FgGyp8 as a putative FgRab1 GAP is required for growth and pathogenesis by regulating FgSnc1-mediated secretory vesicles fusion in Fusarium graminearum

ZHANG Xing-zhi, CHEN Shuang, Yakubu Saddeeq ABUBAKAR,3, MAO Xu-zhao, MIAO Peng-fei,WANG Zong-hua, ZHOU Jie#, ZHENG Hua-wei

1 Fujian Key Laboratory on Conservation and Sustainable Utilization of Marine Biodiversity/Ministerial and Provincial Joint Innovation Centre for Safety Production of Cross-Strait Crops, Minjiang University, Fuzhou 350108, P.R.China

2 Fujian University Key Laboratory for Plant-Microbe Interaction, College of Life Sciences, Fujian Agriculture and Forestry University,Fuzhou 350002, P.R.China

3 Department of Biochemistry, Faculty of Life Sciences, Ahmadu Bello University, Zaria 810211, Nigeria

Abstract Fusarium graminearum is an important plant pathogenic fungus that causes disease and yield reduction in many cereal crops, such as wheat and barley.Gyp8 stimulates GTP hydrolysis on Ypt1 in yeast.However, the functions of Gyp8 in plant pathogenic fungi are still unknown.In this study, we investigated the roles of FgGyp8 in F.graminearum by genetic and pathological analyses.Through gene knockout and phenotypic analyses, we found that FgGyp8 is required for vegetative growth in F.graminearum.The conidiation, conidial size and number of septa per conidium of ΔFggyp8 mutant are significantly reduced when compared to the wild type PH-1.Furthermore, FgGyp8 is crucial for pathogenicity on wheat coleoptiles and wheat heads.FgGyp8 contains a conserved TBC domain.Domain deletion analysis showed that the TBC domain, C- and N-terminal regions of FgGyp8 are all important for its biological functions in F.graminearum.Moreover, we showed that FgGyp8 catalyzes the hydrolysis of the GTP on FgRab1 to GDP in vitro, indicating that FgGyp8 is a GTPase-activating protein (GAP) for FgRab1.In addition, we demonstrated that FgGyp8 is required for FgSnc1-mediated fusion of secretory vesicles with the plasma membrane in F.graminearum.Finally, we showed that FgGyp8 has functional redundancy with another FgRab1 GAP, FgGyp1, in F.graminearum.Taken together, we conclude that FgGyp8 is required for vegetative growth, conidiogenesis, pathogenicity and acts as a GAP for FgRab1 in F.graminearum.

Keywords: Fusarium graminearum, FgGyp8, GTPase-activating protein, FgRab1, conidiogenesis, pathogenicity

1.Introduction

Fusariumgraminearumis the causative agent of Fusarium head blight of wheat and barley around the world, and it was listed as one of the top 10 fungal plant pathogens (Bushnellet al.2003; Deanet al.2012;Gardineret al.2020; Gonget al.2021; Shiet al.2021;Sunet al.2021).Its infection not only results in severe yield losses but also contaminates the host grains with various mycotoxins, especially deoxynivalenol (DON)(Chenet al.2019).The mechanisms for virulence and regulation of trichothecene biosynthesis have been widely studied during the past three decades (Chenet al.2019).In recently years, various studies suggest that vesicle trafficking machineries like Rab GTPases,SNAREs, retromer, ESCRT complex, and polarisome are required for the development and pathogenicity ofF.graminearum(Zhenget al.2015, 2016; Zhanget al.2016; Liet al.2017, 2019; Xieet al.2019; Adnanet al.2020; Zheng Het al.2021).

Rab proteins (designated as Ypt in yeast) represent the largest branch of the Ras-like small GTPase superfamily and there are 66 Rab genes in the human genome (Li and Segev 2012; Li and Marlin 2015).Rab proteins function as molecular switches in membrane trafficking,including coat recruitment, uncoating, fission, motility,target selection and fusion (Stenmark 2009; Li and Segev 2012; Mizuno-Yamasakiet al.2012).In recent years, Rab GTPases have been shown to be important for development and pathogenicity of phytopathogenic fungi, including but not limited toUstilagomaydis,Botrytiscinerea,F.graminearum,Magnaportheoryzae,Fusariumverticillioides(Bielskaet al.2014; Zhanget al.2014; Zhenget al.2015, 2016; Yanget al.2017; Yanet al.2020; Yuanet al.2022).Rab5 is required for longdistance retrograde motility of early endosomes to trigger transcription of effector-encoding genes during plant infection inU.maydis(Bielskaet al.2014).MoSec4 is involved in vegetative development and pathogenicity by regulating extracellular protein secretion, while MoYpt7 is required for membrane fusion during autophagy and for pathogenicity inM.oryzae(Liuet al.2015; Zhenget al.2016).FgRab1 plays vital roles in vegetative growth,conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking inF.graminearum(Zhenget al.2015; Yuanet al.2022).Similarly, FgRab5 and FgRab7 have been demonstrated to mediate the biogenesis of endosomes as well as the recruitment of the retromer complex to the endosomes for cargo sorting inF.graminearum(Abubakaret al.2021).

