Conserved retrograde trafficking mechanisms regulate fungal development and pathogenicity through Rab6-GARP-Retromer-SNARE coordination

Conserved retrograde trafficking mechanisms regulate fungal development and pathogenicity through Rab6-GARP-Retromer-SNARE coordination | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Plant, Cell & Environment This is a preprint and has not been peer reviewed. Data may be preliminary. 12 August 2025 V1 Latest version Share on Conserved retrograde trafficking mechanisms regulate fungal development and pathogenicity through Rab6-GARP-Retromer-SNARE coordination Authors : Yunfei Long , Haoran Zhang , Xingyuan Wu , Xin Chen , Ying Lin , Yakubu Saddeeq Abubakar 0000-0002-5228-0548 , Huawei Zheng , Zonghua Wang 0000-0002-0869-9683 , and Wenhui Zheng [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175497706.67845306/v1 Published Plant, Cell & Environment Version of record Peer review timeline 251 views 172 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Retrograde transport from endosomes to the trans-Golgi network (TGN) is essential for intracellular trafficking, yet its molecular mechanism remains poorly understood. In Fusarium graminearum , ten Rab GTPases associated with the Golgi-associated retrograde protein (GARP) complex were identified through immunoprecipitation followed by mass spectrometry (IP-MS). Among these, only deletion of FgRab6 disrupted the proper localization of the GARP complex to the TGN. FgRab6 directly interacts with the GARP subunit FgVps52 via a conserved Q73 residue, which is critical for fungal growth and pathogenicity. Notably, this Q73-dependent interaction is evolutionarily conserved across eukaryotic species. Upon GTP activation, FgRab6 recruits FgVps52 to the TGN, thereby facilitating the assembly of the GARP complex through the sequential recruitment of additional subunits, including FgVps51, FgVps53, and FgVps54. The fully assembled GARP complex subsequently recruits the retromer complex and mediates the retrograde trafficking of SNARE proteins—FgSnc1, FgTlg1, and FgTlg2—from endosomes to the TGN. Disruption of this pathway severely compromises fungal development and virulence. Collectively, these findings identify a FgRab6–GARP–retromer–coordinated vesicle trafficking pathway that mediates the retrograde transport of SNARE proteins from endosomes to the TGN, which is critical for the pathogenicity of F. graminearum . This work provides new mechanistic insights into vesicular transport and highlights potential targets for antifungal intervention. Introduction Fusarium graminearum , the primary pathogen responsible for Fusarium head blight in wheat worldwide(Bian et al., 2024; Qu et al., 2010), has been recognized as one of the ten most damaging fungal plant pathogens(Chen et al., 2019; Dean et al., 2012; Gardiner et al., 2020; Sun et al., 2021). Its infection results in significant yield losses and introduces a variety of mycotoxins into host grains, of which deoxynivalenol is the most abundant(Hooft and Bureau, 2021; Pinto et al., 2022; Sumarah, 2022). Over the past thirty years, much of the research has focused on understanding the virulence of the pathogen and the mechanisms that regulate trichothecene biosynthesis(Huang et al., 2024; Wang et al., 2022). Recently, numerous studies have shown that components of retrograde transport pathway from endosomes to the trans-Golgi network (TGN), including Rab GTPases, GARP complex, retromer complex, SNARE proteins play a crucial role in the growth and pathogenicity of F. graminearum(Miao et al., 2023; Xing-Zhi et al., 2023; Yunfei Long, 2023; Zheng et al., 2021; Zheng et al., 2015; Zheng et al., 2018c; Zheng et al., 2016) . Retrograde transport pathway from endosomes to the TGN in all eukaryotic cells and facilitates the transport of substances and the transmission of signals(Faini et al., 2013; Raposo and Stoorvogel, 2013). This process is associated with a well-defined and dynamic spatiotemporal coupling, orchestrated by various regulatory proteins. Rab proteins serve as molecular switch points in membrane transport and are involved in processes such as protein coat assembly, uncoating, cell division, movement, target recognition and membrane fusion(Stenmark, 2009). The Rab proteins, known as Ypt in yeast, form the largest subgroup within the Ras-related small GTPase superfamily, and 66 Rab genes have been identified in the human genome(Homma et al., 2021; Li and Marlin, 2015; Reiner and Lundquist, 2018). These proteins are evolutionarily conserved from yeast to humans and act as molecular switches, switching between inactive (GDP-bound) and active (GTP-bound) states to promote the formation, scission, translocation and fusion of vesicles at specific target membrane sites(Bhuin and Roy, 2014; Cherfils and Zeghouf, 2013; Faini et al., 2013; Homma et al., 2021). In recent years, Rab GTPases have been identified as crucial for the development and virulence of various plant pathogenic fungi such as Ustilago maydis , Botrytis cinerea , Magnaporthe oryzae and Fusarium verticillioides (Liu et al., 2015; Schneider et al., 2022; Yan et al., 2020; Zhang et al., 2014). FgRab6 is critical for vegetative growth, sporulation and pathogenicity of F. graminearum(Zheng et al., 2015) . Similarly, FgRab5, FgRab7, FgRab8 and FgRab1 have been shown to control endosomes biogenesis and modulate the growth, development and virulence of F. graminearum via vesicular transport and cargo sorting mechanisms(Abubakar et al., 2021; Yang et al., 2020; Yuan et al., 2022; Zheng et al., 2018a; Zheng et al., 2021; Zheng et al., 2018b). The GARP complex was originally discovered through yeast studies focusing on protein transport mechanisms(Conibear et al., 2003; Conibear and Stevens, 2000). As a member of the multi-subunit tethering complexes (MTCs), GARP is located at the cytosolic periphery of the TGN. This heteromeric protein complex consists of four different subunits: vacuolar protein sorting 51 (VPS51), VPS52, VPS53, and VPS54(Bröcker et al., 2010). The GARP complex is important for the binding of transport vesicles to the TGN, facilitating membrane fusion between the vesicles and their target membranes(Khakurel and Lupashin, 2023). In addition to GARP, other transport machineries, such as the retromer complex, also play a role in the retrograde transport of cargo proteins to their proper location, ensuring the continuity of their biological functions(Abubakar et al., 2017; Ma and Burd, 2020). The retromer complex is responsible for mediating the vesicular transport of certain membrane proteins from endosomes back to the TGN and plasma membrane(Buser and Spang, 2023). It is a heteropentamer composed of two major subcomplexes: a core trimer consisting of Vps35, Vps29, and Vps26, often referred to as the cargo selection complex (CSC), and a dimer of sorting nexin (SNX) proteins (such as Vps5 and Vps17) that associate with phosphatidylinositol 3-phosphate-enriched endosomal membranes during cargo sorting(Abubakar et al., 2021). Our previous studies have shown that in plant pathogenic fungi such as M. oryzae and F. graminearum , the retromer complex plays a crucial role in cargo sorting at the endosomes. This mechanism control fungal growth and virulence by selectively sorting and retrieving specific cargo proteins(Chen et al., 2023; Zheng et al., 2016). In F. graminearum , the GARP complex, located in the trans-Golgi network (TGN), has the ability to recruit the retromer complex, and thus supports its transfer from the endosome to the TGN(Yunfei Long, 2023). SNARE (Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor) proteins are of central importance in membrane fusion processes and ensure the precise targeting and release of cargo molecules (Buser and Spang, 2023) . These proteins belong to an extensive superfamily that is conserved in a variety of species (Burri and Lithgow, 2004; Wang et al., 2017 ) . The defining feature of SNARE proteins is their SNARE motif, which consists of a conserved sequence of 60 