Two pathogenesis-related proteins interact with leucine-rich repeat proteins to promote Alternaria leaf spot resistance in apple

Abstract

Alternaria leaf spot in apple (Malus x domestica), caused by the fungal pathogen Alternaria alternata f. sp. mali (also called A. mali), is a devastating disease resulting in substantial economic losses. We previously established that the resistance (R) protein MdRNL2, containing a coiled-coil, nucleotide-binding, and leucine-rich repeat (CC_R-NB-LRR) domain, interacts with another CC_R-NB-LRR protein, MdRNL6, to form a MdRNL2–MdRNL6 complex that confers resistance to A. mali. Here, to investigate the function of the MdRNL2–MdRNL6 complex, we identified two novel pathogenesis-related (PR) proteins, MdPR10-1 and MdPR10-2, that interact with MdRNL2. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays confirmed that MdPR10-1 and MdPR10-2 interact with MdRNL2 and MdRNL6 at the leucine-rich repeat domain. Transient expression assays demonstrated that accumulation of MdPR10-1 and MdPR10-2 enhanced the resistance of apple to four strains of A. mali that we tested: ALT1, GBYB2, BXSB5, and BXSB7. In vitro antifungal activity assays demonstrated that both the proteins contribute to Alternaria leaf spot resistance by inhibiting fungal growth. Our data provide evidence for a novel regulatory mechanism in which MdRNL2 and MdRNL6 interact with MdPR10-1 and MdPR10-2 to inhibit fungal growth, thereby contributing to Alternaria leaf spot resistance in apple. The identification of these two novel PR proteins will facilitate breeding for fungal disease resistance in apple.

References

[1] Zhang, C. X., Tian, Y. & Cong, P. H. Proteome analysis of pathogen-responsive proteins from apple leaves induced by the Alternaria Blotch Alternaria alternata. PLoS ONE. 10, e0122233 (2015).

[2] Abe, K., Iwanami, H., Kotoda, N., Moriya, S. & Takahashi, S. Evaluation of apple genotypes and Malus species for resistance to Alternaria blotch caused by Alternaria alternata apple pathotype using detached-leaf method. Plant Breed. 129, 208-218 (2010).

[3] Chung, K. R. Stress response and pathogenicity of the necrotrophic fungal pathogen Alternaria alternata. Scientifica. 2012, 635431 (2012).

[4] Zhang, Q. et al. Md-miR156ab and Md-miR395 target WRKY transcription factors to influence apple resistance to leaf spot disease. Front Plant Sci. 8, 526 (2017).

[5] Hugot, K., Aime, S., Conrod, S., Poupet, A. & Galiana, E. Developmental regulated mechanisms affect the ability of a fungal pathogen to infect and colonize tobacco leaves. Plant J. 20, 163-170 (1999).

[6] Johnson, R. D. et al. Cloning and characterization of a cyclic peptide synthetase gene from Alternaria alternata apple pathotype whose product is involved in AM-toxin synthesis and pathogenicity. Mol. Plant Microbe 13, 742-753 (2020).

[7] Moffett, P., Farnham, G., Peart, J. & Baulcombe, D. C. Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death. EMBO J. 21, 4511-4519 (2020).

[8] Jones, J. & Dangl, J. The plant immune system. Nature. 444, 323-329 (2006).

[9] Choi, D. S., Hwang, I. S. & Hwang, B. K. Requirement of the cytosolic interaction between pathogenesis-related protein10 and leucine-rich repeat protein1 for cell death and defense signaling in pepper. Plant Cell. 24, 1675-1690 (2012).

[10] Van Loon, L. C., Rep, M. & Pieterse, C. M. Significance of inducible defense-related proteins in infected plants. Annu Rev. Phytopathol. 44, 135-162 (2006).

[11] Christensen, A. B. et al. The molecular characterization of two barley proteins establishes the novel PR-17 family of pathogenesis-related proteins. Mol. plant Pathol. 3, 135-144 (2002).

[12] McGee, J. D., Hamer, J. E. & Hodges, T. K. Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea. Plant Microbe Interact. 14, 877-886 (2001).

[13] Zhou, X. J., Lu, S., Xu, Y. H., Wang, J. W. & Chen, X. Y. A cotton cDNA (GaPR-10) encoding a pathogenesis-related 10 protein with in vitro ribonuclease activity. Plant Sci. 162, 629-636 (2002).