Rab GTPases are regulated by guanine nucleotide exchange factor (GEF) cascades, GTPase-activating protein (GAP) cascades and effectors.GEFs catalyze the displacement of prebound GDP, converting it to GTP,while GAPs mediate the intrinsic hydrolysis of GTP to GDP (Mizuno-Yamasakiet al.2012).Previous studies identified several regulators of FgRab GTPases (Zheng Het al.2018, 2021; Yanget al.2020; Zheng Qet al.2021).FgSec2A, a GEF of FgRab8, is important for polarized growth, virulence and DON production inF.graminearum(Zheng Het al.2018).FgVps9, a Rab5 GEF, is critical for DON biosynthesis and plant infection inF.graminearum(Yanget al.2020).FgMon1, a GEF of FgRab7, is important for vacuole fusion, autophagy and pathogenicity inF.graminearum(Liet al.2015).

So far, most identified Rab GAPs contain Tre2–Bub2–Cdc16 (TBC) domains in mammalian cells and yeast(Fukuda 2011).In our previous study, we identified 12 TBC domain-containing proteins inF.graminearumgenome (Zheng Het al.2021), among which FgMsb3 is a GAP of FgRab8 and regulates the polarized trafficking,vegetative growth and pathogenicity ofF.graminearum(Zheng Het al.2021), and FgGyp1 is a GAP of FgRab1 and is important for vegetative growth, conidiation, and virulence and negatively regulates DON biosynthesis(Zheng Qet al.2021).However, the roles of other TBC domain-containing proteins are still unknown inFgraminearium.In yeast, Gyp8 encodes a Rab GAP whose preferred substrate is Ypt1 (De Antoniet al.2002;Cooperet al.2006).Overexpression ofGYP8suppresses αSyn toxicity (Cooperet al.2006).In addition, Gyp8 also stimulates GTP hydrolysis on Ypt31, Ypt32, Ypt6,and Sec4, and controls the retrograde trafficking and exocytosis of secretory vesicles (De Antoniet al.2002;Brettet al.2008).The functions of Gyp8 homolog proteins in plant pathogenic fungi are still unclear and whether it has GTP hydrolysis activityinvitroalso awaits future investigations.

In this study, we systematically investigated the roles of Gyp8 in the wheat head blight fungusF.graminearum, and found that FgGyp8 is required for vegetative growth, conidiogenesis and pathogenicity of the fungus.Furthermore, domain deletion analysis showed that TBC domain and the N- and C-terminal regions are important for the functions of FgGyp8.Strikingly, FgGyp8 was found to stimulate GTP hydrolysis on FgRab1invitro.We further demonstrated that FgGyp8 is required for FgSnc1-mediated fusion of secretory vesicles with the plasma membrane inF.graminearum.

2.Materials and methods

2.1.Strains, media and growth conditions

TheF.graminearumwild type PH-1 and the mutants used in this study are listed in Table 1.Growth rates of the various strains were measured on complete media(CM), minimal media (MM) and starch yeast media (SYM)after incubation at 28°C for 3 days, as previously reported(Zhenget al.2015; Zhanget al.2021).

