自噬相关基因FpAtg3参与假禾谷镰孢的生长和致病

doi:10.3864/j.issn.0578-1752.2024.06.005

Abstract

Abstract:

【Background】 Fusarium crown rot (FCR), caused by Fusarium pseudograminearum, is one of the newly-occurred destructive diseases in the main wheat-growing region of China. Autophagy is an evolutionarily conserved cellular process that regulates the growth, development and infection of different phytopathogenic fungi. However, the role of autophagy in F. pseudograminearum is still unknown.【Objective】 To clarify the role of the autophagy gene FpAtg3 in the growth and pathogenicity of F. pseudograminearum, understand the mechanisms of the fungal infection, and to provide a theoretical basis for FCR prevention.【Method】 The known Atg3 proteins of different fungi were downloaded from NCBI, and the MEGA 5.05 was used to construct the phylogenetic tree of Atg3 proteins. The Split-PCR approach was used to generate FpAtg3 gene-replacement constructs, and then the FpAtg3 deletion mutants (ΔFpAtg3) were constructed by polyethylene glycol (PEG)-mediated protoplast fungal transformation, and obtained by hygromycin resistance screening and PCR detection. The sequence of FpAtg3 and its native promoter was amplified and fused in pKNTG vector. The plasmid of pKNTG-FpAtg3 was then introduced into ΔFpAtg3 protoplasts for the complementation assay. The hyphal growth and colony morphology of the wild type, ΔFpAtg3 and the complementary (FpAtg3-C) strains were assayed on PDA, CM and MM plates. The hyphal blocks were cultured on PDA plates for hyphal morphology and hyphal fusion test; hyphal blocks were introduced into liquid CMC medium to assess conidiation and conidia morphology; conidia were cultured on Petri dishes to explore conidial anastomosis tube (CAT)-mediated fusion. The hyphal blocks were inoculated on wheat coleoptiles and barley leaves to explore pathogenicity, and the pot-culture experiment was performed for FCR assay. Congo red, sodium dodecyl sulphate (SDS) and H₂O₂ reagents were added to PDA plates to determine the F. pseudograminearum responses to cell wall, cell membrane and oxidative stresses, respectively.【Result】 Atg3 homologous proteins from different fungi were highly conserved and consistent with the direction of species evolution. The FpAtg3 had a close relationship with Atg3 of Fusarium graminearum and Fusarium oxysporum. The ΔFpAtg3 and FpAtg3-C strains were obtained. The phenotypic measurements showed that compared to wild type and FpAtg3-C strains, ΔFpAtg3 exhibited significantly reduced colony growth rates and aerial hypha, curved hypha, reduced conidiation, shorter in length and fewer in septa; ΔFpAtg3 also showed significantly reduced hyphal fusion rate and CAT-mediated fusion rate. The pathogenicity of ΔFpAtg3 on wheat coleoptiles and barley leaves was significantly reduced comparing to that of the wild type and FpAtg3-C strains. The reduced pathogenicity of ΔFpAtg3 was further examined on FCR. Furthermore, ΔFpAtg3 displayed more sensitive to cell wall, cell membrane and oxidative stress than that of the wild type and FpAtg3-C strains.【Conclusion】 Autophagy-related gene FpAtg3 plays important roles in growth, conidiation, hyphal fusion, pathogenicity and response to abiotic stresses of F. pseudograminearum.

Key words: Fusarium crown rot (FCR), Fusarium pseudograminearum, Atg3, hyphal fusion, pathogenicity

DONG ZaiFang, DING TengTeng, SHAN YiXuan, LI HongLian, CHEN LinLin, XING XiaoPing. Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum[J].Scientia Agricultura Sinica, 2024, 57(6): 1080-1090.