to 70 amino acids. In addition, they contain either a single transmembrane domain or a lipid-modified motif linked to the SNARE motif via a short linker at the C-terminus (Hong, 2005) . Their main role is to facilitate fusion between vesicles and target membranes, which is essential for the transport of intracellular cargos (Jahn et al., 2024) . Consequently, vesicular transport systems are essential for the proper sorting and movement of SNARE proteins. The SNARE complexes Snc1, Vti1, Tlg1 and Tlg2 are involved in retrograde transport from endosomes to the trans-Golgi network (TGN)(Kama et al., 2007; Morvan et al., 2015). In pathogenic fungi, specific vesicle trafficking pathways play a crucial role in the sorting and transport of SNARE proteins. In F. graminearum , for example, the cargo sorting and transport complex FgSnx41-FgSnx4 regulates polarized growth and pathogenicity by sorting and directing the SNARE protein FgSnc1 at the endosomal level(Zheng et al., 2018c). In addition, the Rab protein FgRab1 mediates the membrane fusion functionality of SNARE proteins and thus influences the growth, development and pathogenicity of F. graminearum (Xing-Zhi et al., 2023). In M. oryzae , the MoRab7/retromer sorting pathway controls the secretion of the cytoplasmic effector Pwl2 by sorting and transporting the SNARE protein MoSnc1, thereby influencing the virulence of the pathogen(Chen et al., 2023). Although the retrograde transport pathway from endosomes to the TGN is essential for eukaryotic cells, the mechanisms by which GTPase proteins, the GARP complex, the retromer complex, and SNARE proteins collaborate to regulate this pathway remain unknown. In this study, we provide important insights into the molecular mechanisms underlying retrograde transport from endosomes to the TGN in eukaryotic cells. We initially employed IP-MS to screen and identify ten Rab GTPase family members potentially associated with the GARP complex. Subsequent reverse genetic analyses revealed that among these candidates, only deletion of FgRAB6 disrupted the proper localization of the GARP complex from the TGN, indicating that GARP recruitment to the TGN is specifically mediated by FgRab6. Although the interaction between Rab6 and the GARP complex has been previously reported(Khakurel and Lupashin, 2023). our findings provide important insights which advance the current understanding of this interaction. Notably, we demonstrated that FgRab6 directly interacts with the GARP subunit FgVps52 at the TGN. Moreover, we identified for the first time that the conserved amino acid residue Q73 within the Rab domain of FgRab6 is essential for this interaction. This residue is also required for the recruitment of FgVps52 to the TGN and plays a critical role in regulating the growth and pathogenicity of F. graminearum . Importantly, the Q73-mediated Rab6–Vps52 interaction is evolutionarily conserved across fungi, plants and animals. Furthermore, our study provides the first mechanistic insight into the recruitment and assembly of the GARP complex at the TGN. We propose that GTP-bound FgRab6 initially recruits FgVps52 to the TGN, which subsequently facilitates the recruitment of other GARP subunits—FgVps51, FgVps52, and FgVps53—thereby promoting proper complex assembly. Once assembled, the GARP complex enhances the retrograde trafficking of the retromer complex from the endosomes to the TGN, which in turn regulates the localization of key SNARE complex components, including FgSnc1, FgTlg1 and FgTlg2. These SNARE proteins are indispensable for the growth, development and virulence of F. graminearum . In summary, we propose a novel model of retrograde transport in which GTP-activated FgRab6 recruits the GARP subunit FgVps52 to the TGN, thereby facilitating assembly of GARP complex (heterologous tetramer) and retromer-mediated trafficking from endosomes to the TGN. This process ensures proper SNARE protein trafficking and ultimately governs the growth, development and pathogenicity of F. graminearum . Immunoprecipitation-mass spectrometry analysis to identify potential GARP complex interacting proteins The GARP complex is essential for the growth, development and pathogenicity of F. graminearum (Yunfei Long, 2023). However, the mechanism by which the GARP complex is recruited to the TGN to mediate vesicle transport from endosomes to the TGN to regulate the growth, development and pathogenicity of F. graminearum remains unknown. To investigate this mechanism, we performed immunoprecipitation-mass spectrometry (IP-MS) to identify proteins that potentially interact with the GARP complex core subunits FgVps51 and FgVps53. The results showed that 1864 and 2506 proteins were pulled by the FgVps51-GFP and FgVps53-GFP baits, respectively, while only 140 and 12 proteins were captured by the controls PH-1-GFP (wild type expressing GFP) and PH-1 (wild type), respectively (Fig. S1 and Table S1-S4). Subsequent analysis revealed that 1268 proteins were common in FgVps51-GFP and FgVps53-GFP captured protein candidates after excluding those proteins that appeared in PH-1-GFP and PH-1 pulldown (Fig. S1a). Further enrichment analysis of the 1268 proteins using Gene Ontology (GO) revealed 10 significant molecular function (MF) pathways (P < 0.0001)(Jafari and Ansari-Pour, 2019). The molecular function (MF) enrichment analysis revealed that the third most enriched category was GTP-binding-related proteins, comprising 10 putative Rab GTPases (Fig. S1b, c). Rab GTPases are small GTP-binding proteins whose intrinsic GTPase activity plays a crucial role in vesicle transport(Mizuno-Yamasaki et al., 2012). However, the relationship between Rab GTPases and the GARP complex remains unknown in F. graminearum . To explore this relationship, we first generated the Rab GTPase mutants and expressed FgVps51-GFP and FgVps53-GFP in the mutants to analyze their subcellular localizations. Previous studies have shown that loss of FgRab1 and FgRab11 is lethal in F. graminearum , making it impossible to obtain deletion mutants(Yuan et al., 2022). Interestingly, as shown in Fig.1a, among the ten candidate Rab GTPase proteins, only deletion of FgRAB6 led to alteration in the normal localization of FgVps51-GFP and FgVps53-GFP, which became diffusely distributed in the cytoplasm of the fungal hyphae instead of the usual punctate distribution. The GARP complex is primarily composed of four main subunits: Vps51, Vps52, Vps53 and Vps54(Khakurel and Lupashin, 2023). To investigate whether mutation of FgRab6 affects the localization of the GARP subunits Vps52 and Vps54, we co-transformed FgVPS52 -GFP and FgVPS54 -GFP constructs into the Δ Fgrab6 mutant protoplasts. As shown in Fig S2, the FgVps52-GFP and FgVps54-GFP fusion proteins lost their normal localization and became dispersed in the cytoplasm. The GARP complex is primarily localized to the TGN, where it mediates the retrograde transport of vesicles from the endosomes to the TGN(Khakurel and Lupashin, 2023). To further investigate whether the deletion of FgRAB6 affects the localization of the GARP complex at the TGN, FgVps51-GFP and FgVps53-GFP were co-expressed with the TGN marker FgSec7-mCherry in the Δ Fgrab6 mutant. As shown in Fig1. b and c, FgVps51-GFP and FgVps53-GFP lost their normal distribution at the TGN and appeared dispersed in the cytoplasm. In contrast, when GFP-FgRab6 was expressed in Δ Fgvps51 and Δ Fgvps53 mutants, respectively, the subcellular localization of GFP-FgRab6 was not obviously different from that in the wild type (Fig. S3). These results suggest that FgRab6 regulates the recruitment of the GARP complex to the TGN. FgRab6 interacts with the GARP complex through FgVps52 Although the structure of the GARP complex has already been established(Khakurel and Lupashin, 2023), the mechanism by which the complex interacts with FgRab6 remains unclear. We investigated this interaction by yeast two-hybrid (Y2H) assays. The results demonstrated that FgRab6 directly interacts with the GARP complex subunit FgVps52, but not with