FolVps9, a Guanine Nucleotide Exchange Factor for FolVps21, Is Essential for Fungal Development and Pathogenicity in Fusarium oxysporum f. sp. lycopersici

Abstract

The soil-borne, asexual fungus Fusarium oxysporum f.sp. lycopersici (Fol) is the causal agent of tomato wilt disease. Autophagy plays a crucial role in the development and virulence of Fol. The Fol endosomal system is highly dynamic and has been shown to be associated with conidiogenesis and pathogenicity. Rab GTPases and the regulators are highly conserved in regulating autophagy and endocytosis in most eukaryotes. Identification and characterization of additional Rab regulators in fungal pathogens should facilitate the understanding of the autophagy and endocytosis in different filamentous fungi. Here, we have identified and characterized the yeast VPS9 homolog FolVPS9 in Fol. Targeted gene deletion showed that FolVPS9 is important for growth, conidiation and virulence in Fol. Cytological examination revealed that FolVps9 co-localized with FolVps21 (a marker of early endosome) and played a critical role in endocytosis and autophagosome degradation. Pull-down assays showed that FolVps9 interacted with FolVps21, which was also important for development and plant infection in Fol. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays revealed that FolVps9 specifically interacts with the GDP-bound form of FolVps21. Furthermore, a constitutively active form of FolVps21 (Q72L) was able to rescue defects of ΔFolvps9 and ΔFolvps21 mutants. In summary, our study provides solid evidence that FolVps9 acts as a FolVps21 guanine nucleotide exchange factor (GEFs) to modulate endocytosis and autophagy, thereby controlling vegetative growth, asexual development and pathogenicity in Fol.

FgVps9, a Rab5 GEF, Is Critical for DON Biosynthesis and Pathogenicity in Fusarium graminearum

“…Here, our data have shown that Vps9 domain, rather than CUE domain, in FgVps9 regulates the endosomal localization and physiological functions of the entire protein in F. graminearum. This result is consistent with that observed in Fol (Li et al, 2019), indicating that the function of Vps9 domain is pleiotropic in fungi. We also found that the two GEF activity sites in the FgVps9 domain play an essential role in F. graminearum.…”

“…In our study, FgVPS9 deletion mutant also shows reduced vegetative growth, endocytosis, virulence, and DON production, while autophagy is only partially blocked in Fgvps9 mutant with most autophagosomes labeled by GFP-ATG8 fused to vacuoles in liquid MM-N media. However, in P. oryzae and Fol, remarkably less fluorescence signals of GFP-ATG8 and few autophagosomes were examined in the vacuoles of the Povps9 and Folvps9 mutants when comparing with those of wild-type strains, suggesting that autophagy is seriously blocked in Povps9 and Folvps9 mutants (Zhu et al, 2018;Li et al, 2019). Collectively, these data indicated that, similar with PoVps9 and FolVps9, FgVps9 functions in both endocytosis and autophagy processes, which involved in fungal growth, virulence, and DON production; however, the role of FgVps9 on autophagy is not as important as its homologs in P. oryzae and Fol.…”

“…Deletion of the endocytosis-related gene RAB5 in P. oryzae and F. graminearum results in growth defect and complete loss of the infection to plants (Zheng et al, 2015;Yang et al, 2017). Moreover, Vps9, a Vps21 GEF, colocalizes with the early endosome marker Vps21 and plays an important role in vegetative growth, endocytosis, and pathogenicity in the rice blast fungus P. oryzae and the tomato pathogen Fol (Zhu et al, 2018;Li et al, 2019). These results collectively reveal that endocytosis is closely associated with the growth and pathogenicity of the plant fungal pathogens.…”

“…In budding yeast, Vps9 is predominantly cytosolic and partially localizes to endosomes (Paulsel et al, 2013;Bean et al, 2015). In P. oryzae and Fol, Vps9 colocalize with the early endosome marker Vps21 (Zhu et al, 2018;Li et al, 2019). In order to determine the localization of FgVps9 in F. graminearum, we subjected our previously generated complementation strain harboring the GFP-FgVps9 fusion protein to laser scanning confocal microscopy.…”

“…FolVps9 also shows GEF activity on FolVps21 in Fusarium oxysporum f.sp. lycopersici (Fol) where deletion of FolVPS9 phenocopied the single-and double-deletion mutants lacking Vps9 homologs in P. oryzae (Li et al, 2019).…”