Inducible MdAGG lectins in apple immunity toward fire blight: CRISPR/Cas9 validation and their potential for intragenesis approaches

Abstract

Fire blight, caused by the bacterium Erwinia amylovora, represents a significant threat to apple (Malus domestica) production. Currently, only a limited number of genes effectively involved in resistance to E. amylovora have been identified. Seeking new resistance candidates, we focused on a multigene family encoding amaranthin-like lectins, which are highly upregulated following chemical elicitation by acibenzolar-S-methyl (ASM). These lectins are believed to contribute to downstream defense by promoting bacterial aggregation, which led to their designation as Malus domestica agglutinins (MdAGGs). When loss-of-function editions were introduced into MdAGG genes, the plant’s ability to mount a fully effective defense response against fire blight upon ASM treatment was compromised, confirming the role of MdAGGs in fire blight resistance. Next, we coupled the pPPO16 promoter, endogenous to apple and known to be rapidly induced during E. amylovora infection, with the coding sequence of MdAGG10 to create apple lines with fire blight-inducible MdAGG10 expression. Early MdAGG10 expression in these lines significantly improved resistance to fire blight, and an additional ASM treatment further enhanced this resistance. In summary, we conclude that MdAGGs act as defense genes whose timely expression can provide effective resistance against E. amylovora.

Acknowledgements

The authors are grateful for the technical support provided by the ANAN shared platform of the SFR QUASAV and the PHENOTIC platform (greenhouse facilities). The authors thank their funders: this research was funded by the Agg-FREDI project (INRAE BAP Department 2020-2021) and the French Priority Research Programme ‘Cultiver et Protéger Autrement’ (PPR-CPA) managed by the Agence Nationale de la Recherche (project ANR-20-PCPA-0003 CapZeroPhyto). A PhD grant was also awarded to A.B. by INRAE-BAP Department and the Pays-de-la-Loire Region.

Author contributions

The research was designed by E.V. and A.D. Apple transgenic lines were obtained by A.B., N.D., E.R. and E.V. Plant inoculation and phenotyping were performed by A.B., N.D., E.R., C.H., E.V. and A.D. Genotyping, qPCR analysis and immunodetection were completed by A.B., A.D. and E.V. Data analyses were completed by A.B., A.D. and E.V. Interpretation and writing of the manuscript: the manuscript was drafted by A.B., completed by E.V., A.D. and M.N.B. and reviewed by all the authors. E.V., A.D. and M.N.B. supervised the research project.

Data availability

All data supporting the findings of this study are available in the article and in its supplementary information files.

Conflicts of interest statement

The authors declare no competing interests.

Supplementary material

Supplementary material are available at Horticulture Research online.

References

[1] McNally RR, Zhao Y, Sundin GW.Towards understanding fire blight:virulence mechanisms and their regulation in Erwinia Amylovora. In: Murillo J, Jackson RW, Vinatzer BA. et al., Poole,eds. Bacteria-Plant Interactions:Advanced Research and Future Trends. United Kingdom:Caister Academic Press, 2015,61-82

[2] Ayers AR, Ayers SB, Goodman RN. Extracellular polysaccha-ride of Erwinia amylovora: a correlation with virulence. Appl Environ Microbiol. 1979;38:659-66

[3] Berry MC, McGhee GC, Zhao Y. et al. Effect of a waaL muta-tion on lipopolysaccharide composition, oxidative stress sur-vival, and virulence in Erwinia amylovora. FEMS Microbiol Lett. 2009;291:80-7

[4] Boutrot F, Zipfel C. Function, discovery, and exploitation of plant pattern recognition receptors for broad-spectrum dis-ease resistance. Annu Rev Phytopathol. 2017;55:257-86

[5] Bonn WG,Van der Zwet T. Distribution and economic impor-tance of fire blight. In: Vanneste JL, Erwinia amylovora. Wallingford,ed.Fire Blight:the Dis-ease and its Causative Agent, United Kingdom:CABI Publishing, 2000,37-53

[6] Campa M, Miranda S, Licciardello C. et al. Application of new breeding techniques in fruit trees. Plant Physiol. 2023;194:1304-22

[7] Peil A, Hübert C, Wensing A. et al. Mapping of fire blight resis-tance in Malus ×robusta 5 flowers following artificial inocula-tion. BMC Plant Biol. 2019;19:532

[8] Emeriewen OF, Richter K, Kilian A. et al. Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca. Mol Breed. 2014;34:407-19

