Apple ( Malus × domestica Borkh.) is a perennial woody plant that often suffers from various biological stresses. Many harmful pathogens can infect apple trees and lead to reduced production. We comprehensively identified the WRKY genes in the apple genome and analyzed their expression in response to several biological stressors, including Alternaria alternata , Pythium ultimum , Botryosphaeria dothidea , Erwinia amylovora , Penicillium expansum , Gymnosporangium yamadae , and Apple replant disease. There were 113 MdWRKYs identified in the apple genome. Twenty-two MdWRKYs were differentially expressed in response to at least five pathogens. Promoter sequence analysis showed that these genes carried many defense- and stress-responsive elements, such as MeJA-response elements, salicylic acid-response elements, and W-box elements, in their promoters. Transient expression assays showed that MdWRKY 40 a and MdWRKY 54 h played negative roles in defense against B. dothidea infection. WRKY 40 and WRKY 60 and the MdWKRY 33 s might play important roles in responding to pathogens and are conserved in some plants. These differentially expressed MdWRKYs might play key roles in the apple response to multiple pathogens.
KeywordsApple<i>WRKY</i>Transcription FactorPathogensBig Data
Xu, J., Li, M., Jiao, P., Tao, H., Wei, N., Ma, F. and Zhang, J. (2015) Dynamic Transcription Profiles of “Qinguan” Apple (Malus × Domestica) Leaves in Response to Marssonina coronaria Inoculation. Frontiers in Plant Science, 6, Article No. 842. https://doi.org/10.3389/fpls.2015.00842
Shen, F., Huang, Z., Zhang, B., Wang, Y., Zhang, X., Wu, T., Xu, X., Zhang, X. and Han, Z. (2019) Mapping Gene Markers for Apple Fruit Ring Rot Disease Resistance Using a Multi-Omics Approach. G3: Genes, Genomes, Genetics, 9, 1663-1678. https://doi.org/10.1534/g3.119.400167
Eulgem, T. (2006) Dissecting the WRKY Web of Plant Defense Regulators. PLoS Pathogens, 2, Article No. e126. https://doi.org/10.1371/journal.ppat.0020126
Eulgem, T., Rushton, P.J., Robatzek, S. and Somssich, I.E. (2000) The WRKY Superfamily of Plant Transcription Factors. Trends in Plant Science, 5, 199-206. https://doi.org/10.1016/S1360-1385(00)01600-9
Xu, X., Chen, C., Fan, B. and Chen, Z. (2006) Physical and Functional Interactions between Pathogen-Induced Arabidopsis WRKY18, WRKY40, and WRKY60 Transcription Factors. The Plant Cell, 18, 1310-1326. https://doi.org/10.1105/tpc.105.037523
Birkenbihl, R.P., Kracher, B., Roccaro, M. and Somssich, I.E. (2017) Induced Genome-Wide Binding of Three Arabidopsis WRKY Transcription Factors during Early MAMP-Triggered Immunity. The Plant Cell, 29, 20-38. https://doi.org/10.1105/tpc.16.00681
Wang, D., Amornsiripanitch, N. and Dong, X. (2006) A Genomic Approach to Identify Regulatory Nodes in the Transcriptional Network of Systemic Acquired Resistance in Plants. PLoS Pathogens, 2, Article No. e123. https://doi.org/10.1371/journal.ppat.0020123
Lai, Z., Vinod, K., Zheng, Z., Fan, B. and Chen, Z. (2008) Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens. BMC Plant Biology, 8, Article No. 68. https://doi.org/10.1186/1471-2229-8-68
Journot-Catalino, N., Somssich, I.E., Roby, D. and Kroj, T. (2006) The Transcription Factors WRKY11 and WRKY17 Act as Negative Regulators of Basal Resistance in Arabidopsis Thaliana. The Plant Cell, 18, 3289-3302. https://doi.org/10.1105/tpc.106.044149
Zhang, Q., Li, Y., Zhang, Y., Wu, C., Wang, S., Hao, L., Wang, S. and Li, T. (2017) Md-MiR156ab and Md-MiR395 Target WRKY Transcription Factors to Influence Apple Resistance to Leaf Spot Disease. Frontiers in Plant Science, 8, Article No. 526. https://doi.org/10.3389/fpls.2017.00526
Zhang, F., Wang, F., Yang, S., Zhang, Y., Xue, H., Wang, Y., Yan, S., Wang, Y., Zhang, Z. and Ma, Y. (2019) MdWRKY100 Encodes a Group I WRKY Transcription Factor in Malus domestica That Positively Regulates Resistance to Colletotrichum gloeosporioides Infection. Plant Science, 286, 68-77. https://doi.org/10.1016/j.plantsci.2019.06.001
