The Arabidopsis pattern recognition receptor EFR enhances fire blight resistance in apple

Abstract

Fire blight disease, caused by the bacterium Erwinia amylovora (E. amylovora), is responsible for substantial losses in cultivated apples worldwide. An important mechanism of plant immunity is based on the recognition of conserved microbial molecules, named pathogen-associated or microbe-associated molecular patterns (PAMPs or MAMPs), through pattern recognition receptors (PRRs), leading to pattern-triggered immunity (PTI). The interspecies transfer of PRRs represents a promising strategy to engineer broad-spectrum and durable disease resistance in crops. EFR, the Arabidopsis thaliana PRR for the PAMP elf18 derived from the elongation factor thermal unstable (EF-Tu) proved to be effective in improving bacterial resistance when expressed into Solanaceae and other plant species. In this study, we tested whether EFR can affect the interaction of apple with E. amylovora by its ectopic expression in the susceptible apple rootstock M.26. Stable EFR expression led to the activation of PAMP-triggered immune response in apple leaves upon treatment with supernatant of E. amylovora, as measured by the production of reactive oxygen species and the induction of known defense genes. The amount of tissue necrosis associated with E. amylovora infection was significantly reduced in the EFR transgenic rootstock compared to the wild-type. Our results show that the expression of EFR in apple rootstock may be a valuable biotechnology strategy to improve the resistance of apple to fire blight.

References

[1] Zipfel, C. Plant pattern recognition receptors. Trends Immunol. 35, 345-351 (2014).

[2] Lu, F. et al. Enhancement of innate immune system in monocot rice by transferring the dicotyledonous elongation factor Tu receptor EFR. J. Integrat. Plant Biol. https://doi.org/10.1111/jipb.12306 (2014).

[3] Wu, Y. & Zhou, J. M. Receptor like kinases in plant innate immunity. J. Integr. Plant Biol. 55, 1271-1286 (2013).

[4] Kawano, Y. & Shimamoto, K. Early signaling network in rice PRR-mediated and R-mediated immunity. Curr. Opin. Plant Biol. 16, 496-504 (2013).

[5] Schwessinger, B. & Ronald, P. C. Plant innate immunity: perception of conserved microbial signatures. Annu. Rev. Plant Biol. 63, 451-482 (2012).

[6] Klarzynski, O. et al. Linear beta-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiol. 124, 1027-1038 (2000).

[7] Kaku, H. et al. Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc. Natl Acad. Sci. USA 103, 11086-11091 (2006).

[8] Felix, G., Duran, J. D., Volko, S. & Boller, T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J. 18, 265-276 (1999).

[9] Kunze, G. et al. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell. 16, 3496-3507 (2004).

[10] Zipfel, C. et al. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125, 749-760 (2006).

[11] Lacombe, S. et al. Interfamily transfer of a plant pattern recognition receptor confers broad-spectrum bacterial resistance. Nat. Biotechnol. 28, 365-369 (2009).

[12] Malnoy, M., Venisse, J. S., Faize, M., Geider, K. & Chevreau, E. Expression of viral EPS-depolymearse reduces fire blight susceptibility in transgenic pear. Plant Cell Rep. 23, 632-638 (2005).

[13] Pfeilmeier, S. et al. Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium, but not nitrogen-fixing rhizobial symbiosis. Plant Biotechnol. J. 17, 569-579 (2019).

[14] Boschi, F. et al. Enhanced bacterial wilt resistance in potato through expression of Arabidopsis EFR and introgression of quantitative resistance from Solanum commersonii. Front Plant Sci. 25, 1642 (2017).

[15] Schwessinger, B. et al. Transgenic expression of the dicotyledonous pattern recognition receptor EFR in rice leads to ligand dependent activation of defense responses. PLoS Pathogen 11, e1004809 (2015).

[16] Schoonbeek, H. J. et al. Arabidopsis EF-TU receptor enhances bacterial disease resistance in transgenic wheat. N. Phytologist 206, 606-613 (2015).

[17] Malnoy, M. et al. Fire Blight: applied genomic insights of the pathogen and host. Annu. Rev. Phytopathol. 8, 475-494 (2012).

[18] Sundin, G. W., Castiblanco, L. F., Yuan, X., Zeng, Q. & Yang, C. H. Bacterial disease management: challenges, experience, innovation and future prospects. Mol. Plant Pathol. 17, 1506-1518 (2016).

[19] Broggini, G. A. et al. Engineering fire blight resistance into the apple cultivar ‘Gala’ using the FB_MR5 CC-NBS-LRR resistance gene of Malus x robusta 5. Plant Biotechnol. J. 12, 728-733 (2014).

[20] Durel, C. E., Denancé, C. & Brisset, M. N. 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 52, 139-147 (2009).

[21] Emeriewen, O. et al. Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca. Mol. Breed. 34, 407-419 (2014).

[22] Emeriewen, O. F., Richter, K., Hanke, M. V., Malnoy, M. & Peil, A. The fire blight resistance QTL of Malus fusca (Mfu10) is affected but not broken down by the highly virulent Canadian Erwinia amylovora strain E2002A. Eur. J. Plant Pathol. 141, 631-635 (2015).

[23] Emeriewen, O. F., Richter, K., Hanke, M. V., Malnoy, M. & Peil, A. Further insights into Malus fusca fire blight resistance. J. Plant Pathol. 99, 45-49 (2017a).

[24] Emeriewen, O. F. 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. 148, 1011-1018 (2017b).

[25] Emeriewen, O. F. et al. Towards map-based cloning of FB_Mfu10: identification of a receptor-like kinase candidate gene underlying the Malus fusca fire blight resistance locus on linkage group 10. Mol. Breed. 38, 106 (2018).

