自噬相关蛋白PlAtg3参与荔枝霜疫霉的营养生长，孢子囊割裂，细胞自噬和致病性

doi:10.1016/j.jia.2024.01.009

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (11): 3788-3800.DOI: 10.1016/j.jia.2024.01.009

自噬相关蛋白PlAtg3参与荔枝霜疫霉的营养生长，孢子囊割裂，细胞自噬和致病性

收稿日期:2023-09-25 接受日期:2023-11-09 出版日期:2024-11-20 发布日期:2024-10-10

Autophagy-related protein PlAtg3 participates in vegetative growth, sporangial cleavage, autophagy and pathogenicity of Peronophythora litchii

Chengdong Yang^{1, 2, 3*}, Manfei Luo^{1, 2*}, Xue Zhang^{1, 2}, Linlin Ye^{1, 2}, Ge Yu^{1, 2}, Yi Lü^{1, 2}, Yi Chen^{1, 2}, Taixu Chen^{1, 2}, Xuejian Wang^{1, 2}, Wanzhen Feng^{1, 2#}, Qinghe Chen^{1, 2#}

¹ School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China
² Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests of Ministry of Education, School of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China
³ Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, Fuzhou 350013, China

Received:2023-09-25 Accepted:2023-11-09 Online:2024-11-20 Published:2024-10-10
About author:Chengdong Yang, E-mail: Chengdy@hainanu.edu.cn; #Correspondence Qinghe Chen, E-mail: qhchen@hainanu.edu.cn; Wanzhen Feng, E-mail: fwz@hainanu.edu.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the grants from the Hainan Provincial Natural Science Foundation, China (321QN190 and 321CXTD437), the National Natural Science Foundation of China (32202246 and 32160614), the Open Project Program of Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests, China (MIMCP-202102), and the Scientific Research Foundation of Hainan University, China (KYQD(ZR)-21042 and KYQD(ZR)-20080).

摘要/Abstract

摘要： 由植物病原卵菌荔枝霜疫霉引起的荔枝霜疫病是荔枝上最具破坏性的病害之一，它的发生会给荔枝的生产带来巨大经济损失。研究表明，细胞自噬在病原真菌的生长发育和致病过程中发挥至关重要作用。然而，植物病原卵菌中有关细胞自噬的功能尚不清楚。为了探究细胞自噬在荔枝霜疫霉生长发育和致病过程中的作用。在本研究中，通过生物信息学分析鉴定到了一个酵母自噬相关蛋白Atg3的同源蛋白，命名为PlAtg3。利用CRISPR/Cas9基因编辑技术对其敲除和原位回补发现，PlATG3的缺失削弱了荔枝霜疫霉的营养生长和有性/无性发育。通过特异性自噬体染料单丹黄酰戊二胺（monodansylcadaverine，MDC）对自噬体染色发现，PlATG3突变体中自噬体数量明显低于野生型菌株。FM4-64染料对细胞膜染色发现PlATG3的缺失影响了孢子囊细胞质割裂而导致游动孢子释放率显著降低。Atg8被认为是各种物种中的自噬标记蛋白。Western blot分析显示PlAtg8-PE在PlATG3突变体中的积累较野生型增多，表明PlAtg3参与了PlAtg8-PE的降解。但是，与酵母中ScAtg3能与ScAtg8互作相比，酵母双杂交（yeast two hybrid，Y2H）结果显示荔枝霜疫霉中PlAtg3无法与PlAtg8互作，可能是由于PlAtg3中缺乏Atg8家族相互作用基序（Atg8 family interacting motif，AIM）。此外，致病性实验表明PlATG3的缺失显著降低了荔枝霜疫霉对荔枝叶片和果实的致病力。综上所述，我们的数据揭示了PlAtg3在荔枝霜疫霉的菌丝生长、有性/无性发育、孢子囊割裂和游动孢子释放、自噬体形成和致病过程中发挥至关重要作用。这项研究有助于更好地理解荔枝霜疫霉的致病机制，并为开发更有效的控制卵菌类病害的策略提供见解。

Abstract:

