Ethylene Promotes Expression of the Appressorium- and Pathogenicity-Related Genes via GPCR- and MAPK-Dependent Manners in Colletotrichum gloeosporioides

Abstract

Ethylene (ET) represents a signal that can be sensed by plant pathogenic fungi to accelerate their spore germination and subsequent infection. However, the molecular mechanisms of responses to ET in fungi remain largely unclear. In this study, Colletotrichum gloeosporioides was investigated via transcriptomic analysis to reveal the genes that account for the ET-regulated fungal development and virulence. The results showed that ET promoted genes encoding for fungal melanin biosynthesis enzymes, extracellular hydrolases, and appressorium-associated structure proteins at 4 h after treatment. When the germination lasted until 24 h, ET induced multiple appressoria from every single spore, but downregulated most of the genes. Loss of selected ET responsive genes encoding for scytalone dehydratase (CgSCD1) and cerato-platanin virulence protein (CgCP1) were unable to alter ET sensitivity of C. gloeosporioides in vitro but attenuated the influence of ET on pathogenicity. Knockout of the G-protein-coupled receptors CgGPCR3-1/2 and the MAPK signaling pathway components CgMK1 and CgSte11 resulted in reduced ET sensitivity. Taken together, this study in C. gloeosporioides reports that ET can cause transcription changes in a large set of genes, which are mainly responsible for appressorium development and virulence expression, and these processes are dependent on the GPCR and MAPK pathways.

Keywords: GPCRs; MAPK; anthracnose; appressorium; ethylene; postharvest disease.

Figure 1

The phenotypic responses of C. gloeosporioides to ET during the in vitro culture and host infection processes. (A) Microscopic analysis of control (CK) and ET-treated samples. (B) The percentage of appressorium formation on glass in the CK/ET treatment groups based on calculating three replicates of 300 conidia for each sample. (C) Light and fluorescent microscopic visualization of Cg-gfp spores germinating and infecting Arabidopsis leaves; the DAB panel indicates the inoculation sites after DAB staining.

Figure 2

Macroscopic analysis of the transcriptome of C. gloeosporioides regulated by ET. (A) Heatmap analysis of differential genes between the CK and ET treatment groups at 4 hpi and 24 hpi. (B) UpSetR visualizations of intersections between the upregulated and downregulated differential genes of the CK/ET treatment groups at 4 hpi and 24 hpi. (C) Principal component analysis of the CK-4hpi, ET-4hpi, CK-24hpi, and ET-24hpi transcriptome data. (D) Correlation of gene expression values obtained by RNA-seq and qRT-PCR analysis with 27 genes, and an R² value of 0.9089 was obtained by comparing the results obtained with the two techniques.

Figure 3

Summary of Gene Ontology (GO) terms enriched with the differentially expressed genes (DEGs) regulated by ET at 4 hpi and 24 hpi. (A) GOCircle showing the DEGs of the top 10 upregulated GO terms at 4 hpi. (B) GOCircle displaying the DEGs in the top 10 downregulated GO terms at 4 hpi. (C), GOCircle showing the DEGs of the top 10 GO terms at 24 hpi.

Figure 4

ET can significantly promote the expression of hydrophobic-surface-binding protein A genes and cutinase-related genes. (A) ET can significantly promote the upregulation of genes encoding for hydrophobic-surface-binding protein A (HsbA). (B) ET can significantly upregulate the expression levels of cutinase and its transcription factor (CTF1α and CTF1β) genes. (C) In contrast to the WT and Cg-gfp control strains, ET can induce the expression of GFP driven by the promoters of the two HsbA genes in the P_{CGLO_03844}-gfp and P_{CGLO_13252}-gfp transgenic strains at 24 hpi after incubation on glass slides.

Figure 5

ET can significantly promote the expression of melanin synthesis genes, chitin deacetylase, and apoptosis-related genes. (A) Ethylene can significantly promote the upregulation of genes related to melanin synthesis. (B) Double-stained with Calcofluor targeting mainly chitin (blue) and Eosin Y targeting mainly chitosan (yellow). The bottom right table in the panel shows sample order information for the fungal gene expression heatmaps.

Figure 6

Phenotypic characterization of three mutants with disruption of the ET-responsive genes including CgCAP22, CgSCD1, and CgCP1. (A) The left two rows of photos demonstrate the front (F) and reverse (R) sides of the fungal colonies grown on PDA for 5 days; the right bar chart indicates that the colony diameters of the mutants showed no significant difference in comparison with the WT. (B) ET significantly promoted the appressorium formation of WT and the mutants in vitro at 4 h after incubation on glass slides. (C) ET treatment enhanced ROS accumulation of WT and ΔCgcap22 in host leaves, but caused no effect on ΔCgscd1 and ΔCgcp1 at 24 h after inoculation. For (B,C): p < 0.05 (*).

Figure 7

MAPK and GPCRs were involved in mediating ET signaling to promote appressorium development and pathogenicity. (A,C) The front (F) and reverse (R) sides of colony morphology and expansion rates of the strains grown on PDA for 5 days. (B,D,E) The effect of ET on spore germination and appressorium development at 4 h after incubation on glass slides. (F) The influence of ET on ROS accumulation of the plant leaves infected with WT, ΔCgste11, and ΔCggpcr3-1/2 strains at 24 h after inoculation. For (C–F): p < 0.05 (*).

Figure 8

Comparative expression analysis of the ET-induced genes in the WT and ΔCgmk1, ΔCgste11, and ΔCggpcr3-1/2 mutants.