条纹病菌效应蛋白Pg00778调控对大麦致病力的作用

doi:10.3864/j.issn.0578-1752.2025.15.007

摘要/Abstract

摘要：

【目的】大麦条纹病是由大麦条纹病菌（Pyrenophora graminea）引起的重大农业病害，严重影响大麦的产量和品质。条纹病菌侵染寄主过程中分泌多种效应蛋白调控植物防御系统，增强其致病力。明确条纹病菌效应蛋白Pg00778的功能及作用机理，为条纹病菌致病机制研究提供理论参考。【方法】基于本课题组前期测序的大麦条纹病强致病力菌株QWC基因组数据，结合大麦条纹病菌与高感大麦互作的转录组分析结果得到一个候选效应蛋白Pg00778。以大麦条纹病菌野生株QWC为试材克隆Pg00778，并分析其序列特征，使用MEGA11.0构建大麦条纹病菌与其他病原菌蛋白的系统发育树。在本氏烟叶片细胞中瞬时表达验证Pg00778的毒性功能，运用烟草亚细胞定位试验研究Pg00778蛋白的表达位置。采用CaCl₂-PEG4000介导的原生质体转化法获得Pg00778基因干扰突变菌株及过表达转化菌株，通过菌株营养生长和致病力测定，对Pg00778在大麦条纹病菌侵染大麦中的功能进行分析。【结果】 Pg00778不含信号肽、无跨膜区域且无已知的功能结构域，属于非经典的效应蛋白；系统发育分析表明Pg00778与小麦黄斑叶枯病菌（XM_066107220.1）进化关系最近；在本氏烟叶片中瞬时表达Pg00778既不能诱导细胞坏死，也不能抑制由BAX引起的细胞坏死；Pg00778主要定位于细胞核、细胞膜和细胞质；与野生株相比，Pg00778-RNAi突变株基因相对表达量分别降低41.6%和54.0%，Pg00778-OE转化株基因相对表达量分别升高290.0%、397.4%和263.7%；干扰Pg00778导致大麦条纹病菌的生长速率显著降低21.4%—30.1%，侵染大麦后叶片叶绿素相对含量升高34.1%—39.1%；Pg00778-OE转化株菌落生长速率较野生株明显加快7.3%—12.6%，侵染大麦叶片后叶绿素相对含量较野生组显著降低20.0%—23.0%；菌丝显微形态并无明显差别；致病力检测发现Pg00778-RNAi突变株发病率较野生株分别下降51.5%和49.0%，Pg00778-OE转化株发病率分别升高19.0%、20.3%和21.2%；菌株侵染大麦后，与野生型植株相比，Pg00778-OE转化株侵染植株叶片中的条纹病症状更加严重，台盼蓝染色更深、更蓝，活性氧的积累量显著增加；Pg00778-RNAi突变株侵染叶片条纹症状明显减少，细胞膜较完整，活性氧的积累量显著降低。【结论】效应蛋白Pg00778正调控大麦条纹病菌的致病力，是影响大麦条纹病菌侵染寄主的重要效应蛋白。

关键词: 大麦条纹病菌, 大麦条纹病, 效应蛋白, Pg00778, RNA干扰, 过表达, 致病力

Abstract:

