Graft incompatibility between pepper and tomato elicits an immune response and triggers localized cell death

Abstract

Graft compatibility is the capacity of two plants to form cohesive vascular connections. Tomato and pepper are incompatible graft partners; however, the underlying cause of graft rejection between these two species remains unknown. We diagnosed graft incompatibility between tomato and diverse pepper varieties based on weakened biophysical stability, decreased growth, and persistent cell death using viability stains. Transcriptomic analysis of the junction was performed using RNA sequencing, and molecular signatures for incompatible graft response were characterized based on meta-transcriptomic comparisons with other biotic processes. We show that tomato is broadly incompatible with diverse pepper cultivars. These incompatible graft partners activate prolonged transcriptional changes that are highly enriched for defense processes. Amongst these processes was broad nucleotide-binding and leucine-rich repeat receptors (NLR) upregulation and genetic signatures indicative of an immune response. Using transcriptomic datasets for a variety of biotic stress treatments, we identified a significant overlap in the genetic profile of incompatible grafting and plant parasitism. In addition, we found over 1000 genes that are uniquely upregulated in incompatible grafts. Based on NLR overactivity, DNA damage, and prolonged cell death, we hypothesize that tomato and pepper graft incompatibility is characterized by an immune response that triggers cell death which interferes with junction formation.

Acknowledgements

H.R.T. was supported by a United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA) Predoctoral Fellowship (2020-67011-31882); M.H.F., A.G., S.P., and M.N. were supported by the National Science Foundation (NSF) (CAREER IOS-1942437) and by a U.S.-Israel Binational Science Foundation grant (2019192). Imaging data was acquired through the Cornell Institute of Biotechnology’s Imaging Facility, with NIH (S10OD018516) funding for the shared Zeiss LSM880 confocal/multiphoton microscope. Access to the Instron Universal testing stand was supported by the Delaware Center for Musculoskeletal Research from the National Institute of Health’s National Institute of General Medical Sciences (P20GM139760). Thank you to Noor AlBader for assistance with data transfer.

Author contributions

H.R.T. and M.H.F. designed the study. H.R.T., A.G., A.H., S.Y., L.E., M.N., and E.S. carried out the experimentation. H.R.T. analyzed the data. H.R.T. wrote the first draft of the manuscript. All authors contributed critically to the final draft and approval publication.

Data availability

RNA reads collected for this project have been deposited on NCBI GEO (GSE256079). Previously published RNA-seq data used in this research can be accessed on NCBI at PRJNA628162, PRJNA687611, PRJNA756681, PRJNA600385, and PRJNA773605.

Conflict of interest statement

None to declare.

Supplementary Data

Supplementary data is available at Horticulture Research online.

References

[1] Harrison DJ, Burgess PG. Use of rootstock resistance for controlling fusarium wilt of tomatoes. Plant Pathol. 1962; 11 :23-5

[2] Mudge K, Janick J, Scofield S. et al. A History of Grafting. In: Hortic Rev. 2009,437-93

[3] Warschefsky EJ, Klein LL, Frank MH. et al. Rootstocks: diversity, domestication, and impacts on shoot phenotypes. Trends Plant Sci. 2016; 21 :418-37

[4] Williams B, Ahsan MU, Frank MH. Getting to the root of grafting-induced traits. Curr Opin Plant Biol. 2021; 59 :101988

[5] Moore R. A model for graft compatibility-incompatibility in higher plants. Am J Bot. 1984; 71 :752-8

[6] Argles GK. A review of the literature on stock-scion incompatibility in fruit trees: with particular reference to pome and stone fruits. Imperial Bureau of Fruit Production. 1937.

[7] Mendel K. The anatomy and histology of the bud-union in citrus. Palest J Bot Hortic Sci. 1936; 1 :13-46

[8] Andrews PK, Marquez CS. Graft incompatibility. In: Hortic Rev. 2010,183-232

[9] Eames AJ, Cox LG. A remarkable tree-fall and an unusual type of graft-union failure. Am J Bot. 1945; 32 :331-5

[10] Moore R, Walker DB. Studies of vegetative compatibility-incompatibility in higher plants. Ii. A structural study of an incompatible heterograft between sedum telephoides (crassulaceae) and Solanum pennellii (solanaceae). Am J Bot. 1981; 68 :831-42

[11] Webster AD Vigour mechanisms in dwarfing rootstocks for temperate fruit trees.Acta Horticulturae 658. 2004

[12] King SR, Davis AR, Liu W. et al. Grafting for disease resistance. HortScience. 2008; 43 :1673-6

[13] Gur A. The role of the cyanogensic glycoside of the quince in the incompatibility between pear cultivars and quince rootstocks. Hortic Res. 1968; 8 :113-34