the other subunits (Fig. 2a). Consistently, FgVps52-GFP and mCherry-FgRab6 were observed to colocalize (Fig. 2b). Furthermore, we conducted a bimolecular fluorescence complementation (BiFC) assay to validate this dynamic interaction. The resulting YFP fluorescence signal confirmed the positive interaction of FgVps52 and FgRab6 in vivo (Fig. S4). To determine the subcellular localization for this interaction, we included the TGN marker FgSec7-mCherry in the BiFC experiment. The punctate YFP signal significantly co-localized with the TGN marker, indicating that FgRab6 and FgVps52 interact at the TGN (Fig. 2c). To further validate the association of FgRab6 and FgVps52, an in-silico interaction model of FgRab6 and FgVps52 was analyzed using AlphaFold3. This model revealed that the amino acid residues Q23, D40, Y43, I47, G72, Q73, R75 and Y83 of FgRab6 are involved in its interaction with FgVps52 through hydrogen bonds (Fig. 2d). These residues were found to be located within the RAB domain of FgRab6 (Fig. 2e), which is known to be the main platform for Rab6 interaction with specific binding partners, including GEFs, GAPs and effector proteins(Pylypenko et al., 2018). To further understand the role of the RAB domain in the FgRab6–FgVps52 interaction, we generated a domain deletion mutant of FgRab6 (FgRab6 ΔRAB ), which lacks residues 15–178. Y2H assays demonstrated that FgRab6 ΔRAB lost its ability to interact with FgVps52 (Fig. 2f). Consistently, we observed that deletion of FgRab6 RAB domain also disrupted the recruitment of FgVps52 to the TGN (Fig. 2g, h). In contrast, expression of GFP-FgRab6 in Δ Fgvps52 mutant did not result in any significant changes to the subcellular localization of GFP-FgRab6 compared to the wild type (Fig. S5). In summary, our findings indicate that, through its RAB domain, FgRab6 interacts with FgVps52 to recruit the latter to the TGN. FgRab6 Q73 is essential for FgVps52 binding and fungal virulence Bioinformatic analysis revealed that the RAB domain of FgRab6 is highly conserved from yeast to mammals (Fig. S6a). Based on this observation, we hypothesized that the interaction between FgRab6 and FgVps52 is also evolutionarily conserved. To test this hypothesis, we used AlphaFold3 to predict the structural models of Rab6-Vps52 interactions across multiple species, including F. graminearum (FgRab6 and FgVps52), Fusarium oxysporum (FoRab6 and FoVps52), Neurospora crassa (NcRab6 and NcVps52), Saccharomyces cerevisiae (ScRab6 and ScVps52), Oryza sativa (OsRab6 and OsVps52), and Rattus norvegicus (RnRab6 and RnVps52). These predictions revealed that specific amino acid residues in Rab6 form hydrogen bonds with Vps52 (Fig.3a). Y2H assays subsequently validated these interactions, confirming that the Rab6-Vps52 interaction is conserved across divergent taxa (Fig. 3b). Interestingly, further structural analysis indicated that the glutamine residue at position 73 (Q73) in Rab6 is conserved among filamentous fungi . In addition, conserved residues (Q69, Q68, and Q72) were identified in yeast, plants and animals (Fig.3a). To investigate the conservation of this residue, we performed multiple sequence alignments of Rab6 orthologs from these species, and this analysis revealed the evolutionary conservation of Q73 (Fig. 3c and S6b). Given its conservation and predicted role in protein-protein interaction, we hypothesized that Q73 is critical for the interaction between FgRab6 and FgVps52. To test this, we generated a point mutation substituting glutamine at position 73 with alanine (Q73A), thereby disrupting its side-chain-mediated hydrogen bonding. Y2H assays showed that the Q73A mutation markedly impaired the interaction between FgRab6 and FgVps52 (Fig. 3d). Moreover, as shown in Fig. 3e, the Q73 residue is also required for the proper recruitment of FgVps52 to the TGN. Furthermore, phenotypic analysis showed that the Q73A mutation significantly reduced hyphal growth and virulence of F. graminearum on wheat spikes compared to the wild-type strain (Fig. 3f, and S6c). The conserved Q73 residue in Rab6 is critical for Vps52 binding across eukaryotes Previous studies have indicated that the conserved Q73 and T46 residues in Rab6 are essential for the activation of its GTPase activity(Stenmark and Olkkonen, 2001; Wakade et al., 2020).To test whether the T46 residue in FgRab6 is also important for its interaction with FgVps52, we introduced a T46A point mutation since the T46 residue is known to regulate GTP binding. However, as shown in Fig. 3d, the T46A mutation showed no obvious effect on the FgRab6–FgVps52 interaction. On the other hand, we investigated the evolutionary conservation of the Q73-dependent interaction by examining its functional role across diverse species, ranging from filamentous fungi to plants and mammals. Notably, substitution of this residue with alanine (Q73A) in both rice ( O. sativa ) and rat ( R. norvegicus ) homologs significantly impaired the binding affinity between Rab6 and Vps52, mirroring the effects observed in fungal systems. This finding underscores the critical and conserved role of Q73 in mediating this protein-protein interaction (Fig. 3g). Taken together, these results demonstrated that the Q73 residue in Rab6 plays an evolutionarily conserved role in Vps52 binding across eukaryotes. The constitutively active form of FgRab6 is responsible for the recruitment of FgVps52 to the TGN Rab GTPases serve as essential molecular switches, cycling between inactive GDP-bound and active GTP-bound states to regulate vesicle and membrane transports(Homma et al., 2021). As presented earlier, the conserved amino acid residue Q73 in FgRab6 is crucial for its interaction with FgVps52 (Fig. 3d). Specifically, the Q73 residue is a key site for the activation of the GTPase activity of FgRab6, and this residue is highly conserved across different species (Fig. S7)(Makaraci et al., 2019; Wakade et al., 2020). Based on this, we hypothesized that the GTP-bound form of FgRab6 recruits the GARP complex to the TGN to coordinate the vesicle transport process. To test this hypothesis, we generated two different forms of overexpressed FgRab6: the GDP-bound inactive form (dominant negative, T28N: FgRab6-DN) and the GTP-bound active form (constitutively active, Q73L: FgRab6-CA) (Fig. 4a). Quantitative reverse transcription PCR (RT-qPCR) analysis revealed that the expression levels of FgRab6-DN and FgRab6-CA increased more than 20-fold compared to the wild type (Fig. 4b). Next, we examined the effect of the overexpression of FgRab6-CA and FgRab6-DN on the subcellular localization of FgVps52-GFP. In the FgRab6-CA overexpressed strain, FgVps52-GFP was localized in the TGN, similar to the wild-type strain. In contrast, in the FgRab6-DN overexpressed strain, FgVps52-GFP was erroneously scattered throughout the cytoplasm and lost its colocalization with the TGN (Fig. 4c). Considering that Rab proteins switch between their GDP-bound inactive and GTP-bound active states, shuttling between the cytoplasm and membranes(Hutagalung and Novick, 2011), we speculated that the cytoplasmic dispersion of FgVps52-GFP in the FgRab6-DN strain was because FgRab6 does not associate with the TGN membrane. To investigate this further, we labeled both FgRab6-DN and FgRab6-CA with GFP and observed their subcellular localizations by confocal microscopy. As speculated, the GFP-FgRab6-DN predominantly localized to the cytoplasm, while the GFP-FgRab6-CA localized to the TGN (Fig. 4d, e). Collectively, these results demonstrate that FgVps52 is recruited to the TGN by the active GTP-bound form of FgRab6. FgVps52 serves as a key FgRab6 effector at the TGN The mislocalization of FgVps52 observed in the FgRab6-DN overexpressed strain prompted us to compare the phenotypic characteristics of this strain with those of the Δ Fgvps52 mutant. We found that the growth and pathogenicity of the two strains were similar but significantly lower than those of the wild type. In contrast, no significant decrease in either growth or pathogenicity