College of Horticulture, Shenyang Agricultural University/Key Laboratory of Protected Horticulture, Ministry of Education/Northern National & Local Joint Engineering Research Center of Horticultural Facilities Design and Application Technology (Liaoning), Shenyang 110866, China

Search for more papers by this author

, and

Yushi Luan

^†Corresponding authors: Y. Luan;

E-mail Address: ysluan@dlut.edu.cn

, and H. Qi;

E-mail Address: qihongyan@syau.edu.cn

https://orcid.org/0000-0001-9397-9171

School of Bioengineering, Dalian University of Technology, Dalian 116024, China

Search for more papers by this author

Affiliations

Authors and Affiliations

Yuhui Hong¹
Jun Meng²
Xiaoli He¹
Yuanyuan Zhang¹
Yarong Liu¹
Chengwei Zhang³
Hongyan Qi⁴^†
Yushi Luan¹^†

¹School of Bioengineering, Dalian University of Technology, Dalian 116024, China
²School of Computer Science and Technology, Dalian University of Technology, Dalian 116024, China
³Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry Sciences, Beijing 100000, China
⁴College of Horticulture, Shenyang Agricultural University/Key Laboratory of Protected Horticulture, Ministry of Education/Northern National & Local Joint Engineering Research Center of Horticultural Facilities Design and Application Technology (Liaoning), Shenyang 110866, China

Published Online:2 Aug 2021https://doi.org/10.1094/PHYTO-08-20-0360-R

View article

Abstract

Late blight, caused by Phytophthora infestans, is severely damaging to the global tomato industry. Micro-RNAs (miRNAs) have been widely demonstrated to play vital roles in plant resistance by repressing their target genes. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) method has been continuously improved and extensively applied to edit plant genomes. However, editing multiplex miRNAs by CRISPR/Cas9 in tomato has not been studied yet. We knocked out miR482b and miR482c simultaneously in tomato through the multiplex CRISPR/Cas9 system. Two transgenic plants with silenced miR482b and miR482c simultaneously and one transgenic line with silenced miR482b alone were obtained. Compared with wild-type plants, the disease symptoms of three transgenic plants upon infection were reduced, accompanied by increased expression of their common target nucleotide binding site-leucine-rich repeat genes and decreased levels of reactive oxygen species. Furthermore, silencing miR482b and miR482c simultaneously was more resistant than silencing miR482b alone in tomato. More importantly, we found that knocking out miR482b and miR482c can elicit expression perturbation of other miRNAs, suggesting cross-regulation between miRNAs. Our study demonstrated that editing miR482b and miR482c simultaneously with CRISPR/Cas9 is an efficient strategy for generating pathogen-resistant tomatoes, and cross-regulation between miRNAs may reveal the novel mechanism in tomato–P. infestans interactions.