[14] Moore R. Graft incompatibility between pear and quince: the influence of metabolites of Cydonia oblonga on suspension cultures of Pyrus communis. Am J Bot. 1986; 73 :1-4

[15] Mosse B, Herrero J. Studies on incompatibility between some pear and quince grafts. J Hortic Sci. 1951; 26 :238-45

[16] Burbridge E, Diamond M, Dix PJ. et al. Use of cell morphology to evaluate the effect of a peroxidase gene on cell death induction thresholds in tobacco. Plant Sci. 2007; 172 :853-60

[17] Hirsch T, Marchetti P, Susin SA. et al. The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene. 1997; 15 :1573-81

[18] Lockshin RA, Zakeri Z. Apoptosis, autophagy, and more. Int J Biochem Cell Biol. 2004; 36 :2405-19

[19] Janeway C, Travers P, Walport M. et al. Immunobiology: The Immune System in Health and Disease Vol. 2. New York: Garland Pub; 2001:154

[20] Amarante-Mendes GP, Adjemian S, Branco LM. et al. Pattern recognition receptors and the host cell death molecular machinery. Front Immunol. 2018; 9 :2379

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

[22] Steinbrenner AD, Muñoz-Amatriaín M, Chaparro AF. et al. A receptor-like protein mediates plant immune responses to herbivore-associated molecular patterns. Proc Natl Acad Sci USA. 2020; 117 :31510-8

[23] Boller T, He SY. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science. 2009; 324 :742-4

[24] Stergiopoulos I, de Wit PJGM. Fungal effector proteins. Annu Rev Phytopathol. 2009; 47 :233-63

[25] Wu L, Chen H, Curtis C. et al. Go in for the kill: how plants deploy effector-triggered immunity to combat pathogens. Virulence. 2014; 5 :710-21

[26] Thomas HR, Gevorgyan A, Frank MH. Anatomical and biophysical basis for graft incompatibility within the Solanaceae. J Exp Bot. 2023; 74 :4461-70

[27] Thomas H, Van den Broeck L, Spurney R. et al. Gene regulatory networks for compatible versus incompatible grafts identify a role for SlWOX4 during junction formation. Plant Cell. 2022; 34 :535-56

[28] Deloire A, Hébant C. Peroxidase activity and lignification at the Interface between stock and Scion of compatible and incompatible grafts of capsicum on Lycopersicum. Ann Bot. 1982; 49 :887-91

[29] Ives L, Brathwaite R, Barclay G. et al. Graft compatibility of scotch bonnet (Capsicum chinense Jacq ) with selected salt-tolerant solanaceous. J Agric Sci Technol B. 2012; 2 :81-92

[30] Kawaguchi M, Taji A, Backhouse D. et al. Anatomy and physiology of graft incompatibility in solanaceous plants. J Hortic Sci Biotechnol. 2008; 83 :581-8

[31] Zeist AR, Giacobbo CL, da Silva Neto GF. et al. Compatibility of tomato cultivar Santa Cruz Kada grafted on different Solanaceae species and control of bacterial wilt. Hortic Bras. 2018; 36 :377-81

[32] Moore R. Studies of vegetative compatibility-incompatibility in higher plants. Iv. The development of tensile strength in a compatible and an incompatible graft. Am J Bot. 1983; 70 :226-31

[33] Melnyk CW, Schuster C, Leyser O. et al. A developmental framework for graft formation and vascular reconnection in Arabidopsis thaliana. Curr Biol. 2015; 25 :1306-18

[34] Moore R. Graft compatibility and incompatibility in higher plants. Dev Comp Immunol. 1981; 5 :377-89

[35] Tiedemann R. Graft union development and symplastic phloem contact in the heterograft Cucumis sativus on Cucurbita ficifolia. J Plant Physiol. 1989; 134 :427-40

[36] Brutus A, Sicilia F, Macone A. et al. A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA. 2010; 107 :9452-7

[37] Ferrari S, Savatin DV, Sicilia F. et al. Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development. Front Plant Sci. 2013; 4 :49

[38] Nothnagel EA, McNeil M, Albersheim P. et al. Host-pathogen interactions. XXII. A galacturonic acid oligosaccharide from plant cell walls elicits phytoalexins. Plant Physiol. 1983; 71 :916-26

[39] Bashir S, Rehman N, Fakhar Zaman F. et al. Genome-wide characterization of the gene family in tomato and their relatedness to disease resistance. Front Genet. 2022; 13 :931580

[40] Lee H, Mang H, Choi E. et al. Genome-wide functional analysis of hot pepper immune receptors reveals an autonomous NLR clade in seed plants. New Phytol. 2021; 229 :532-47

