BAR domain is essential for early endosomal trafficking and dynamics in Ascochyta rabiei

Abstract

Ascochyta blight disease is a devastating disease caused by the fungal pathogen Ascochyta rabiei that threatens chickpea production around the globe. Endocytic mechanism has a significant role in fungal growth and virulence. The underlying biology of biogenesis of central component of endocytosis viz Rab5 vesicles, is not completely understood. The involvement of F-BAR domain containing protein (ArF-BAR) in various cellular processes that collectively make ArF-BAR as an important virulence determinant. Here, we report that ArF-BAR is involved in biogenesis and motility of early endosome. In the absence of ArF-BAR gene (Δarf-bar), fungal mutants exhibited reduced number of EGFP coated ArRab5 vesicles, along with the considerable reduction in their dynamics. Here, we show that ArF-BAR interacts with clathrin light chain (ArCLC), specifically with its F-BAR domain. These findings suggests the novel role of ArF-BAR in biogenesis and dynamics of early endosome. Additionally, ArF-BAR is involved in clathrin-mediated mechanism of endocytosis which is required for host infection and disease development. Identification of this pathway offers new impending targets for disease intervention in plants.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-022-03451-5.

Keywords: Chickpea; Clathrin; Endocytosis; Fungal pathogens; Rab5.

Fig. 1

ArF-BAR regulates the biogenesis and motility of early endosome. a Confocal image showing EGFP-ArRab5 coated early endosomes in WT and Δarf-bar. Difference in the number of GFP coated Rab5 vesicles were observed in hyphae of two different fungal transformants. More number of ArRab5 punctate were observed in WT as compared to Δarf-bar. Images were optically sectioned, Z-stack images having 1 µm step size were captured. b Confocal images of migrating ArRab5 decorated with EGFP, in WT and Δarf-bar. Arrow head indicates the temporal localization of early endosomes. c Kymographs of moving Rab5 endosomes in WT and Δarf-bar. Sale Bar represents 10 µm in length

Fig. 2

Evolutionary relationships of ArCLC with other homologous proteins among different organisms. The phylogenetic tree obtained through the neighborhood-joining method, based on the analysis of protein sequences isolated from various pathogenic fungi, yeast, D. melanogaster, and H. sapiens, constructed using MEGAX. Protein IDs are included in parentheses, whose sequence were used to construct phylogeny

Fig. 3

Multiple sequence alignment of ArCLC protein with its homologs from various organisms. a Schematic representation of ArCLC protein. The 5–250 amino-acid protein has Clathrin-light chain (CLC) domain (pink), 150–179 amino-acid has Integrase core domain (rve_3) (black), and 137–221 amino acid has putative nuclear localization signal (yellow). b Multiple sequence alignment shows that CLC protein is highly conserved in different fungal species. The integrase core domain is marked in the box (black). c Multiple sequence alignment showing the conservation of ArCLC protein with well characterized CLC protein from S. cerevisiae, D. melanogaster, and H. sapiens. The alignment of the protein was determined by multiple sequence and structure alignment program PROMALS3D using default parameters. The alignment was visualized with visualization software Jalview. Color code for sequence conservation varies from blue (least conserved) to red (highly conserved). Shaded box below the alignment indicates the consensus sequence or the degree of conservation among the proteins. d Domain organization of CLC protein from S. cerevisiae, D. melanogaster, and H. sapiens

Fig. 4

ArF-BAR interacts ArCLC via F-BAR domain. a Split-ubiquitin based yeast two-hybrid assay to check protein–protein interaction between ArF-BAR and ArCLC in yeast cytoplasm. The yeast strain NMY-51 was co-transformed with bait and prey. Growth of yeast cells on interaction selective media QDO (SD/-L-W-A-H), suggests positive interaction between ArF-BAR and ArCLC. LargeT and Δp53 are used as positive control, while the empty bait and prey vector was used as negative control. pAI-Alg5 and pDL2-Alg5 are used to check the transactivation property. b Yeast cells failed to grow on QDO, as compared to DDO (SD/-L-W), when they were co-transformed with ArCLC and SH₃ domains [SH₃(ArF-BAR); 508–760 amino acids] of ArF-BAR, suggesting SH₃ of ArF-BAR has no role in interaction with ArCLC. c Yeast cells was observed on QDO when they were co-transformed with F-BAR domain of ArF-BAR and ArCLC, suggesting ArF-BAR interacts ArCLC via F-BAR domain. The representative images were captured after 48 h of spotting of yeast cells