Litchi downy blight, caused by the plant pathogenic oomycete Peronophythora litchii, is one of the most devastating diseases on litchi and resulted in huge economic losses. Autophagy plays an essential role in the development and pathogenicity of the filamentous fungi. However, the function of autophagy in oomycetes remain elusive. Here, an autophagy-related protein Atg3 homolog PlAtg3 was identified and characterized in P. litchii. The absence of PlATG3 through the CRISPR/Cas9 gene replacement strategy compromised vegetative growth and sexual/asexual development. Cytological analyses revealed that the deletion of PlATG3 impaired autophagosome formation with monodansylcadaverine (MDC) staining and significantly disrupted zoospore release due to defects of sporangial cleavage with FM4-64 staining. Atg8 is considered to be an autophagy marker protein in various species. Western blot analysis indicated that PlAtg3 is involved in degradation of PlAtg8-PE. Interestingly, PlAtg3 was unable to interact with PlAtg8 in yeast two hybrid (Y2H) assays, possibly due to the absence of the Atg8 family interacting motif (AIM) in PlAtg3. Furthermore, pathogenicity assays revealed that the deletion of PlATG3 considerably reduced the virulence of P. litchii. Taken together, our data reveal that PlAtg3 plays an important role in radial growth, asexual/sexual development, sporangial cleavage and zoospore release, autophagosome formation, and pathogenicity in P. litchii. This study contributes to a better understanding of the pathogenicity mechanisms of P. litchii and provides insights for the development of more effective strategies to control oomycete diseases.

Key words: PlAtg3 , sporangial cleavage , autophagy , pathogenicity , Peronophythora litchii

Chengdong Yang, Manfei Luo, Xue Zhang, Linlin Ye, Ge Yu, Yi Lü, Yi Chen, Taixu Chen, Xuejian Wang, Wanzhen Feng, Qinghe Chen. 自噬相关蛋白PlAtg3参与荔枝霜疫霉的营养生长，孢子囊割裂，细胞自噬和致病性[J]. Journal of Integrative Agriculture, 2024, 23(11): 3788-3800.

Chengdong Yang, Manfei Luo, Xue Zhang, Linlin Ye, Ge Yu, Yi Lü, Yi Chen, Taixu Chen, Xuejian Wang, Wanzhen Feng, Qinghe Chen. Autophagy-related protein PlAtg3 participates in vegetative growth, sporangial cleavage, autophagy and pathogenicity of Peronophythora litchii[J]. Journal of Integrative Agriculture, 2024, 23(11): 3788-3800.

参考文献

Asakura M, Ninomiya S, Sugimoto M, Oku M, Yamashita S, Okuno T, Sakai Y, Takanoa Y. 2009. Atg26-mediated pexophagy is required for host invasion by the plant pathogenic fungus Colletotrichum orbiculare. The Plant Cell, 21, 1291–1304.

Blanco F A, Judelson H S. 2005. A bZIP transcription factor from Phytophthora interacts with a protein kinase and is required for zoospore motility and plant infection. Molecular Microbiology, 56, 638–648.

Chen L, Zhang X, Wang W, Geng X J, Shi Y, Na R, Dou D L, Li H L. 2017. Network and role analysis of autophagy in Phytophthora sojae. Scientific Reports, 7, 1879.

Fang Y F, Cui L K, Gu B, Arredondo F, Tyler B M. 2017. Efficient genome editing in the oomycete Phytophthora sojae using CRISPR/Cas9. Current Protocols in Microbiology, 44, 21A.1.1–21A.1.26.

Farré J C, Subramani S. 2016. Mechanistic insights into selective autophagy pathways: lessons from yeast. Nature Reviews Molecular Cell Biology, 17, 537–552.

Fukuda T, Furukawa K, Maruyama T, Noda N N, Kanki T. 2023a. Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy. Autophagy, 19, 3019–3021.

Fukuda T, Furukawa K, Maruyama T, Yamashita S I, Noshiro D, Song C, Ogasawara Y, Okuyama K, Alam J M, Hayatsu M, Saigusa T, Inoue K, Ikeda K, Takai A, Chen L, Lahiri V, Okada Y, Shibata S, Murata K, Klionsky D J, et al. 2023b. The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy. Molecular Cell, 83, 2045–2058.e9.