Table 1 Wild type (PH-1) and mutant strains of the fungus used in this study

2.2.Fungal transformation, gene deletion and complementation

Protoplast preparation andF.graminearumtransformation were performed following standard protocols as previously reported (Houet al.2002).A split-marker approach was adopted to generate gene replacement mutants ofFgGYP8(Catlettet al.2003).The primers used to amplify the flanking sequences ofFgGYP8gene are listed in Table 2, and the transformants were screened by PCR and further confirmed by Southern blotting.For complementation, we amplified the coding sequence ofFgGYP8together with its native promoter(totaling 3 159-bp in length) from PH-1 genome using the primers FgGYP8-CF/FgGYP8-GR, and then cloned it into pKNTG2 vector after digesting it with the restriction enzymesHindIII andKpnI using the ClonExpress?II One Step Cloning Kit (Vazyme Biotech Co., Ltd.,Nanjing, China).Finally, the pKNTG-FgGyp8 vector was sequenced for verification.FgGYP1andFgGYP8double deletion mutants were constructed by deletingFgGYP1gene in the ΔFggup8mutant using neomycinresistant marker (G418) and putative ΔFggyp1ΔFggyp8double deletion mutants were screened by PCR using the primers listed in Table 2.

Table 2 PCR primers used in this study

2.3.Construction of pKNTG-FgGyp8ΔN, pKNTG-Fg-Gyp8ΔTBC and pKNTG-FgGyp8ΔC vectors

The domain deletion construct pKNTG-FgGyp8ΔNwas generated by PCR amplification of the sequences from pKNTG-FgGyp8 plasmid using FgGYP8CF/FgGYP8ZR-N,FgGYP8NF/FgGYP8GR primer pairs (Table 2)respectively, and cloned into pKNTG2 vector using ClonExpress?II One Step Cloning Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and verified by sequence analysis.For pKNTG-FgGyp8ΔTBCconstruct, FgGYP8CF/FgGYP8TBC1R and FgGYP8TBC2F/FgGYP8GR primer pairs were used to amplify FgGyp8 sequence from pKNTG-FgGyp8 plasmid respectively, and cloned into pKNTG2 vector and verified by sequence analysis.For pKNTG-FgGyp8ΔCvector, the sequence from pKNTGFgGyp8 plasmid was amplified using FgGYP8CF/FgGYP8ZR-C primer pairs and then cloned into pKNTG2 vector and further verified by sequencing.

2.4.Pathogenicity and DON production assays

Conidial suspension was prepared for inoculation on young wheat coleoptiles as previously reported (Yinet al.2018), and disease symptoms were observed 7 days after inoculation.Infection assays on flowering wheat heads were performed as previously described (Zhenget al.2015) and symptoms were observed and photographed 14 days after inoculation.For DON production assays,the wild type PH-1, mutants and complemented strains were grown in liquid trichothecene biosynthesis induction(TBI) media incubated at 28°C for 7 d in the dark.DON was then assayed as previously described (Zheng Het al.2018).All experiments were repeated three times.

2.5.GTPase activity assay

pMBP-FgGyp8 vector was constructed by amplifyingFgGYP8coding sequence from the cDNA of PH-1 using the primer pairs listed in Table 2.The amplicon was then cloned into the Maltose-binding protein (MBP)vector pMAL-c2X.pMBP-FgRab1 vector was similarly constructed as reported previously (Zheng Qet al.2021).MBP-FgGyp8, MBP-FgRab1 and MBP proteins were expressed inEscherichiacoliBL21 strain and purified by Amylose resin (Sangon Biotech, Shanghai, China, No.C500096), respectively.Next, these proteins were used for GAP activity assay using a GTPase Assay Kit (Sigma-Aldrich, St.Louis, MO, USA, Catalog Number MAK113)according to the manufacturer’s protocols.

2.6.Live cell imaging of F.graminearum

For hyphal tip localization assay, a mycelial block from SYM agar was excised around the growing edge of the hyphae and placed upside down on a coverslip and observed directly under a laser confocal microscope.The calcofluor white (CFW) excitation used was 405 nm light.GFP excitation was performed at 488 nm light.

2.7.Accession numbers

All the Gyp8 protein sequences used in this study can be found at the National Center for Biotechnology Information(NCBI, https://www.ncbi.nlm.nih.gov/) database.The detailed protein sequence accession numbers are as follows:

FgGyp8 (Fusariumgraminearum, XP_011317808.1),FvGyp8 (Fusariumverticillioides, XP_018753244.1),FoGyp8 (Fusariumoxysporum, EGU78277.1), VdGyp8(Verticilliumdahlia, RBQ90076.1), MoGyp8 (Magnaporthe oryzae, XP_003711868.1), NcGyp8 (Neurospora crassa, XP_961909.1), ScGyp8 (Saccharomyces cerevisiae, NP_444296.1), BcGyp8 (Botrytiscinerea,XP_001551510.1), SsGyp8 (Sclerotiniasclerotiorum,XP_001597680.1), UmGyp8 (Ustilagomaydis,XP_011391424.1).