[9] Durel CE, Denancé C, Brisset MN. Two distinct major QTL for resistance to fire blight co-localize on linkage group 12 in apple genotypes ‘Evereste’ and Malus floribunda clone 821. Genome. 2009;52:139-47

[10] Emeriewen OF, Peil A, Richter K. et al. Fire blight resistance of Malus ×arnoldiana is controlled by a quantitative trait locus located at the distal end of linkage group 12. Eur J Plant Pathol. 2017;148:1011-8

[11] Fahrentrapp J, Broggini GAL, Kellerhals M. et al. A candidate gene for fire blight resistance in Malus × robusta 5 is coding for a CC-NBS-LRR. Tree Genet Genomes. 2013;9:237-51

[12] Emeriewen OF, Richter K, Piazza S. et al. Towards map-based cloning of FB Mfu10: identification of a receptor-like kinase candidate gene underlying the Malus fusca fire blight resis-tance locus on linkage group 10. Mol Breed. 2018;38:106

[13] Parravicini G, Gessler C, Denancé C. et al. Identification of serine/threonine kinase and nucleotide-binding site-leucine-rich repeat (NBS-LRR) genes in the fire blight resistance quan-titative trait locus of apple cultivar ‘Evereste’: candidate fire blight resistance genes. Mol Plant Pathol. 2011;12:493-505

[14] Emeriewen OF, Flachowsky H, Peil A. Characterization of genomic DNA sequence of the candidate gene for FB Mfu10 associated with fire blight resistance in Malus species. BMC Res Notes. 2021;14:291

[15] Emeriewen OF, Wöhner T, Flachowsky H. et al. Malus hosts-Erwinia amylovora interactions: strain pathogenicity and resistance mechanisms. Front Plant Sci. 2019;10:551

[16] Kost TD, Gessler C, Jänsch M. et al. Development of the first cisgenic apple with increased resistance to fire blight. PLoS One. 2015;10:e0143980

[17] Joshi SG, Schaart JG, Groenwold R. et al. Functional analysis and expression profiling of HcrVf1 and HcrVf2 for development of scab resistant cisgenic and intragenic apples. Plant Mol Biol. 2011;75:579-91

[18] Campa M, Piazza S, Righetti L. et al. HIPM is a suscepti-bility gene of Malus spp.: reduced expression reduces sus-ceptibility to Erwinia amylovora. Mol Plant-Microbes Interact. 2019;32:167-75

[19] Pompili V, Dalla Costa L, Piazza S. et al. Reduced fire blight sus-ceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system. Plant Biotechnol J. 2020;18:845-58

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

[21] Chavonet E, Gaucher M, Warneys R.et al. Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance. Plant Physiol. 2018;2022;188:1350–68

[22] Warneys R, Gaucher M, Robert P. et al. Acibenzolar-S-methyl reprograms apple transcriptome toward resistance to rosy apple aphid. Front Plant Sci. 2018;9:1795

[23] Peumans WJ,Van Damme E.Lectins as plant defense proteins. Plant Physiol. 1995;109:347-52

[24] Bellande K, Bono JJ, Savelli B. et al. Plant lectins and lectin receptor-like kinases: how do they sense the outside? Int J Mol Sci. 2017;18:1164-88

[25] De Coninck T, Van Damme EJM. Review: the multiple roles of plant lectins. Plant Sci. 2021;313:111096

[26] Van Damme EJM, Lannoo N, Peumans WJ.Plant lectins. In: Kuete V, Amsterdam,ed. Advances in Botanical Research.Netherlands:Elsevier, 2008,107-209

[27] Bodelot A, Chavonet E, Brisset MN. et al. Overexpression of an apple broad range agglutinating lectin does not promote in planta resistance to fire blight and bacterial wilt. J Plant Pathol. 2023;106:1011-22

[28] Gaucher M, Righetti L, Aubourg S. et al. An Erwinia amylovora inducible promoter for improvement of apple fire blight resis-tance. Plant Cell Rep. 2022;41:1499-513

[29] Charrier A, Vergne E, Dousset N. et al. Efficient targeted muta-genesis in apple and first time edition of pear using the CRISPR-Cas9 system.Front Plant Sci. 2019;10:40-51

[30] Sun X, Jiao C, Schwaninger H. et al. Phased diploid genome assemblies and pan-genomes provide insights into the genetic history of apple domestication. Nat Genet. 2020;52:1423-32