【Objective】 Barley leaf stripe is a significant agricultural disease caused by Pyrenophora graminea, which severely affects the yield and quality of barley. During the process of infecting its host, P. graminea secretes a variety of effector proteins to modulate the plant’s defense system, thereby enhancing its pathogenicity. The objective of this study is to clarify the function and mechanism of action of the P. graminea effector protein Pg00778, and to provide a theoretical reference for the study of P. graminea.【Method】 Based on the previously sequenced genome data of the highly virulent P. graminea strain QWC from our research group, combined with transcriptomic analysis of the P. graminea-highly susceptible barley interaction, a candidate effector protein, Pg00778, was identified. Using the P. graminea wild-type strain QWC as the experimental material, the Pg00778 was cloned, sequence characteristics were analyzed, and a phylogenetic tree was constructed using MEGA11.0 with homologous proteins from P. graminea and other pathogenic fungi. The virulence function of Pg00778 was validated through transient expression in Nicotiana benthamiana leaf cells, while its subcellular localization was determined using tobacco leaf localization assays. Both RNAi mutants and overexpression transformants of Pg00778 were generated via CaCl₂-PEG4000-mediated protoplast transformation. Subsequent assessments of mycelial growth and pathogenicity were conducted to elucidate the function of Pg00778 during P. graminea infection in barley.【Result】 Pg00778 lacks signal peptides, transmembrane domains, and known functional domains, categorizing it as a non-classical effector protein. Phylogenetic analysis indicated that Pg00778 has the closest evolutionary relationship with the P. tritici-repentis (XM_066107220.1). The transient expression of Pg00778 in N. benthamian leaves was ineffective in inducing cell death and inhibiting cell death caused by BAX. Pg00778 is mainly localized in the nucleus, cell membrane and cytoplasm. Compared with the wild-type strain QWC, the relative gene expression levels of the Pg00778 in RNAi mutants were reduced by 41.6% and 54.0%, respectively, while the relative gene expression levels of the Pg00778 in OE mutants increased by 290.0%, 397.4%, and 263.7%, respectively. A significant reduction was detected in the growth rate of the RNAi mutants by 21.4% to 30.1%, and an increase in the relative content of chlorophyll in barley leaf by 34.1% to 39.1% after infection. The colony growth rate of the Pg00778-OE mutants was significantly faster than that of the wild-type strain by 7.3% to 12.6%, and the relative content of chlorophyll in barley leaf infected by the OE mutants was significantly lower than that of the wild-type strain by 20.0% to 23.0%. There was no significant difference in the microscopic morphology of mycelium. Pathogenicity test revealed that the incidence of Pg00778-RNAi mutants decreased by 51.5% and 49.0% compared to the QWC, while the Pg00778-OE mutants increased by 19.0%, 20.3%, and 21.2%. Compared with ‘Alexis’ infected by wild-type strain QWC, ‘Alexis’ infected by Pg00778-OE mutants showed more severe stripe disease symptoms, deeper and bluer trypan blue staining, and significantly increased accumulation of reactive oxygen species in the leaves; the stripe invasion symptoms of leaf infected by Pg00778-RNAi mutants were significantly reduced, the cell membrane was more intact, and the accumulation of reactive oxygen species was significantly reduced.【Conclusion】 Effector protein Pg00778 positively regulates the pathogenicity of P. graminea and serves as a crucial effector protein influencing its infection to the host.

Key words: Pyrenophora graminea, barley leaf stripe, effector protein, Pg00778, RNAi, overexpression, pathogenicity

杨文娟, 高嘉诚, 王艳婷, 李妍, 郭铭, 汪军成, 孟亚雄, 王化俊, 司二静. 条纹病菌效应蛋白Pg00778调控对大麦致病力的作用[J]. 中国农业科学, 2025, 58(15): 3020-3035.

YANG WenJuan, GAO JiaCheng, WANG YanTing, LI Yan, GUO Ming, WANG JunCheng, MENG YaXiong, WANG HuaJun, SI ErJing. Function of Effector Pg00778 Regulation on the Pathogenicity of Pyrenophora graminea to Barley[J]. Scientia Agricultura Sinica, 2025, 58(15): 3020-3035.

