Function identification of miR482b, a negative regulator during tomato resistance to Phytophthora infestans

Abstract

Tomato is an important horticultural and economic crop cultivated worldwide. As Phytophthora infestans becomes a huge threat to tomato production, it is necessary to study the resistance mechanisms of tomato against P. infestans. Our previous research has found that miR482 might be involved in tomato– P. infestans interaction. In this study, miR482b precursor was cloned from Solanum pimpinellifolium “L3708” and miR482b was shown to decrease in abundance in tomato following P. infestans infection. Compared to wild-type tomato plants, tomato plants that overexpressed miR482b displayed more serious disease symptoms after P. infestans infection, with more necrotic cells, longer lesion diameters, and increased P. infestans abundance. Meanwhile, silencing of miR482b was performed by short tandem target mimic (STTM), resulting in enhancement of tomato resistance to P. infestans. Using miRNA and degradome data sets, NBS–LRR disease-resistance genes targeted by miR482b were validated. Negative correlation between the expression of miR482b and its target genes was found in all miR482b-overexpressing and -silencing tomato plants. Our results provide insight into tomato miR482b involved in the response to P. infestans infection, and demonstrate that miR482b–NBS–LRR is an important component in the network of tomato– P. infestans interaction.

References

[1] Baysal-Gurel, E., Li, R., Ling, K. S. & Miller, S. A. First report of tomato chlorotic spot virus infecting tomatoes in ohio. Plant Dis. 99, 163-164 (2015).

[2] Balmant, K. M. et al. Redox proteomics of tomato in response to Pseudomonas syringae infection. Hortic. Res 2, 15043 (2015).

[3] Bhattarai, K., Louws, F. J., Williamson, J. D. & Panthee, D. R. Differential response of tomato genotypes to Xanthomonas-specific pathogen-associated molecular patterns and correlation with bacterial spot (Xanthomonas perforans) resistance. Hortic. Res. 3, 16035 (2016).

[4] Zhang, C. Z., Liu, L. & Wang, X. X. et al. The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans. Theor. Appl. Genet. 127, 1353-1364 (2014).

[5] Fry, W. E., Birch, P. R. & Judelson, H. S. et al. Five reasons to consider Phytophthora infestans a reemerging pathogen. Phytopathology 105, 966-981 (2015).

[6] Zhang, C., Liu, L. & Zheng, Z. et al. Fine mapping of the Ph-3 gene conferring resistance to late blight (Phytophthora infestans) in tomato. Theor. Appl. Genet. 126, 2643-2653 (2013).

[7] Nowicki, M., Fooled, M. R., Nowakowska, M. & Kozik, E. U. Potato and tomato late blight caused by Phytophthora infestans: an overview of pathology and resistance breeding. Plant Dis. 96, 4-17 (2012).

[8] Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233 (2009).

[9] Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microRNA targets. Cell 115, 787-798 (2003).

[10] Tang, G. & Tang, X. Short tandem target mimic: a long journey to the engineered molecular landmine for selective destruction/blockage of microRNAs in plants and animals. J. Genet. Genom. 40, 291-296 (2013).

[11] Zhang, X., Zou, Z. & Gong, P. et al. Over-expression of microRNA169 confers enhanced drought tolerance to tomato. Biotechnol. Lett. 33, 403-409 (2011).

[12] Mallory, A. C., Bartel, D. P. & Bartel, B. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell 17, 1360-1375 (2005).

[13] Luan, Y. et al. Effective enhancement of resistance to Phytophthora infestans by overexpression of miR172a and b in Solanum lycopersicum. Planta 247, 127-138 (2018).

[14] Luan, Y., Cui, J., Wang, W. & Meng, J. MiR1918 enhances tomato sensitivity to Phytophthora infestans infection. Sci. Rep. 6, 35858 (2016).

[15] Franco-Zorrilla, J. M., Valli, A. & Todesco, M. et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet. 39, 1033-1037 (2007).

[16] Tang, G., Yan, J. & Gu, Y. et al. Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods 58, 118-125 (2012).

[17] Yan, J., Gu, Y. & Jia, X. et al. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415-427 (2012).

[18] Jia, X., Ding, N. & Fan, W. et al. Functional plasticity of miR165/166 in plant development revealed by small tandem target mimic. Plant Sci. 233, 11-21 (2015).

[19] Gu, Z., Huang, C., Li, F. & Zhou, X. A versatile system for functional analysis of genes and microRNAs in cotton. Plant Biotechnol. J. 12, 638-649 (2014).

[20] Nizampatnam, N. R., Schreier, S. J., Damodaran, S., Adhikari, S. & Subramanian, S. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development. Plant J. 84, 140-153 (2015).

[21] Zhao, J., Liu, Q. & Hu, P. et al. An efficient Potato virus X-based microRNA silencing in Nicotiana benthamiana. Sci. Rep. 6, 20573 (2016).