[26] Gardiner, S. E. et al. Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus ‘Robusta 5’ accessions. BMC Genet. 13, 1-20 (2012).

[27] Kost, T. D. et al. Development of the first cisgenic apple with increased resistance to fire blight. PLoS ONE 10, e0143980 (2015).

[28] Malnoy, M., Xu, M., Borejsza-Wysocka, E. E., Korban, S. S. & Aldwinckle, H. S. Two receptor-like genes, Vfa1 and Vfa2, confer resistance to the fungal pathogen Venturia inaequalis inciting apple scab disease. Mol. Plant-Microbe Interact. 21, 448-458 (2008).

[29] Malnoy, M., Reynoird, J. P., Borejsza-Wysocka, E. E. & Aldwinckle, H. S. Activation of the pathogen-inducible Gst1 promoter of potato after elicitation by Venturia ineaqualis and Erwinia amylovora in transgenic apple (Malus X domestica). Transgenic Res. 15, 83-93 (2006).

[30] Pitzschke, A., Djamei, A., Bitton, F. & Hirt, H. A major role of the MEKK1-MKK1/2-MPK4 pathway in ROS signaling. Mol. Plant 2, 120-137 (2009).

[31] Volk, G. M. et al. Ex situ conservation of vegetatively propagated species: development of a seed-based core collection for Malus sieversii. J. Am. Soc. Hort. Sci. 130, 203-210 (2005).

[32] Volk, G. M. et al. Genetic diversity and disease resistance of wild Malus orientalis from Turkey and Southern Russia. J. Am. Soc. Hort. Sci. 133, 383-389 (2008).

[33] Malnoy, M., Jin, Q., Borejsza-Wysocka, E., He, S. Y. & Aldwinckle, H. S. Overexpression of the Apple Gene MpNPR1 causes increased disease resistance in Malus X domestica. Mol. Plant-Microbe Interact. 20, 1568-1580 (2007).

[34] Malnoy, M., Venisse, J. S. & Chevreau, E. Expression of a bacterial effector, Harpin N_Ea, causes increased resistance to fire blight in Pyrus communis . Tree Genet. Genomes 1, 41-49 (2005).

[35] Vogt, I. et al. Gene-for-gene relationship in the host-pathogen system Malus x robusta 5-Erwinia amylovora. N. Phytol. 197, 1262-1275 (2013).

[36] Haroldsen, V. M. et al. Mobility of transgenic nucleic acids and proteins within grafted rootstocks for agricultural improvement. Front. Plant Sci. 3, 39 (2012).

[37] Mudge, K. et al. A history of grafting. Hortic. Rev. 35, 437-493 (2009).

[38] Pouget, R. Histoire de la Lutte contre le Phylloxera de la Vigne en France (Institut National de la Recherche Agronomique, Paris, 1990).

[39] Kasai, A., Bai, S., Li, T. & Harada, T. Graft-transmitted siRNA signal from the root induces visual manifestation of endogenous post-transcriptional gene silencing in the scion. PLoS ONE 6, e16895 (2011).

[40] Zhao, D. & Song, G. Rootstock-to-scion transfer of transgene derived small interfering RNAs and their effect on virus resistance in non transgenic sweet cherry. Plant Biotechnol. J. 12, 1319-1328 (2014).

[41] Kumar, S. et al. Transgenic expression of EFR and Ds2 genes for field management of bacterial wilt and bacterial spot of tomato. Phytopathology 108, 1042-1411 (2018).

[42] Larkin, M. et al. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007).

[43] Bolar, J. P., Norelli, J. L., Aldwinckle, H. S. & Hanke, V. An efficient method for rooting and acclimation of micropropagated apple cultivar. HortScience 33, 1251-1252 (1998).

[44] Borejsza-Wysocka, E., Norelli, J. L., Aldwinckle, H. S. & Malnoy, M. Long-tern stability of attacin E expression in transformed apple after 12 years in the field and effect of the expression of this gene in the fruit characteristics. BMC Biotechnol. 10, 41 (2010).

[45] Müller, K. et al. In vivo cell wall loosening by hydroxyl radicals during cress seed germination and elongation growth. Plant Physiol. 150, 1855-1865 (2009).

[46] Venisse, J. S., Gullner, G. & Brisset, M. N. Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora. Plant Physiol. 125, 2164-2172 (2001).

[47] Dalla Costa, L. et al. Development of a Taqman Real-time PCR method to quantify nptII in apple lines obtained with ‘established’ or ‘new breeding’ techniques of genetic modification. Food Anal. Methods 245, 643-652 (2019).

[48] Campa, M. et al. HIPM is a susceptibility gene of Malus: reduced expression reduces susceptibility to Erwinia amylovora. Mol. Plant-Microbe Interact. 32, 167-175 (2019).

[49] Pompili, V., Dalla Costa, L., Piazza, S., Pindo, M. & Malnoy, M. Reduced fire blight susceptibility in apple cultivars using a high-efficiency CRISPR/Cas9-FLP/FRT-based gene editing system. Plant Biotechnol. J. 18, 845-858 (2020).

[50] Beranger, R. et al. Agricultural and domestic pesticides in house dust from different agricultural areas in France. Environ. Sci. Pollut. Res. 26, 19632-19645 (2019).

[51] Holtappels, M., Noben, J. P. & Valcke, R. Virulence of Erwinia amylovora a prevalent apple pathogen; Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses. Proteomics 16, 2377-2390 (2016).

[52] Steinberg, E. M. & Beer, S. V. Creation and complementation of pathogenicity mutants of Erwinia amylovora. Mol. Plant Microbe Interact. 1, 135-144 (1988).