An effector of Erysiphe necator translocates to chloroplasts and plasma membrane to suppress host immunity in grapevine

Abstract

The powdery mildew (Erysiphe necator) is a prevalent pathogen hampering grapevine growth in the vineyard. An arsenal of candidate secreted effector proteins (CSEPs) was encoded in the E. necator genome, but it is largely unclear what role CSEPs plays during the E. necator infection. In the present study, we identified a secreted effector CSEP080 of E. necator, which was located in plant chloroplasts and plasma membrane. Transient expressing CSEP080 promotes plant photosynthesis and inhibits INF1-induced cell death in tobacco leaves. We found that CSEP080 was a necessary effector for the E. necator pathogenicity, which interacted with grapevine chloroplast protein VviB6f (cytochrome b6-f complex iron–sulfur subunit), affecting plant photosynthesis. Transient silencing VviB6f increased the plant hydrogen peroxide production, and the plant resistance to powdery mildew. In addition, CSEP080 manipulated the VviPE (pectinesterase) to promote pectin degradation. Our results demonstrated the molecular mechanisms that an effector of E. necator translocates to host chloroplasts and plasma membrane, which suppresses with the grapevine immunity system by targeting the chloroplast protein VviB6f to suppress hydrogen peroxide accumulation and manipulating VviPE to promote pectin degradation.

Acknowledgements

We would like to give great thanks to Dr Weixing Shan, Dr Jun Guo and Dr Yu Du from Northwest A&F University for kindly providing the vectors. We gratefully acknowledge all members in the 4103 group of the State Key Laboratory of Crop Stress Biology for Arid Areas, NWAFU. This work was supported by the National Natural Science Foundation of China (Grant No. 31972986, 32272670) and the Key Research and Development Program of Shaanxi province (2023-YBNY-059).

Author contributions

Y.-Q.W. – conceptualization, funding acquisition and writing draft. B.M. – writing improving and edition, experiment conduction. Z.T. and R.T. – experiment conduction. M.L., J.C. and X.X.– formal analysis.

Data availability

All relevant data in this study are provided in the article and its supplementary data.

Conflict of interest statement

The authors declare no conflict of interest.

References

[1] Jones JD, Dangl JL . The plant immune system. Nature. 2006; 444: 323-9

[2] Dodds PN, Rathjen JP . Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet. 2010; 11: 539-48

[3] Couto D, Zipfel C . Regulation of pattern recognition receptor signalling in plants. Nat Rev Immunol. 2016; 16: 537-52

[4] Cui HT, Tsuda K, Parker JE . Effector-triggered immunity: from pathogen perception to robust defense. Annu Rev Plant Biol. 2015; 66: 487-511

[5] Bi G, Zhou Z, Wang W et al. Receptor-like cytoplasmic kinases directly link diverse pattern recognition receptors to the activation of mitogen-activated protein kinase cascades in Arabidopsis. Plant Cell. 2018; 30: 1543-61

[6] Chisholm ST, Coaker G, Day B et al. Host-microbe interactions: shaping the evolution of the plant immune response. Cell. 2006; 124: 803-14

[7] Ngou BPM, Ding P, Jones JDG . Thirty years of resistance: zig-zag through the plant immune system. Plant Cell. 2022; 34: 1447-78

[8] Zhai K, Liang D, Li H et al. NLRs guard metabolism to coordinate pattern- and effector-triggered immunity. Nature. 2022; 601: 245-51

[9] Yuan M, Jiang Z, Bi G et al. Pattern-recognition receptors are required for NLR-mediated plant immunity. Nature. 2021; 592: 105-9

[10] Jiang C, Hei R, Yang Y et al. An orphan protein of Fusarium graminearum modulates host immunity by mediating proteasomal degradation of TaSnRK1α. Nat Commun. 2020; 11: 4382

[11] Ma SC, Lapin D, Liu L et al. Direct pathogen-induced assembly of an NLR immune receptor complex to form a holoenzyme. Science. 2020; 370: eabe3069

[12] Toruno TY, Stergiopoulos I, Coaker G . Plant-pathogen effectors: cellular probes interfering with plant defenses in spatial and temporal manners. Annu Rev Phytopathol. 2016; 54: 419-41