Fan, H., Wang, F., Gao, H., Wang, L., Xu, J. and Zhao, Z. (2011) Pathogen-Induced MdWRKY1 in ‘Qinguan’ Apple Enhances Disease Resistance. Journal of Plant Biology, 54, 150-158. https://doi.org/10.1007/s12374-011-9151-1
Zhao, X., Qi, C., Jiang, H., Zhong, M., You, C., Li, Y. and Hao, Y. (2020) MdWRKY15 Improves Resistance of Apple to Botryosphaeria dothidea via the Salicylic Acid-Mediated Pathway by Directly Binding the MdICS1 Promoter. Journal of Integrative Plant Biology, 62, 527-543. https://doi.org/10.1111/jipb.12825
Zhao, X., Qi, C., Jiang, H., Zhong, M., Zhao, Q., You, C., Li, Y. and Hao, Y. (2019) MdWRKY46-Enhanced Apple Resistance to Botryosphaeria dothidea by Activating the Expression of MdPBS3.1 in the Salicylic Acid Signaling Pathway. Molecular Plant-Microbe Interactions ® , 32, 1391-1401. https://doi.org/10.1094/MPMI-03-19-0089-R
Dong, J., Chen, C. and Chen, Z. (2003) Expression Profiles of the Arabidopsis WRKY Gene Superfamily during Plant Defense Response. Plant Molecular Biology, 51, 21-37. https://doi.org/10.1023/A:1020780022549
Zhu, X., Liu, S., Meng, C., Qin, L., Kong, L. and Xia, G. (2013) WRKY Transcription Factors in Wheat and Their Induction by Biotic and Abiotic Stress. Plant Molecular Biology Reporter, 31, 1053-1067. https://doi.org/10.1007/s11105-013-0565-4
Guo, C., Guo, R., Xu, X., Gao, M., Li, X., Song, J., Zheng, Y. and Wang, X. (2014) Evolution and Expression Analysis of the Grape (Vitis vinifera L.) WRKY Gene Family. Journal of Experimental Botany, 65, 1513-1528. https://doi.org/10.1093/jxb/eru007
Jiang, Y., Duan, Y., Yin, J., Ye, S., Zhu, J., Zhang, F., Lu, W., Fan, D. and Luo, K. (2014) Genome-Wide Identification and Characterization of the Populus WRKY Transcription Factor Family and Analysis of Their Expression in Response to Biotic and Abiotic Stresses. Journal of Experimental Botany, 65, 6629-6644. https://doi.org/10.1093/jxb/eru381
Chen, P. and Liu, Q. (2019) Genome-Wide Characterization of the WRKY Gene Family in Cultivated Strawberry (Fragaria × Ananassa Duch.) and the Importance of Several Group III Members in Continuous Cropping. Scientific Reports, 9, Article No. 8423. https://doi.org/10.1038/s41598-019-44479-7
Meng, D., Li, Y., Bai, Y., Li, M. and Cheng, L. (2016) Genome-Wide Identification and Characterization of WRKY Transcriptional Factor Family in Apple and Analysis of Their Responses to Waterlogging and Drought Stress. Plant Physiology and Biochemistry, 103, 71-83. https://doi.org/10.1016/j.plaphy.2016.02.006
Lui, S., Luo, C., Zhu, L., Sha, R., Qu, S., Cai, B. and Wang, S. (2017) Identification and Expression Analysis of WRKY Transcription Factor Genes in Response to Fungal Pathogen and Hormone Treatments in Apple (Malus Domestica). Journal of Plant Biology, 60, 215-230. https://doi.org/10.1007/s12374-016-0577-3
Daccord, N., Celton, J., Linsmith, G., Becker, C., Choisne, N., Schijlen, E., et al. (2017) High-Quality de Novo Assembly of the Apple Genome and Methylome Dynamics of Early Fruit Development. Nature Genetics, 49, 1099-1106. https://doi.org/10.1038/ng.3886
Mount, D.W. (2007) Using the Basic Local Alignment Search Tool (BLAST). Cold Spring Harbor Protocols, 2007, pdb.top17. https://doi.org/10.1101/pdb.top17
Finn, R.D., Bateman, A., Clements, J., Coggill, P., Eberhardt, R.Y., Eddy, S.R., et al. (2014) Pfam: The Protein Families Database. Nucleic Acids Research, 42, D222-D230. https://doi.org/10.1093/nar/gkt1223
Lu, S., Wang, J., Chitsaz, F., Derbyshire, M.K., Geer, R.C., Gonzales, N.R., et al. (2020) CDD/SPARCLE: The Conserved Domain Database in 2020. Nucleic Acids Research, 48, D265-D268. https://doi.org/10.1093/nar/gkz991
Chen, C., Chen, H., Zhang, Y., Thomas, H.R., Frank, M.H., He, Y. and Xia, R. (2020) TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Molecular Plant, 13, 1194-1202. https://doi.org/10.1016/j.molp.2020.06.009