[22] Jia, X., Shen, J. et al. Small tandem target mimic-mediated blockage of microRNA858 induces anthocyanin accumulation in tomato. Planta 242, 283-293 (2015).

[23] Akdogan, G., Tufekci, E. D., Uranbey, S. & Unver, T. miRNA-based drought regulation in wheat. Funct. Integr. Genom. 16, 221-233 (2016).

[24] Li, W., Wang, T., Zhang, Y. & Li, Y. Overexpression of soybean miR172c confers tolerance to water deficit and salt stress, but increases ABA sensitivity in transgenic Arabidopsis thaliana. J. Exp. Bot. 67, 175-194 (2016).

[25] Luan, Y. et al. High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans. Planta 241, 1405-1416 (2015).

[26] Jin, H. Endogenous small RNAs and antibacterial immunity in plants. FEBS Lett. 582, 2679-2684 (2008).

[27] Navarro, L. et al. A plant miRNA contributes to antibacterial resistance by repressing auxin signaling. Science 312, 436-439 (2006).

[28] Boccara, M. et al. The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP- and effector-triggered immunity through the post-transcriptional control of disease resistance genes. PLoS Pathog. 10, e1003883 (2014).

[29] Li, F., Pignatta, D. & Bendix, C. et al. MicroRNA regulation of plant innate immune receptors. Proc. Natl Acad. Sci. USA 109, 1790-1795 (2012).

[30] Ouyang, S. et al. MicroRNAs suppress NB domain genes in tomato that confer resistance to Fusarium oxysporum. PLoS Pathog. 10, e1004464 (2014).

[31] Yang, L. et al. Overexpression of potato miR482e enhanced plant sensitivity to Verticillium dahliae infection. J. Integr. Plant Biol. 57, 1078-1088 (2015).

[32] Li, W., Xu, Y. P. & Cai, X. Z. Transcriptional and posttranscriptional regulation of the tomato leaf mould disease resistance gene Cf-9. Biochem. Biophys. Res. Commun. 470, 163-167 (2016).

[33] Bao, D. et al. Downregulation of genes coding for core RNAi components and disease resistance proteins via corresponding microRNAs might be correlated with successful SMV infection in soybean. Mol. Plant Pathol. https://doi.org/10.1111/mpp.12581 (2017).

[34] Shivaprasad, P. V. et al. A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs. Plant Cell 24, 859-874 (2012).

[35] Tsushima, D., Adkar-Purushothama, C. R., Taneda, A. & Sano, T. Changes in relative expression levels of viroid-specific small RNAs and microRNAs in tomato plants infected with severe and mild symptom-inducing isolates of Potato spindle tuber viroid. J. Gen. Plant Pathol. 81, 49-62 (2015).

[36] Feng, J., Liu, S., Wang, M., Lang, Q. & Jin, C. Identification of microRNAs and their targets in tomato infected with Cucumber mosaic virus based on deep sequencing. Planta 240, 1335-1352 (2014).

[37] Zhang, Y. et al. Identification of miRNAs and their targets in cotton inoculated with Verticillium dahliae by high-throughput sequencing and degradome analysis. Int J. Mol. Sci. 16, 14749-14768 (2015).

[38] Li, X. et al. Identification of soybean microRNAs involved in soybean cyst nematode infection by deep sequencing. PLoS ONE 7, e39650 (2012).

[39] Zhao, C. Z., Xia, H. & Cao, T. J. et al. Small RNA and degradome deep sequencing reveals peanut microRNA roles in response to pathogen infection. Plant Mol. Biol. Rep. 33, 1013-1029 (2015).

[40] Zhu, Q. H. et al. MiR482 regulation of NBS-LRR defense genes during fungal pathogen infection in cotton. PLoS ONE 8, e84390 (2013).

[41] Marone, D., Russo, M. A., Laido, G., De Leonardis, A. M. & Mastrangelo, A. M. Plant nucleotide binding site-leucine-rich repeat (NBS-LRR) genes: active guardians in host defense responses. Int. J. Mol. Sci. 14, 7302-7326 (2013).

[42] Mao, Z. C. et al. Cloning and functional analyses of pepper CaRKNR involved in Meloidogyne incognita resistance. Euphytica 205, 903-913 (2015).

[43] Ma, J. et al. Pi64, encoding a novel CC-NBS-LRR protein, confers resistance to leaf and neck blast in rice. Mol. Plant Microbe Interact. 28, 558-568 (2015).

[44] Wang, X. M. et al. Characterization of a novel NBS-LRR gene involved in bacterial blight resistance in rice. Plant Mol. Biol. Rep. 31, 649-656 (2013).