was observed in the FgRab6-CA overexpressed strain compared to the wild type (Fig. S8). Next, we hypothesized that FgVps52 may function as an effector of FgRab6 to influence the growth and pathogenicity of F. graminearum . To test this, we cloned FgVPS52 into a pGADT7 (AD) vector and FgRAB6-CA and FgRAB6-DN genes into a pGBKT7 (BD) vector, respectively, and then co-transformed these constructs into S. cerevisiae AH109 cells for Y2H assays. The results showed that FgVps52 directly interacts with FgRab6-CA, but not with FgRab6-DN (Fig. 4f). This interaction was further confirmed by BiFC in which the TGN marker FgSec7-mCherry was included. The TGN marker clearly co-localized with the punctate YFP signals, indicating that FgRab6-CA and FgVps52 interact in the TGN in F. graminearum (Fig. 4g). Taken together, we conclude that FgVps52 functions as an effector of FgRab6 at the TGN. FgVps52 mediates the assembly of the GARP complex In a previous study, we demonstrated that the four subunits of the GARP complex form a tetrameric complex which localizes to the TGN where they regulate the retrograde trafficking of transport vesicles(Yunfei Long, 2023). However, the mechanism underlying the spatio-temporal assembly and recruitment of the GARP complex to the TGN remains unclear. To explore whether FgVps52 regulates the assembly and recruitment of the GARP complex, we analyzed the interactions among the GARP complex subunits in Δ Fgvps52 mutant strain. The results revealed that deletion of FgVPS52 disrupted the interactions among the GARP subunits which compromised the assembly of the complex (Fig. S9). Additionally, we co-transformed the TGN marker FgSec7-mCherry with each of the other three subunits FgVps51-GFP, FgVps53-GFP and FgVps54-GFP into the Δ Fgvps52 mutant strain and investigated their possible localization to the TGN. The results showed that the absence of FgVps52 resulted in the disruption of the normal subcellular localization of FgVps51-GFP, FgVps53-GFP and FgVps54-GFP on the TGN, causing them to be abnormally dispersed in the cytoplasm (Fig. 5a-c). In contrast, when FgVps52-GFP was expressed in the Δ Fgvps51 , Δ Fgvps53 and Δ Fgvps54 mutants, the subcellular localization of FgVps52-GFP remained unchanged compared to the wild type (Fig. 5d-g). Conversely, deletion of any of the three subunits FgVPS51 , FgVPS53 and FgVPS54 does not affect the subcellular localization of the other two subunits (Fig. S10). These findings suggest that FgVps52 plays a central role in regulating the assembly and recruitment of the GARP subunits to the TGN. The SNARE complex subunits FgTlg1 and FgTlg2 are critical for the growth, development and pathogenicity of F. graminearum Our previous studies have shown that the GARP complex regulates the growth, development and pathogenicity of F. graminearum by mediating the sorting and transport of the SNARE protein FgSnc1(Long et al., 2025). However, it remains unclear whether the GARP complex interacts with other SNARE proteins. To bridge this knowledge gap, we revisited our FgVps51-GFP and FgVps53-GFP IP-MS data, searching for any SNARE proteins as potential GARP interacting partners. Interestingly, we found FgSnc1 (FGSG_08537), FgVti1 (FGSG_13906), FgTlg1 (FGSG_07007) and FgTlg2 (FGSG_01890) as potential interactome which were significantly enriched in the IP-MS assays (Table S5). In fungi, Snc1, Vti1, Tlg1 and Tlg2 interact to form a SNARE complex(Adnan et al., 2023). Consistently, an in-silico analysis using AlphaFold3 revealed the possibility of interaction among these proteins in F. graminearum (Fig. 6a). This was experimentally validated by Y2H assays which showed positive interaction among FgSnc1, FgVti1 and FgTlg1, each of which directly interacts with FgTlg2 (Fig. 6b). While our earlier studies highlighted the essential roles of FgSnc1 in the growth, development and pathogenicity of F. graminearum (Zheng et al., 2018c), the functions of FgVti1, FgTlg1 and FgTlg2 remain poorly understood in filamentous fungi. To uncover the roles of these proteins in F. graminearum , we attempted to delete the genes encoding the proteins and successfully generated the null gene mutants Δ Fgtlg1 and Δ Fgtlg2 (Fig. S11). However, despite repeated attempts, we were unable to generate the ΔFgvti1 mutant, suggesting that FgVTI1 is a lethal gene. Phenotypic analysis of the Δ Fgtlg1 and Δ Fgtlg2 mutants revealed significantly reduced growth on CM medium compared to PH-1 and complemented strains (Fig. 6c, d). Additionally, we observed a marked reduction in the quantity of spores produced by the mutants. Moreover, when cultured in liquid carboxymethylcellulose (CMC) medium, the wild type and complemented strains produced morphologically normal conidia. In contrast, the Δ Fgtlg1 and Δ Fgtlg2 mutants produced conidia with fewer septa, as evidenced by CFW staining of the conidia (Fig. 6e, f). Furthermore, the mutant conidia were significantly shorter (Fig. 6g). Also, the conidia produced by the mutant strains were significantly fewer than those of PH-1 and complemented strains at different time points (Fig. 6h). Collectively, these findings underscore the critical roles of FgTlg1 and FgTlg2 in vegetative growth and asexual reproduction of F. graminearum . With respect to the fungal pathogenicity, the Δ Fgtlg1 and Δ Fgtlg2 mutants caused significantly milder wilt symptoms on wheat spikes than the control strains (Fig. 6i). Similarly, when the Δ Fgtlg1 and Δ Fgtlg2 strains were inoculated on coleoptiles, the disease symptoms induced by the mutants were markedly reduced after 7 days of incubation under moist conditions, relative to the PH-1 and complemented strains (Fig. 6j). To investigate the role of FgTlg1 and FgTlg2 in deoxynivalenol (DON) production, we measured the DON levels produced by the strains after 7 days of incubation in the dark at 28°C in liquid TBI medium. We found that the mutants had significantly reduced DON production compared to PH-1 and complemented strains (Fig. 6k). To corroborate these findings, we assessed the expression levels of the DON biosynthesis genes ( FgTRI1 , FgTRI4 and FgTRI12 ) in the mutant strains using qRT-PCR. As shown in Fig. 6l, the expressions of these genes were significantly downregulated in the mutants. In conclusion, the SNARE complex subunits FgTlg1 and FgTlg2 are essential for the growth, development and pathogenicity of F. graminearum . The GARP/retromer pathway transports the SNARE complex, regulating F. graminearum ’s development and pathogenicity To further investigate the possible mechanisms by which the SNARE complex subunits FgTlg1 and FgTlg2 regulate the growth, development and pathogenicity of F. graminearum , we generated GFP-FgTlg1 and GFP-FgTlg2 strains and performed subcellular localization analyses. The results showed that the GFP signals of GFP-FgTlg1 and GFP-FgTlg2 showed punctate distribution throughout the fungal cells (Fig. 7a, b). Additionally, co-localization analysis demonstrated that GFP-FgTlg1 and GFP-FgTlg2 co-localized with the TGN and also partially co-localized with endosomes (Fig. 7c, d, e, f). Notably, time-lapse microscopy further captured a continuous movement of GFP-FgTlg1 and GFP-FgTlg2 towards the TGN (Videos S1, S2). Our previous research has shown that the GARP complex recruits the retromer complex from the endosome to the TGN to establish a GARP/retromer transport pathway that mediates the transport of the cargo protein FgSnc1(Long et al., 2025) , We hypothesized that the GARP/retromer transport pathway also mediates the trafficking of the SNARE complex subunits FgTlg1 and FgTlg2 from endosomes to the TGN, thereby regulating growth and pathogenicity of F. graminearum . To analyze this speculation, we examined the direct interaction between FgVps52 and FgTlg1 or FgTlg2. Co-localization and co-immunoprecipitation (Co-IP) analyses showed that GFP-FgTlg1 and GFP-FgTlg2 significantly co-localized with FgVps52-mCherry and interacted in vivo (Fig. 8a-d). To further validate these results, we analyzed the subcellular localizations