[14] Hashimoto, M. et al. A novel rice PR10 protein, RSOsPR10, specififically induced in roots by biotic and abiotic stresses, possibly via the jasmonic acid signaling pathway. Plant Cell Physiol. 45, 550-559 (2004).

[15] Park, C. J. et al. Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J. 37, 186-198 (2004).

[16] Liu, J. J. & Ekramoddoullah, A. K. M. The family 10 of plant pathogenesis-related proteins: Their structure, regulation, and function in response to biotic and abiotic stresses. Physiol. Mol. Plant Pathol. 68, 3-13 (2006).

[17] Breiteneder, H. et al. The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene. EMBO J. 8, 1935-1938 (1989).

[18] Hashimoto, M. et al. The Role of alpha-synuclein assembly and metabolism in the pathogenesis of Lewy body disease. J. Mol. Neurosci. 24, 343-352 (2004).

[19] Lo, S. C., Hipskind, J. D. & Nicholson, R. L. cDNA cloning of a sorghum pathogenesis-related protein (PR-10) and differential expression of defense-related genes following inoculation with Cochliobolus heterostrophus or Colletotrichum sublineolum. Mol. plant-microbe Interact. 12, 479-489 (1999).

[20] Matton, D. P. & Brisson, N. Cloning, expression, and sequence conservation of pathogenesis-related gene transcripts of potato. Mol. plant-microbe Interact. 2, 325-331 (1989).

[21] Pagliarani, G. et al. Genomic organisation of the Mal d 1 gene cluster on linkage group 16 in apple. Mol. Breed. 29, 759-778 (2012).

[22] Somssich, I. E., Schmelzer, E., Bollmann, J. & Hahlbrock, K. Rapid activation by fungal elicitor of genes encoding “pathogenesis-related” proteins in cultured parsley cells. Proc. Natl Acad. Sci. USA. 83, 2427-2430 (1986).

[23] Walter, M. H., Liu, J. W., Grand, C., Lamb, C. J. & Hess, D. Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class of conserved PR proteins including pollen allergens. Mol. Gen. Genet. 222, 353-360 (1990).

[24] Warner, S. A., Scott, R. & Draper, J. Characterisation of a wound-induced transcript from the monocot asparagus that shares similarity with a class of intracellular pathogenesis-related (PR) proteins. Plant Mol. Biol. 19, 555-561 (1992).

[25] Jung, Y. H. et al. The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways. Plant Physiol. Biochem. 43, 397-406 (2005).

[26] Jung, Y. H. et al. Differential expression of defense/stressrelated marker proteins in leaves of a unique rice blast lesion mimic mutant (blm). J. Proteome Res. 5, 2586-2598 (2005).

[27] Besbes, F., Habegger, R. and Schwab, W. Induction of PR-10 genes an metabolites in strawberry plants in response to Verticillium dahliae infection. BMC Plant Biol. 19, 128 (2019).

[28] Somssich, I. E., Schmelzer, E., Kawalleck, P. & Hahlbrock, K. Gene structure and in situ transcript localization of pathogenesis-related protein 1 in parsley. Mol. Gen. Genet 213, 93-98, https://doi.org/10.1007/BF00333403 (1988).

[29] Breiteneder, H. et al. Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens. Eur. J. Biochem 233, 484-489, https://doi.org/10.1111/j.1432-1033.1995.484_2.x (2010).

[30] Vanek-Krebitz, M. et al. Cloning and sequencing of Mal d 1, the major allergen from apple (Malus domestica), and its immunological relationship to Bet v 1, the major birch pollen allergen. Biochem Biophys. Res Commun. 214, 538-551, https://doi.org/10.1006/bbrc.1995.2320 (1995).

[31] Puhringer, H. et al. The promoter of an apple Ypr10 gene, encoding the major allergen Mal d 1, is stress- and pathogen-inducible. Plant Sci. 152, 35-50 (2000).

[32] Jung, H. W. & Hwang, B. K. The leucine-rich repeat (LRR) protein, CaLRR1, interacts with the hypersensitive induced reaction (HIR) protein, CaHIR1, and suppresses cell death induced by the CaHIR1 protein. Mol. Plant Pathol. 8, 503-514 (2007).