Fukuda T, Kanki T. 2021. Atg43, a novel autophagy-related protein, serves as a mitophagy receptor to bridge mitochondria with phagophores in fission yeast. Autophagy, 17, 826–827.

Guan B, Xue H. 2022. Arabidopsis autophagy-related 3 (ATG3) facilitates the liquid–liquid phase separation of ATG8e to promote autophagy. Science Bulletin, 67, 350–354.

Han S, Wang Y, Zheng X, Jia Q, Zhao J, Bai F, Hong Y, Liu Y. 2015. Cytoplastic glyceraldehyde-3-phosphate dehydrogenases interact with ATG3 to negatively regulate autophagy and immunity in Nicotiana benthamiana. The Plant Cell, 27, 1316–1331.

Hanada T, Satomi Y, Takao T, Ohsumi Y. 2009. The amino-terminal region of Atg3 is essential for association with phosphatidylethanolamine in Atg8 lipidation. Federation of European Biochemical Societies Letters, 583, 1078–1083.

Hardham A R. 2007. Cell biology of plant–oomycete interactions. Cell Microbiology, 9, 31–39.

Huang J M, Xi P G, Deng Y Z, Huang W X, Wang J R, Zhao Q Q, Yang W S, Li W, Situ J J, Jiang L Q, Guan T F, Li M H, Jiang Z D, Kong G H. 2021. The mitogen-activated protein kinase PlMAPK2 is involved in zoosporogenesis and pathogenicity of Peronophythora litchii. International Journal of Molecular Sciences, 22, 3524.

Hurley J H, Schulman B A. 2014. Atomistic autophagy: The structures of cellular self-digestion. Cell, 157, 300–311.

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.

Jiang Y M, Wang Y, Song L, Liu H, Lichter A, Kerdchoechuen O, Joyce D C, Shi J. 2006. Postharvest characteristics and handling of litchi fruit - an overview. Australian Journal of Experimental Agriculture, 46, 1541–1556.

Judelson H S, Blanco F A. 2005. The spores of Phytophthora: Weapons of the plant destroyer. Nature Reviews Microbiology, 3, 47–58.

Kao C W, Leu L S. 1980. Sporangium germination of Peronophythora litchii, the causal organism of litchi downy blight. Mycologia, 72, 737–748.

Kershaw M J, Talbot N J. 2009. Genome-wide functional analysis reveals that infection-associated fungal autophagy is necessary for rice blast disease. The Proceedings of the National Academy of Sciences of the United States of America, 106, 15967–15972.

Khalid A R, Lv X L, Naeem M, Mehmood K, Shaheen H, Dong P, Qiu D, Ren M Z. 2019. Autophagy related gene (ATG3) is a key regulator for cell growth, development, and virulence of Fusarium oxysporum. Genes, 10, 658.

Kikuma T, Kitamoto K. 2011. Analysis of autophagy in Aspergillus oryzae by disruption of AoATG13, AoATG4, and AoATG15 genes. Federation of European Microbiological Societies Microbiology Letters, 316, 61–69.

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. Eukaryotic Cell, 5, 1328–1336.

Laboratories C. 2007. Matchmaker Gal4 Two-Hybrid System 3 & Libraries User Manual. [2007-4-3]. http://www.clontech.com/images/pt/PT3247-1.PDF

Li Y T, Yi C, Chen C C, Lan H, Pan M, Zhang S J, Huang Y C, Guan C J, Li Y M, Yu L, Liu L. 2017. A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation. Nature Communications, 8, 14846.

Lin L, Ye W W, Wu J W, Xuan M R, Li Y F, Gao J, Wang Y L, Wang Y, Dong S M, Wang Y C. 2018. The MADS-box transcription factor PsMAD1 is involved in zoosporogenesis and pathogenesis of Phytophthora sojae. Frontiers in Microbiology, 9, 2259.

Liu D D, Li Z X, Hou M L. 2022. Silencing the autophagy-related genes ATG3 and ATG9 promotes SRBSDV propagation and transmission in Sogatella furcifera. Insects, 13, 394.