3.Results

3.1.ldentification of FgGyp8 in Fusarium graminearum

To identify the FgGyp8 protein inF.graminearumgenome, we used theS.cerevisiaeGyp8 amino acid sequence as a reference to carry out a BLAST search against the available fungal genomes (https://fungidb.org/fungidb/app).We identified a homolog of Gyp8 at the FGSG_01953 locus ofF.graminearumgenome.FGSG_01953 encodes a protein of 420 amino acid residues and it is 25.98% similar to theS.cerevisiaeGyp8.It covers 58.00% of the total length of FgGyp8 and ScGyp8.Phylogenetic analysis of Gyp8 homologs suggest that the protein is conserved in phytopathogenic fungi, especially inF.oxysporum,F.verticillioides, andM.oryzae(Appendix A).

3.2.Generation and characterization of FgGYP8 deletion mutant

To investigate the roles of FgGyp8 inF.graminearum,targeted gene replacement strategy was used to deleteFgGYP8gene in the wild type PH-1 (Appendix B-a).Three mutants were identified by PCR and subjected to Southern blot for confirmation, after which ΔFggyp8-3was identified as the correct mutant (Appendix B-b).Furthermore, we performed gene complementation by reintroducingFgGYP8gene along with its native promoter into the ΔFggyp8mutant, and we succeeded in generating the complemented strain (ΔFggyp8-C) of ΔFggyp8mutant in which the defects observed in the mutant were restored.

3.3.FgGyp8 plays important roles in vegetative growth and conidiogenesis of F.graminearum

To determine the function of FgGyp8 in vegetative growth ofF.graminearum, the wild type PH-1,FgGYP8gene deletion mutant (ΔFggyp8) and complemented strain(ΔFggyp8-C) were inoculated on complete media (CM),minimal media (MM) and starch yeast media (SYM) and incubated at 28°C.After 3 days of incubation, colony morphology and diameters of each strain were measured and analyzed.As shown in Fig.1-A and B, we found that the colony morphology of ΔFggyp8mutant was similar to that of the PH-1, but the colony growth was significantly reduced as compared to the PH-1 and ΔFggyp8-C.We therefore conclude that FgGyp8 is required for normal vegetative growth ofF.graminearum.

Conidia formed byF.graminearumare believed to be the main inoculums infecting flowering wheat heads (Franclet al.1999).To know whether FgGyp8 is required for conidiogenesis inF.graminearum, the PH-1,ΔFggyp8and ΔFggyp8-Cstrains were inoculated in liquid carboxymethylcellulose (CMC) media for conidia production and incubated at 28°C for 3 days.The conidia produced by the strains were harvested and observed under a microscope.We found that ΔFggyp8mutant could produce conidia but the sizes of the conidia were smaller than those produced by the PH-1 and complemented strain (Fig.2-A).The average length of the conidia produced by ΔFggyp8mutant is only 22.86 μm while those from the PH-1 and complemented strain have average lengths of 36.48 and 33.01 μm, respectively (Fig.2-B).Similarly, the average width of ΔFggyp8mutant conidia is 5.21 μm while those of PH-1 and complemented strain are 4.77 and 4.65 μm, respectively (Fig.2-C).The ratio of conidia length to width of ΔFggyp8mutant is significantly smaller (4.39) than PH-1 (7.65) and ΔFggyp8-Cstrain(7.10).We further stained the conidia with CFW (10 μg mL–1) to visualize their septa under a fluorescence microscope.We observed that different mutants’ conidia had different numbers of septa as showed in Fig.2-D.From the results, it is obvious that more than 60% of the conidia produced by the ΔFggyp8mutant have less than 2 septa per conidium (Fig.2-D), while only 20% of the conidia produced by the wild type and the complemented strain have this number of septa (Fig.2-D).Quantitative statistical analyses showed that the average number of septa per conidium and the number of conidia produced by the ΔFggyp8mutant were significantly less than those of the wild type PH-1 and complemented strain ΔFggyp8-C(Fig.2-E and F).Taken together, these results indicate that FgGyp8 is required for conidiogenesis and conidial morphology inF.graminearum.