Pertea, M., Kim, D., Pertea, G.M., Leek, J.T. and Salzberg, S.L. (2016) Transcript-Level Expression Analysis of RNA-Seq Experiments with HISAT, StringTie and Ballgown. Nature Protocols, 11, 1650-1667. https://doi.org/10.1038/nprot.2016.095
Love, M.I., Huber, W. and Anders, S. (2014) Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biology, 15, Article No. 550. https://doi.org/10.1186/s13059-014-0550-8
Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P. and Rombauts, S. (2002) PlantCARE, a Database of Plant CIS-Acting Regulatory Elements and a Portal to Tools for in Silico Analysis of Promoter Sequences. Nucleic Acids Research, 30, 325-327. https://doi.org/10.1093/nar/30.1.325
Jiang, Y., Liu, C., Yan, D., Wen, X., Liu, Y., Wang, H., Dai, J., Zhang, Y., Liu, Y., Zhou, B. and Ren, X. (2017) MdHB1 Down-Regulation Activates Anthocyanin Biosynthesis in the White-Fleshed Apple Cultivar ‘Granny Smith.’ Journal of Experimental Botany, 68, 1055-1069. https://doi.org/10.1093/jxb/erx029
Zhu, L., Ni, W., Liu, S., Cai, B., Xing, H. and Wang, S. (2017) Transcriptomics Analysis of Apple Leaves in Response to Alternaria alternata Apple Pathotype Infection. Frontiers in Plant Science, 8, Article No. 22. https://doi.org/10.3389/fpls.2017.00022
Weiß, S., Liu, B., Reckwell, D., Beerhues, L. and Winkelmann, T. (2017) Impaired Defense Reactions in Apple Replant Disease-Affected Roots of Malus domestica “M26.” Tree Physiology, 37, 1672-1685. https://doi.org/10.1093/treephys/tpx108
Weiß, S. and Winkelmann, T. (2017) Transcriptome Profiling in Leaves Representing Aboveground Parts of Apple Replant Disease Affected Malus domestica ‘M26’ Plants. Scientia Horticulturae, 222, 111-125. https://doi.org/10.1016/j.scienta.2017.05.012
Shin, S., Zheng, P., Fazio, G., Mazzola, M., Main, D. and Zhu, Y. (2016) Transcriptome Changes Specifically Associated with Apple (Malus domestica) Root Defense Response during Pythium ultimum Infection. Physiological and Molecular Plant Pathology, 94, 16-26. https://doi.org/10.1016/j.pmpp.2016.03.003
Zhu, Y., Shao, J., Zhou, Z. and Davis, R.E. (2019) Genotype-Specific Suppression of Multiple Defense Pathways in Apple Root during Infection by Pythium ultimum. Horticulture Research, 6, Article No. 10. https://doi.org/10.1038/s41438-018-0087-1
Singh, J., Fabrizio, J., Desnoues, E., Silva, J.P., Busch, W. and Khan, A. (2019) Root System Traits Impact Early Fire Blight Susceptibility in Apple (Malus × Domestica). BMC Plant Biology, 19, Article No. 579. https://doi.org/10.1186/s12870-019-2202-3
Ballester, A.-R., Norelli, J., Burchard, E., Abdelfattah, A., Levin, E., González-Candelas, L., Droby, S. and Wisniewski, M. (2017) Transcriptomic Response of Resistant (PI613981-Malus Sieversii) and Susceptible (“Royal Gala”) Genotypes of Apple to Blue Mold (Penicillium Expansum) Infection. Frontiers in Plant Science, 8, Article No. 1981. https://doi.org/10.3389/fpls.2017.01981
Tao, S., Auer, L., Morin, E., Liang, Y. and Duplessis, S. (2019) Transcriptome Analysis of Apple Leaves Infected by the Rust Fungus Gymnosporangium Yamadae at Two Sporulation Stages. Molecular Plant-Microbe Interactions ® , 33, 444-461. https://doi.org/10.1094/MPMI-07-19-0208-R
Chen, H., Lai, Z., Shi, J., Xiao, Y., Chen, Z. and Xu, X. (2010) Roles of Arabidopsis WRKY18, WRKY40 and WRKY60 Transcription Factors in Plant Responses to Abscisic Acid and Abiotic Stress. BMC Plant Biology, 10, Article No. 281. https://doi.org/10.1186/1471-2229-10-281
Zipfel, C., Robatzek, S., Navarro, L., Oakeley, E.J., Jones, J.D.G., Felix, G. and Boller, T. (2004) Bacterial Disease Resistance in Arabidopsis through Flagellin Perception. Nature, 428, 764-767. https://doi.org/10.1038/nature02485
Choura, M., Rebaï, A. and Masmoudi, K. (2015) Unraveling the WRKY Transcription Factors Network in Arabidopsis thaliana by Integrative Approach. Network Biology, 5, 55-61.