LITERATURE CITED

Ahuja, I., Kissen, R., and Bones, A. M. 2012. Phytoalexins in defense against pathogens. Trends Plant Sci. 17:73-90. https://doi.org/10.1016/j.tplants.2011.11.002 Crossref, Medline, ISI, Google Scholar
Bednarek, P. 2012. Chemical warfare or modulators of defence responses: The function of secondary metabolites in plant immunity. Curr. Opin. Plant Biol. 15:407-414. https://doi.org/10.1016/j.pbi.2012.03.002 Crossref, Medline, ISI, Google Scholar
Belkhadir, Y., Subramaniam, R., and Dangl, J. L. 2004. Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr. Opin. Plant Biol. 7:391-399. https://doi.org/10.1016/j.pbi.2004.05.009 Crossref, Medline, ISI, Google Scholar
Bi, H., Fei, Q., Li, R., Liu, B., Xia, R., Char, S. N., Meyers, B. C., and Yang, B. 2020. Disruption of miRNA sequences by TALENs and CRISPR/Cas9 induces varied lengths of miRNA production. Plant Biotechnol. J. 18:1526-1536. https://doi.org/10.1111/pbi.13315 Crossref, Medline, ISI, Google Scholar
Bigeard, J., Colcombet, J., and Hirt, H. 2015. Signaling mechanisms in pattern-triggered immunity (PTI). Mol. Plant 8:521-539. https://doi.org/10.1016/j.molp.2014.12.022 Crossref, Medline, ISI, Google Scholar
Bonnet, E., He, Y., Billiau, K., and Van de Peer, Y. 2010. TAPIR, a web server for the prediction of plant microRNA targets, including target mimics. Bioinformatics 26:1566-1568. https://doi.org/10.1093/bioinformatics/btq233 Crossref, Medline, ISI, Google Scholar
Brigelius-Flohé, F. R., and Flohé, L. 2020. Regulatory phenomena in the glutathione peroxidase superfamily. Antioxid. Redox Signal. 33:498-516. https://doi.org/10.1089/ars.2019.7905 Crossref, Medline, ISI, Google Scholar
Canto-Pastor, A., Bruno, A. M. C. S., Adrian, A. V., William, S., Sebastian, S., and David, C. B. 2019. Enhanced resistance to bacterial and oomycete pathogens by short tandem target mimic RNAs in tomato. Proc. Natl. Acad. Sci. USA 116:2755-2760. https://doi.org/10.1073/pnas.1814380116 Crossref, Medline, ISI, Google Scholar
Chang, H., Yi, B., Ma, R., Zhang, X., Zhao, H., and Xi, Y. 2016. CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo. Sci. Rep. 6:22312. https://doi.org/10.1038/srep22312 Crossref, Medline, ISI, Google Scholar
Chen, K., Wang, Y., Zhang, R., Zhang, H., and Gao, C. 2019a. CRISPR/Cas genome editing and precision plant breeding in agriculture. Annu. Rev. Plant Biol. 70:667-697. https://doi.org/10.1146/annurev-arplant-050718-100049 Crossref, Medline, ISI, Google Scholar
Chen, L., Meng, J., He, X. L., Zhang, M., and Luan, Y. S. 2019b. Solanum lycopersicum microRNA1916 targets multiple target genes and negatively regulates the immune response in tomato. Plant Cell Environ. 42:1393-1407. https://doi.org/10.1111/pce.13468 Crossref, Medline, ISI, Google Scholar
Chuck, G., Cigan, A. M., Saeteurn, K., and Hake, S. 2007. The heterochronic maize mutant Corngrass1 results from overexpression of a tandem microRNA. Nat. Genet. 39:544-549. https://doi.org/10.1038/ng2001 Crossref, Medline, ISI, Google Scholar
Cohen, Y., Rubin, A. E., and Galperin, M. 2018. Oxathiapiprolin-based fungicides provide enhanced control of tomato late blight induced by mefenoxam-insensitive Phytophthora infestans. PLoS One 13:e0204523. https://doi.org/10.1371/journal.pone.0204523 Crossref, Medline, ISI, Google Scholar
Dai, X., Zhuang, Z., and Zhao, P. X. 2018. psRNATarget: A plant small RNA target analysis server (2017 release). Nucleic Acids Res. 46:W49-W54. https://doi.org/10.1093/nar/gky316 Crossref, Medline, ISI, Google Scholar
de Vries, S., Kloesges, T., and Rose, L. E. 2015. Evolutionarily dynamic, but robust, targeting of resistance genes by the miR482/2118 gene family in the Solanaceae. Genome Biol. Evol. 7:3307-3321. https://doi.org/10.1093/gbe/evv225 Crossref, Medline, ISI, Google Scholar
de Vries, S., Kukuk, A., von Dahlen, J. K., Schnake, A., Kloesges, T., and Rose, L. E. 2018. Expression profiling across wild and cultivated tomatoes supports the relevance of early miR482/2118 suppression for Phytophthora resistance. Proc. Biol. Sci. 285:20172560. Crossref, Medline, ISI, Google Scholar