In addition to conidia, ascospores also act as primary inoculums inF.graminearumduring overwintering (Sunet al.2021).Therefore, we checked the possibility of forming asci and ascospores by ΔFggyp8mutant.As shown in Fig.2-G, the results indicate that PH-1,ΔFggyp8and ΔFggyp8-Cstrains produced similar sizes of perithecia on carrot agar plates.Furthermore, the number and morphology of the asci and ascospores produced by ΔFggyp8mutant are obviously similar to those produced by the wild type and complemented strain (Fig.2-H).These data suggest that FgGyp8 is not required for sexual reproduction ofF.graminearum.

Fig.2 Involvement of FgGyp8 in Fusarium graminearum conidiogenesis and conidial morphology.A, morphologies of wild type PH-1, FgGYP8 deletion mutant (ΔFggyp8) and complemented strain (ΔFggyp8-C) conidia harvested from liquid CMC after incubation at 28°C for 3 days.The conidia were stained with 10 μg mL–1 calcofluor white (CFW) before microscopy.Bar=10 μm.B and C,lengths and widths of the conidia from the various strains.About 100 conidia from each strain were counted in each experiment.D, statistical analysis of the distribution of septa number in the conidia.E, average number of septa in the conidial.F, number of conidia produced by the indicated strains.G, FgGyp8 is not required for perithecia formation by F.graminearium.H, FgGyp8 is not important for ascospores formation by F.graminearium.Error bars represent mean±SD from three replicates (＊＊, P<0.01).

3.4.FgGyp8 is important for plant infection

To investigate the role of FgGyp8 in the pathogenesis ofF.graminearum, wheat coleoptiles were infected with conidia suspensions of similar concentrations from the wild type PH-1, ΔFggyp8and the complemented strain ΔFggyp8-Cand kept under moist condition for 7 days.After this period of time, we observed reduced disease symptoms on ΔFggyp8mutant compared to the wild type and complemented strain (Fig.3-A).Furthermore,the PH-1, ΔFggyp8and ΔFggyp8-Cstrains were also inoculated on flowering wheat heads and the plants kept alive for 14 days.As shown in Fig.3-B and C, the PH-1 and complemented strain ΔFggyp8-Ccaused typical head blight symptoms in the inoculated kernels which spread to nearby spikelets on the same heads at similar rates,whereas the blight symptoms caused by ΔFggyp8mutant spread to the nearby spikelets at much slower rate under the same condition.Collectively, these results suggest that FgGyp8 plays an important role in the virulence ofF.graminearum.

DON is one of the important virulence factors inF.graminearum(Proctoret al.1995).To find out whether FgGyp8 is required for DON production, the PH-1, ΔFggyp8and ΔFggyp8-Cstrains were grown in liquid trichothecene biosynthesis induction (TBI) media at 28°C for 7 d in the dark.As shown in Fig.3-D, the results indicate that the quantity of DON produced by ΔFggyp8mutant was slightly more than those recorded in PH-1 and ΔFggyp8-Cstrains, though the difference was not significant.This result indicates that FgGyp8 is not important for DON production inF.graminearum.

3.5.Functional characterization of the TBC domain and N- and C-terminal regions of FgGyp8 in F.graminearum

FgGyp8 has a conserved TBC domain.To dissect the roles of this domain in relation to the functions of FgGyp8, we generated pFgGYP8ΔTBC(lacking the TBC domain, 70–288 aa), pFgGYP8ΔN(lacking the N-terminal,1–69 aa), pFgGYP8ΔC(lacking the C-terminal, 289–420 aa)constructs (Fig.4-A) and transformed them into the protoplasts of ΔFggyp8to obtain FgGyp8ΔTBC, FgGyp8ΔNand FgGyp8ΔCmutants, and then analyzed the phenotypes of each of the mutants.As shown in Fig.4-B–D, phenotypic analysis of these mutants in comparison with the wild type revealed that FgGyp8ΔTBC, FgGyp8ΔNand FgGyp8ΔCmutants exhibited small-colony phenotypes with reduced vegetative growths and conidiations, similar to the defects observed in ΔFggyp8mutant, respectively.

Fig.3 Pathogenicity and deoxynivalenol (DON) production analyses of FgGYP8 deletion mutant (ΔFggyp8).A, pathogenicity assay of ΔFggyp8 mutant on wheat coleoptiles at 7 days post infection.B, pathogenicity assay of ΔFggyp8 mutant on flowering wheat heads.Photographs were taken at 14 days post-inoculation.C, disease indices of the indicated strains rated by the number of symptomatic spikelets 14 days post-inoculation.D, the DON production of wild type PH-1, ΔFggyp8 and complemented strain(ΔFggyp8-C) strains.Error bars represent mean±SD from three replicates.＊＊, statistically significant differences P<0.01; ns, no significant difference.

