Abstract
Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. But the role of Rab GTPases in Fusarium verticillioides, one of the key corn pathogens worldwide, remains elusive. These Small GTPases in fungi, particularly those homologous to Saccharomyces cerevisiae Sec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. In this study, our aim was to investigate the molecular functions of FvSec4 in F. verticillioides associated with physiology and virulence. Interestingly, the FvSec4 null mutation did not impair the expression of key conidiation-related genes. Also, the mutant did not show any defect in sexual development, including perithecia production. Meanwhile, GFP-FvSec4 localized to growing hyphal tips and raised the possibility that FvSec4 is involved in protein trafficking and endocytosis. The mutant exhibited defect in corn stalk rot virulence and also significant alteration of fumonisin B1 production. The mutation led to higher sensitivity to oxidative and cell wall stress agents, and defects in carbon utilization. Gene complementation fully restored the defects in the mutant demonstrating that FvSec4 plays important roles in these functions. Taken together, our data indicate that FvSec4 is critical in F. verticillioides hyphal development, virulence, mycotoxin production and stress responses.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alexander NJ, Proctor RH, McCormick SP (2009) Genes, gene clusters, and biosynthesis of trichothecenes and fumonisins in Fusarium. Toxin Rev 28(2–3):198–215
Bartnicki-Garcia S, Hergert F, Gierz G (1989) Computer simulation of fungal morphogenesis and the mathematical basis for hyphal (tip) growth. Protoplasma 153(1–2):46–57
Chanda A, Roze LV, Kang S, Artymovich KA, Hicks GR, Raikhel NV, Calvo AM, Linz JE (2009) A key role for vesicles in fungal secondary metabolism. Proc Natl Acad Sci USA 106(46):19533–19538. https://doi.org/10.1073/pnas.0907416106
Chanda A, Roze LV, Linz JE (2010) A possible role for exocytosis in aflatoxin export in Aspergillus parasiticus. Eukaryot Cell 9(11):1724–1727. https://doi.org/10.1128/EC.00118-10
Chen X, Ebbole DJ, Wang Z (2015) The exocyst complex: delivery hub for morphogenesis and pathogenesis in filamentous fungi. Curr Opin Plant Biol 28:48–54. https://doi.org/10.1016/j.pbi.2015.09.003
Christensen S, Borrego E, Shim WB, Isakeit T, Kolomiets M (2012) Quantification of fungal colonization, sporogenesis, and production of mycotoxins using kernel bioassays. J Vis Exp. https://doi.org/10.3791/3727
Desjardins AE, Munkvold GP, Plattner RD, Proctor RH (2002) FUM1–a gene required for fumonisin biosynthesis but not for maize ear rot and ear infection by Gibberella moniliformis in field tests. Mol Plant Microb Interact 15(11):1157–1164. https://doi.org/10.1094/MPMI.2002.15.11.1157
Dumas B, Borel C, Herbert C, Maury J, Jacquet C, Balsse R, Esquerre-Tugaye MT (2001) Molecular characterization of CLPT1, a SEC4-like Rab/GTPase of the phytopathogenic fungus Colletotrichum lindemuthianum which is regulated by the carbon source. Gene 272(1–2):219–225
Fischer-Parton S, Parton RM, Hickey PC, Dijksterhuis J, Atkinson HA, Read ND (2000) Confocal microscopy of FM4-64 as a tool for analysing endocytosis and vesicle trafficking in living fungal hyphae. J Microsc 198(Pt 3):246–259
Goud B, Salminen A, Walworth NC, Novick PJ (1988) A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 53(5):753–768. https://doi.org/10.1016/0092-8674(88)90093-1
Guo W, Roth D, Walch-Solimena C, Novick P (1999) The exocyst is an effector for Sec4p, targeting secretory vesicles to sites of exocytosis. EMBO J 18(4):1071–1080. https://doi.org/10.1093/emboj/18.4.1071
He B, Guo W (2009) The exocyst complex in polarized exocytosis. Curr Opin Cell Biol 21(4):537–542. https://doi.org/10.1016/j.ceb.2009.04.007
Johansen J, Alfaro G, Beh CT (2016) Polarized exocytosis induces compensatory endocytosis by Sec4p-regulated cortical actin polymerization. PLoS Biol 14(8):e1002534. https://doi.org/10.1371/journal.pbio.1002534
