SummaryEndocytosis and exocytosis are strictly segregated at the ends of hyphal cells of filamentous fungi, with a collar of endocytic activity encircling the growing cell tip, which elongates through directed membrane fusion. It has been proposed that this separation supports an endocytic recycling pathway that maintains polar localization of proteins at the growing apex. In a search for proteins in the filamentous fungus Aspergillus nidulans that possess an NPFxD motif, which signals for endocytosis, a Type 4 P-Type ATPase was identified and named DnfA. Interestingly, NPFxD is at a different region of DnfA than the same motif in the Saccharomyces cerevisiae ortholog, although endocytosis is dependent on this motif for both proteins. DnfA is involved in asexual sporulation and polarized growth. Additionally, it is segregated within the Spitzenkörper from another Type 4 P-type ATPase, DnfB. Next, the phosphatidylserine marker GFP::Lact-C2 was expressed in growing hyphae, which revealed that this phospholipid is enriched on the cytosolic face of secretory vesicles. This distribution is affected by deleting either dnfA or dnfB. These findings provide evidence for the spatial and temporal segregation of Type4-ATPases in filamentous fungi, and the asymmetric distribution of phosphatidylserine to the Spitzenkörper in A. nidulans.

Summary Fusarium verticillioides is a fungal pathogen that is responsible for maize ear rot and stalk rot diseases worldwide. The fungus also produces carcinogenic mycotoxins, fumonisins on infested maize. Unfortunately, we still lack clear understanding of how the pathogen responds to host and environmental stimuli to trigger fumonisin biosynthesis. The heterotrimeric G protein complex, consisting of canonical Gα, Gβ and Gγ subunits, is involved in transducing signals from external stimuli to regulate downstream signal transduction pathways. Previously, we demonstrated that Gβ protein FvGbb1 directly impacts fumonisin regulation but not other physiological aspects in F. verticillioides. In this study, we identified and characterized a RACK1 (Receptor for Activated C Kinase 1) homolog FvGbb2 as a putative Gβ‐like protein in F. verticillioides. The mutant exhibited severe defects not only in fumonisin biosynthesis but also vegetative growth and conidiation. FvGbb2 was positively associated with carbon source utilization and stress agents but negatively regulated general amino acid control. While FvGbb2 does not interact with canonical G protein subunits, it may associate with diverse proteins in the cytoplasm to regulate vegetative growth, virulence, fumonisin biosynthesis and stress response in F. verticillioides.

Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. But the role of Rab GTPases in Fusarium verticillioides, one of the key corn pathogens worldwide, remains elusive. These Small GTPases in fungi, particularly those homologous to Saccharomyces cerevisiae Sec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. Here, we characterized the molecular functions of FvSec4 by generating a null mutant and learned that it is important for vegetative growth, hyphal branching, and conidiation. Interestingly, the mutation did not impair the expression of key conidiation-related genes. Meanwhile, the mutant did not show any defect in sexual development, including perithecia production. GFP-FvSec4 localized to growing hyphal tips, and raised the possibility that FvSec4 is involved in protein trafficking and endocytosis. The mutant exhibited defect in corn stalk rot virulence and also significant alteration of fumonisn B1 production. The mutation led to more sensitivity to oxidative and cell wall stress agents, and defects in carbon utilization. Gene complementation fully restored the defects in the mutant demonstrating that FvSec4 plays important role in these functions. Taken together, our data indicate that FvSec4 plays important roles in F. verticillioides hyphal development, virulence, mycotoxin production and stresses response. Further study is needed to characterize whether the mutation in FvSec4 leads to altered vesicle trafficking and protein secretion, which ultimately impact F. verticillioides physiology and virulence.

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Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of Ga, Gb, and Gg subunits, plays an important role in transducing signals under environmental stress. Furthermore, regulators of G-protein signaling (RGS) proteins act as negative regulators in heterotrimeric G protein signaling. Earlier studies demonstrated that Ga and Gb subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gb deletion mutant DFvgbb1. Saccharomyces cerevisiae and Aspergillus nidulans contain a single copy of FlbA, but F. verticillioides has two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. Importantly, FvFlbA2 has a negative role in FB1 regulation. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While DFvflbA1 deletion mutant exhibited no significant defects, DFvflbA2 and DFvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays suggest that FvFlbA paralogs interact with key heterotrimeric G protein components to impact F. verticillioides FB1 production and asexual development.