[45] Lanfermeijer, F. C., Dijkhuis, J., Sturre, M. J. G., de Haan, P. & Hille, J. Cloning and characterization of the durable tomato mosaic virus resistance gene Tm-2² from Lycopersicon esculentum . Plant Mol. Biol. 52, 1039-1051 (2003).

[46] Gutierrez, J. R. et al. Prf immune complexes of tomato are oligomeric and contain multiple Pto-like kinases that diversify effector recognition. Plant J. 61, 507-518 (2010).

[47] Luan, Y., Wang, W. & Liu, P. Identification and functional analysis of novel and conserved microRNAs in tomato. Mol. Biol. Rep. 41, 5385-5394 (2014).

[48] Sun, G., Luan, Y. & Cui, J. Mining and characterization of miRNAs closely associated with the pathogenicity in tomato. Yi Chuan 36, 69-76 (2014).

[49] Cui, J., Luan, Y., Jiang, N., Bao, H. & Meng, J. Comparative transcriptome analysis between resistant and susceptible tomato allows the identification of lncRNA16397 conferring resistance to Phytophthora infestans by co-expressing glutaredoxin. Plant J. 89, 577-589 (2017).

[50] Li, J., Luan, Y. & Liu, Z. SpWRKY1 mediates resistance to Phytophthora infestans and tolerance to salt and drought stress by modulating reactive oxygen species homeostasis and expression of defense-related genes in tomato. Plant Cell Tissue Organ Cult. 123, 67-81 (2015).

[51] Jiang, Y. et al. Overexpression of poplar PtrWRKY89 in transgenic Arabidopsis leads to a reduction of disease resistance by regulating defense-related genes in salicylate- and jasmonate-dependent signaling. PLoS ONE 11, e0149137 (2016).

[52] Addo-Quaye, C., Eshoo, T. W., Bartel, D. P. & Axtell, M. J. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr. Biol. 18, 758-762 (2008).

[53] Addo-Quaye, C., Miller, W. & Axtell, M. J. CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 25, 130-131 (2009).

[54] Fan, C., Wei, Q., Hao, Z. & Li, G. Prediction and functional analysis of lincRNAs targeted by miRNAs. Yi Chuan 36, 1226-1234 (2014).

[55] Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F. & Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 12 (2007).

[56] Wi, S. J., Ji, N. R. & Park, K. Y. Synergistic biosynthesis of biphasic ethylene and reactive oxygen species in response to hemibiotrophic Phytophthora parasitica in tobacco plants. Plant Physiol. 159, 251-265 (2012).

[57] Ye, W. & Ma, W. Filamentous pathogen effectors interfering with small RNA silencing in plant hosts. Curr. Opin. Microbiol. 32, 1-6 (2016).

[58] Qiao, Y., Liu, L. & Xiong, Q. et al. Oomycete pathogens encode RNA silencing suppressors. Nat. Genet. 45, 330-333 (2013).

[59] Qiao, Y., Shi, J., Zhai, Y., Hou, Y. & Ma, W. Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection. Proc. Natl Acad. Sci. USA 112, 5850-5855 (2015).

[60] Wang, J. et al. Genome-wide analysis of tomato long non-coding RNAs and identification as endogenous target mimic for microRNA in response to TYLCV infection. Sci. Rep. 5, 16946 (2015).

[61] Gao, C. et al. MicroRNA profiling analysis throughout tomato fruit development and ripening reveals potential regulatory role of RIN on microRNAs accumulation. Plant Biotechnol. J. 13, 370-382 (2015).

[62] Zhang, H. et al. Short tandem target mimic rice lines uncover functions of miRNAs in regulating important agronomic traits. Proc. Natl Acad. Sci. USA 114, 5277-5282 (2017).

[63] Cao, D. et al. Regulations on growth and development in tomato cotyledon, flower and fruit via destruction of miR396 with short tandem target mimic. Plant Sci. 247, 1-12 (2016).

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

[65] Li, X., Zhang, Y., Yin, L. & Lu, J. Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1 enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana. Protoplasma 254, 957-969 (2017).

[66] Gu, K. et al. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature 435, 1122-1125 (2005).

[67] Radwan, O. et al. Genetic diversity and genomic distribution of homologs encoding NBS-LRR disease resistance proteins in sunflower. Mol. Genet. Genom. 280, 111-125 (2008).

[68] Li, X. et al. Comparative transcriptome analysis reveals defense-related genes and pathways against downy mildew in Vitis amurensis grapevine. Plant Physiol. Biochem. 95, 1-14 (2015).

[69] Zhang, C. et al. Overexpression of a novel peanut NBS-LRR gene AhRRS5 enhances disease resistance to Ralstonia solanacearum in tobacco. Plant Biotechnol. J. 15, 39-55 (2017).

[70] Yang, J. et al. Molecular cloning and functional analysis of GbRVd, a gene in Gossypium barbadense that plays an important role in conferring resistance to Verticillium wilt. Gene 575, 687-694 (2016).