[13] Mukhtar MS, Carvunis AR, Dreze M et al. Independently evolved virulence effectors converge onto hubs in a plant immune system network. Science. 2011; 333: 596-601

[14] de Torres Zabala M, Littlejohn G, Jayaraman S et al. Chloroplasts play a central role in plant defence and are targeted by pathogen effectors. Nat Plants. 2015; 1: 15074

[15] Serrano I, Audran C, Rivas S . Chloroplasts at work during plant innate immunity. J Exp Bot. 2016; 67: 3845-54

[16] Yokochi Y, Fukushi Y, Wakabayashi KI et al. Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2021; 118: e2114952118

[17] Littlejohn GR, Breen S, Smirnoff N et al. Chloroplast immunity illuminated. New Phytol. 2021; 229: 3088-107

[18] Kretschmer M, Damoo D, Djamei A et al. Chloroplasts and plant immunity: where are the fungal effectors? Pathogens. 2019; 9: 19

[19] Medina-Puche L, Tan H, Dogra V et al. A defense pathway linking plasma membrane and chloroplasts and co-opted by pathogens. Cell. 2020; 182: 1109-1124.e25

[20] Yang X, Lu Y, Wang F et al. Involvement of the chloroplast gene ferredoxin 1 in multiple responses of Nicotiana benthamiana to potato virus X infection. J Exp Bot. 2020; 71: 2142-56

[21] Li Z, Li C, Fu S et al. NSvc4 encoded by rice stripe virus targets host chloroplasts to suppress chloroplast-mediated defense. Viruses. 2021; 14: 36

[22] Toufexi A, Duggan C, Pandey P et al. Chloroplasts navigate towards the pathogen interface to counteract infection by the Irish potato famine pathogen. bioRxiv. 2019; 516443.

[23] Liu R, Chen T, Yin X et al. A Plasmopara viticola RXLR effector targets a chloroplast protein PsbP to inhibit ROS production in grapevine. Plant J. 2021; 106: 1557-70

[24] Xu Q, Tang C, Wang X et al. An effector protein of the wheat stripe rust fungus targets chloroplasts and suppresses chloroplast function. Nat Commun. 2019; 10: 5571

[25] Wang XD, Zhai T, Zhang X et al. Two stripe rust effectors impair wheat resistance by suppressing import of host Fe-S protein into chloroplasts. Plant Physiol. 2021; 187: 2530-43

[26] Wang D, Kanyuka K, Papp-Rupar M . Pectin: a critical component in cell-wall-mediated immunity. Trends Plant Sci. 2023; 28: 10-3

[27] Bethke G, Thao A, Xiong G et al. Pectin biosynthesis is critical for cell wall integrity and immunity in Arabidopsis thaliana. Plant Cell. 2016; 28: 537-56

[28] Zhang J, Yu X, Zhang C et al. Pectin lyase enhances cotton resistance to Verticillium wilt by inducing cell apoptosis of Verticillium dahliae. J Hazard Mater. 2021; 404: 124029

[29] Zhang L, Liu J, Cheng J et al. lncRNA7 and lncRNA2 modulate cell wall defense genes to regulate cotton resistance to Verticillium wilt. Plant Physiol. 2022; 189: 264-84

[30] Zhang WJ, Pedersen C, Kwaaitaal M et al. Interaction of barley powdery mildew effector candidate CSEP0055 with the defence protein PR17c. Mol Plant Pathol. 2012; 13: 1110-9

[31] Liao WL, Nielsen ME, Pedersen C et al. Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity. J Exp Bot. 2023; 74: 118-29

[32] Zhao JH, Huang YY, Wang H et al. Golovinomyces cichoracearum effector-associated nuclear localization of RPW8.2 amplifies its expression to boost immunity in Arabidopsis. New Phytol. 2023; 238: 367-82

[33] Pennington HG, Gheorghe DM, Damerum A et al. Interactions between the powdery mildew effector BEC1054 and barley proteins identify candidate host targets. J Proteome Res. 2016; 15: 826-39

[34] Jaillon O, Aury JM, Noel B et al. The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla. Nature. 2007; 449: 463-7