[31] García-Almodóvar RC, Clemente-Moreno MJ, Díaz-Vivancos P. et al. Greenhouse evaluation confirms in vitro sharka resis-tance of genetically engineered h-UTR/P1 plum plants. Plant Cell Tissue Org Cult. 2015;120:791-6

[32] Malnoy M, Faize M, Venisse JS. et al. Expression of viral EPS-depolymerase reduces fire blight susceptibility in transgenic pear. Plant Cell Rep. 2005;23:632-8

[33] Malabarba J, Chevreau E, Dousset N. et al. New strategies to overcome present CRISPR/Cas9 limitations in apple and pear: efficient dechimerization and base editing. Int J Mol Sci. 2020;22:319-37

[34] Billing E. Fire blight. Why do views on host invasion by Erwinia amylovora differ? Plant Pathol. 2011;60:178-89

[35] Cabrales-Orona G, Martínez-Gallardo N, Délano-Frier JP. Functional characterization of an Amaranth Natterin-4-Like-1 gene in Arabidopsis thaliana. Front Sustain Food Syst. 2022;5: 814188

[36] Wu J, Luo X, Guo H. et al. Transgenic cotton, expressing Ama-ranthus caudatus agglutinin, confers enhanced resistance to aphids. Plant Breed. 2006;125:390-4

[37] Wang X, Yang F,Von Bodman SB.The genetic and structural basis of two distinct terminal side branch residues in stew-artan and amylovoran exopolysaccharides and their potential role in host adaptation. Mol Microbiol. 2012;83:195-207

[38] Schaart JG, Krens FA, Wolters AA. et al. Transformation meth-ods for obtaining marker-free genetically modified plants. In: Stewart, Touraev CN A, Citovsky V. et al., Plant Trans-formation Technologies. Hoboken, New Jersey,eds.United States:Wiley, 2010,229-42

[39] King EO, Ward MK, Raney DE. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954;44:301-7

[40] Chow CN, Yang CW, Wu NY. et al. PlantPAN 4.0: updated database for identifying conserved non-coding sequences and exploring dynamic transcriptional regulation in plant promoters. Nucleic Acids Res.2024;52:D1569-78

[41] Marshallsay C, Kiss T, Filipowicz W. Amplification of plant U3 and U6 snRNA gene sequences using primers specific for an upstream promoter element and conserved intragenic regions. Nucleic Acids Res. 1990;18:3459-66

[42] Haeussler M, Schönig K, Eckert H. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 2016; 17:148

[43] Daccord N, Celton JM, Linsmith G. et al. High-quality de novo assembly of the apple genome and methylome dynamics of early fruit development. Nat Genet. 2017;49: 1099-106

[44] Karimi M, Inzé D, Depicker A. GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 2002;7:193-5

[45] Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473-97

[46] Shaner G, Finney RE. The effect of nitrogen fertilization on the expression of slow-mildewing resistance in Knox wheat. Phytopathology. 1977;77:1051-5

[47] Fulton TM, Chunwongse J, Tanksley SD. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep. 1995;13:207-9

[48] Corpet F. Multiple sequence alignment with hierarchical clus-tering. Nucleic Acids Res. 1988;16:10881-90

[49] Vandesompele J, Preter KD, Roy NV. et al. Accurate normal-ization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:0034.1

[50] R Core Team.2023. R: A Language and Environment for Statis-tical Computing. Vienna: R Foundation for Statistical Comput-ing. https://www.R-project.org/

[51] Wickham H.ggplot2:Elegant Graphics for Data Analysis. Cham:Springer International; 2016:

[52] Kassambara A.2023a. ggpubr: ’ggplot2’ based publication ready plots. R package version 0.6.0. https://CRAN.R-project.org/package=ggpubr

[53] Canty A, Ripley BD.boot: bootstrap R (S-plus) functions. 2024; R package version 1.3-31. https://cran.r-project.org/web/packages/boot/boot.pdf

[54] Kassambara A.2023b. rstatix: pipe-friendly framework for basic statistical tests. R package version 0.7.2, https://rpkgs.datanovia.com/rstatix/

[55] Ogle DH, Doll JC, Wheeler AP, Dinno A.2023. FSA: simple fisheries stock assessment methods. R package version 0.9.5, https://CRAN.R-project.org/package=FSA

[56] Mangiafico SS.2024 rcompanion: Functions to Support Extension Education Program Evaluation. Rutgers Cooperative Extension, New Brunswick, New Jersey. version 2.4.36. https://CRAN.R-project.org/package=rcompanion/