[41] Freh M, Gao J, Petersen M. et al. Plant autoimmunity-fresh insights into an old phenomenon. Plant Physiol. 2022; 188 :1419-34

[42] Gantner J, Ordon J, Kretschmer C. et al. An EDS1-SAG101 complex is essential for TNL-mediated immunity. Plant Cell. 2019; 31 :2456-74

[43] Rietz S, Stamm A, Malonek S. et al. Different roles of enhanced disease Susceptibility1 (EDS1) bound to and dissociated from phytoalexin Deficient4 (PAD4) in Arabidopsis immunity. New Phytol. 2011; 191 :107-19

[44] Zhu S, Jeong R-D, Venugopal SC. et al. SAG101 forms a ternary complex with EDS1 and PAD4 and is required for resistance signaling against turnip crinkle virus. PLoS Pathog. 2011; 7 :e1002318

[45] Enyedi AJ, Yalpani N, Silverman P. et al. Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc Natl Acad Sci USA. 1992; 89 :2480-4

[46] Koornneef A, Pieterse CMJ. Cross talk in defense signaling. Plant Physiol. 2008; 146 :839-44

[47] Lorenzo O, Piqueras R, Sánchez-Serrano JJ. et al. Ethylene response factor1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell. 2003; 15 :165-78

[48] Wang J, Li D, Chen N. et al. Plant grafting relieves asymmetry of jasmonic acid response induced by wounding between scion and rootstock in tomato hypocotyl. PLoS One. 2020; 15 :e0241317

[49] Cárdenas PD, Sonawane PD, Pollier J. et al. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway. Nat Commun. 2016; 7 :10654

[50] Itkin M, Heinig U, Tzfadia O. et al. Biosynthesis of antinutritional alkaloids in solanaceous crops is mediated by clustered genes. Science. 2013; 341 :175-9

[51] Milner SE, Brunton NP, Jones PW. et al. Bioactivities of glycoalkaloids and their aglycones from Solanum species. J Agric Food Chem. 2011; 59 :3454-84

[52] Panda S, Jozwiak A, Sonawane PD. et al. Steroidal alkaloids defence metabolism and plant growth are modulated by the joint action of gibberellin and jasmonate signalling. New Phytol. 2022; 233 :1220-37

[53] Nakayasu M, Shioya N, Shikata M. et al. JRE4 is a master transcriptional regulator of defense-related steroidal glycoalkaloids in tomato. Plant J. 2018; 94 :975-90

[54] Abdelkareem A, Thagun C, Nakayasu M. et al. Jasmonate-induced biosynthesis of steroidal glycoalkaloids depends on COI 1 proteins in tomato. Biochem Biophys Res Commun. 2017; 489 :206-10

[55] Li X, Li Y, Ahammed GJ. et al. RBOH1-dependent apoplastic H2O2 mediates epigallocatechin-3-gallate-induced abiotic stress tolerance in Solanum lycopersicum L. Environ Exp Bot. 2019; 161 :357-66

[56] Raziq A, Wang Y, Mohi Ud Din A. et al. A comprehensive evaluation of salt tolerance in tomato (Var. Ailsa Craig): responses of physiological and transcriptional changes in RBOH’s and ABA biosynthesis and signalling genes. Int J Mol Sci. 2022; 23 :1603

[57] Nisa M-U, Huang Y, Benhamed M. et al. The plant DNA damage response: signaling pathways leading to growth inhibition and putative role in response to stress conditions. Front Plant Sci. 2019; 10 :653

[58] Rodriguez E, Chevalier J, El Ghoul H. et al. DNA damage as a consequence of NLR activation. PLoS Genet. 2018; 14 :e1007235

[59] Olvera-Carrillo Y, Van Bel M, Van Hautegem T. et al. A conserved core of programmed cell death indicator genes discriminates developmentally and environmentally induced programmed cell death in plants. Plant Physiol. 2015; 169 :2684-99

[60] Asahina M, Azuma K, Pitaksaringkarn W. et al. Spatially selective hormonal control of RAP2.6L and ANAC 071 transcription factors involved in tissue reunion in Arabidopsis. Proc Natl Acad Sci USA. 2011; 108 :16128-32

[61] Lakehal A, Dob A, Rahneshan Z. et al. Ethylene response factor 115 integrates jasmonate and cytokinin signaling machines to repress adventitious rooting in Arabidopsis. New Phytol. 2020; 228 :1611-26

[62] Matsuoka K, Yanagi R, Yumoto E. et al. RAP2.6L and jasmonic acid-responsive genes are expressed upon Arabidopsis hypocotyl grafting but are not needed for cell proliferation related to healing. Plant Mol Biol. 2018; 96 :531-42