Kean LS, Fuller RS, Nichols JW (1993) Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane. J Cell Biol 123(6 Pt 1):1403–1419
Kim MS, Zhang H, Yan H, Yoon B-J, Shim WB (2018) Characterizing co-expression networks underpinning maize stalk rot virulence in Fusarium verticillioides through computational subnetwork module analyses. Sci Rep 8(1):8310
Lazar T, Gotte M, Gallwitz D (1997) Vesicular transport: how many Ypt/Rab-GTPases make a eukaryotic cell? Trends Biochem Sci 22(12):468–472
Lehmann S, Serrano M, L’Haridon F, Tjamos SE, Metraux JP (2015) Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112:54–62. https://doi.org/10.1016/j.phytochem.2014.08.027
Leslie JF, Summerell BA (2008) The Fusarium laboratory manual. Wiley, Amsterdam
Ma LJ, Geiser DM, Proctor RH, Rooney AP, O’Donnell K, Trail F, Gardiner DM, Manners JM, Kazan K (2013) Fusarium pathogenomics. Annu Rev Microbiol 67:399–416. https://doi.org/10.1146/annurev-micro-092412-155650
Mao YX, Kalb VF, Wong B (1999) Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans. J Bacteriol 181(23):7235–7242
Novick P (2016) Regulation of membrane traffic by Rab GEF and GAP cascades. Small GTPases 7(4):252–256. https://doi.org/10.1080/21541248.2016.1213781
Powers-Fletcher MV, Feng X, Krishnan K, Askew DS (2013) Deletion of the sec4 homolog srgA from Aspergillus fumigatus is associated with an impaired stress response, attenuated virulence and phenotypic heterogeneity. PLos One 8(6):e66741. https://doi.org/10.1371/journal.pone.0066741
Proctor RH, Hohn TM, McCormick SP (1995) Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microb Interact 8(4):593–601
Proctor RH, Desjardins AE, Plattner RD, Hohn TM (1999) A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. Fungal Genet Biol 27(1):100–112. https://doi.org/10.1006/fgbi.1999.1141
Riquelme M (2013) Tip growth in filamentous Fungi: a road trip to the apex. In: Gottesman S (ed) Annual review of microbiology, vol 67, pp 587–609. https://doi.org/10.1146/annurev-micro-092412-155652
Riquelme M, Sanchez-Leon E (2014) The Spitzenkorper: a choreographer of fungal growth and morphogenesis. Curr Opin Microbiol 20:27–33. https://doi.org/10.1016/j.mib.2014.04.003
Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucl Acids Res 42(Web Server issue):W320–W324. https://doi.org/10.1093/nar/gku316
Sagaram US, Shim WB (2007) Fusarium verticillioides GBB1, a gene encoding heterotrimeric G protein beta subunit, is associated with fumonisin B biosynthesis and hyphal development but not with fungal virulence. Mol Plant Pathol 8(4):375–384. https://doi.org/10.1111/j.1364-3703.2007.00398.x
Salminen A, Novick PJ (1987) A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 49(4):527–538. https://doi.org/10.1016/0092-8674(87)90455-7
Sambrook J, Russell DW (2001) Molecular cloning-Sambrook & Russel-Vol. 1, 2, 3, 3rd edn. Cold Spring Harbor Laboratory Press
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675
Schultzhaus ZS, Shaw BD (2015) Endocytosis and exocytosis in hyphal growth. Fungal Biol Rev 29(2):43–53. https://doi.org/10.1016/j.fbr.2015.04.002
Schultzhaus Z, Yan H, Shaw BD (2015) Aspergillus nidulans flippase DnfA is cargo of the endocytic collar and plays complementary roles in growth and phosphatidylserine asymmetry with another flippase, DnfB. Mol Microbiol 97(1):18–32. https://doi.org/10.1111/mmi.13019
Seo JA, Proctor RH, Plattner RD (2001) Characterization of four clustered and coregulated genes associated with fumonisin biosynthesis in Fusarium verticillioides. Fungal Genet Biol 34(3):155–165. https://doi.org/10.1006/fgbi.2001.1299
Shim WB, Woloshuk CP (1999) Nitrogen repression of fumonisin B1 biosynthesis in Gibberella fujikuroi. FEMS Microbiol Lett 177(1):109–116. https://doi.org/10.1111/j.1574-6968.1999.tb13720.x