Eamens, A. L., Agius, C., Smith, N. A., Waterhouse, P. M., and Wang, M. B. 2011. Efficient silencing of endogenous microRNAs using artificial microRNAs in Arabidopsis thaliana. Mol. Plant 4:157-170. https://doi.org/10.1093/mp/ssq061 Crossref, Medline, ISI, Google Scholar
Farmer, E. E., and Mueller, M. J. 2013. ROS-mediated lipid peroxidation and RES-activated signaling. Annu. Rev. Plant Biol. 64:429-450. https://doi.org/10.1146/annurev-arplant-050312-120132 Crossref, Medline, ISI, Google Scholar
Fry, W. E. 2016. Phytophthora infestans: New tools (and old ones) lead to new understanding and precision management. Annu. Rev. Phytopathol. 54:529-547. https://doi.org/10.1146/annurev-phyto-080615-095951 Crossref, Medline, ISI, Google Scholar
Fry, W. E., Birch, P. R., Judelson, H. S., Grunwald, N. J., Danies, G., Everts, K. L., Gevens, A. J., Gugino, B. K., Johnson, D. A., Johnson, S. B., McGrath, M. T., Myers, K. L., Ristaino, J. B., Roberts, P. D., Secor, G., and Smart, C. D. 2015. Five reasons to consider Phytophthora infestans a reemerging pathogen. Phytopathology 105:966-981. https://doi.org/10.1094/PHYTO-01-15-0005-FI Link, ISI, Google Scholar
Guzman, A. R., Kim, J. G., Taylor, K. W., Lanver, D., and Mudgett, M. B. 2020. Tomato atypical receptor kinase1 is involved in the regulation of preinvasion defense. Plant Physiol. 183:1306-1318. https://doi.org/10.1104/pp.19.01400 Crossref, Medline, ISI, Google Scholar
Ha, M., and Kim, V. N. 2014. Regulation of microRNA biogenesis. Nat. Rev. Mol. Cell Biol. 15:509-524. https://doi.org/10.1038/nrm3838 Crossref, Medline, ISI, Google Scholar
Hofacker, I. L. 2003. Vienna RNA secondary structure server. Nucleic Acids Res. 31:3429-3431. https://doi.org/10.1093/nar/gkg599 Crossref, Medline, ISI, Google Scholar
Hong, Y. H., Meng, J., He, X. L., Zhang, Y. Y., and Luan, Y. S. 2019. Overexpression of MiR482c in tomato induces enhanced susceptibility to late blight. Cells 8:822. https://doi.org/10.3390/cells8080822 Crossref, ISI, Google Scholar
Hong, Y. H., Meng, J., Zhang, M., and Luan, Y. S. 2020. Identification of tomato circular RNAs responsive to Phytophthora infestans. Gene 746:144652. https://doi.org/10.1016/j.gene.2020.144652 Crossref, Medline, ISI, Google Scholar
Hou, X., Cui, J., Liu, W., Jiang, N., Zhou, X., Qi, H., Meng, J., and Luan, Y. 2020. LncRNA39026 enhances tomato resistance to Phytophthora infestans by decoying miR168a and inducing PR gene expression. Phytopathology 110:873-880. https://doi.org/10.1094/PHYTO-12-19-0445-R Link, ISI, Google Scholar
Jiang, N., Cui, J., Meng, J., and Luan, Y. 2018a. A tomato nucleotide binding sites-leucine-rich repeat gene is positively involved in plant resistance to Phytophthora infestans. Phytopathology 108:980-987. https://doi.org/10.1094/PHYTO-12-17-0389-R Link, ISI, Google Scholar
Jiang, N., Cui, J., Shi, Y., Yang, G., Zhou, X., Hou, X., Meng, J., and Luan, Y. 2019. Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato–Phytophthora infestans interaction. Hortic. Res. 6:28. https://doi.org/10.1038/s41438-018-0096-0 Crossref, Medline, ISI, Google Scholar
Jiang, N., Meng, J., Cui, J., Sun, G., and Luan, Y. 2018b. Function identification of miR482b, a negative regulator during tomato resistance to Phytophthora infestans. Hortic. Res. 5:9. https://doi.org/10.1038/s41438-018-0017-2 Crossref, Medline, ISI, Google Scholar
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., and Charpentier, E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816-821. https://doi.org/10.1126/science.1225829 Crossref, Medline, ISI, Google Scholar
Kamoun, S., Furzer, O., Jones, J. D., Judelson, H. S., Ali, G. S., Dalio, R. J., Roy, S. G., Schena, L., Zambounis, A., Panabières, F., Cahill, D., Ruocco, M., Figueiredo, A., Chen, X. R., Hulvey, J., Stam, R., Lamour, K., Gijzen, M., Tyler, B. M., Grünwald, N. J., Mukhtar, M. S., Tomé, D. F., Tör, M., Van Den Ackerveken, G., McDowell, J., Daayf, F., Fry, W. E., Lindqvist-Kreuze, H., Meijer, H. J., Petre, B., Ristaino, J., Yoshida, K., Birch, P. R., and Govers, F. 2015. The top 10 oomycete pathogens in molecular plant pathology. Mol. Plant Pathol. 16:413-434. https://doi.org/10.1111/mpp.12190 Crossref, Medline, ISI, Google Scholar