[33] Choi, H. W., Kim, Y. J. & Hwang, B. K. The hypersensitive induced reaction and leucine-rich repeat proteins regulate plant cell death associated with disease and plant immunity. Mol. Plant Microbe Interact. 24, 68-78 (2011).

[34] Fernandes, H., Michalska, K., Sikorski, M. and Jaskolski, M. Structural and functional aspects of PR-10 proteins. FEBS J. 280, 1169-1199 (2013).

[35] Fujimoto, Y. et al. Purifification and cDNA cloning of cytokinin-specifific binding protein from mung bean (Vigna radiata). Eur. J. Biochem. 258, 794-802 (1998).

[36] Mogensen, J. E., Wimmer, R., Larsen, J. N., Spangfort, M. D. & Otzen, D. E. The major birch allergen, Bet v 1, shows affifinity for a broad spectrum of physiological ligands. J. Biol. Chem. 277, 23684-23692 (2002).

[37] Markovic´-Housley, Z. et al. Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier. J. Mol. Biol. 325, 123-133 (2003).

[38] Koistinen, K. M. et al. Birch PR-10c interacts with several biologically important ligands. Phytochemistry 66, 2524-2533 (2005).

[39] Harteveld, D., Akinsanmi, O., Chandra, K. & Drenth, A. Timing of infection and development of alternaria diseases in the canopy of apple trees. Plant Dis. 98, 401-408 (2014).

[40] Sawamura, K. Studies on apple Alternaria blotch caused by Alternaria mali Roberts. Bull. Fac. Agric. Hirosaki Univ. 18, 152-235 (1972).

[41] Li, Y. et al. Interactions of apple and the Alternaria alternata apple pathotype. Crit. Rev. Plant Sci. 32, 141-150 (2013).

[42] Hwang, B., Yun, J. & Kim, Z. Geographic variation of esterase isozymes in populations of Alternaria mali on apple leaves. J. Phytopathol. 119, 225-231 (1987).

[43] Filajdic, N. & Sutton, T. Identification and distribution of Alternaria mali on apples in North Carolina and susceptibility of different varieties of apples to Alternaria blotch. Plant Dis. 75, 1045-1048 (1991).

[44] Gagkaeva, T. & Levitin, M. Identification of the pathogen causing leaf spots on apple trees in Krasnodar province orchards. Mikol. Fitopatol. 34, 58-62 (2000).

[45] Bulajic, A., Filajdic, N., Babovic, M. & Sutton, T. First report of Alternaria mali on apples in Yugoslavia. Plant Dis. 80, 709 (1996).

[46] Soleimani, M. & Esmailzadeh, M. First report of Alternaria mali causing apple leaf blotch disease in Iran. Australas. Plant Dis. Notes. 2, 57-58 (2007).

[47] Ozgonen, H. & Karaca, G. First report of Alternaria mali causing necrotic leaf spot of apples in Turkey. Plant Pathol. 55, 578 (2006).

[48] Rollemberg, C., Fayad, F., Hamada, N. & De Mio, L. First report of Alternaria mali on apples in Brazil. Phytopathology 101, 155 (2011).

[49] Wang, M., Weiberg, A., Dellota, E., Yamane, D. & Jin, H. Botrytis small RNA Bc-siR37 suppresses plant defense genes by cross-kingdom RNAi. RNA Biol. 14, 421-428 (2017).

[50] Gachon, C. & Saindrenan, P. Real-time PCR monitoring of fungal development in Arabidopsis thaliana infected by Alternaria brassicicola and Botrytis cinerea. Plant Physiol. Biochem. 42, 367-371 (2004).

[51] Livak, K. & Schmittgen, T. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT Method . Methods 25, 402-408 (2001).

[52] Bai, S., Kasai, A., Yamada, K., Li, T. & Harada, T. A mobile signal transported over a long distance induces systemic transcriptional gene silencing in a grafted partner. J. Exp. Bot. 62, 4561-4570 (2011).

[53] Song, N., et al. An erf2-like transcription factor regulates production of the defense sesquiterpene capsidiol upon alternaria alternata infection. J. Exp Bot. 20, 5895-5908 (2019).