Siriputthaiwan P, Jauneau A, Herbert C, Garcin D, Dumas B (2005) Functional analysis of CLPT1, a Rab/GTPase required for protein secretion and pathogenesis in the plant fungal pathogen Colletotrichum lindemuthianum. J Cell Sci 118(2):323–329. https://doi.org/10.1242/jcs.01616
Stroupe C, Brunger AT (2000) Crystal structures of a Rab protein in its inactive and active conformations. J Mol Biol 304(4):585–598. https://doi.org/10.1006/jmbi.2000.4236
TerBush DR, Maurice T, Roth D, Novick P (1996) The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 15(23):6483–6494
van der Does HC, Rep M (2017) Adaptation to the host environment by plant-pathogenic Fungi. Annu Rev Phytopathol 55:427–450. https://doi.org/10.1146/annurev-phyto-080516-035551
Woloshuk CP, Shim WB (2013) Aflatoxins, fumonisins, and trichothecenes: a convergence of knowledge. FEMS Microbiol Rev 37(1):94–109. https://doi.org/10.1111/1574-6976.12009
Wu F, Groopman JD, Pestka JJ (2014) Public health impacts of foodborne mycotoxins. Annu Rev Food Sci Technol 5:351–372. https://doi.org/10.1146/annurev-food-030713-092431
Yang P, Chen Y, Wu H, Fang W, Liang Q, Zheng Y, Olsson S, Zhang D, Zhou J, Wang Z, Zheng W (2018) The 5-oxoprolinase is required for conidiation, sexual reproduction, virulence and deoxynivalenol production of Fusarium graminearum. Curr Genet 64(1):285–301. https://doi.org/10.1007/s00294-017-0747-y
Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Dominguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41(11):973–981. https://doi.org/10.1016/j.fgb.2004.08.001
Yun Y, Zhou X, Yang S, Wen Y, You H, Zheng Y, Norvienyeku J, Shim WB, Wang Z (2019) Fusarium oxysporum f. sp. lycopersici C2H2 transcription factor FolCzf1 is required for conidiation, fusaric acid production, and early host infection. Curr Genet 65(3):773–783. https://doi.org/10.1007/s00294-019-00931-9
Zhang Z, Qin G, Li B, Tian S (2014) Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence. Mol Plant Microb Interact 27(6):590–600. https://doi.org/10.1094/mpmi-10-13-0314-r
Zhang H, Kim M, Huang J, Shim W (2017) FvLcp1, a novel LysM/Chitin-binding protein, is important for ear rot virulence and fumonisin biosynthesis in Fusarium verticillioides. Phytopathology, vol 12. American Phytopathological Society, New York, p 87
Zhang H, Mukherjee M, Kim JE, Yu W, Shim WB (2018) Fsr1, a striatin homologue, forms an endomembrane-associated complex that regulates virulence in the maize pathogen Fusarium verticillioides. Mol Plant Pathol 19(4):812–826. https://doi.org/10.1111/mpp.12562
Zheng H, Zheng W, Wu C, Yang J, Xi Y, Xie Q, Zhao X, Deng X, Lu G, Li G, Ebbole D, Zhou J, Wang Z (2015) Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in Fusarium graminearum. Environ Microbiol 17(11):4580–4599. https://doi.org/10.1111/1462-2920.12982
Zheng HK, Chen SM, Chen XF, Liu SY, Dang X, Yang CD, Giraldo MC, Oliveira-Garcia E, Zhou J, Wang ZH, Valent B (2016) The small GTPase MoSec4 is involved in vegetative development and pathogenicity by regulating the extracellular protein secretion in Magnaporthe oryzae. Front Plant Sci. https://doi.org/10.3389/fpls.2016.01458
Acknowledgements
We thank Dr. Brian Shaw, Ms. Blake Commer and Mr. Joe Vasselli (Department of Plant Pathology and Microbiology, Texas A&M University) for help and discussion in microscopy. This research was supported in part by the Agriculture and Food Research Initiative Competitive Grants Program Grant (2013-68004-20359) from the USDA National Institute of Food and Agriculture. The authors declare no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M. Kupiec.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Yan, H., Huang, J., Zhang, H. et al. A Rab GTPase protein FvSec4 is necessary for fumonisin B1 biosynthesis and virulence in Fusarium verticillioides. Curr Genet 66, 205–216 (2020). https://doi.org/10.1007/s00294-019-01013-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-019-01013-6