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参考文献 53

[1]	吴文平. 河北省丝孢菌的研究I、禾本科植物上蠕形菌的初步研究. 河北省科学院学报, 1990(1): 52-64.
	WU W P. Study on hyphomycetes in Hebei I. Some graminicolous species of Genera Drechslera, Bipolaris and Exserohilum. Journal of the Hebei Academy of Sciences, 1990(1): 52-64. (in Chinese)
[2]	JAT V P, SHARM P, SHAH R. Patho-physiological studies on culture filtrates of Drechslera graminea causing leaf stripe of barley. Annals of Plant Protection Sciences, 2015, 23(1): 115-119.
[3]	ARNST B J, SHERIDAN J E, GRBAVAC N. Two important fungal seed-borne diseases of barley in New Zealand: Net blotch caused by Drechslera teres (Sacc.) Shoemaker and leaf stripe caused by Drechslera graminea (Rabenh. ex Schlecht.) Shoemaker. New Zealand Journal of Agricultural Research, 1978, 21(4): 697-701.
[4]	PORTA-PUGLIA A, DELOGU G, VANNACCI G. Pyrenophora graminea on winter barley seed: Effect on disease incidence and yield losses. Journal of Phytopathology, 1986, 117(1): 26-33.
[5]	JONES J D, DANGL J L. The plant immune system. Nature, 2006, 444(7117): 323-329.
[6]	YU X, FENG B M, HE P, SHAN L B. From chaos to harmony: Responses and signaling upon microbial pattern recognition. Annual Review of Phytopathology, 2017, 55: 109-137. doi: 10.1146/annurev-phyto-080516-035649 pmid: 28525309
[7]	ZHANG Z, WU Y, GAO M H, ZHANG J, KONG Q, LIU Y N, BA H P, ZHOU J M, ZHANG Y L. Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2. Cell Host & Microbe, 2012, 11(3): 253-263.
[8]	MACHO A P, ZIPFEL C. Targeting of plant pattern recognition receptor-triggered immunity by bacterial type-III secretion system effectors. Current Opinion in Microbiology, 2015, 23: 14-22. doi: 10.1016/j.mib.2014.10.009 pmid: 25461568
[9]	KOECK M, HARDHAM A R, DODDS P N. The role of effectors of biotrophic and hemibiotrophic fungi in infection. Cellular Microbiology, 2011, 13(12): 1849-1857. doi: 10.1111/j.1462-5822.2011.01665.x pmid: 21848815
[10]	ESSE H P, BOLTON M D, STERGIOPOULOS I, WIT P, THOMMA B. The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor. Molecular Plant-Microbe Interactions, 2007, 20(9): 1092-1101.
[11]	SONG J, WIN J, TIAN M, SCHORNACK S, KASCHANI F, ILYAS M, HOORN R A, KAMOUN S. Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defense protease Rcr3. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(5): 1654-1659.
[12]	DOEHLEMANN G, VAN DER LINDE K, AßMANN D, SCHWAMMBACH D, HOF A, MOHANTY A, JACKSON D, KAHMANN R. Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells. PLoS Pathogens, 2009, 5(2): e1000290.
[13]	WEI J P, WANG X D, HU Z Y, WANG X J, WANG J L, WANG J F, HUANG X L, KANG Z S, TANG C L. The Puccinia striiformis effector Hasp98 facilitates pathogenicity by blocking the kinase activity of wheat TaMAPK4. Journal of Integrative Plant Biology, 2023, 65(1): 249-264.
[14]	MCLELLAN H, BOEVINK P C, ARMSTRONG M R, PRITCHARD L, GOMEZ S, MORALES J, WHISSON S C, BEYNON J L, BIRCH P R. An RxLR effector from Phytophthora infestans prevents re-localisation of two plant NAC transcription factors from the endoplasmic reticulum to the nucleus. PLoS Pathogens, 2013, 9(10): e1003670.
[15]	LORRAIN C, GONCALVES DOS SANTOS K C, GERMAIN H, HECKER A, DUPLESSIS S. Advances in understanding obligate biotrophy in rust fungi. New Phytologist, 2019, 222(3): 1190-1206. doi: 10.1111/nph.15641 pmid: 30554421
[16]	LOWE R G T, HOWLETT B J. Indifferent, affectionate, or deceitful: Lifestyles and secretomes of fungi. PLoS Pathogens, 2012, 8(3): e1002515.
[17]	SONAH H, DESHMUKH R K, BELANGER R R. Computational prediction of effector proteins in fungi: Opportunities and challenges. Frontiers in Plant Science, 2016, 7: 126. doi: 10.3389/fpls.2016.00126 pmid: 26904083
[18]	DODDS P N, RAFIQI M, GAN P H, HARDHAM A R, JONES D A, ELLIS J G. Effectors of biotrophic fungi and oomycetes: Pathogenicity factors and triggers of host resistance. New Phytologist, 2009, 183(4): 993-1000. doi: 10.1111/j.1469-8137.2009.02922.x pmid: 19558422