Furthermore, these mutants were inoculated on flowering wheat heads to assess their pathogenicity.At 14 days post inoculation, we observed that the pathogenicity of TBC domain and the N- and C-terminals deletion mutants are similar to those observed in ΔFggyp8mutant,which is significantly reduced compared to PH-1 (Fig.5-A and B).Taken together, we conclude from these results that the TBC domain and the N- and C-terminal regions are all important for the functions of FgGyp8 inF.graminearum.

3.6.FgGyp8 functions as a GAP for FgRab1 and is required for plasma membrane localization of the v-SNARE FgSnc1 in F.grminearum

A previous study indicated that Gyp8 is a GTPaseactivating protein of Ypt1 (Rab1) and stimulates GTP hydrolysis on Ypt1 in yeast (De Antoniet al.2002).To analyze whether FgGyp8 possesses the ability to hydrolysis the GTP bound to FgRab1 inF.graminearum,we performedinvitroRab GAP activity assay.As shown in Fig.6-A, we found that FgGyp8 has the ability to hydrolyze FgRab1-GTP to FgRab1-GDP, suggesting that FgGyp8 is a GAP for FgRab1 inF.graminearum.

Fig.4 Functional characterization of the Tre2–Bub2–Cdc16 (TBC) domain and N- and C-terminal regions of FgGyp8.A, schematic diagram of FgGyp8-full length, TBC domain deletion, N-terminal and C-terminal deletion mutants.B, the colony morphologies of wild type PH-1, FgGYP8 deletion mutant (ΔFggyp8), Fggyp8ΔN (ΔN), Fggyp8ΔTBC (ΔTBC) and Fggyp8ΔC (ΔC) on CM, MM and SYM after 3 days.C, the colony diameters of the indicated strains on CM, MM and SYM after 3 days.D, conidiations of the indicated strains.Error bars represent mean±SD from three replicates, and bars with same letters are not significantly different at P<0.05.

FgSnc1 regulates the fusion of secreted vesicles with fungal growing apex and plasma membrane inF.grminearum(Zheng Wet al.2018).In yeast, the v-SNARE Snc1 mediates the fusion of vesicles from Golgi to plasma membrane (Lewiset al.2000).In our previous study, we found that the dominant-negative state of FgRab1 disrupts the exocytosis of FgSnc1 to the plasma membrane inF.grminearum(Yuanet al.2022).To investigate if FgGyp8 is required for the exocytosis of FgSnc1 inF.grminearum, we transformed pGFP-FgSNC1 plasmid into the protoplasts of PH-1 and ΔFggyp8, respectively,and subsequently observed their subcellular localizations.As shown in Fig.6-B and C, GFP-FgSnc1 localizes to the plasma membrane and hyphal apex in the growing hyphal cells of PH-1.However, GFP-FgSnc1 does not accumulate at the plasma membrane in the ΔFggyp8mutant.This result indicates that FgGyp8 is required for the plasma membrane localization of the v-SNARE FgSnc1 inF.grminearumand is therefore involved in polarized vesicle trafficking and secretion during FgSnc1-mediated exocytosis.

FgGyp1 was identified as a GAP of FgRab1 in our previous study (Zheng Qet al.2021), to determine the relationship of FgGyp1 with FgGyp8 inF.graminearum,we knocked outFgGYP1gene in the ΔFggyp8mutant,and obtained theFgGYP1FgGYP8double deletion mutant(ΔFggyp1ΔFggyp8).As shown in Fig.7, the vegetative growth of ΔFggyp1ΔFggyp8was significantly decreased compared to that in ΔFggyp1or ΔFggyp8single gene deletion mutants, respectively.This result indicates that FgGyp1 and FgGyp8 have functional redundancy inF.graminearum.