Karim, A., Jiang, Y., Guo, L., Ling, Z., Ye, S., Duan, Y., Li, C. and Luo, K. (2015) Isolation and Characterization of a Subgroup IIa WRKY Transcription Factor PtrWRKY40 from Populus trichocarpa. Tree Physiology, 35, 1129-1139. https://doi.org/10.1093/treephys/tpv084
Cui, X., Yan, Q., Gan, S., Xue, D., Wang, H., Xing, H., Zhao, J. and Guo, N. (2019) GmWRKY40, a Member of the WRKY Transcription Factor Genes Identified from Glycine max L., Enhanced the Resistance to Phytophthora sojae. BMC Plant Biology, 19, Article No. 598. https://doi.org/10.1186/s12870-019-2132-0
Birkenbihl, R.P., Diezel, C. and Somssich, I.E. (2012) Arabidopsis WRKY33 Is a Key Transcriptional Regulator of Hormonal and Metabolic Responses toward Botrytis Cinerea Infection. Plant Physiology, 159, 266-285. https://doi.org/10.1104/pp.111.192641
Zheng, X.-Y., Spivey, N.W., Zeng, W., Liu, P. P., Fu, Z.Q., Klessig, D.F., He, S.Y. and Dong, X. (2012) Coronatine Promotes Pseudomonas syringae Virulence in Plants by Activating a Signaling Cascade That Inhibits Salicylic Acid Accumulation. Cell Host & Microbe, 11, 587-596. https://doi.org/10.1016/j.chom.2012.04.014
Liu, F., Li, X., Wang, M., Wen, J., Yi, B., Shen, J., Ma, C., Fu, T. and Tu, J. (2018) Interactions of WRKY15 and WRKY33 Transcription Factors and Their Roles in the Resistance of Oilseed Rape to Sclerotinia Infection. Plant Biotechnology Journal, 16, 911-925. https://doi.org/10.1111/pbi.12838
Yu, Y., Liu, Z., Wang, L., Kim, S.G., Seo, P.J., Qiao, M., Wang, N., Li, S., Cao, X., Park, C.M. and Xiang, F. (2016) WRKY71 Accelerates Flowering via the Direct Activation of FLOWERING LOCUS T and LEAFY in Arabidopsis thaliana. The Plant Journal, 85, 96-106. https://doi.org/10.1111/tpj.13092
Guo, D., Zhang, J., Wang, X., Han, X., Wei, B., Wang, J., Li, B., Yu, H., Huang, Q., Gu, H., Qu, L. J. and Qin, G. (2015) The WRKY Transcription Factor WRKY71/EXB1 Controls Shoot Branching by Transcriptionally Regulating RAX Genes in Arabidopsis. The Plant Cell, 27, 3112-3127. https://doi.org/10.1105/tpc.15.00829
Cheng, H., Liu, H., Deng, Y., Xiao, J., Li, X. and Wang, S. (2015) The WRKY45-2 WRKY13 WRKY42 Transcriptional Regulatory Cascade Is Required for Rice Resistance to Fungal Pathogen. Plant Physiology, 167, 1087-1099. https://doi.org/10.1104/pp.114.256016
Bostock, R.M. (2005) Signal Crosstalk and Induced Resistance: Straddling the Line between Cost and Benefit. Annual Review of Phytopathology, 43, 545-580. https://doi.org/10.1146/annurev.phyto.41.052002.095505
Durrant, W.E. and Dong, X. (2004) Systemic Acquired Resistance. Annual Review of Phytopathology, 42, 185-209. https://doi.org/10.1146/annurev.phyto.42.040803.140421
Li, J., Brader, G. and Palva, E.T. (2004) The WRKY70 Transcription Factor: A Node of Convergence for Jasmonate-Mediated and Salicylate-Mediated Signals in Plant Defense. The Plant Cell, 16, 319-331. https://doi.org/10.1105/tpc.016980
Kim, K.C., Lai, Z., Fan, B. and Chen, Z. (2008) Arabidopsis WRKY38 and WRKY62 Transcription Factors Interact with Histone Deacetylase 19 in Basal Defense. The Plant Cell, 20, 2357-2371. https://doi.org/10.1105/tpc.107.055566