Kim, B. S. 2012. Evaluation of tomato genetic resources for the development of resistance breeding lines against late blight. Res. Plant Dis. 18:35-39. https://doi.org/10.5423/RPD.2012.18.1.035 Crossref, Google Scholar
Kozomara, A., Birgaoanu, M., and Griffiths-Jones, S. 2019. miRBase: from microRNA sequences to function. Nucleic Acids Res. 47:D155-D162. https://doi.org/10.1093/nar/gky1141 Crossref, Medline, ISI, Google Scholar
Leesutthiphonchai, W., Vu, A. L., Ah-Fong, A. M. V., and Judelson, H. S. 2018. How does Phytophthora infestans evade control efforts? Modern insight into the late blight disease. Phytopathology 108:916-924. https://doi.org/10.1094/PHYTO-04-18-0130-IA Link, ISI, Google Scholar
Li, J. B., Luan, Y. S., and Liu, Z. 2015. Overexpression of SpWRKY1 promotes resistance to Phytophthora nicotianae and tolerance to salt and drought stress in transgenic tobacco. Physiol. Plant. 155:248-266. https://doi.org/10.1111/ppl.12315 Crossref, Medline, ISI, Google Scholar
Li, N. Y., Ma, X. F., Short, D. P. G., Li, T. G., Zhou, L., Gui, Y. J., Kong, Z. Q., Zhang, D. D., Zhang, W. Q., Li, J. J., Subbarao, K. V., Chen, J. Y., and Dai, X. F. 2018a. The island cotton NBS-LRR gene GbaNA1 confers resistance to the non-race 1 Verticillium dahliae isolate Vd991. Mol. Plant Pathol. 19:1466-1479. https://doi.org/10.1111/mpp.12630 Crossref, Medline, ISI, Google Scholar
Li, R., Li, R., Li, X., Fu, D., Zhu, B., Tian, H., Luo, Y., and Zhu, H. 2018b. Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum. Plant Biotechnol. J. 16:415-427. https://doi.org/10.1111/pbi.12781 Crossref, Medline, ISI, Google Scholar
Li, T., Yang, X., Yu, Y., Si, X., Zhai, X., Zhang, H., Dong, W., Gao, C., and Xu, C. 2018c. Domestication of wild tomato is accelerated by genome editing. Nat. Biotechnol. 36:1160-1163. https://doi.org/10.1038/nbt.4273 Crossref, ISI, Google Scholar
Liu, Q., Wang, C., Jiao, X., Zhang, H., Song, L., Li, Y., Gao, C., and Wang, K. 2019. Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Sci. China Life Sci. 62:1-7. https://doi.org/10.1007/s11427-018-9402-9 Crossref, Medline, ISI, Google Scholar
Livak, K. J., and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^{(-Delta C(T))}. Methods 25:402-408. https://doi.org/10.1006/meth.2001.1262 Crossref, Medline, ISI, Google Scholar
Luan, Y., Cui, J., Li, J., Jiang, N., Liu, P., and Meng, J. 2018. Effective enhancement of resistance to Phytophthora infestans by overexpression of miR172a and b in Solanum lycopersicum. Planta 247:127-138. https://doi.org/10.1007/s00425-017-2773-x Crossref, Medline, ISI, Google Scholar
Luan, Y., Cui, J., Zhai, J., Li, J., Han, L., and Meng, J. 2015. High-throughput sequencing reveals differential expression of miRNAs in tomato inoculated with Phytophthora infestans. Planta 241:1405-1416. https://doi.org/10.1007/s00425-015-2267-7 Crossref, Medline, ISI, Google Scholar
Mascia, T., Santovito, E., Gallitelli, D., and Cillo, F. 2010. Evaluation of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in infected tomato plants. Mol. Plant Pathol. 11:805-816. Medline, ISI, Google Scholar
Melotto, M., Underwood, W., and He, S. Y. 2008. Role of stomata inplant innate immunity and foliar bacterial diseases. Annu. Rev. Phytopathol. 46:101-122. https://doi.org/10.1146/annurev.phyto.121107.104959 Crossref, Medline, ISI, Google Scholar
Montague, T. G., Cruz, J. M., Gagnon, J. A., Church, G. M., and Valen, E. 2014. CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42:W401-W407. https://doi.org/10.1093/nar/gku410 Crossref, Medline, ISI, Google Scholar
Mur, L. A., Kenton, P., Lloyd, A. J., Ougham, H., and Prats, E. 2008. The hypersensitive response; the centenary is upon us but how much do we know? J. Exp. Bot. 59:501-520. https://doi.org/10.1093/jxb/erm239 Crossref, Medline, ISI, Google Scholar