4.Discussion

Rab proteins constitute the largest family of small Raslike GTPases with 11 members identified in yeast and 66 members in humans, with a variety of regulatory functions in membrane trafficking (Stenmark 2009; Hutagalung and Novick 2011).As a molecular switch, Rab GTPases are controlled by accessory factors: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) (Mizuno-Yamasakiet al.2012).GAPs are negative regulators of Rab GTPases; to date, the functions of Rab GAPs in plant pathogens are still largely unknown.In this study, we identified and functionally characterized a TBC domain-containing protein FgGyp8 in the phytopathogenic fungusF.graminearumfor the first time.Our results showed that FgGyp8 is involved in vegetative growth, conidiogenesis and pathogenicity in the wheat pathogen (Fig.8).Furthermore, the TBC domain, N- and C-terminal regions of FgGyp8 are all required for the full functions of the protein.

Fig.5 Pathogenicity analysis of ΔN, ΔTBC and ΔC mutants of FgGyp8.A, infection assays of wild type PH-1, FgGYP8 deletion mutant (ΔFggyp8), ΔN, ΔTBC and ΔC strains on flowering wheat heads, photographs were taken at 14 days post-inoculation.B,disease index was rated by the number of symptomatic spikelets at 14 days post-inoculation.Error bars represent mean±SD from three replicates, and bars with same letters are not significantly different at P<0.05.

Fig.6 FgGyp8 is a GTPase-activating protein (GAP) of FgRab1 and is required for plasma membrane localization of the v-SNARE protein FgSnc1.A, in vitro GAP activity assay of FgGyp8.Control, MBP.One unit is the amount of enzyme (GTPase) that catalyzes the production of 1 μmol free phosphate group per minute under the assay conditions.Error bars represent mean±SD from three replicates, and two-tailed Student’s t-test was used for paired comparison of the GAP activity between MBP control and MBP-FgGyp8 (＊, P<0.05).B, the localization of GFP-FgSnc1 in the wild type PH-1 and ΔFggyp8 mutant.GFP-FgSnc1 localizes to the plasma membrane of the wild type PH-1, while this plasma membrane localization almost disappeared in the ΔFggyp8 mutant.C, line scans of GFP-FgSnc1 signals in the wild type PH-1 and ΔFggyp8 mutant.

Fig.7 Colony morphologies of the wild type (PH-1), ΔFggyp1, ΔFggyp8, FgGYP1-FgGYP8 double deletion mutant (ΔFggyp1ΔFggyp8)grown on CM at 28°C for 3 days.

Fig.8 A proposed operational model of how FgGyp8 regulates FgRab1 for polarized exocytosis, growth, conidiogenesis and pathogenicity in Fusarium graminearum.GEF, guanine nucleotide exchange factor.

Several GAPs have been identified in the model fungusS.cerevisiae, including Gyp1, Gyp2, Msb3/Msb4,Gyp5, Gyp6, Gyp7 and Gyp8 (Duet al.1998; Albert and Gallwitz 1999; Will and Gallwitz 2001; Chesneauet al.2004), but only little is known about GAPs in other fungi.In our previous studies, FgMsb3 was identified as a GAP for FgRab8, and it regulates polarized trafficking, growth and pathogenicity inF.graminearum(Zheng Het al.2021).FgGyp1 catalytically converts the GTP on FgRab1 to its GDP form through its TBC domain, thus regulating the vegetative growth, conidiation, virulence and DON biosynthesis ofF.graminearum(Zheng Qet al.2021).In addition, the Msb3 homolog protein Gyp3 regulates cellular morphogenesis inNeurosporacrassa(Callejas-Negrete and Castro-Longoria 2019).In this study,FgGyp8 was demonstrated to act as a GAP for FgRab1 inF.graminearum.

