Abstract
In eukaryotes, the ubiquitin-like (UBL) protein-activating enzymes play a crucial role in autophagy process, however, it is poorly characterized in filamentous fungi. Here, we investigated the functions of two UBL activating enzymes, BcAtg3 (E2) and BcAtg7 (E1) in the plant pathogenic fungus Botrytis cinerea. The physical interaction of BcAtg3 with BcAtg7 was demonstrated by yeast two-hybrid system. Subcellular localization assays showed that BcAtg3 diffused in cytoplasm, and BcAtg7 localized in cytoplasm as pre-autophagosomal structures (PAS). Target gene deletion experiments revealed that both BcATG3 and BcATG7 are essential for autophagy pathway. Notably, the single deletion mutant of BcATG3 and BcATG7 displayed similar biological phenotypes, including the defects in mycelial growth, conidiation and sclerotial formation. Infection tests showed that both BcATG3 and BcATG7 were required for full virulence of B. cinerea. All of these defective phenotypes were rescued by gene complementation. These results indicate that BcATG3 and BcATG7 are necessary for autophagy to regulate fungal development and pathogenesis in B. cinerea.
Similar content being viewed by others
References
Abeliovich H, Klionsky DJ (2001) Autophagy in yeast: mechanistic insights and physiological function. Microbiol Mol Biol R 65:463–479. https://doi.org/10.1128/MMBR.65.3.463-479.2001
Amselem J, Cuomo CA, van Kan JA, Viaud M, Benito EP, Couloux A, Coutinho PM, de Vries RP, Dyer PS, Fillinger S, Fournier E, Gout L, Hahn M, Kohn L, Lapalu N, Plummer KM, Pradier JM, Quevillon E, Sharon A, Simon A, ten Have A, Tudzynski B, Tudzynski P, Wincker P, Andrew M, Anthouard V, Beever RE, Beffa R, Benoit I, Bouzid O, Brault B, Chen Z, Choquer M, Collemare J, Cotton P, Danchin EG, Da Silva C, Gautier A, Giraud C, Giraud T, Gonzalez C, Grossetete S, Guldener U, Henrissat B, Howlett BJ, Kodira C, Kretschmer M, Lappartient A, Leroch M, Levis C, Mauceli E, Neuveglise C, Oeser B, Pearson M, Poulain J, Poussereau N, Quesneville H, Rascle C, Schumacher J, Segurens B, Sexton A, Silva E, Sirven C, Soanes DM, Talbot NJ, Templeton M, Yandava C, Yarden O, Zeng Q, Rollins JA, Lebrun MH, Dickman M (2011) Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet 7:e1002230. https://doi.org/10.1371/journal.pgen.1002230
Boya P, Reggiori F, Codogno P (2013) Emerging regulation and functions of autophagy. Nat Cell Biol 15:713–720. https://doi.org/10.1038/ncb2788
Chu X, Feng M, Ying S (2016) Qualitative ubiquitome unveils the potential significances of protein lysine ubiquitination in hyphal growth of Aspergillus nidulans. Curr Genet 62:191–201. https://doi.org/10.1007/s00294-015-0517-7
Cui Z, Gao N, Wang Q, Ren Y, Wang K, Zhu T (2015) BcMctA, a putative monocarboxylate transporter, is required for pathogenicity in Botrytis cinerea. Curr Genet 61:545–553. https://doi.org/10.1007/s00294-015-0474-1
Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13:414–430. https://doi.org/10.1111/j.1364-3703.2011.00783.x
Elad Y, Williamson B, Tudzynski P, Delen N (2007) Botrytis: biology, pathology and control. Springer, Netherlands
Fujita N, Itoh T, Omori H, Fukuda M, Noda T, Yoshimori T (2008) The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy. Mol Biol Cell 19:2092–2100. https://doi.org/10.1091/mbc.E07-12-1257
Geng J, Klionsky DJ (2008) The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. EMBO Rep 9:859–864. https://doi.org/10.1038/embor.2008.163
Glick D, Barth S, Macleod KF (2010) Autophagy: cellular and molecular mechanisms. J Pathol 221:3–12. https://doi.org/10.1002/path.2697