of GFP-FgTlg1 and GFP-FgTlg2 in Δ Fgvps52 mutant hyphae by confocal microscopy. The results indicated that deletion of FgVps52 caused GFP-FgTlg1 and GFP-FgTlg2 to be mis-localized to the vacuoles as confirmed by FM4-64 staining (Fig. 8e). Interestingly, we also observed that FgVps35 (the core subunit of the retromer complex) interacts with FgTlg1 and FgTlg2 in vivo (Fig. 9a, b, c, d). Deletion of FgVps35 similarly disrupted the normal localizations of GFP-FgTlg1 and GFP-FgTlg2, leading to their incorrect sorting to the vacuoles (Fig. 9e). These results suggest that the GARP/retromer-mediated vesicle trafficking pathway regulates the transport of the SNARE proteins FgTlg1 and FgTlg2 from endosomes to the TGN. Discussion Retrograde transport from endosomes to the TGN is a vital process in eukaryotic cells. Disruption of cargo sorting or transport within this pathway can lead to severe cellular dysfunction and has been implicated in the development of various diseases(Gallon and Cullen, 2015; Pei et al., 2023). Despite its importance, the molecular mechanisms underlying this complex process remain incompletely understood. In this study, we demonstrate that the GTP-bound active form of Rab6 interacts directly with the GARP complex subunit Vps52, recruiting it from the cytoplasm to the TGN. Vps52 subsequently mediates the recruitment of the remaining GARP subunits namely Vps51, Vps53 and Vps54, thereby promoting the assembly of the functional tetrameric GARP complex at the TGN. Once assembled, the GARP complex facilitates the recruitment of the retromer complex, which in turn mediates the sorting and transport of a SNARE proteins Snc1, Tlg1 and Tlg2s at the TGN. This Rab6-GARP-retromer pathway is critical for deoxynivalenol (DON) toxin production, as well as for the growth, development and pathogenicity of F. graminearum (Fig. 10). In eukaryotic cells, vesicular transport plays a crucial role in coordinating various cellular functions(Ma and Burd, 2020). The exchange of macromolecules like proteins and lipids between organelles is mainly regulated through vesicular transport(Abubakar et al., 2023). This intricate process encompasses several key stages, including vesicle budding, transport, tethering, docking and membrane fusion(Bonifacino and Glick, 2004). Tethering factors are critical for initiating the interaction between transport vesicles and their target membranes(Chou et al., 2016; Ma and Burd, 2020). To date, ten distinct tethering factor complexes have been identified in eukaryotes, each playing a unique regulatory role in various transport pathways(Desfougères et al., 2015; McDermott and Kim, 2015). Among these, the GARP complex is primarily localized to the TGN, where it functions as a tethering factor in endosomes-to-TGN retrograde transport pathway(Khakurel and Lupashin, 2023; Yu and Hughson, 2010). Earlier studies have emphasized the role of small GTPases, particularly Rab GTPases, in regulating vesicle transport between organelles(Stenmark, 2009). Rab GTPases act as molecular switches that coordinate vesicle budding, transport and fusion(Hutagalung and Novick, 2011). In this study, we used immunoprecipitation mass spectrometry (IP-MS) and identified 1,268 proteins that may interact with the GARP complex. Further bioinformatic analyses identified ten candidates Rab GTPases (Fig. S1). We found that FgRab6 is a key regulatory factor for the proper localization of the GARP complex to the TGN (Fig. 1), consistent with a previous report that Rab GTPases regulate the localizations and functions of tethering complexes(Gilleron et al., 2024). Studies also showed that Rab GTPases, including Rab6, interact with tethering factors such as the GARP complex to ensure efficient transport of proteins to target membranes(Homma and Fukuda, 2021; Liewen et al., 2005). In this study, we extended this understanding by demonstrating that FgRab6 specifically interacts with the GARP subunit FgVps52 through a conserved Q73 residue within its Rab domain. Notably, the Q73 residue is indispensable for the recruitment of FgVps52 to the TGN and is crucial for regulating the growth, development and pathogenicity of F. graminearum , suggesting a role unique to pathogenic fungi. Interestingly, the Q73-mediated interaction between FgRab6 and FgVps52 is evolutionarily conserved across fungi, yeast, plants and animals (Fig. 3). However, given the physiological divergence among these organisms, the functional consequences of Rab GTPase–GARP interactions may differ. Future research could explore how this conserved Rab6–Vps52 interaction contributes to species-specific cellular processes. Studies have shown that Rab proteins regulate intracellular transport pathways by interacting with a variety of downstream effectors(Hutagalung and Novick, 2011). The GTP-bound active form of the Rab proteins interacts with effector factors, recruiting them to specific sites(Aivazian et al., 2006; Grosshans et al., 2006). In this study, we elucidated that only the constitutively active form of Rab6 directly interacts with Vps52 to recruit it to the TGN (Fig. 4). We also highlighted that FgVps52 acts as an effector for FgRab6 in the TGN where it further regulates the recruitment and assembly of other GARP subunits (Fig. 4, 5, S9). Additionally, overexpression of FgRab6 in its GDP-bound form compromises the recruitment of FgVps52 to the TGN (Fig. 4 c), leading to phenotypic defects similar to those observed in the Δ Fgvps52 mutant strain (Fig. S8). This generally holds true in pathogenic fungi in which the active forms of Rab GTPases regulate the fungal pathogenicity by recruiting downstream effectors to the TGN(Wu et al., 2021). Therefore, targeting this form of the Rab GTPases could be a promising strategy for controlling the spread and impact of pathogenic fungi. While our study provided valuable new insights into the GARP/retromer retrograde transport pathway, several limitations must be acknowledged. It is still unclear how the active (GTP-bound) and inactive (GDP-bound) states of FgRab6 regulate the FgRab6-GARP complex. Although we have shown that the GTP-bound form of FgRab6 interacts with FgVps52 and recruits it to the TGN to regulate the recruitment and assembly of the GARP complex, the precise molecular details of this activation process, as well as the roles of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) in regulating this process remain unclear. Future studies utilizing advanced techniques such as cryo-electron microscopy (cryo-EM) or single-molecule imaging could provide further insights into the dynamic activation and regulation of Rab proteins during vesicular transport. Also, the biological role of the SNARE complex in the retrograde transport pathway is not well understood. While our results suggest that the GARP/retromer transport pathway is crucial for the transport of SNARE complex from endosomes to the TGN and for their proper localization at the TGN, the exact mechanism of SNARE-mediated membrane fusion in the context of retrograde transport remains to be elucidated. Future research could focus on the temporal and spatial regulation of SNARE complex assembly and membrane fusion events, employing live-cell imaging techniques to track SNARE dynamics in real time. Additionally, the inability to generate viable mutants for FgVti1 suggests that this protein is likely essential for fungal survival, necessitating further investigation into its specific role in retrograde transport. Future studies could utilize conditional mutation or RNA interference technologies to dissect the roles of FgVti1 in retrograde transport. Materials and Methods Fungal strains and culture conditions The F. graminearum wild type (WT) strain PH-1 was used as a control and as a background strain from which all strains (Table S6). All fungal strains were incubated at 28°C for two days on solid or liquid complete medium (CM), unless otherwise specified. Growth rates on CM plates and conidiation in liquid carboxymethylcellulose (CMC) medium were assessed