Retrograde trafficking from the endosome to the trans‐Golgi network mediated by the retromer is required for fungal development and pathogenicity in Fusarium graminearum

Abstract

SummaryIn eukaryotes, the retromer is an endosome-localized complex involved in protein retrograde transport. However, the role of such intracellular trafficking events in pathogenic fungal development and pathogenicity remains unclear.The role of the retromer complex in Fusarium graminearum was investigated using cell biological and genetic methods.We observed the retromer core component FgVps35 (Vacuolar Protein Sorting 35) in the cytoplasm as fast-moving puncta. FgVps35-GFP co-localized with both early and late endosomes, and associated with the trans-Golgi network (TGN), suggesting that FgVps35 functions at the donor endosome membrane to mediate TGN trafficking. Disruption of microtubules with nocodazole significantly restricted the transportation of FgVps35-GFP and resulted in severe germination and growth defects. Mutation of FgVPS35 not only mimicked growth defects induced by pharmacological treatment, but also affected conidiation, ascospore formation and pathogenicity. Using yeast two-hybrid assays, we determined the interactions among FgVps35, FgVps26, FgVps29, FgVps17 and FgVps5 which are analogous to the yeast retromer complex components. Deletion of any one of these genes resulted in similar phenotypic defects to those of the DFgvps35 mutant and disrupted the stability of the complex.Overall, our results provide the first clear evidence of linkage between the retrograde transport mediated by the retromer complex and virulence in F. graminearum.

The endosomal recycling of FgSnc1 by FgSnx41–FgSnx4 heterodimer is essential for polarized growth and pathogenicity in Fusarium graminearum

“…The punctate cytoplasmic distribution of FgSnx41‐GFP was reminiscent of our previous studies of the retromer protein complex that localizes to endosomes (Zheng H. et al ., ; Zheng W. et al ., ; Zheng et al . ). To determine whether FgSnx41‐GFP localizes to the endosomes, the fluorescent dye FM4‐64, which shows membrane internalization and successive transport to the EEs and late endosomes (Vida & Emr, ; Fischer‐Parton et al ., ; Wedlich‐Soldner et al ., ), was used to trace FgSnx41‐GFP by pulse‐chase experiments.…”

“…a,b). To further confirm whether the FgSnx41‐GFP‐labeled vesicles are EEs, we co‐expressed FgSnx41‐GFP or FgSnx41‐RFP with several cellular organelle markers in F. graminearum cells, including the early endosomal marker mCherry‐FgRab52 (Zheng H. et al ., ; Zheng W. et al ., ), late endosomal marker GFP‐FgRab7 (Zheng H. et al ., ; Zheng W. et al ., ), trans‐Golgi network marker FgKex2‐mCherry (Zheng et al ., ), peroxisome marker FgPex14‐RFP (Li et al ., ) and mitochondrial marker FgAtp1‐RFP (Li et al ., ). In the resulting transformants, FgSnx41‐GFP co‐localized with the early endosomal marker, mCherry‐FgRab52, but not with the other organelle‐specific markers (Figs c,d, S5).…”

“…For BiFC assay, different pairs of constructs and single constructs were transformed into the protoplasts of PH‐1. A pair of known physically interacting proteins (FgVps35/FgVps29) (Zheng et al ., ) was used as positive control, whereas four kinds of NYFP‐ and/or CYFP‐expressed strains, including FgSnx41−NYFP, FgSnx41−NYFP+CYFP, FgSnx4−CYFP and FgSnx4−CYFP+NYFP, were used as negative controls. Transformants that were resistant to hygromycin and/or neomycin were isolated and confirmed by PCR.…”

“…Recently, we reported that the retromer is associated with endosomal sorting, through the collection and recycling of cargo proteins, and the regulation of development and pathogenesis in plant‐pathogenic fungi (Zheng H. et al ., ; Zheng W. et al ., ; Zheng et al . ; Abubakar et al ., ). Proteins in the retromer complex are evolutionarily conserved from budding yeast to human, where it is composed of two sub‐complexes: a cargo recognition sub‐complex containing Vps26, Vps29 and Vps35, and a membrane deformation sub‐complex consisting of Vps5 and Vps17 (Zheng et al ., ; Lucas & Hierro, ).…”

“…; Abubakar et al ., ). Proteins in the retromer complex are evolutionarily conserved from budding yeast to human, where it is composed of two sub‐complexes: a cargo recognition sub‐complex containing Vps26, Vps29 and Vps35, and a membrane deformation sub‐complex consisting of Vps5 and Vps17 (Zheng et al ., ; Lucas & Hierro, ). Both Vps5 and Vps17 contain two characteristic membrane‐binding modules: a phosphoinositide‐binding PX (phox homology) domain and a membrane‐curvature‐sensing Bin/amphiphysin/Rvs (BAR) domain, which are members of the SNX‐BAR subfamily (Carlton et al ., ; Teasdale & Collins, ).…”