Liu Y, Schiff M, Czymmek K, Tallóczy Z, Levine B, Dinesh-Kumar S P. 2005. Autophagy regulates programmed cell death during the plant innate immune response. Cell, 121, 567–577.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods, 25, 402–408.

Luo Q, Wang F X, Zhong N Q, Wang H Y, Xia G X. 2014. The role of autophagy during development of the oomycete pathogen Phytophthora infestans. Jouanal of Genetics Genomics, 41, 225–228.

Lv W Y, Wang C Y, Yang N, Que Y W, Talbot N J, Wang Z Y. 2017. Genome-wide functional analysis reveals that autophagy is necessary for growth, sporulation, deoxynivalenol production and virulence in Fusarium graminearum. Scientific Reports, 7, 11062.

Meng S, Xiong M, Jagernath J S, Wang C C, Qiu J H, Shi H B, Kou Y J. 2020. UvAtg8-mediated autophagy regulates fungal growth, stress responses, conidiation, and pathogenesis in Ustilaginoidea virens. Rice, 13, 56.

Mizushima N, Komatsu M. 2011. Autophagy: Renovation of cells and tissues. Cell, 147, 728–741.

Murrow L, Debnath J. 2018. Atg12–Atg3 coordinates basal autophagy, endolysosomal trafficking, and exosome release. Molecular & Cellular Oncology, 5, e1039191.

Murrow L, Malhotra R, Debnath J. 2015. ATG12–ATG3 interacts with Alix to promote basal autophagic flux and late endosome function. Nature Cell Biology, 17, 300–310.

Nadal M, Gold S E. 2010. The autophagy genes ATG8 and ATG1 affect morphogenesis and pathogenicity in Ustilago maydis. Molecular Plant Pathology, 11, 463–478.

Nair U, Yen W L, Mari M, Cao Y, Xie Z P, Baba M, Reggiori F, Klionsky D J. 2012. A role for Atg8-PE deconjugation in autophagosome biogenesis. Autophagy, 8, 780–793.

Pollack J K, Harris S D, Marten M R. 2009. Autophagy in filamentous fungi. Fungal Genetics and Biology, 46, 1–8.

Popelka H, Klionsky D J. 2021. Multiple structural rearrangements mediated by high-plasticity regions in Atg3 are key for efficient conjugation of Atg8 to PE during autophagy. Autophagy, 17, 1805–1808.

Qiu M, Li Y, Ye W W, Zheng X B, Wang Y C. 2021. A CRISPR/Cas9-mediated in situ complementation method for Phytophthora sojae mutants. Molecular Plant Pathology, 22, 373–381.

Radoshevich L, Murrow L, Chen N, Fernandez E, Roy S, Fung C, Debnath. J. 2010. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell, 142, 590–600.

Sakoh-Nakatogawa M, Matoba K, Asai E, Kirisako H, Ishii J, Noda N N, Inagaki F, Nakatogawa H, Ohsumi Y. 2013. Atg12–Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site. Nature Structural & Molecular Biology, 20, 433–439.

Sun J H, Gao Z Y, Zhang X C, Zou X X, Cao L L, Wang J B. 2017. Transcriptome analysis of Phytophthora litchii reveals pathogenicity arsenals and confirms taxonomic status. PLoS ONE, 12, e0178245.

Suzuki K, Ohsumi Y. 2010. Current knowledge of the pre-autophagosomal structure (PAS). Federation of European Biochemical Societies Letters, 584, 1280–1286.

Talibi I, Boubaker H, Boudyach E H, Ben Aoumar A A. 2014. Alternative methods for the control of postharvest citrus diseases. Journal of Applied Microbiology, 117, 1–17.

Tanida I, Tanida-Miyake E, Komatsu M, Ueno T, Kominami E. 2002. Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p. Journal of Biological Chemistry, 277, 13739–13744.

Tyler B M. 2007. Phytophthora sojae: Root rot pathogen of soybean and model oomycete. Molecular Plant Pathology, 8, 1–8.

Veneault-Fourrey C, Barooah M, Egan M, Wakley G, Talbot N J. 2006. Autophagic fungal cell death is necessary for infection by the rice blast fungus. Science, 312, 580–583.

