Activation of disease resistance against Botryosphaeria dothidea by downregulating the expression of MdSYP121 in apple

Abstract

In plants, the vesicle fusion process plays a vital role in pathogen defence. However, the importance of the vesicle fusion process in apple ring rot has not been studied. Here, we isolated and characterised the apple syntaxin gene MdSYP121. Silencing the MdSYP121 gene in transgenic apple calli increased tolerance to Botryosphaeria dothidea infection; this increased tolerance was correlated with salicylic acid (SA) synthesis-related and signalling-related gene transcription. In contrast, overexpressing MdSYP121 in apple calli resulted in the opposite phenotypes. In addition, the results of RNA sequencing (RNA-Seq) and quantitative real-time PCR (qRT-PCR) assays suggested that MdSYP121 plays an important role in responses to oxidation–reduction reactions. Silencing MdSYP121 in apple calli enhanced the expression levels of reactive oxygen species (ROS)-related genes and the activity of ROS-related enzymes. The enhanced defence response status in MdSYP121-RNAi lines suggests that syntaxins are involved in the defence response to B. dothidea. More importantly, we showed that MdSYP121 forms a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex with MdSNAP33, and the complex may participate in regulating resistance to B. dothidea. In conclusion, by regulating the interaction of SA pathway and oxidation–reduction process, MdSYP121 can influence the pathogen infection process in apple.

References

[1] Nebenführ, A. Vesicle traffic in the endomembrane system: a tale of COPs, Rabs and SNAREs. Curr. Opin. Plant Biol. 5, 507-512 (2002).

[2] Carter, C. J., Bednarek, S. Y. & Raikhel, N. V. Membrane trafficking in plants: new discoveries and approaches. Curr. Opin. Plant Biol. 7, 701-707 (2004).

[3] Lipka, V., Kwon, C. & Panstruga, R. SNARE-ware: the role of SNARE-domain proteins in plant biology. Annu. Rev. Cell Dev. Biol. 23, 147-174 (2007).

[4] Wang, D., Weaver, N. D., Kesarwani, M. & Dong, X. Induction of protein secretory pathway is required for systemic acquired resistance. Science 308, 1036-1040 (2005).

[5] Carr, J. P., Dixon, D. C., Nikolau, B. J., Voelkerding, K. V. & Klessig, D. F. Synthesis and localization of pathogenesis-related proteins in tobacco. Mol. Cell Biol. 7, 1580-1583 (1987).

[6] Sanderfoot, A. Increases in the number of SNARE genes parallels the rise of multicellularity among the green plants. Plant Physiol. 144, 6-17 (2007).

[7] Fasshauer, D., Sutton, R. B., Brunger, A. T. & Jahn, R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl Acad. Sci. USA 95, 15781-15786 (1998).

[8] Bock, J. B., Matern, H. T., Peden, A. A. & Scheller, R. H. A genomic perspective on membrane compartment organization. Nature 409, 839-841 (2001).

[9] Jahn, R. & Scheller, R. H. SNAREs-engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631-643 (2006).

[10] Collins, N. C. et al. SNARE-protein-mediated disease resistance at the plant cell wall. Nature 425, 973-977 (2003).

[11] Kalde, M., Nühse, T. S., Findlay, K. & Peck, S. C. The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1. Proc. Natl Acad. Sci. USA 104, 11850-11855 (2007).

[12] Kwon, C. et al. Co-option of a default secretory pathway for plant immune responses. Nature 451, 835-840 (2008).

[13] Nühse, T. S., Boller, T. & Peck, S. C. A plasma membrane syntaxin is phosphorylated in response to the bacterial elicitor flagellin. J. Biol. Chem. 278, 45248-45254 (2003).

[14] Schmelzer, E. Cell polarization, a crucial process in fungal defence. Trends Plant Sci. 7, 411-415 (2002).

[15] Reichardt, I. et al. Mechanisms of functional specificity among plasma-membrane syntaxins in Arabidopsis. Traffic 12, 1269-1280 (2011).

[16] Assaad, F. F. et al. The PEN1 Syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. Mol. Biol. Cell 15, 5118-5129 (2004).

[17] Nielsen, M. E., Feechan, A., Böhlenius, H., Ueda, T. & Thordal-Christensen, H. Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1. Proc. Natl Acad. Sci. USA 109, 11443-11448 (2012).

[18] Zhang, Z. et al. A SNARE-protein has opposing functions in penetration resistance and defence signalling pathways. Plant J. 49, 302-312 (2007).

[19] Eschen-Lippold, L. et al. Activation of defense against phytophthora infestans in potato by down-regulation of syntaxin gene expression. New Phytol. 193, 985-996 (2012).

[20] Tang, W., Ding, Z., Zhou, Z. Q., Wang, Y. Z. & Guo, L. Y. Phylogenetic and pathogenic analyses show that the causal agent of apple ring rot in China is Botryosphaeria dothidea. Plant Dis. 96, 486-496 (2012).

[21] Guo, L., Li, J. & Li, B. Investigations on the occurrence and chemical control of Botryosphaeria canker of apple in China. Plant Prot. 35, 120-123 (2009).