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References 37

[1]	BRAGARD C, BAPTISTA P, CHATZIVASSILIOU E, DI SERIO F, GONTHIER P, MIRET J A, JUSTESEN A F, MACLEOD A, MAGNUSSON C S, MILONAS P, et al. Pest categorisation of Fusarium pseudograminearum. EFSA Journal, 2022, 20(6): e07399.
[2]	KAZAN K, GARDINER D M. Fusarium crown rot caused by Fusarium pseudograminearum in cereal crops: Recent progress and future prospects. Molecular Plant Pathology, 2018, 19(7): 1547-1562. doi: 10.1111/mpp.2018.19.issue-7
[3]	LI H L, YUAN H X, FU B, XING X P, SUN B J, TANG W H. First report of Fusarium pseudograminearum causing crown rot of wheat in Henan, China. Plant Disease, 2012, 96(7): 1065.
[4]	ZHOU H F, HE X L, WANG S, MA Q Z, SUN B J, DING S L, CHEN L L, ZHANG M, LI H L. Diversity of the Fusarium pathogens associated with crown rot in the Huanghuai wheat-growing region of China. Environmental Microbiology, 2019, 21(8): 2740-2754. doi: 10.1111/emi.2019.21.issue-8
[5]	李怡文, 李桂香, 黄中乔, 苗建强, 刘西莉. 假禾谷镰孢引起的小麦茎基腐病发生危害与防控研究进展. 农药学学报, 2022, 24(5): 949-961.
	LI Y W, LI G X, HUANG Z Q, MIAO J Q, LIU X L. Research progress on the occurrence, damage and prevention of Fusarium crown rot caused by Fusarium pseudograminearum. Chinese Journal of Pesticide Science, 2022, 24(5): 949-961. (in Chinese)
[6]	俞慧友. 30个!中国科协发布2022年科技领域重大问题难题. 科技日报, (2022-06-28) [2023-10-25].
	YU H Y. China Association for Science and Technology released 30 major problems in the field of science and technology in 2022. Science and Technology Daily, (2022-06-28) [2023-10-25]. (in Chinese)
[7]	FENG Y C, HE D, YAO Z Y, KLIONSKY D J. The machinery of macroautophagy. Cell Research, 2014, 24(1): 24-41. doi: 10.1038/cr.2013.168 pmid: 24366339
[8]	KLIONSKY D J, BAEHRECKE E H, BRUMELL J H, CHU C T, CODOGNO P, CUERVO A M, DEBNATH J, DERETIC V, ELAZAR Z, ESKELINEN E L, et al. A comprehensive glossary of autophagy- related molecules and processes (2nd edition). Autophagy, 2011, 7(11): 1273-1294. doi: 10.4161/auto.7.11.17661
[9]	ASIF N, LIN F C, LI L, ZHU X M, NAWAZ S. Regulation of autophagy machinery in Magnaporthe oryzae. International Journal of Molecular Sciences, 2022, 23(15): 8366. doi: 10.3390/ijms23158366
[10]	ZHU X M, LI L, WU M, LIANG S, SHI H B, LIU X H, LIN F C. Current opinions on autophagy in pathogenicity of fungi. Virulence, 2019, 10(1): 481-489. doi: 10.1080/21505594.2018.1551011
[11]	CEBOLLERO E, VAN DER VAART A, ZHAO M, RIETER E, KLIONSKY D J, HELMS J B, REGGIORI F. Phosphatidylinositol- 3-phosphate clearance plays a key role in autophagosome completion. Current Biology, 2012, 22(17): 1545-1553. doi: 10.1016/j.cub.2012.06.029
[12]	XIE Z, KLIONSKY D J. Autophagosome formation: Core machinery and adaptations. Nature Cell Biology, 2007, 9(10): 1102-1109. doi: 10.1038/ncb1007-1102 pmid: 17909521
[13]	VOIGT O, POGGELER S. Self-eating to grow and kill: Autophagy in filamentous ascomycetes. Applied Microbiology and Biotechnology, 2013, 97(21): 9277-9290. pmid: 24077722
[14]	LIU X H, LU J P, LIN F C. Autophagy during conidiation, conidial germination and turgor generation in Magnaporthe grisea. Autophagy, 2007, 3(5): 472-473. doi: 10.4161/auto.4339
[15]	LIU X H, GAO H M, XU F, LU J P, DEVENISH R J, LIN F C. Autophagy vitalizes the pathogenicity of pathogenic fungi. Autophagy, 2012, 8(10): 1415-1425. doi: 10.4161/auto.21274
[16]	DONG B, LIU X H, LU J P, ZHANG F S, GAO H M, WANG H K, LIN F C. MgAtg9 trafficking in Magnaporthe oryzae. Autophagy, 2009, 5(7): 946-953. doi: 10.4161/auto.5.7.9161
[17]	KERSHAW M J, TALBOT N J. Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(37): 15967-15972.
[18]	VENEAULT-FOURREY C, BAROOAH M, EGAN M, WAKLEY G, TALBOT N J. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science, 2006, 312(5773): 580-583. doi: 10.1126/science.1124550
[19]	ZHU X M, LI L, CAI Y Y, WU X Y, SHI H B, LIANG S, QU Y M, NAQVI N I, DEL POETA M, DONG B, LIN F C, LIU X H. A VASt-domain protein regulates autophagy, membrane tension, and sterol homeostasis in rice blast fungus. Autophagy, 2021, 17(10): 2939-2961. doi: 10.1080/15548627.2020.1848129
[20]	LV W Y, WANG C Y, YANG N, QUE Y W, TALBOT N J, WANG Z Y. Genome-wide functional analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol production and virulence in Fusarium graminearum. Scientific Reports, 2017, 7(1): 11062. doi: 10.1038/s41598-017-11640-z
[21]	陈琳琳, 耿雪晶, 马宇明, 王利民, 施艳, 李洪连. 假禾谷镰孢菌细胞自噬相关基因的鉴定与表达分析. 植物病理学报, 2018, 48(3): 357-364.
