Fusarium verticillioidesis recognized as an important stalk rot pathogen of maize worldwide, but our knowledge of genetic mechanisms underpinning this pathosystem is limited. Previously, we identified a striatin-like protein Fsr1 that plays an important role in stalk rot. To further characterize transcriptome networks downstream of Fsr1, we performed next-generation sequencing (NGS) to investigate relative read abundance and also to infer co-expression networks utilizing the preprocessed expression data through partial correlation. We used a probabilistic pathway activity inference strategy to identify functional subnetwork modules likely involved in virulence. Each subnetwork modules consisted of multiple correlated genes with coordinated expression patterns, but the collective activation levels were significantly different inF. verticillioideswild type versusfsr1mutant. We also identified putative hub genes from predicted subnetworks for functional validation and network robustness studies through mutagenesis, virulence and qPCR assays. Our results suggest that these genes are important virulence genes that regulate the expression of closely correlated genes, demonstrating that these are important hubs of their respective subnetworks. Lastly, we used keyF. verticillioidesvirulence genes to computationally predict a subnetwork of maize genes that potentially respond to fungal genes by applying cointegration-correlation-expression strategy.

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 .

Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B₁ (FB₁) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB₁ biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB₁ biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB₁ biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.

Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rot. While stalk rot pathogens are prevalent worldwide, our understanding of the stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified the F. verticillioides FvSYN1 gene, which was demonstrated to play an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of the FvSyn1 protein, namely how different motifs contribute to development and virulence in F. verticillioides by generating motif-specific deletion mutants. Microscopic observation showed that the Delta Fvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild-type progenitor. Moreover, the Delta Fvsyn1 mutant was sensitive to cell wall stress agents, resulting in vegetative growth reduction. We showed that the FvSyn1:: GFP protein is associated with the endomembrane, but this did not clarify why the deletion of FvSyn1 led to stress sensitivity and aberrant hyphal development. Characterization of the FvSyn1 domains indicated that both the syntaxin N-terminus (SynN) domain and the SNARE C-terminus domain play distinct roles in fungal development, but also function collectively in the context of virulence. We also determined that two domains in FvSyn1 are not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization, including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 domains in hyphal growth, cell wall stress response and virulence in F. verticillioides.