Gronover CS, Kasulke D, Tudzynski P, Tudzynski B (2001) The role of G protein alpha subunits in the infection process of the gray mold fungus Botrytis cinerea. Mol Plant Microbe Interact 14:1293–1302. https://doi.org/10.1094/MPMI.2001.14.11.1293
Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y (2000) A ubiquitin-like system mediates protein lipidation. Nature 408:488–492. https://doi.org/10.1038/35044114
Josefsen L, Droce A, Sondergaard TE, Sørensen JL, Bormann J, Schäfer W, Giese H, Olsson S (2012) Autophagy provides nutrients for nonassimilating fungal structures and is necessary for plant colonization but not for infection in the necrotrophic plant pathogen Fusarium graminearum. Autophagy 8:326–337. https://doi.org/10.4161/auto.18705
Jung B, Kim S, Lee J (2014) Microcyle conidiation in filamentous fungi. Mycobiology 42:1–5. https://doi.org/10.5941/MYCO.2014.42.1.1
Khan IA, Lu J, Liu X, Rehman A, Lin F (2012) Multifunction of autophagy-related genes in filamentous fungi. Microbiol Res 167:339–345. https://doi.org/10.1016/j.micres.2012.01.004
Kikuma T, Ohneda M, Arioka M, Kitamoto K (2006) Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot Cell 5:1328–1336. https://doi.org/10.1128/EC.00024-06
Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y (2000) The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151:263–276. https://doi.org/10.1083/jcb.151.2.263
Komatsu M, Tanida I, Ueno T, Ohsumi M, Ohsumi Y, Kominami E (2001) The C-terminal region of an Apg7p/Cvt2p is required for homodimerization and is essential for its E1 activity and E1–E2 complex formation. J Biol Chem 276:9846–9854. https://doi.org/10.1074/jbc.M007737200
Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N (2004) The role of autophagy during the early neonatal starvation period. Nature 432:1032–1036. https://doi.org/10.1038/nature03029
Levine B, Klionsky DJ (2004) Development by self-digestion. Dev Cell 6:463–477. https://doi.org/10.1016/S1534-5807(04)00099-1
Liu Y, Schiff M, Czymmek K, Tallóczy Z, Levine B, Dinesh-Kumar SP (2005) Autophagy regulates programmed cell death during the plant innate immune response. Cell 121:567–577. https://doi.org/10.1016/j.cell.2005.03.007
Liu X, Lu J, Zhang L, Dong B, Min H, Lin F (2007) Involvement of a Magnaporthe grisea Serine/Threonine kinase gene, MgATG1, in appressorium turgor and pathogenesis. Eukaryot Cell 6:997–1005. https://doi.org/10.1128/EC.00011-07
Liu X, Gao H, Xu F, Lu J, Devenish RJ, Lin F (2012) Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy 8:1415–1425. https://doi.org/10.4161/auto.21274
Lu J, Liu X, Feng X, Min H, Lin F (2009) An autophagy gene, MgATG5, is required for cell differentiation and pathogenesis in Magnaporthe oryzae. Curr Genet 55:461–473. https://doi.org/10.1007/s00294-009-0259-5
Lv W, Wang C, Yang N, Que Y, Talbot NJ, Wang Z (2017) Genome-wide functional analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol production and virulence in Fusarium graminearum. Sci Rep 7:11062. https://doi.org/10.1038/s41598-017-11640-z
Mcdonald BA, Martinez JP (1990) Restriction fragment length polymorphisms in Septoria tritici occur at a high frequency. Curr Genet. https://doi.org/10.1007/BF00312858
Mizushima N, Noda T, Yoshimori T, Tanaka Y, Ishii T, George MD, Klionsky DJ, Ohsumi M, Ohsumi Y (1998) A protein conjugation system essential for autophagy. Nature 395:395–398. https://doi.org/10.1038/26506
Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 152:657–668. https://doi.org/10.1083/jcb.152.4.657
Nolting N, Bernhards Y, Pöggeler S (2009) SmATG7 is required for viability in the homothallic ascomycete Sordaria macrospora. Fungal Genet Biol 46:531–542. https://doi.org/10.1016/j.fgb.2009.03.008