	CHEN L L, GENG X J, MA Y M, WANG L M, SHI Y, LI H L. Identification and expression analysis of autophagy-related gene in Fusarium pseudograminearum. Acta Phytopathologica Sinica, 2018, 48(3): 357-364. (in Chinese)
[22]	CHEN L L, SHAN Y X, DONG Z F, ZHANG Y K, PENG M Y, YUAN H X, SHI Y, LI H L, XING X P. A potential hyphal fusion protein complex with an important role in development and virulence interacts with autophagy related proteins in Fusarium pseudograminearum. Journal of Integrative Agriculture, doi: 10.1016/j.jia.2023.09.005.
[23]	FANG D M, XIE H Z, HU T, SHAN H, LI M. Binding features and functions of ATG3. Frontiers in Cell and Developmental Biology, 2021, 9: 685625. doi: 10.3389/fcell.2021.685625
[24]	LIU K, ZHAO Q, LIU P L, CAO J N, GONG J Q, WANG C Q, WANG W X, LI X Y, SUN H Y, ZHANG C, et al. ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance. Autophagy, 2016, 12(11): 2000-2008. pmid: 27575019
[25]	赵静雅, 夏荟清, 彭梦雅, 凡卓, 殷悦, 徐赛博, 张楠, 陈文波, 陈琳琳. 假禾谷镰孢转录因子FpAPSES的鉴定与功能分析. 中国农业科学, 2021, 54(16): 3428-3439. doi: 10.3864/j.issn.0578-1752.2021.16.006.
	ZHAO J Y, XIA H Q, PENG M Y, FAN Z, YIN Y, XU S B, ZHANG N, CHEN W B, CHEN L L. Identification and functional analysis of transcription factors FpAPSES in Fusarium pseudograminearum. Scientia Agricultura Sinica, 2021, 54(16): 3428-3439. doi: 10.3864/j.issn.0578-1752.2021.16.006. (in Chinese)
[26]	WANG L M, ZHANG Y F, DU Z L, KANG R J, CHEN L L, XING X P, YUAN H X, DING S L, LI H L. FpPDE 1 function of Fusarium pseudograminearum on pathogenesis in wheat. Journal of Integrative Agriculture, 2017, 16(11): 2504-2512. doi: 10.1016/S2095-3119(17)61689-7
[27]	VANGALIS V, PAPAIOANNOU I A, MARKAKIS E A, KNOP M, TYPAS M A. The NADPH oxidase A of Verticillium dahliae is essential for pathogenicity, normal development, and stress tolerance, and it interacts with Yap1 to regulate redox homeostasis. Journal of Fungi, 2021, 7(9): 740. doi: 10.3390/jof7090740
[28]	LV W Y, XU Z, TALBOT N J, WANG Z Y. The sorting nexin FgAtg20 is involved in the Cvt pathway, non-selective macroautophagy, pexophagy and pathogenesis in Fusarium graminearum. Cellular Microbiology, 2020, 22(8): e13208.
[29]	WANG Y J, LIU X, XU Y J, GU Y Y, ZHANG X Y, ZHANG M X, WEN W, LEE Y W, SHI J R, MOHAMED S R, GODA A A, WU H J, GAO X W, GU Q. The autophagy-related proteins FvAtg4 and FvAtg8 are involved in virulence and fumonisin biosynthesis in Fusarium verticillioides. Virulence, 2022, 13(1): 764-780. doi: 10.1080/21505594.2022.2066611
[30]	AOKI T, O’DONNELL K. Morphological and molecular characterization of Fusarium pseudograminearum sp. nov., formerly recognized as the group 1 population of F. graminearum. Mycologia, 1999, 91: 597. doi: 10.1080/00275514.1999.12061058
[31]	CLARK-COTTON M R, JACOBS K C, LEW D J. Chemotropism and cell-cell fusion in fungi. Microbiology and Molecular Biology Reviews, 2022, 86(1): e0016521. doi: 10.1128/MMBR.00165-21
[32]	FLEIßNER A, HERZOG S. Signal exchange and integration during self-fusion in filamentous fungi. Seminars in Cell & Developmental Biology, 2016, 57: 76-83.
[33]	CORRAL-RAMOS C, ROCA M G, DI PIETRO A, RONCERO M I, RUIZ-ROLDAN C. Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum. Autophagy, 2015, 11(1): 131-144. doi: 10.4161/15548627.2014.994413
[34]	NUTA G C, GILAD Y, GOLDBERG N, MERIL S, BAHLSEN M, CARVALHO S, KOZER N, BARR H, FRIDMANN S Y, HERCIK K, BREHOVA P, NENCKA R, BIALIK S, EISENSTEIN M, KIMCHI A. Identifying a selective inhibitor of autophagy that targets ATG12- ATG3 protein-protein interaction. Autophagy, 2023, 19(8): 2372-2385. doi: 10.1080/15548627.2023.2178159
[35]	ZHENG Y M, QIU Y, GRACE C R R, LIU X, KLIONSKY D J, SCHULMAN B A. A switch element in the autophagy E2 Atg3 mediates allosteric regulation across the lipidation cascade. Nature Communications, 2019, 10(1): 3600. doi: 10.1038/s41467-019-11435-y pmid: 31399562
[36]	MA K, FU W, TANG M, ZHANG C H, HOU T Y, LI R, LU X P, WANG Y N, ZHOU J Y, LI X, ZHANG L Y, WANG L N, ZHAO Y, ZHU W G. PTK2-mediated degradation of ATG3 impedes cancer cells susceptible to DNA damage treatment. Autophagy, 2017, 13(3): 579-591. doi: 10.1080/15548627.2016.1272742 pmid: 28103122
[37]	YIN Z Y, CHEN C, YANG J, FENG W Z, LIU X Y, ZUO R F, WANG J Z, YANG L N, ZHONG K L, GAO C Y, ZHANG H F, ZHENG X B, WANG P, ZHANG Z G. Histone acetyltransferase MoHat1 acetylates autophagy-related proteins MoAtg3 and MoAtg9 to orchestrate functional appressorium formation and pathogenicity in Magnaporthe oryzae. Autophagy, 2019, 15(7): 1234-1257. doi: 10.1080/15548627.2019.1580104

