (D) protein concentration. (E) FDA staining assay. (F) PI staining assay. Data are mean ± standard error of three replicate samples. Vertical bars represent standard errors of the means. Scale bars: (E,F) 60 μm.

Figure 4

GO and KEGG analyses of DEGs between anthocyanin-treated and control groups. (A) Venn diagram showing the overlap of expressed genes. Pink represents anthocyanin treatment, green represents control. (B) Volcano plot of DEGs. Splashes represent different genes, where gray indicates the mean number of genes without significant differential expression, red indicates the mean of significantly upregulated genes, and blue indicates the mean of significantly downregulated genes. (C) GO enrichment analysis of DEGs. (D) KEGG enrichment analysis of DEGs.

Figure 5

Observation of leaf tissues infected with rust. (A) Phenotypes of leaf tissues of M. ‘Profusion’ and M. micromalus infected by apple rust. I, II, and III represent the rust infection development stages. (B) Sections of disease-infected tissue. PY, pycnium; AE; aecidium; AES; aeciospores. (C) Morphological differences between peridial cells in different colored rust spots. (D) Morphological differences between aeciospore cells in different colored rust spots. Scale bars: (A) 1 cm; (B) 1.5 mm (I-II), 6 mm (III); (C) 20 μm; (D) 30 μm.

Figure 6

Changes in gene expression in M. ‘Profusion’ and M. micromalus leaves during rust infection. Each PCR reaction was carried out in triplicate and repeated thrice. Columns and bars represent the means and standard errors (n = 3), respectively.

Figure 7

Correlations among anthocyanins and essential genes in three pathways of the cell wall and membrane metabolism, oxidoreductase activity, and antibiotic biosynthesis at rust infection development stages of RITs in M. ‘Profusion’. All data are representative of at least three biological replicate.