using established methods(Zheng et al., 2016). Generation of Gene Deletion Mutants and Complementation To generate the mutants, a gene deletion strategy utilizing homologous recombination was employed(Yu et al., 2004). In brief, we amplified approximately 1 kb of the flanking sequences situated upstream and downstream of a target gene using the primers listed in Table S7. These fragments were subsequently fused with a hygromycin resistance gene cassette by overlap extension (SOE) PCR. The resulting construct was then transformed into the protoplasts of the wild type PH-1 strain. Initial screening of the transformants was conducted by PCR using the primer pairs detailed in Table S7, and further confirmation was performed by quantitative real-time PCR (qRT-PCR)(Xu et al., 2024). To complement the deleted genes, the full-length open reading frames (ORFs) of the targeted genes, along with their native promoters, were amplified and inserted into pKNTG vectors using ligation-independent cloning (C112-01, Vazyme, Nanjing, China), which ensured efficient and precise integration. To confirm the integrity and accuracy of the cloned sequences, the complementation vectors were sequenced before being transformed into the corresponding mutant protoplasts by PEG-mediated transformation. Pathogenicity and Deoxynivalenol (DON) Production Assays Following a method outlined in a previous study(Zheng et al., 2021), we assessed the pathogenicity of the fungal strains on wheat spikelets, coleoptiles. Disease symptoms were evaluated on wheat heads at the 14 days post-inoculation (dpi) and on coleoptiles at the 7 dpi. The levels of deoxynivalenol (DON) produced by the various fungal strains were quantified using a DON detection kit (Wiseste Biotech Co. Ltd, China) after a 7-day incubation in liquid trichothecene biosynthesis induction (TBI) media. For the analysis of TRI gene expressions, qRT-PCR was carried out using ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme, China). The primers used for the qRT-PCR analysis are provided in Table S7. Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescence Complementation (BiFC) Assays A yeast two-hybrid (Y2H) assay was conducted following standard protocols(Zheng et al., 2021). In brief, full-length cDNAs of the target genes were inserted into two vectors: pGADT7 (AD), which contains a GAL4 activation domain, and pGBKT7 (BD), which harbors a GAL4-binding domain. These constructs, representing the prey and bait, respectively, were introduced into Saccharomyces cerevisiae AH109 strain. All transformants were initially inoculated onto SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade media plates and incubated at 30 °C for 3 days. After colony selection, cell densities were adjusted to appropriate concentrations and spotted onto SD/-Trp/-Leu and SD/-Trp/-Leu/-His/-Ade plates supplemented with 20 mg/mL X-α-Gal, followed by incubation at 30 °C for another 3 days. The interaction between the BD-Lam and AD-T served as the negative control, while BD-53 and AD-T interaction was used as a positive control. For BiFC assays, FgVPS51 , FgVPS52 and FgVPS53 gene fragments were respectively cloned into pKNT-NYFP (N-terminal portion of the YFP fluorescent protein) vector, while FgRAB6 , FgRAB6-CA, FgVPS53 and FgVPS54 fragments were cloned into a pKNT-CYFP (C-terminal portion of the YFP fluorescent protein) vector, respectively. Strains expressing NYFP and/or CYFP, including FgVps52-NYFP+CYFP, NYFP+FgRab6-CYFP, FgVps51-NYFP+CYFP, NYFP+FgVps53-CYFP, NYFP+FgVps54-CYFP, FgVps53-NYFP+CYFP, NYFP+FgVps54-CYFP were used as negative controls. Hygromycin- and/or neomycin-resistant transformants were isolated and confirmed by PCR. YFP signals were visualized using a Nikon CUS-W1 spinning-disk confocal microscope (Nikon, Japan). Co-Immunoprecipitation (Co-IP) assay Total proteins were extracted from the tested strains and incubated with 30 μL magnetic anti-GFP agarose beads (Smart-Life Sciences, China) at 4°C for 4 hours to allow the GFP-tagged proteins to bind to the beads. After incubation, the beads were collected and washed three times with 500 μL cold wash buffer (50 mM Tris, 0.15 M NaCl, pH 7.4) to remove any unbound proteins or impurities. The washing procedure was repeated three more times with a magnetic frame and fresh cold buffer. After washing, the beads were resuspended in 100 μL wash buffer containing SDS-loading buffer. To elute the bound proteins, the samples were heated at 100°C for 15 minutes. The eluted proteins were then analyzed by immunoblot using anti-GFP antibodies (Abmart, China) and subjected to mass spectrometry analysis (Fujian Agriculture and Forestry University Analysis and Testing Center, China). For the Co-IP assay, strains expressing fusion proteins were cultured in liquid complete medium (CM) with continuous shaking at 110 rpm for 3 days. The mycelial biomass was filtered, washed with sterile distilled water, and ground to a fine powder using liquid nitrogen. The powder was then lysed in extraction buffer containing protease inhibitors (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40), and the total cell lysates were incubated with GFP-Trap A beads at 4°C for 4 hours. After addition of protein loading buffer (Cat. LT103; Epizyme Biotech), the bound proteins were eluted by heating to 100°C for 10 minutes and analyzed by Western blot as previously described(Chen et al., 2023). Staining and Live Cell Imaging of F. graminearum To observe the Spitzenkörper, plasma membrane, septa and vacuolar membranes, FM4-64 (Molecular Probes, Eugene, OR, USA) was used at a final concentration of 8 μM. Freshly collected conidia were stained with 0.1 mg/ml calcofluor white (CFW) (Sigma-Aldrich, USA) for 30 seconds to visualize septa. For subcellular localization studies on mycelia, a block of actively growing hyphae was excised from CM or SYM agar and placed upside down on a glass slide for direct observation using a confocal microscope. The excitation wavelengths used were 488 nm for GFP, 561 nm for mCherry and FM4-64, and 405 nm for CFW. Live cell fluorescence imaging was performed with a Nikon A1R laser scanning confocal microscope (Nikon, Japan) (Zheng et al., 2016). Images were captured within a single focal plane unless otherwise specified, and sequential images were exported as AVI files.Ten hyphae of comparable lengths were randomly chosen for analysis. The overall number of fluorescent puncta, including both GFP- and YFP-labeled signals, was quantified. Co-localized puncta were subsequently identified, and the co-localization rate was calculated as the percentage of co-localized spots relative to the total number of fluorescent signals, using the formula: (co-localized spots / total fluorescent spots) × 100. Protein structure prediction and molecular docking The protein sequences used in this study were retrieved from the national center for biotechnology information (NCBI) database (https://www.ncbi.nlm.nih.gov/). Protein structure predictions and molecular docking simulations were performed using AlphaFold3, with structural visualization carried out in PyMOL software. Additionally, molecular docking between FgRab6-DN and guanosine diphosphate (GDP) as well as between FgRab6-CA and guanosine triphosphate (GTP) was conducted and the results were visualized using PyMOL. Acknowledgements We appreciate Prof. Jie Zhou, Drs Lili Lin, Jiexiong Hu, Yingzi Yun as well as Jia Chen, Shuyuan Cheng, and Dingyang Zhang for their helpful suggestions. We also appreciate Dr. Qiurong Xie of the Fujian University of Traditional Chinese Medicine for providing us with the cDNA of Rattus norvegicus . We would like to thank the Instrumental Analysis Center of Fujian Agriculture and Forestry University for mass spectrometric analysis. This research was supported by the National Natural Science Foundation of China (32272481, 32122071). Conflicts of Interest The authors declare no conflicts of interest. Data availability The data supporting the findings of this study are accessible within the main paper and its Supplemental Information files. Source data are provided in this paper. 