[19]	RIDOUT C J, SKAMNIOTI P, PORRITT O, SACRISTAN S, JONES J D G, BROWN J K M. Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance. The Plant Cell, 2006, 18(9): 2402-2414.
[20]	RABOUILLE C. Pathways of unconventional protein secretion. Trends in Cell Biology, 2017, 27(3): 230-240. doi: S0962-8924(16)30205-7 pmid: 27989656
[21]	LIU T L, SONG T Q, ZHANG X, YUAN H B, SU L M, LI W L, XU J, LIU S H, CHEN L L, CHEN T Z, ZHANG M X, GU L C, ZHANG B L, DOU D L. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nature Communications, 2014, 5: 4686. doi: 10.1038/ncomms5686 pmid: 25156390
[22]	靳生杰, 洪平, 张丽琼. 玉门市大麦条纹病的发生与防治. 农业科技与信息, 2008(13): 35-36.
	JIN S J, HONG P, ZHANG L Q. Occurrence and control of barley stripe disease in Yumen City. Agricultural Science-Technology and Information, 2008(13): 35-36. (in Chinese)
[23]	牛小霞. 河西地区大麦条纹病的发病规律及防治措施. 现代化农业, 2014(2): 2-4.
	NIU X X. The epidemic pattern and control measures of barley stripe disease in the Hexi Corridor Region. Modernizing Agriculture, 2014(2): 2-4. (in Chinese)
[24]	TAKKEN F L, GOVERSE A. How to build a pathogen detector: Structural basis of NB-LRR function. Current Opinion in Plant Biology, 2012, 15(4): 375-384. doi: 10.1016/j.pbi.2012.05.001 pmid: 22658703
[25]	DUPLESSIS S, CUOMO C A, LIN Y C, AERTS A, TISSERANT E, VENEAULT-FOURREY C, JOLY D L, HACQUARD S, AMSELEM J, CANTAREL B L, et al. Obligate biotrophy features unraveled by the genomic analysis of rust fungi. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(22): 9166-9171.
[26]	SI E J, MENG Y X, MA X L, LI B C, WANG J C, YAO L R, YANG K, ZHANG Y, SHANG X W, WANG H J. Genome resource for barley leaf stripe pathogen Pyrenophora graminea. Plant Disease, 2020, 104(2): 320-322.
[27]	CEULEMANS E, IBRAHIM H M, CONINCK B, GOOSSENS A. Pathogen effectors: Exploiting the promiscuity of plant signaling hubs. Trends in Plant Science, 2021, 26(8): 780-795. doi: 10.1016/j.tplants.2021.01.005 pmid: 33674173
[28]	许丹洁, 林巧霞, 李正康, 庄小倩, 凌宇, 赖美玲, 陈晓婷, 鲁国东. OsOPR10正调控水稻对稻瘟病和白叶枯病的抗性. 中国水稻科学, 2024, 38(4): 364-374. doi: 10.16819/j.1001-7216.2024.231215
	XU D J, LIN Q X, LI Z K, ZHUANG X Q, LING Y, LAI M L, CHEN X T, LU G D. OsOPR10 positively regulates rice blast and bacterial blight resistance. Chinese Journal of Rice Science, 2024, 38(4): 364-374. (in Chinese)
[29]	赵彬森, 高承宇, 张健, 冯浩, 黄丽丽. Vm-milR21调控苹果黑腐皮壳侵染致病的功能和机理. 微生物学报, 2023, 63(12): 4738-4751.
	ZHAO B S, GAO C Y, ZHANG J, FENG H, HUANG L L. Function and mechanism study of Vm-milR21 in the infection of apple tree Valsa canker pathogen. Acta Microbiologica Sinica, 2023, 63(12): 4738-4751. (in Chinese)
[30]	DUVAUD S, GABELLA C, LISACEK F, STOCKINGER H, IOANNIDIS V, DURINX C. Expasy, the Swiss bioinformatics resource portal, as designed by its users. Nucleic Acids Research, 2021, 49(W1): W216-W227.
[31]	TEUFEL F, ARMENTEROS J J, JOHANSEN A R, GISLASON M H, PIHL S I, TSIRIGOS K D, WINTHER O, BRUNAK S, VON HEIJNE G, NIELSEN H. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nature Biotechnology, 2022, 40(7): 1023-1025. doi: 10.1038/s41587-021-01156-3 pmid: 34980915
[32]	ARAI M, MITSUKE H, IKEDA M, XIA J X, KIKUCHI T, SATAKE M, SHIMIZU T. ConPred II: A consensus prediction method for obtaining transmembrane topology models with high reliability. Nucleic Acids Research, 2004, 32: W390-W393. doi: 10.1093/nar/gkh380 pmid: 15215417
[33]	SPERSCHNEIDER J, DODDS P. EffectorP 3.0: Prediction of apoplastic and cytoplasmic effectors in fungi and oomycetes. Molecular Plant-Microbe Interactions, 2022, 35(2): 146-156.
[34]	WANG J Y, CHITSAZ F, DERBYSHIRE M K, GONZALES N R, GWADZ M, LU S N, MARCHLER G H, SONG J S, THANKI N, YAMASHITA R A, et al. The conserved domain database in 2023. Nucleic Acids Research, 2023, 51(D1): D384-D388.
[35]	DAUDI A, O’BRIEN J A. Detection of hydrogen peroxide by DAB staining in Arabidopsis leaves. Bio-Protocol, 2012, 2(18): e263.