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引物Primer	序列Sequence (5′-3′)
FpAtg3-F1	TGATAAGAAAGAGCAAGCT
FpAtg3-R1	TTGACCTCCACTAGCTCCAGCCGATGGCTTCGGGATTGATAA
FpAtg3-F2	ATAGAGTAGATGCCGACCGCGGGTTCACTGCATGGCTTATC
FpAtg3-R2	GCCTGGTTCCTGATCTAGAT
FpAtg3-PF	CTTCCAAAGACCTGACTAC
FpAtg3-PR	GGCCCCTTCAACGTTCTAG
FpAtg3nj-F	GCCAAATCACACCCGAAGAG
FpAtg3nj-R	GAATCGGCGCCAGAATCTC
HYG/F	GTCGACAGAAGATGATATTG
HYG/R	GAACCCGCGGTCGGCATCTACTCTAT
YG/F	GATGTAGGAGGGCGTGGATATGTCCT
HY/R	GTATTGACCGATTCCTTGCGGTCCGAA
H850F	TTCCTCCCTTTATTTCAGATTCAA
H852R	ATGTTGGCGACCTCGTATTGG
H855R	GCTGATCTGACCAGTTGC
H856F	GTCGATGCGACGCAATCGT
PKNTG-F	CTATAGGGCGAATTGGGTACCGACCTACCCATGTTTACTTG
PKNTG-R	GCAGGCATGCAAGCTTATCGATGGAGCTTTTGCTGCTCTTG
GFP-R	GATGCCCTTCAGCTCGATGCGGTTCA

菌株 Strain	孢子浓度 Conidia concentration (×10⁶/m<BOLD>L</BOLD>)	孢子长度 Conidia length (μm)	隔膜数 Septa per conidium	菌丝融合率 Hyphal fusion rate (%)	孢子融合芽管率 CAT-mediated fusion rate (%)
WT	2.25±0.23	38.84±1.53	3.97±0.80	42.0±7.3	36.1±3.7
ΔFpAtg3	1.03±0.27**	23.72±2.03**	2.86±0.55**	22.2±5.4**	17.8±3.1**
FpAtg3-C	2.18±0.15	38.58±1.27	3.82±0.72	39.8±4.6	35.8±3.9

Autophagy-Related Gene FpAtg3 Involves in Growth and Pathogenicity of Fusarium pseudograminearum

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