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The endosomal recycling of FgSnc1 by FgSnx41-FgSnx4 heterodimer is essential for polarized growth and pathogenicity in Fusarium graminearum . New Phytol 219, 654-671.Zheng, W., Zheng, H., Zhao, X., Zhang, Y., Xie, Q., Lin, X., Chen, A., Yu, W., Lu, G., Shim, W.B. , et al. (2016). Retrograde trafficking from the endosome to the trans-Golgi network mediated by the retromer is required for fungal development and pathogenicity in Fusarium graminearum . New Phytol 210, 1327-1343. Figure legend Fig. 1. The Rab GTPase FgRab6 is essential for the punctate localization of the GARP complex subunits FgVps51 and FgVps53 in F. graminearum . (a) The GARP complex subunits FgVps51-GFP and FgVps53-GFP were expressed in various mutant strains, including FgRab1-DN (dominant-negative, S22N), Δ Fgrab8, Δ Fgrab7, Δ Fgrab6, Δ Fgrabx, Δ Fgrab52, Δ Fgrab51, FgRab11-DN (dominant-negative, I121N), Δ Fgrab2 and Δ Fgrab4 . FgRab1-DN and FgRab11-DN serve as essential genes in F. graminearum . (b) Deletion of FgRAB6 disrupts the colocalization of the GARP complex subunit FgVps51 with the TGN marker FgSec7-mCherry, resulting in its mislocalization to the cytoplasm. (c) Deletion of FgRAB6 hinders the colocalization of the GARP complex subunit FgVps53 with FgSec7-mCherry, leading to its accumulation in the cytoplasm. Scale bar = 10 µm. DIC, differential interference contrast. Fig. 2. Analysis of the interaction of the GARP complex with FgRab6. (a) A yeast two-hybrid (Y2H) assay was used to test the interaction between FgRab6 and each of the components of the GARP complex. pGBKT7-53/pGADT7-T (labelled P) and pGBKT7-Lam/pGADT7-T (labelled N) were used as positive and negative controls, respectively. Yeast transformants expressing the indicated constructs were screened for growth on selective media containing SD-Leu-Trp and SD-Trp-Leu-His-Ade, and tested for α-galactosidase (LacZ) activity. (b) FgVps52-GFP colocalizes with mCherry-FgRab6 in F. graminearum . White arrow shows the colocalization sites. (c) A bimolecular fluorescence complementation (BiFC) assay confirming the interaction of FgVps52-NYFP with CYFP-FgRab6 in vivo . The punctate YFP signals of FgVps52-NYFP and CYFP-FgRab6 colocalized significantly with the trans-Golgi network (TGN) marker FgSec7-mCherry, respectively. The negative control used for the assay is shown in Fig. S2. (d) Molecular docking analysis for FgVps52 (cyan) and FgRab6 (green) proteins in F. graminearum . (e) Schematic diagram showing eight specific residues in FgRab6 protein. The RAB domain of FgRab6 is highlighted in green, with specific amino acid residues (Q23, D40, Y43, I47, G72, Q73, R75, and Y83) that are involved in the interaction with FgVps52. (f) The interaction of FgRab6 with FgVps52 occurs through the RAB domain of FgRab6. (g-h) Deletion of the RAB domain in FgRab6 impairs the recruitment of FgVps52 to the TGN, resulting in its cytoplasmic accumulation. Scale bar = 10 µm. DIC, differential interference contrast. Fig. 3 Functional analysis of the conserved amino acid residue Q73 in FgRab6. (a) Molecular docking analysis of FgVps52 (cyan) and FgRab6 (green) proteins. Molecular docking analysis of NcVps52 (magenta) and NcRab6 (cyan) proteins from Neurospora crassa . Molecular docking analysis of FoVps52 (green) and FoRab6 (magenta) proteins from Fusarium oxysporum . Molecular docking analysis of OsVps52 (cyan) and OsRab6 (orange) proteins from Oryza sativa . Molecular docking analysis of ScVps52 (magenta) and ScRab6 (blue) proteins from Saccharomyces cerevisiae . Molecular docking analysis of RnVps52 (orange) and RnRab6 (yellow) proteins from Rattus norvegicus . (b) Yeast two-hybrid (Y2H) assays revealed positive interactions between Vps52 and Rab6 homologs in different species, including F. graminearum (FgVps52–FgRab6), F.oxysporum (FoVps52–FoRab6), N.crassa (NcVps52–NcRab6), S.cerevisiae (ScVps52–ScRab6), O.sativa (OsVps52–OsRab6), and R.norvegicus (RnVps52–RnRab6). (c) Sequence alignment analysis showed that the Q73 residue of FgRab6 is conserved across multiple species. (d) Y2H assays were performed to examine the impact of point mutations on the interaction between FgRab6 and FgVps52. The Q73 residue was essential for their interaction. In the constructs FgRab6 Q73A and FgRab6 T46A , glutamine (Q) and threonine (T) were substituted with alanine (A), respectively. (e) The FgRab6 Q73A mutant blocks FgVps52-GFP localization to the TGN. (f) Pathogenicity assays on wheat heads demonstrated that the FgRab6 Q73A mutant exhibited significantly reduced virulence. (g) Y2H assays were further used to validate the role of the conserved Q73 residue in mediating Rab6–Vps52 interactions in both animal ( R. norvegicus ) and plant ( O. sativa ) systems. Statistical analysis was processed by one-way ANOVA for multiple comparisons using GraphPad Prism 9 (****p 0.05). Scale bar = 10 µm. DIC, differential interference contrast. Fig. 4 Effects of the GDP- and GTP-bound states of FgRab6 on the recruitment of FgVps52 to the TGN. (a) The 3D structure of the GDP-bound T28N mutant of FgRab6 was predicted using AlphaFold3 and molecular docking was performed with GDP. Similarly, the 3D structure of the GTP-bound Q73L mutant of FgRab6 was predicted using AlphaFold3 and molecular docking was conducted with GTP. (b) The expression levels of FgRab6-CA (constitutively active, Q73L) and FgRab6-DN (dominant negative, T28N) were quantified. Standard deviations were calculated from three biological replicates. (c) FgRab6-CA is essential for the proper localization of FgVps52 to the TGN. In the wild-type PH-1 strain, FgVps52-GFP co-localized with the TGN marker FgSec7. Similarly, in the FgRab6-CA overexpressed strain, FgVps52-GFP also co-localized with FgSec7 in the TGN. In contrast, overexpression of FgRab6-DN resulted in mis-localization of FgVps52-GFP to the cytoplasm, thereby preventing its proper localization to the TGN. (d) GFP-FgRab6-CA co-localized with the TGN marker FgSec7-mCherry. Zoomed (boxed) regions represent regions of partial co-localization (white) of GFP-FgRab6-CA with FgSec7-mCherry. (e) GFP-FgRab6-DN was dispersed throughout the cytoplasm in PH-1 mycelia, resulting in loss of co-localization with the TGN marker FgSec7-mCherry. (f) Yeast two-hybrid (Y2H) assay confirming the interaction between FgVps52 and the constitutively active form of FgRab6 (FgRab6-CA). (g) Bimolecular fluorescence complementation (BiFC) assay further verifying the interaction of FgVps52-NYFP with CYFP-FgRab6-CA in vivo . The punctate YFP signals colocalized significantly with the TGN marker FgSec7-mCherry. Statistical analysis was conducted using one-way ANOVA for multiple comparisons using GraphPad Prism 9 (****p < 0.0001). Scale bar = 10 µm. DIC, differential interference contrast. Fig. 5. FgVps52 is essential for the recruitment of the GARP complex to the TGN in F. graminearum . (a) FgVps51-GFP co-localized with FgSec7 in the growing hyphae of the wild-type strain (PH-1), but failed to localize to the TGN in the Δ FgVps52 strain. (b) Similarly, FgVps53-GFP co-localized with FgSec7 in PH-1 strain but not in Δ Fgvps52 mutant strain. (c) FgVps54-GFP also co-localized with the TGN marker FgSec7 in PH-1 strain but not in Δ Fgvps52 mutant strain. (d-g) Deletion of FgVPS51 , FgVPS53 or FgVPS54 does not affect the TGN localization of FgVps52. In PH-1 hyphae, FgVps52-GFP co-localized with FgSec7-mCherry. FgVps52-GFP localized to the TGN in Δ Fgvps51 , Δ Fgvps53 and Δ Fgvps54 mutants, respectively. White arrows highlight the sites of co-localization. Scale bar = 10 µm. DIC, differential interference contrast. Fig. 6. The SNARE complex subunits FgTlg1 and FgTlg2 are critical for the virulence of F. graminearum . (a) AlphaFold3-generated model illustrating the complex formed by FgSnc1, FgTlg1, FgVti1 and FgTlg2. (b) Yeast two-hybrid (Y2H) assays showing the interaction dynamics between FgSnc1, FgTlg1, FgTlg2 and FgVti1. (c) Colony morphology of the wild-type strain (PH-1), gene deletion mutants (Δ Fgtlg1 and Δ Fgtlg2 ) and their complemented strains (Δ Fgtlg1-C and Δ Fgtlg2-C ) cultured on CM media for two days. (d) The colony diameters of the tested strains cultured on CM media under same experimental conditions. (e) Conidial septation of the indicated strains after calcofluor white (CFW) staining. (f) Analysis of the percentage of conidia with different number of septa in the tested strains. (g) Analysis of conidial lengths of the tested strains. (h) A significant reduction in conidia production was observed in Δ Fgtlg1 and Δ Fgtlg2 mutants at various time points. (i) Wheat heads inoculated with mycelial plugs from PH-1, Δ Fgtlg1 , Δ Fgtlg2 and their respective complemented strains. The number of infected spikelets per wheat head was recorded and photographed at 14 days post-infection (dpi). (j) Assessment of pathogenicity on wheat coleoptiles, with lesion lengths measured at 7 dpi. (k) Bar graphs representing DON production by the indicated strains in liquid trichothecene biosynthesis induction (TBI) medium. (l) Bar graphs showing the relative expression levels of FgTRI1, FgTRI4 and FgTRI12 genes in the tested strains. Values represent means of independent experiments. Statistical analysis was processed by one-way ANOVA for multiple comparisons using GraphPad Prism 9 (****p 0.05). Scale bar = 10 µm. DIC, differential interference contrast. Fig. 7. Subcellular localizations of the SNARE complex subunits FgTlg1 and FgTlg2 (a, b) GFP-labeled FgTlg1 and FgTlg2 exhibit punctate localization at the tips and bases of vegetative hyphae in F. graminearum . (c, d) GFP-FgTlg1 and GFP-FgTlg2 show partial co-localization with the TGN marker FgSec7-mCherry. Zoomed (boxed) regions represent regions of partial co-localization (white) of GFP-FgTlg1 and GFP-FgTlg2 with FgSec7-mCherry, with co-localization rates of 61.01 ± 4.78% for FgTlg1 and 58.75 ± 5.36% for FgTlg2. (e, f) GFP-FgTlg1 and GFP-FgTlg2 also showed partial co-localization with the early endosome marker mCherry-FgRab52. The zoomed areas represent regions of partial co-localization (white) of GFP-FgTlg1 and GFP-FgTlg2 with mCherry-FgRab52, with co-localization rates of 41.07 ± 8.13% for FgTlg1 and 42.29 ± 7.63% for FgTlg2. The line scans were generated at the positions indicated by the arrows in the zoomed areas, respectively, to depict the relative fluorescence intensity of the co-localization. Scale bar = 10 µm. DIC, differential interference contrast. Fig. 8. Interaction and regulation of the TGN-Associated SNARE proteins FgTlg1 and FgTlg2 by the GARP complex subunit FgVps52. (a, b) Co-localization of GFP-FgTlg1 and GFP-FgTlg2 with FgVps52-mCherry, respectively, was observed in F. graminearum hyphae. The zoomed (boxed) regions represent the regions of partial co-localization (white) of GFP-FgTlg1 and GFP-FgTlg2 with FgVps52-mCherry, respectively, as indicated by the white arrows. Line scan analyses were performed at the positions indicated by the arrows to visualize the relative fluorescence intensity of FgVps52-mCherry alongside GFP-FgTlg1 and GFP-FgTlg2. (c, d) Co-immunoprecipitation assays demonstrating positive interactions of FgVps52 with FgTlg1 and FgTlg2, respectively. Immunoprecipitation was performed on the strains co-expressing these proteins using GFP-trap beads. Immunoblot analysis involving GFP- and Myc-specific antibodies confirmed the presence of immunoprecipitated signals (GFP-FgTlg1 and GFP-FgTlg2) and co-immunoprecipitated FgVps52-Myc signal. (e) Deletion of FgVps52 disrupted the punctate localization of FgTlg1 and FgTlg2, resulting in their mis-sorting and subsequent transport to the vacuolar membrane. Scale bar = 10 µm. DIC, differential interference contrast. Fig. 9. Interaction and regulation of the TGN-Associated SNARE proteins FgTlg1 and FgTlg2 by the retromer complex subunit FgVps35. (a, b) Co-localization of GFP-FgTlg1 and GFP-FgTlg2 with FgVps35-mCherry, respectively, was observed in F. graminearum hyphae. The zoomed (boxed) regions represent the regions of partial co-localization (marked by the white arrows) of GFP-FgTlg1, GFP-FgTlg2 and FgVps35-mCherry. Line scan analysis, performed at the arrow-marked positions, shows the relative fluorescence intensity of FgVps35-mCherry alongside GFP-FgTlg1 and GFP-FgTlg2. (c, d) Co-immunoprecipitation experiments revealed positive interactions of FgVps35 with FgTlg1 and FgTlg2, respectively. These interactions were confirmed by immunoprecipitation on the strains co-expressing these proteins, using GFP-trap beads. The immunoprecipitated GFP-tagged proteins (GFP-FgTlg1 and GFP-FgTlg2) and the co-immunoprecipitated FgVps35-Myc protein were detected via immunoblot analysis, involving GFP- and Myc-specific antibodies. (e) Deletion of FgVps35 disrupted the punctate distribution of FgTlg1 and FgTlg2, leading to their mis-sorting and transport to the vacuolar membrane. This mis-localization impaired the proper transport of FgTlg1 and FgTlg2 from early endosomes to the trans-Golgi network (TGN). Scale bar = 10 µm. DIC, differential interference contrast. Fig. 10. A working model illustrating the relationship between Rab6-GARP-retromer -mediated transport of TGN-associated SNARE proteins from endosomes to the TGN in Fusarium graminearum (a) Under normal conditions, Rab6 interacts with the GARP complex subunit Vps52 through its amino acid residue Q73 when in its active GTP-bound state (Rab6-GTP). This interaction facilitates the recruitment of Vps52 to the TGN, which subsequently enables the recruitment of the entire GARP subunits to the TGN. This process promotes the retrograde transport of the retromer complex from early endosomes to the TGN, thereby ensuring proper sorting and transport of TGN-associated SNARE proteins (including FgSnc1, FgTlg1 and FgTlg2). This mechanism is essential for the effective delivery of these cargo proteins from the early endosomes to the TGN. (b) In the absence of Vps52, the GARP complex fails to be recruited to the TGN, thereby disrupting cargo delivery to the TGN. This disruption impedes the retrograde transport of the retromer complex from the endosomes to the TGN. Consequently, the TGN-associated SNARE proteins (FgSnc1, FgTlg1, and FgTlg2) are misrouted to the vacuolar degradation pathway. This misrouting compromises the growth, development and pathogenicity of F. graminearum . Information & Authors Information Version history V1 Version 1 12 August 2025 Peer review timeline Published Plant, Cell & Environment Version of Record 11 Jan 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Plant, Cell & Environment Keywords fusarium graminearum cargo sorting garp rab signaling Authors Affiliations Yunfei Long Fujian Agriculture and Forestry University View all articles by this author Haoran Zhang Fujian Agriculture and Forestry University View all articles by this author Xingyuan Wu Fujian Agriculture and Forestry University View all articles by this author Xin Chen Fujian Agriculture and Forestry University View all articles by this author Ying Lin Jingdezhen People's Government View all articles by this author Yakubu Saddeeq Abubakar 0000-0002-5228-0548 Fujian Agriculture and Forestry University View all articles by this author Huawei Zheng Minjiang University View all articles by this author Zonghua Wang 0000-0002-0869-9683 Fujian Agriculture and Forestry University View all articles by this author Wenhui Zheng [email protected] Fujian Agriculture and Forestry University View all articles by this author Metrics & Citations Metrics Article Usage 251 views 172 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yunfei Long, Haoran Zhang, Xingyuan Wu, et al. Conserved retrograde trafficking mechanisms regulate fungal development and pathogenicity through Rab6-GARP-Retromer-SNARE coordination. Authorea . 12 August 2025. DOI: https://doi.org/10.22541/au.175497706.67845306/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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