[36]	LEHMANN S, SERRANO M, L’HARIDON F, TJAMOS S E, METRAUX J P. Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry, 2015, 112: 54-62. doi: 10.1016/j.phytochem.2014.08.027 pmid: 25264341
[37]	KALDERON D, RICHARDSON W D, MARKHAM A F, SMITH A E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature, 1984, 311(5981): 33-38.
[38]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method. Methods, 2001, 25(4): 402-408.
[39]	SUKA I E, CHEW B L, GOH H H, ISA N M. Transformation of PRT6 RNAi construct into tomato (Solanum lycopersicum) cv. Micro-Tom//AIP Conference Proceedings, 2018, 1940(1): 020059.
[40]	ARABI M I E, JAWHAR M, MIRALI N. Storage protein (hordein) patterns of barley-Pyrenophora graminea interaction. Seed Science and Technology, 2005, 33(2): 409-418.
[41]	张宝成, 白艳芬, 陈骥. 病虫害侵染对植物光合作用影响研究进展. 河南师范大学学报(自然科学版), 2015, 43(2): 119-125.
	ZHANG B C, BAI Y F, CHEN J. Effects of photosynthesis by pests and diseases: A review. Journal of Henan Normal University (Natural Science Edition), 2015, 43(2): 119-125. (in Chinese)
[42]	高芬, 褚建梅, 李静虹, 王梦亮. 植物病原真菌致病机理研究进展. 江苏农业学报, 2014, 30(5): 1174-1179.
	GAO F, CHU J M, LI J H, WANG M L. Research progress in the pathogenesis of plant pathogenic fungi. Jiangsu Journal of Agricultural Sciences, 2014, 30(5): 1174-1179. (in Chinese)
[43]	SUGIO A, MACLEAN A M, KINGDOM H N, GRIEVE V M, MANIMEKALAI R, HOGENHOUT S A. Diverse targets of phytoplasma effectors: From plant development to defense against insects. Annual Review of Phytopathology, 2011, 49: 175-195. doi: 10.1146/annurev-phyto-072910-095323 pmid: 21838574
[44]	GUO M, SI E J, HOU J, YAO L R, WANG J C, MENG Y X, MA X L, LI B C, WANG H J. Pgmiox mediates stress response and plays a critical role for pathogenicity in Pyrenophora graminea, the agent of barley leaf stripe. Plant Science, 2025, 350: 112308.
[45]	LIANG Q Q, LI B C, WANG J C, REN P R, YAO L R, MENG Y X, SI E J, SHANG X W, WANG H J. PGPBS, a mitogen-activated protein kinase kinase, is required for vegetative differentiation, cell wall integrity, and pathogenicity of the barley leaf stripe fungus Pyrenophora graminea. Gene, 2019, 696: 95-104.
[46]	刘海颖. 大麦条纹病菌多聚半乳糖醛酸酶基因Pgpg1功能分析[D]. 兰州: 甘肃农业大学, 2022.
	LIU H Y. Functional analysis of polygalacturonase gene Pgpg1 of Pyrenophora graminea[D]. Lanzhou: Gansu Agricultural University, 2022. (in Chinese)
[47]	王建锋, 成嘉欣, 舒伟学, 张艳茹, 王晓杰, 康振生, 汤春蕾. 小麦条锈菌效应蛋白Hasp83在条锈菌致病性中的功能分析. 中国农业科学, 2023, 56(5): 866-878. doi: 10.3864/j.issn.0578-1752.2023.05.005.
	WANG J F, CHENG J X, SHU W X, ZHANG Y R, WANG X J, KANG Z S, TANG C L. Functional analysis of effector Hasp83 in the pathogenicity of Puccinia striiformis f. sp. tritici. Scientia Agricultura Sinica, 2023, 56(5): 866-878. doi: 10.3864/j.issn.0578-1752.2023.05.005. (in Chinese)
[48]	JONES K, KHANG C H. Visualizing the movement of Magnaporthe oryzae effector proteins in rice cells during infection. Methods in Molecular Biology, 2018, 1848: 103-117.
[49]	MOSQUERA G, GIRALDO M C, KHANG C H, COUGHLAN S, VALENT B. Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as biotrophy-associated secreted proteins in rice blast disease. The Plant Cell, 2009, 21(4): 1273-1290.
[50]	KHANG C H, BERRUYER R, GIRALDO M C, KANKANALA P, PARK S Y, CZYMMEK K, KANG S, VALENT B. Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement. The Plant Cell, 2010, 22(4): 1388-1403.
[51]	ZHANG L S, NI H, DU X, WANG S, MA X W, NURNBERGER T, GUO H S, HUA C L. The Verticillium-specific protein VdSCP7 localizes to the plant nucleus and modulates immunity to fungal infections. New Phytologist, 2017, 215: 368-381.
[52]	AVERYANOV A. Oxidative burst and plant disease resistance. Frontiers in Bioscience, 2009, 1: 142-152.
[53]	HUANG J, HE Z, WANG J, ZHA X, XIAO Q, LIU G, LI Y, KANG J. A novel effector FlSp1 inhibits the colonization of endophytic Fusarium lateritium and increases the resistance to Ralstonia solanacearum in tobacco. Journal of Fungi, 2023, 9: 519.