In yeast, one GAP may have multiple substrates,for example, Gyp8 is a GAP for Ypt1, Ypt6, and Sec4,respectively (De Antoniet al.2002; Brettet al.2008).In contrast, one Ypt protein may also have multiple GAPs,such as Ypt1, Sec4, Gyp1, Gyp5 and Gyp8, whose common substrate is Ypt1 (De Antoniet al.2002).They are known to be potent GAPsinvitrofor Ypt1, a GTPase with an essential function in ER-to-Golgi transport (De Antoniet al.2002).In yeast, all the single deletion mutants ofGYP1,GYP5andGYP8were viable and grew like the wild type, while theGYP1/GYP5/GYP8 triple deletion mutant had retarded growth at low temperatures in comparison to the single and double deletion mutants, suggesting that the functions ofGYPgenes in yeast are redundant (De Antoniet al.2002).In addition, deletion of eitherMSB3orMSB4does not show any obvious phenotype but theMSB3/MSB4double deletion mutant grows slowly and displays partial disorganization of actin cytoskeleton in yeast (Biet al.2000).However, the homologue proteins of Gyp1, Gyp8, Msb3/Msb4 inF.graminearum, FgGyp1, FgGyp8 and FgMsb3 are all important for vegetative growth and development(Zheng Het al.2021; Zheng Qet al.2021), suggesting an evolution divergence of these proteins in plant pathogen.Furthermore, compared with FgMsb3 and FgGyp1,FgGyp8 shows gentle phenotype in vegetative growth,suggesting a divergence function of these GAP proteins inF.graminearum.SinceRAB1/YPT1is an essential gene(Schmittet al.1986, 1988; Yuanet al.2022), consistently with the vegetative growth of ΔFggyp1ΔFggyp8double deletion mutant was significantly decreased compared to that in ΔFggyp1or ΔFggyp8single gene deletion mutants,respectively.We speculate that Gyp1 and Gyp8 may have functional redundancy inF.graminearum.

Human TBC1D20, the homologue of yeast Gyp8(Haaset al.2007), blocks protein transport at the level of ER exit by inactivating Rab1 (Haaset al.2007), and this restricts hepatitis C virus replication (Sklanet al.2007)and regulates autophagosome maturation (Sidjaninet al.2016).Loss-of-function mutations in TBC1D20 cause cataracts and male infertility in blind sterile mice and Warburg micro syndrome in humans (Liegelet al.2013).Furthermore, TBC1D20 is essential for mouse bloodtestis barrier integrity through maintaining the phenotype of epithelial cells and modulating the maturation of Sertoli cells (Cuiet al.2020).In mice, TBC1D20 deficiency induces Sertoli cell apoptosis by triggering irreversible endoplasmic reticulum stress (Changet al.2019).The above results suggest that Gyp8 homolog proteins in mammalian cells are involved in multiple development processes.Herein, we demonstrated that FgGyp8 is also required for multiple developmental processes inF.graminearum, such as vegetative growth,conidiogenesis and pathogenicity.

In yeast, Gyp8-GFP localizes to punctate structures(De Antoniet al.2002) in the cell.Further studies demonstrated that it localizes to peroxisomes and is regulated by AAA ATPase Msp1 (Nickersonet al.2020).In this study, FgGyp8-GFP shows weak cytoplasm localization signals inF.graminearum(Appendix C),suggesting a divergent localization pattern of Gyp8 homologue proteins.Like the TBC domains in FgMsb3 and FgGyp1 (Zheng Het al.2021; Zheng Qet al.2021), the TBC domain of FgGyp8 is also required for the full functions ofF.graminearum.FgSnc1 mediates the fusion of secreted vesicles with the fungal growing apex and plasma membrane inF.grminearum(Zheng Wet al.2018).Previous studies showed that FgGyp1 and FgRab1 are both required for FgSnc1-mediated exocytosis (Zheng Qet al.2021; Yuanet al.2022).Consistently, FgGyp8 is required for the plasma membrane localization of FgSnc1 inF.graminearum,suggesting that FgGyp8 is also involved in FgSnc1-mediated exocytosis inF.graminearum(Fig.8).

DON production is considered one of the major virulence factors inF.graminearum(Proctoret al.1995; Jianget al.2016; Maet al.2021).In this study,the virulence of ΔFggyp8is significantly reduced in comparison to that of the wild type PH-1.However,we found that DON production in this mutant is slightly increased when compared with the wild type, suggesting that there are other virulence factors maybe contribute to the reduction virulence of ΔFggyp8mutant, such as slow vegetative growth, conidiation, effectors and nonribosomal octapeptide (Jiaet al.2019; Jianget al.2020).In addition, previous studies also showed reduced virulence but with increased DON production in some mutants ofF.graminearum(Zheng Het al.2021; Wuet al.2022).

5.Conclusion

In summary, results of this study demonstrated that FgGyp8, a putative FgRab1 GAP is required for vegetative growth, conidiogenesis and pathogenesis by regulating FgSnc1-mediated secretory vesicles fusion with the plasma membrane inFusariumgraminearum.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (31970141), the Natural Science Foundation of Fujian Province, China (2020J06047), the Foundation of Minjiang University, China (MJY19019),and the Foundation of Fujian Agriculture and Forestry University, China (KFb22050XA).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2023.04.005