[63] Zhang A, Matsuoka K, Kareem A. et al. Cell-wall damage activates DOF transcription factors to promote wound healing and tissue regeneration in Arabidopsis thaliana. Curr Biol. 2022; 32 :1883-1894.e7

[64] Canher B, Lanssens F, Zhang A. et al. The regeneration factors ERF114 and ERF115 regulate auxin-mediated lateral root development in response to mechanical cues. Mol Plant. 2022; 15 :1543-57

[65] Jhu M-Y, Farhi M, Wang L. et al. Investigating host and parasitic plant interaction by tissue-specific gene analyses on tomato and Interface at three Haustorial developmental stages. Front Plant Sci. 2021; 12 :764843

[66] Ke L, Wang Y, Schäfer M. et al. Transcriptomic profiling reveals shared signalling networks between flower development and herbivory-induced responses in tomato. Front Plant Sci. 2021; 12 :722-810

[67] Srivastava DA, Arya GC, Pandaranayaka EP. et al. Transcriptome profiling data of Botrytis cinerea infection on whole plant Solanum lycopersicum. Mol Plant-Microb Interact. 2020; 33 :1103-7

[68] Zeng Z, Liu Y, Feng X-Y. et al. The RNAome landscape of tomato during arbuscular mycorrhizal symbiosis reveals an evolving RNA layer symbiotic regulatory network. Plant Commun. 2023; 4 :100429

[69] Zhang B, Van Aken O, Thatcher L. et al. The mitochondrial outer membrane AAA ATPase at OM66 affects cell death and pathogen resistance in Arabidopsis thaliana. The Plant Journal. 2014; 80 :709-27

[70] Li J, Brader G, Kariola T. et al. WRKY70 modulates the selection of signaling pathways in plant defense. Plant J. 2006; 46 :477-91

[71] Assunção M, Santos C, Brazão J. et al. Understanding the molecular mechanisms underlying graft success in grapevine. BMC Plant Biol. 2019; 19 :396

[72] Notaguchi M, Kurotani KI, Sato Y. et al. Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases. Science. 2020; 369 :698-702

[73] Kostoff D. Studies on callus tissue. Am J Bot. 1928; 15 :565-76

[74] Jones JDG, Vance RE, Dangl JL. Intracellular innate immune surveillance devices in plants and animals. Science. 2016; 354 :1117-25

[75] Bomblies K, Lempe J, Epple P. et al. Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants. PLoS Biol. 2007; 5 :e236

[76] Tran DTN, Chung E-H, Habring-Müller A. et al. Activation of a plant NLR complex through Heteromeric association with an autoimmune risk variant of another NLR. Curr Biol. 2017; 27 :1148-60

[77] Hollingshead L. A lethal factor in Crepis effective only in an interspecific hybrid. Genetics. 1929; 15 :114-40

[78] Bomblies K, Weigel D. Hybrid necrosis: autoimmunity as a potential gene-flow barrier in plant species. Nat Rev Genet. 2007; 8 :382-93

[79] Tian D, Traw MB, Chen JQ. et al. Fitness costs of R-gene-mediated resistance in Arabidopsis thaliana. Nature. 2003; 423 :74-7

[80] Lai Y, Eulgem T. Transcript-level expression control of plant NLR genes. Mol Plant Pathol. 2018; 19 :1267-81

[81] Barragan AC, Collenberg M, Wang J. et al. A truncated singleton NLR causes hybrid necrosis in Arabidopsis thaliana. Mol Biol Evol. 2021; 38 :557-74

[82] Kostoff D. Acquired immunity in plants. Genetics. 1929; 14 :37-77

[83] Kurotani KI, Wakatake T, Ichihashi Y. et al. Host-parasite tissue adhesion by a secreted type of β-1,4-glucanase in the parasitic plant Phtheirospermum japonicum. Commun Biol. 2020; 3 :407

[84] Ennos AR, Crook MJ, Grimshaw C. The anchorage mechanics of maize, Zea mays, J Exp Bot. 1993; 44 :147-53

[85] Goodman AM, Ennos AR. The effects of mechanical stimulation on the morphology and mechanics of maize roots grown in an aerated nutrient solution. Int J Plant Sci. 2001; 162 :691-6

[86] Hostetler AN, Erndwein L, Ganji E. et al. Maize brace root mechanics vary by whorl, genotype, and reproductive stage. Ann Bot. 2022; 126 :657-68

[87] Fernández-Bautista N, Domínguez-Núñez JA, Moreno MMC. et al. Plant tissue trypan blue staining during phytopathogen infection. Bio-protocol. 2016; 6 :e2078-8

[88] Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15 :550

[89] Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using diamond. Nat Methods. 2014; 12 :59-60

[90] Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 2019; 20 :238

[91] R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing; 2021: