Functional characterization of WRKY46 in grape and its putative role in the interaction between grape and phylloxera (Daktulosphaira vitifoliae)

Abstract

WRKY transcription factors are involved in defense responses caused by biotic stresses. Phylloxera (Daktulosphaira vitifoliae Fitch), a pest widespread in viticulture, elicits transcriptional reprogramming of plant defense-associated components, such as regulons related to WRKYs and salicylic acid (SA) signaling. In this study, we characterized WRKY46, a WRKY transcription factor responsible for phylloxera attack, and revealed the molecular mechanism for WRKY-mediated defense responses to phylloxera. qRT-PCR and GUS staining analyses revealed that WRKY46 is induced in response to phylloxera damage and mechanical wounding. VvWRKY46 is a nuclear-localized transcription factor that activates its downstream target VvCHIB by direct protein–DNA interaction. Regulons involved in the SA-mediated defense response were regulated during incompatible interactions between “1103 Paulsen” rootstock and phylloxera. In addition, WRKY46 exhibited a higher transcript abundance in “1103 Paulsen” than in “Crimson Seedless”, regardless of whether the plants were infected with phylloxera. Furthermore, the enhanced expression of VvWRKY46 significantly attenuated phylloxera attack and delayed nymph development of composite grape plants. In summary, we demonstrated that WRKY46 plays a role in the SA-mediated defense-regulatory network by directly binding to the downstream structural gene VvCHIB. The phylloxera-responsive gene WRKY46 was identified, which could improve the understanding of the basic mechanism of grapevine in response to phylloxera.

References

[1] Chisholm, S. T., Coaker, G., Day, B. & Staskawicz, B. J. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124, 803-814 (2006).

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

[3] Tsuda, K., Katagiri, F., Parker, J. E. & Ellis, J. G. Comparing signaling mechanisms engaged in pattern-triggered & effector-triggered immunity. Curr. Opin. Plant Biol. 13, 459-465 (2010).

[4] Zarate, S. I., Kempema, L. A. & Walling, L. L. Silverleaf whitefly induces salicylic acid defenses and suppresses effectual jasmonic acid defenses. Plant Physiol. 143, 866-875 (2007).

[5] Kawazu, K. et al. Different expression profiles of jasmonic acid and salicylic acid inducible genes in the tomato plant against herbivores with various feeding modes. Arthropod-Plant Interact. 10, 87- 87 (2016).

[6] Liu, Y. & Zhang, S. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16, 3386-3399 (2004).

[7] Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205-227 (2005).

[8] Lin, J. et al. Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode. Plant Biotechnol. J. 11, 1135-1145 (2013).

[9] Leitner, M. & Boland, W. A. Direct and indirect defences induced by piercing-sucking and chewing herbivores in Medicago truncatula. New Phytol. 167, 597-606 (2005).

[10] An, C. & Mou, Z. Salicylic acid and its function in plant immunity. J. Integr. Plant Biol. 53, 412-428 (2011).

[11] Claverie, M. et al. The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region. Plant Physiol. 156, 779-792 (2011).

[12] Du, B. et al. Identification and characterization of Bph14, a gene conferring resistance to brown plant hopper in rice. Proc. Natl Acad. Sci. USA 106, 22163-22168 (2009).

[13] Branch, C., Hwang, C. F., Navarre, D. A. & Williamson, V. M. Salicylic acid is part of the Mi-1-mediated defense response to root-knot nematode in tomato. Mol. Plant Microbe. Inter. 17, 351-356 (2004).

[14] Molinari, S., Fanelli, E. & Leonetti, P. Expression of tomato salicylic acid (SA)-responsive pathogenesis-related genes in Mi-1-mediated and SA-induced resistance to root-knot nematodes. Mol. Plant Pathol. 15, 255-264 (2014).

[15] Du, Y. P., Jiang, E. S., Wang, F. P., Zhang, S. Z. & Zhai, H. Gene expression profiling of rootstock ‘140Ru’ and Vitis vinifera L. cv. ‘Crimson Seedless’ grape roots infected with grape phylloxera. Plant Growth Regul. 73, 1-8 (2014).

[16] Elhamahmy, M. A. M., Mahmoud, M. F. & Bayoumi, T. Y. The effect of applying exogenous salicylic acid on aphid infection and its influence on histophysiological traits and thermal imaging of canola. Cercet. Agron. Mold. 49, 67-85 (2016).

[17] Eulgem, T. & Somssich, I. E. Networks of WRKY transcription factors in defense signaling. Curr. Opin. Plant Biol. 10, 366-371 (2007).

[18] Wang, H. et al. Overexpression of rice WRKY89 enhances ultraviolet B tolerance and disease resistance in rice plants. Plant Mol. Biol. 65, 799-815 (2007).

[19] Li, P. et al. The over-expression of a chrysanthemum WRKY transcription factor enhances aphid resistance. Plant Physiol. Biochem. 95, 26-34 (2015).

[20] Wim, G. et al. A role for AtWRKY23 in feeding site establishment of plant-parasitic nematodes. Plant Physiol. 148, 358-368 (2008).

[21] Chinnapandi, B., Bucki, P. & Braun, S. M. SlWRKY45, nematode-responsive tomato WRKY gene, enhances susceptibility to the root knot nematode; M. javanica infection. Plant Signal. Behav. 12, e1356530 (2017).

[22] Verk, M. C. V., Bol, J. F. & Linthorst, H. J. WRKY transcription factors involved in activation of SA biosynthesis genes. BMC Plant Biol. 11, 89- 89 (2011).

[23] Petersen, K., Fiil, B. K., Mundy, J. & Petersen, M. Downstream targets of WRKY33. Plant Signal Behav. 3, 1033-1034 (2008).

[24] Qiu, J. L. et al. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus. Embo J. 27, 2214-2221 (2014).

[25] Mukhtar, M. S., Nishimura, M. T. & Dangl, J. NPR1 in plant defense: it’s not over ‘til it’s turned over. Cell 137, 804-806 (2009).

[26] Yu, D. & Chen, Z. Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13, 1527-1540 (2001).

[27] Wang, D., Amornsiripanitch, N. & Dong, X. A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. Plos Pathog. 2, e123 (2006).

[28] Kim, K. C., Lai, Z., Fan, B. & Chen, Z. Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell 20, 2357-2371 (2008).

[29] Verk, M. C. V., Neeleman, L., Bol, J. F. & Linthorst, H. J. M. Tobacco transcription factor NtWRKY12 interacts with TGA2.2 in vitro and in vivo. Front Plant Sci. 2, 1085-1091 (2011).

[30] Raman, A., Beiderbeck, R. & Herth, W. Early subcellular responses of susceptible and resistant Vitis taxa to feeding by grape phylloxera Daktulosphaira vitifoliae. Bot. Helv. 119, 31-39 (2009).

[31] Dietrich, A., Wolf, T., Eimert, K. & Schröder, M. B. Activation of gene expression during hypersensitive response (HR) induced by auxin in the grapevine rootstock cultivar ‘Börner’. Vitis. J. Grapevine Res. 49, 15-21 (2010).

[32] Blank, L., Wolf, T., Eimert, K. & Schröder, M. Differential gene expression during hypersensitive response in -resistant rootstock ‘Börner’ using custom oligonucleotide arrays. J. Plant Interact. 4, 261-269 (2009).

[33] Granett, J., Goheen, A. C., Lider, L. A. & White, J. J. Evaluation of grape rootstocks for resistance to type A and type B grape phylloxera. Am. J. Enol. Vitic. 38, 298-300 (1987).

[34] Kellow, A. V., Corrie, A. M. & Heeswjck, R. Surface sterilisation of phylloxera eggs for investigating grapevine-phylloxera interactions in tissue culture. Australian J. Grape and Wine Res. 5, 27-28 (1999).

[35] Gambino, G., Perrone, I. & Gribaudo, I. (2010) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal. 19, 520-525 (1999).

[36] Du, Y. P., Wang, Z. S., Sun, Q. H., Zhai, H. & Wang, Z. Y. Evaluating resistance of some grape varieties and rootstocks to grape phylloxera (in chanese). Acta Èntomol. Sin. 51, 33-39 (2008).

[37] Eulgem, T., Rushton, P. J., Robatzek, S. & Somssich, I. E. The WRKY superfamily of plant transcription factors. Trends Plant Sci. 5, 199-206 (2000).

[38] Nabity, P. D., Haus, M. J., Berenbaum, M. R. & Delucia, E. H. Leaf-galling phylloxera on grapes reprograms host metabolism and morphology. Proc Natl Acad Sci USA.Proc. Natl Acad. Sci. USA 110, 16663-16668 (2013).

[39] Lescot, M. et al. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 30, 325-327 (2002).

[40] Shah, J. The salicylic acid loop in plant defense. Curr. Opin. Plant Biol. 6, 365-371 (2003).

[41] Kim, H. S. & Delaney, T. P. Over-expression of TGA5, which encodes a bZIP transcription factor that interacts with NIM1/NPR1, confers SAR-independent resistance in Arabidopsis thaliana to Peronospora parasitica. Plant J. 32, 151-163 (2010).

[42] Hu, Y., Dong, Q. & Yu, D. Arabidopsis WRKY46 coordinates with WRKY70 and WRKY53 in basal resistance against pathogen Pseudomonas syringae. Plant Sci. 185-6, 288-297 (2012).

[43] Guo, C. et al. Evolution and expression analysis of the grape (Vitis vinifera L.) WRKY gene family. J. Exp. Bot. 65, 1513-1528 (2014).

[44] Zhao, J. et al. Over-expression of a grape WRKY transcription factor gene, VlWRKY48, in Arabidopsis thaliana increases disease resistance and drought stress tolerance. Plant Cell Tissue Organ Cult. 132, 359-370 (2018).

[45] Park, C. J. et al. A hot pepper gene encoding WRKY transcription factor is induced during hypersensitive response to Tobacco mosaic virus and Xanthomonas campestris. Planta 223, 168-179 (2006).

[46] Zhang, Q., Zhu, J., Ni, Y., Cai, Y. & Zhang, Z. Expression profiling of HbWRKY1, an ethephon-induced WRKY gene in latex from Hevea brasiliensis in responding to wounding and drought. Trees 26, 587-595 (2012).

[47] Felton, G. W. & Tumlinson, J. H. Plant-insect dialogs: complex interactions at the plant-insect interface. Curr. Opin. Plant Biol. 11, 457-463 (2008).

[48] Reymond, P., Weber, H., Damond, M. & Farmer, E. E. Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 12, 707-719 (2000).

[49] Mithöfer, A. & Boland, W. Recognition of herbivory-associated molecular patterns. Plant Physiol. 146, 825-831 (2008).

[50] Uehara, T., Sugiyama, S., Matsuura, H., Arie, T. & Masuta, C. Resistant and susceptible responses in tomato to cyst nematode are differentially regulated by salicylic acid. Plant Cell Physiol. 51, 1524-1536 (2010).

[51] Kumari, C., Dutta, T. K., Banakar, P. & Rao, U. Comparing the defence-related gene expression changes upon root-knot nematode attack in susceptible versus resistant cultivars of rice. Sci. Rep. 6, 22846 (2016).

[52] Hamamouch, N., Li, C., Seo, P. J., Park, C. M. & Davis, E. L. Expression of Arabidopsis pathogenesis-related genes during nematode infection. Mol. Plant Pathol. 12, 355-364 (2011).