[22] Wang, N. et al. MYB12 and MYB22 play essential roles in proanthocyanidin and flavonol synthesis in red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant J. 90, 276-292 (2017).

[23] Wang, X. et al. Isolation of high-quality RNA from Reaumuria soongorica, a desert plant rich in secondary metabolites. Mol. Biotechnol. 48, 165-172 (2011).

[24] Ashburner, M. et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25, 25-29 (2000).

[25] Tatusov, R. L. et al. The COG database: an updated version includes eukaryotes. BMC Bioinformatics. 4, 41 (2003).

[26] Kanehisa, M., Goto, S., Kawashima, S., Okuno, Y. & Hattori, M. The KEGG resource for deciphering the genome. Nucleic Acids Res. 32, D277-D280 (2004).

[27] Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).

[28] Li, S. J. et al. The citrus transcription factor, CitERF13, regulates citric acid accumulation via a protein-protein interaction with the vacuolar proton pump, CitVHA-c4. Sci. Rep. 6, 20151 (2016).

[29] Hayashi, T. et al. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 5051-5061 (1994).

[30] Durrant, W. E. & Dong, X. Systemic acquired resistance. Annu. Rev. Phytopathol. 42, 185-209 (2004).

[31] Söllner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318-324 (1993).

[32] Heese, M. et al. Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis. J. Cell Biol. 155, 239-249 (2001).

[33] Ungar, D. & Hughson, F. M. SNARE protein structure and function. Annu. Rev. Cell Dev. Biol. 19, 493-517 (2003).

[34] Spoel, S. H. & Dong, X. Making sense of hormone crosstalk during plant immune responses. Cell Host Microbe 3, 348-351 (2008).

[35] Rietz, S. et al. Different roles of enhanced disease susceptibility1 (EDS1) bound to and dissociated from phytoalexin deficient4 (PAD4) in Arabidopsis immunity. New Phytol. 191, 107-119 (2011).

[36] Diaz-Vivancos, P., Bernal-Vicente, A., Cantabella, D., Petri, C. & Hernández, J. A. Metabolomics and biochemical approaches link salicylic acid biosynthesis to cyanogenesis in peach plants. Plant Cell Physiol. 58, 2057-2066 (2017).

[37] Cui, H. et al. A core function of EDS1 with PAD4 is to protect the salicylic acid defense sector in Arabidopsis immunity. New Phytol. 213, 1802-1817 (2017).

[38] Zhang, L. et al. Cotton GhMKK5 affects disease resistance, induces HR-like cell death, and reduces the tolerance to salt and drought stress in transgenic Nicotiana benthamiana. J. Exp. Bot. 63, 3935-3951 (2012).

[39] Herrera-Vásquez, A., Salinas, P. & Holuigue, L. Salicylic acid and reactive oxygen species interplay in the transcriptional control of defense genes expression. Front. Plant Sci. 6, 171 (2015).

[40] Cao, H., Glazebrook, J., Clarke, J. D., Volko, S. & Dong, X. The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88, 57-63 (1997).

[41] Sircar, D. & Mitra, A. Evidence for p-hydroxybenzoate formation involving enzymatic phenylpropanoid side-chain cleavage in hairy roots of Daucus carota. J. Plant Physiol. 165, 407-414 (2008).

[42] Kneer, R., Poulev, A. A., Olesinski, A. & Raskin, I. Characterization of the elicitor-induced biosynthesis and secretion of genistein from roots of Lupinus luteus L.. J. Exp. Bot. 50, 1553-1559 (1999).

[43] Stratmann, J., Scheer, J. & Ryan, C. A. Suramin inhibits initiation of defense signaling by systemin, chitosan, and a beta-glucan elicitor in suspension-cultured Lycopersicon peruvianum cells. Proc. Natl Acad. Sci. USA 97, 8862-8867 (2000).

[44] An, J. P. et al. The bZIP transcription factor MdHY5 regulates anthocyanin accumulation and nitrate assimilation in apple. Hort. Res. 4, 17023 (2017).

[45] Barna, B., Fodor, J., Harrach, B. D., Pogány, M. & Király, Z. The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic pathogens. Plant Physiol. Biochem. 59, 37-43 (2012).

[46] Taheri, P. & Kakooee, T. Reactive oxygen species accumulation and homeostasis are involved in plant immunity to an opportunistic fungal pathogen. J. Plant Physiol. 216, 152-163 (2017).

[47] Chen, Y.-J., Lyngkjær, M. F. & Collinge, D. B. Future prospects for genetically engineering disease resistance plants. In: Molecular Plant Immunity (ed Sessa, G.) 251-275 (Wiley-Blackwell Press, 2013).

[48] Chen, Z., Silva, H. & Klessig, D. F. Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262, 1883-1886 (1993).

[49] Ochsenbein, C. et al. The role of EDS1 (enhanced disease susceptibility) during singlet oxygen-mediated stress responses of Arabidopsis. Plant J. 47, 445-456 (2006).

[50] Ślesak, I. et al. Phytoalexin Deficient 4 affects reactive oxygen species metabolism, cell wall and wood properties in hybrid aspen (Populus tremula L. × tremuloides). Plant Cell Environ. 38, 1275-1284 (2015).