Ohsumi Y (2001) Molecular dissection of autophagy: two ubiquitin-like systems. Nat Rev Mol Cell Biol 2:211–216. https://doi.org/10.1038/35056522
Ohsumi Y, Mizushima N (2004) Two ubiquitin-like conjugation systems essential for autophagy. Semin Cell Dev Biol 15:231–236. https://doi.org/10.1016/j.semcdb.2003.12.004
Pinan-Lucarré B, Balguerie A, Clavé C (2005) Accelerated cell death in Podospora autophagy mutants. Eukaryot Cell 4:1765–1774. https://doi.org/10.1128/EC.4.11.1765-1774.2005
Pollack JK, Harris SD, Marten MR (2009) Autophagy in filamentous fungi. Fungal Genet Biol 46:1–8. https://doi.org/10.1016/j.fgb.2008.10.010
Ren W, Zhang Z, Shao W, Yang Y, Zhou M, Chen C (2017) The autophagy-related gene BcATG1 is involved in fungal development and pathogenesis in Botrytis cinerea. Mol Plant Pathol 18:238–248. https://doi.org/10.1111/mpp.12396
Schiestl RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet 16:339–346. https://doi.org/10.1007/BF00340712
Schumacher J (2012) Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches. Fungal Genet Biol 49:483–497. https://doi.org/10.1016/j.fgb.2012.03.005
Shintani T, Mizushima N, Ogawa Y, Matsuura A, Noda T, Ohsumi Y (1999) Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. EMBO J 18:5234–5241. https://doi.org/10.1093/emboj/18.19.5234
Shoji JY, Arioka M, Kitamoto (2006) Possible involvement of pleiomorphic vacuolar networks in nutrient recycling in filamentous fungi. Autophagy 2:226–227. https://doi.org/10.4161/auto.2695
Slobodkin MR, Elazar Z (2013) The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy. Essays Biochem 55:51–64. https://doi.org/10.1042/bse0550051
Taherbhoy AM, Tait SW, Kaiser SE, Williams AH, Deng A, Nourse A, Hammel M, Kurinov I, Rock CO, Green DR, Schulman BA (2011) Atg8 transfer from Atg7 to Atg3: a distinctive E1–E2 architecture and mechanism in the autophagy pathway. Mol Cell 44:451–461. https://doi.org/10.1016/j.molcel.2011.08.034
Tanida I, Mizushima N, Kiyooka M, Ohsumi M, Ueno T, Ohsumi Y, Kominami E (1999) Apg7p/Cvt2p: A novel arotein-activating enzyme essential for autophagy. Mol Biol Cell 10:1367–1379. https://doi.org/10.1091/mbc.10.5.1367
Tsukada M, Ohsumi Y (1993) Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett 333:169–174. https://doi.org/10.1016/0014-5793(93)80398-E
Wani W, Boyer-Guittaut M, Dodson M, Chatham J, Darley-Usmar V, Zhang J (2015) Regulation of autophagy by protein post-translational modification. Lab Invest 95:14–25. https://doi.org/10.1038/labinvest.2014.131
Williamson B, Tudzynski B, Tudzynski P, Van Kan JA (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580. https://doi.org/10.1111/j.1364-3703.2007.00417.x
Xiong J (2015) Atg7 in development and disease: panacea or Pandora’s Box? Protein Cell 6:722–734. https://doi.org/10.1007/s13238-015-0195-8
Yu J, Hamari Z, Han K, Seo JA, Reyes-Domínguez Y, Scazzocchio C (2004) Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi. Fungal Genet Biol 41:973–981. https://doi.org/10.1016/j.fgb.2004.08.001
Acknowledgements
This research was supported by the National Science Foundation of China (31672065), the Special Fund for Agro-scientific Research in the Public Interest (201303023 and 201303025) and Jiangsu Provincial Agricultural Plans [CX (14) 2054 and SXGC (2016) 154].
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by M. Kupiec.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Ren, W., Sang, C., Shi, D. et al. Ubiquitin-like activating enzymes BcAtg3 and BcAtg7 participate in development and pathogenesis of Botrytis cinerea. Curr Genet 64, 919–930 (2018). https://doi.org/10.1007/s00294-018-0810-3
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00294-018-0810-3