Voigt O, Poggeler S. 2013. Autophagy genes SmATG8 and SmATG4 are required for fruiting-body development, vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora. Autophagy, 9, 33–49.

Wang H C, Sun H Y, Stammler G, Ma J X, Zhou M G. 2009. Baseline and differential sensitivity of Peronophythora litchii (lychee downy blight) to three carboxylic acid amide fungicides. Plant Pathology, 58, 571–576.

Wang J R, Zhou G Q, Huang W X, Li W, Feng D L, Liu L C, Xi P G, Jiang Z D, Kong G H. 2022. Autophagy-related gene PlATG6a is involved in mycelial growth, asexual reproduction and tolerance to salt and oxidative stresses in Peronophythora litchii. International Journal of Molecular Sciences, 23, 1839.

Wang S, Li Y, Ma C. 2020. Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement. Protein Science, 29, 1511–1523.

Wei Y Z, Zhang H N, Li W C, Xie J H, Wang Y C, Liu L Q, Shi S Y. 2013. Phenological growth stages of lychee (Litchi chinensis Sonn.) using the extended BBCH-scale. Scientia Horticulturae, 161, 273–277.

Xu L X, Xue J H, Wu P, Wang D D, Lin L J, Jiang Y M, Duan X W, Wei X Y. 2013. Antifungal activity of hypothemycin against Peronophythora Litchii in vitro and in vivo. Journal of Agricultural and Food Chemistry, 61, 10091–10095.

Yamada Y, Suzuki N N, Hanada T, Ichimura Y, Kumeta H, Fujioka Y, Ohsumi Y, Inagaki F. 2007. The crystal structure of Atg3, an autophagy-related ubiquitin carrier protein (E2) enzyme that mediates Atg8 lipidation. Journal of Biological Chemistry, 282, 8036–8043.

Yamaguchi M, Noda N N, Nakatogawa H, Kumeta H, Ohsumi Y, Inagaki F. 2010. Autophagy-related protein 8 (Atg8) family interacting motif in Atg3 mediates the Atg3-Atg8 interaction and is crucial for the cytoplasm-to-vacuole targeting pathway. Journal of Biological Chemistry, 285, 29599–29607.

Ye C L, Feng Y L, Yu F F, Jiao Q Q, Wu J G, Ye Z H, Zhang P J, Sun C X, Pang K, Hao P Y, Yu X P. 2021. RNAi‐mediated silencing of the autophagy‐related gene NlATG3 inhibits survival and fecundity of the brown planthopper, Nilaparvata lugens. Pest Management Science, 77, 4658–4668.

Yi C, Ma M S, Ran L L, Zheng J X, Tong J J, Zhu J, Ma C Y, Sun Y F, Zhang S J, Feng W Z, Zhu L Y, Le Y, Gong X Q, Yan X H, Hong B, Jiang F J, Xie Z P, Miao D, Deng H T, Yu L. 2012. Function and molecular mechanism of acetylation in autophagy regulation. Science, 336, 474–477.

Yu G, Li W Q, Yang C D,Zhang X, Luo M F, Chen T X, Wang X J, Wang R B, Chen Q H. 2024. PlAtg8-mediated autophagy regulates vegetative growth, sporangial cleavage, and pathogenesis in Peronophythora litchii. Microbiology Spectrum, 12, e0353123.

Yue J Y, Wang Y J, Jiao J L, Wang H Z. 2021. Silencing of ATG2 and ATG7 promotes programmed cell death in wheat via inhibition of autophagy under salt stress. Ecotoxicology Environmental Safety, 225, 112761.

Zhao X R, Feng W J, Zhu X Y, Li C X, Ma X Y, Li X, Zhu X D, Wei D S. 2019. Conserved autophagy pathway contributes to stress tolerance and virulence and differentially controls autophagic flux upon nutrient starvation in Cryptococcus neoformans. Frontiers in Microbiology, 10, 2690.

自噬相关蛋白PlAtg3参与荔枝霜疫霉的营养生长，孢子囊割裂，细胞自噬和致病性

Autophagy-related protein PlAtg3 participates in vegetative growth, sporangial cleavage, autophagy and pathogenicity of Peronophythora litchii

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