Aspergillus flavus is a saprophytic fungus and opportunistic pathogen that is widely distributed in the soil and air in the form of mycelium and conidia (Liang et al., 2024; Shabeer et al., 2022). This fungus can induce corn ear rot and peanut yellow mold disease, and it has the capacity to produce aflatoxins (AFs) (Amaike and Keller, 2011; Wang et al., 2019). AFs, a group of difurocoumarin derivatives, are primarily classified into four types, with aflatoxin B1 (AFB1) being the most toxic (Klich, 2007). AFB1 is a potent liver carcinogen, and continuous consumption by animals or humans can cause diseases such as growth failure, malnutrition, and immune deficiency (Shabeer et al., 2022; Thakaew and Chaiklangmuang, 2023). In addition, A. flavus can induce human aspergillosis through invasive growth, causing diseases such as keratitis, sinusitis, and skin infections, posing a major threat to people with compromised immune systems (Rudramurthy et al., 2019). Due to the significant impact of A. flavus on agriculture and public health, innovative strategies must be formulated to mitigate these adverse effects. The biosynthesis of AFs involves a complex enzyme cascade that is not only regulated by genes within the cluster, but also indirectly affected by genes outside the cluster (Caceres et al., 2020; Rushing and Selim, 2019). AflR is the main activator of the AF gene cluster, interacting with aflS, which functions as an enhancer in regulating the biosynthesis process (Caceres et al., 2020; Khan et al., 2021). The velvet complex consists of the proteins VeA, LaeA, and VelB, forming heterotrimers in the nucleus to coordinate and govern fungal development and secondary metabolism (Gao et al., 2023). Among these, VeA and LaeA are essential for AF production (Amaike and Keller, 2009; Bayram et al., 2008). Other genes related to energy utilization (Khan et al., 2021; Wang et al., 2019), oxidative stress (Li et al., 2022; Tsitsigiannis and Keller, 2007), and growth and development (Frisvad et al., 2019; Keller, 2019) may also interact directly or indirectly with the AF gene cluster to regulate AF biosynthesis (Caceres et al., 2020). In addition, genes related to cell signal transduction have also been gradually studied (Amare and Keller, 2014; Lohmar et al., 2019).

Rho GTPases function as molecular switches that play essential roles in fungal development and pathogenesis through signal transduction processes (Li et al., 2011; Vicente-Soler et al., 2021; Zhang et al., 2018b). The loss of Rho3 in Magnaporthe grisea leads to delayed conidia germination, abnormal morphology, impaired appressorium formation, and compromised plant penetration (Zheng et al., 2007). RacA is a significant member of the Rho GTPase family, and its deletion has been shown to decrease conidia production, AFB1 synthesis, and seed infestation capacity in A. flavus (Qin et al., 2022). The biological activity of Rho GTPases relies on their ability to alternate between an inactive GDP-bound conformation and an active GTP-bound state (Vicente-Soler et al., 2021). Rho guanine nucleotide exchange factor (GEF) is responsible for the activation of the Rho GTPase, facilitating its transition from the Rho GDP conformation to the Rho GTP conformation (Zhang et al., 2018a). Typically, the activation of Rho GEFs is mediated by the Dbl homology (DH) catalytic domain, which interacts with the flexible switch region of GTPases, inducing remodeling and fostering nucleotide exchange (Samantaray et al., 2013; Buchsbaum, 2007). Various Rho GEFs have been identified in filamentous fungi (García et al., 2022; Vicente-Soler et al., 2021). In Saccharomyces cerevisiae and Aspergillus fumigatus, Rom1 and Rom2 activate Rho1 and its downstream effector mitogen-activated protein (MAP) kinase modules in response to signals from cell surface stress sensors. Deletion of Rom1 and Rom2 results in cell lysis and arrest of polar growth, and Δrom2 mutant exhibits a temperature-sensitive phenotype (Ozaki et al., 1996; Samantaray et al., 2013). In Fission yeast and Aspergillus nidulans, Rgf1, Rgf2, and Rgf3 have been shown to activate Rho1, with Rgf1 controlling polarized growth signals, Rgf2 essential for sporulation, and Rgf3 managing signals for septum synthesis (García et al., 2022; Justa-Schuch et al., 2010; Vicente-Soler et al., 2021). Bud3, functioning as a GEF, plays a significant role in promoting Rho4 activation, regulating septum formation, and localizing the contractile actin ring (CAR) in Neurospora crassa and A. nidulans (Justa-Schuch et al., 2010; Si et al., 2010). In budding yeast, Bud3 serves as a GEF for Cdc42 during the initial growth phase, a crucial process for axial budding (Kang et al., 2014). In Fusarium graminearum, Bud3, a GEF that interacts with Rho4, significantly impacts the pathogenicity of the fungus on wheat (Zhang et al., 2018a). However, the functions of Rho GEFs and their involvement in mycotoxin synthesis regulation have not been explored in A. flavus.

In a previous study (Xu et al., 2024), a Rho GEF GerA (AFLA_033630), closely regulated by VeA, was identified under various culture conditions using chromatin immunoprecipitation and sequencing (ChIP-seq) experiments. It is characterized by a DH domain. Bioinformatics analysis revealed that GerA is relatively conserved among Aspergillus species, less conserved in other filamentous fungi, and no homologue had been identified in S. cerevisiae. The CRISPR/Cas9 gene editing system was used to efficiently generate mutants, elucidating the regulatory role of gerA in the growth, reproduction, AF synthesis, and pathogenicity of A. flavus. The gerA gene is a potential target for developing strategies to control A. flavus infection and AF biosynthesis. Additionally, this study establishes a basis for further exploration of other GEFs in filamentous fungi.