[35] Gadoury DM, Cadle-Davidson L, Wilcox WF et al. Grapevine powdery mildew (Erysiphe necator): a fascinating system for the study of the biology, ecology and epidemiology of an obligate biotroph. Mol Plant Pathol. 2012; 13: 1-16

[36] Jones L, Riaz S, Morales-Cruz A et al. Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator. BMC Genomics. 2014; 15: 1081

[37] Dry IB, Feechan A, Anderson C et al. Molecular strategies to enhance the genetic resistance of grapevines to powdery mildew. Aust J Grape Wine Res. 2010; 16: 94-105

[38] Spanu PD, Abbott JC, Amselem J et al. Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism. Science. 2010; 330: 1543-6

[39] Qiu W, Feechan A, Dry I . Current understanding of grapevine defense mechanisms against the biotrophic fungus (Erysiphe necator), the causal agent of powdery mildew disease. Hortic Res. 2015; 2: 15020

[40] Mu B, Chen J, Wang H et al. An effector CSEP087 from Erysiphe necator targets arginine decarboxylase VviADC to regulate host immunity in grapevine. Sci Hortic. 2022; 303: 111205

[41] Du Y, Chen X, Guo Y et al. Phytophthora infestans RXLR effector PITG20303 targets a potato MKK1 protein to suppress plant immunity. New Phytol. 2021; 229: 501-15

[42] Yoo YJ, Lee HK, Han W et al. Interactions between transmembrane helices within monomers of the aquaporin AtPIP2;1 play a crucial role in tetramer formation. Mol Plant. 2016; 9: 1004-17

[43] Savage Z, Duggan C, Toufexi A et al. Chloroplasts alter their morphology and accumulate at the pathogen interface during infection by Phytophthora infestans. Plant J. 2019; 107: 1771-87

[44] Torres MA, Jones JDG, Dangl JL . Reactive oxygen species signaling in response to pathogens. Plant Physiol. 2006; 141: 373-8

[45] Gao MJ, He Y, Yin X et al. Ca2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector. Cell. 2021; 184: 5391-5404.e17

[46] Muller MC, Praz CR, Sotiropoulos AG et al. A chromosome-scale genome assembly reveals a highly dynamic effector repertoire of wheat powdery mildew. New Phytol. 2019; 221: 2176-89

[47] Pirrello C, Mizzotti C, Tomazetti TC et al. Emergent ascomycetes in viticulture: an interdisciplinary overview. Front Plant Sci. 2019; 10: 1394

[48] Wang J, Yao W, Wang L et al. Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis. Plant Sci. 2017; 263: 142-55

[49] Hu Y, Cheng Y, Yu X et al. Overexpression of two CDPKs from wild Chinese grapevine enhances powdery mildew resistance in Vitis vinifera and Arabidopsis. New Phytol. 2021; 230: 2029-46

[50] Wang L, Xie X, Yao W et al. RING-H2-type E3 gene VpRH2 from Vitis pseudoreticulata improves resistance to powdery mildew by interacting with VpGRP2A. J Exp Bot. 2017; 68: 1669-87

[51] Glawe DA . The powdery mildews: a review of the world’s most familiar (yet poorly known) plant pathogens. Annu Rev Phytopathol. 2008; 46: 27-51

[52] Gao YR, Han YT, Zhao FL et al. Identification and utilization of a new Erysiphe necator isolate NAFU1 to quickly evaluate powdery mildew resistance in wild Chinese grapevine species using detached leaves. Plant Physiol Biochem. 2016; 98: 12-24

[53] Lee SJ, Kim BD, Rose JK . Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen. Nat Protoc. 2006; 1: 2439-47

[54] Fang A, Han Y, Zhang N et al. Identification and characterization of plant cell death-inducing secreted proteins from Ustilaginoidea virens. Mol Plant-Microbe Interact. 2016; 29: 405-16

[55] Cui KC, Liu M, Ke GH et al. Transient silencing of VvCSN5 enhances powdery mildew resistance in grapevine (Vitis vinifera). Plant Cell Tissue Organ Cult. 2021; 146: 621-33

[56] Zhao FL, Li YJ, Hu Y et al. A highly efficient grapevine mesophyll protoplast system for transient gene expression and the study of disease resistance proteins. Plant Cell Tissue Organ Cult. 2016; 125: 43-57