Nekrasov, V., Wang, C., Win, J., Lanz, C., Weigel, D., and Kamoun, S. 2017. Rapid generation of a transgene-free powdery mildew resistant tomato by genome deletion. Sci. Rep. 7:482. https://doi.org/10.1038/s41598-017-00578-x Crossref, Medline, ISI, Google Scholar
Ng, K. L., and Mishra, S. K. 2007. Unique folding of precursor microRNAs: quantitative evidence and implications for de novo identification. RNA 13:170-187. Medline, ISI, Google Scholar
Reichel, M., Li, Y., Li, J., and Millar, A. A. 2015. Inhibiting plant microRNA activity: molecular SPONGEs, target MIMICs and STTMs all display variable efficacies against target microRNAs. Plant Biotechnol. J. 13:915-926. https://doi.org/10.1111/pbi.12327 Crossref, Medline, ISI, Google Scholar
Song, X., Li, Y., Cao, X., and Qi, Y. 2019. MicroRNAs and their regulatory roles in plant–environment interactions. Annu. Rev. Plant Biol. 70:489-525. https://doi.org/10.1146/annurev-arplant-050718-100334 Crossref, Medline, ISI, Google Scholar
Torres, M. A. 2010. ROS in biotic interactions. Physiol. Plant 138:414-429. https://doi.org/10.1111/j.1399-3054.2009.01326.x Crossref, Medline, ISI, Google Scholar
Tsuzuki, M., Futagami, K., Shimamura, M., Inoue, C., Kunimoto, K., Oogami, T., Tomita, Y., Inoue, K., Kohchi, T., Yamaoka, S., Araki, T., Hamada, T., and Watanabe, Y. 2019. An early arising role of the MicroRNA156/529-SPL module in reproductive development revealed by the liverwort Marchantia polymorpha. Curr. Biol. 29:3307-3314.e5. https://doi.org/10.1016/j.cub.2019.07.084 Crossref, Medline, ISI, Google Scholar
Vleeshouwers, V. G., van Dooijeweert, W., Govers, F., Kamoun, S., and Colon, L. T. 2000. The hypersensitive response is associated with host and nonhost resistance to Phytophthora infestans. Planta 210:853-864. https://doi.org/10.1007/s004250050690 Crossref, Medline, ISI, Google Scholar
Wang, H., Yang, H., Shivalila, C. S., Dawlaty, M. M., Cheng, A. W., Zhang, F., and Jaenisch, R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153:910-918. https://doi.org/10.1016/j.cell.2013.04.025 Crossref, Medline, ISI, Google Scholar
Wang, R., Wang, G. L., and Ning, Y. 2019. PALs: Emerging key players in broad-spectrum disease resistance. Trends Plant Sci. 24:785-787. https://doi.org/10.1016/j.tplants.2019.06.012 Crossref, Medline, ISI, Google Scholar
Xun, H., Yang, X., He, H., Wang, M., Guo, P., Wang, Y., Pang, J., Dong, Y., Feng, X., Wang, S., and Liu, B. 2019. Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean. Plant Mol. Biol. 99:95-111. https://doi.org/10.1007/s11103-018-0804-z Crossref, Medline, ISI, Google Scholar
Yan, J., Gu, Y., Jia, X., Kang, W., Pan, S., Tang, X., Chen, X., and Tang, G. 2012. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24:415-427. https://doi.org/10.1105/tpc.111.094144 Crossref, Medline, ISI, Google Scholar
Yu, Y., Jia, T., and Chen, X. 2017. The “how” and “where” of plant microRNAs. New Phytol. 216:1002-1017. https://doi.org/10.1111/nph.14834 Crossref, Medline, ISI, Google Scholar
Zhou, J., Deng, K., Cheng, Y., Zhong, Z., Tian, L., Tang, X., Tang, A., Zheng, X., Zhang, T., Qi, Y., and Zhang, Y. 2017. CRISPR-Cas9 based genome editing reveals new insights into MicroRNA function and regulation in rice. Front. Plant Sci. 8:1598. https://doi.org/10.3389/fpls.2017.01598 Crossref, Medline, ISI, Google Scholar
Zhou, X., Cui, J., Meng, J., and Luan, Y. 2020. Interactions and links among the noncoding RNAs in plants under stresses. Theor. Appl. Genet. 133:3235-3248. https://doi.org/10.1007/s00122-020-03690-1 Crossref, Medline, ISI, Google Scholar

Vol. 111, No. 6
June 2021

ISSN:0031-949X
e-ISSN:1943-7684

Download

Caption

Warts, cankers, and canker warts caused by Botryosphaeria dothidea on apple branches (Xue et al.). Photo credit: Bao-hua Li

Metrics

Downloaded 202 times

Article History

Issue Date: 18 Oct 2021
Published: 2 Aug 2021
First Look: 1 Dec 2020
Accepted: 26 Nov 2020

Pages: 1008-1016

Information

Funding

National Natural Science Foundation of China
Grant/Award Number: 31872116
Grant/Award Number: 32072592
Grant/Award Number: 61872055

Keywords

The author(s) declare no conflict of interest.