Tomato DCL2b is required for the biosynthesis of 22-nt small RNAs, the resulting secondary siRNAs, and the host defense against ToMV

Abstract

The tomato encode four functional DCL families, of which DCL2 is poorly studied. Here, we generated loss-of-function mutants for a tomato DCL2 gene, dcl2b, and we identified its major role in defending against tomato mosaic virus in relation to both natural and manual infections. Genome-wide small RNA expression profiling revealed that DCL2b was required for the processing 22-nt small RNAs, including a few species of miRNAs. Interestingly, these DCL2b-dependent 22-nt miRNAs functioned similarly to the DCL1-produced 22-nt miRNAs in Arabidopsis and could serve as triggers to generate a class of secondary siRNAs. In particular, the majority of secondary siRNAs were derived from plant defense genes when the plants were challenged with viruses. We also examined differentially expressed genes in dcl2b through RNA-seq and observed that numerous genes were associated with mitochondrial metabolism and hormone signaling under virus-free conditions. Notably, when the loss-of-function dcl2b mutant was challenged with tomato mosaic virus, a group of defense response genes was activated, whereas the genes related to lipid metabolism were suppressed. Together, our findings provided new insights into the roles of tomato DCL2b in small RNA biogenesis and in antiviral defense.

References

[1] Achkar, N. P., Cambiagno, D. A. & Manavella, P. A. miRNA biogenesis: a dynamic pathway. Trends Plant. Sci. 21, 1034-1044 (2016).

[2] Li, S., Castillo-Gonzalez, C., Yu, B. & Zhang, X. The functions of plant small RNAs in development and in stress responses. Plant J. 90, 654-670 (2017).

[3] Holoch, D. & Moazed, D. RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71-84 (2015).

[4] Blevins, T. et al. Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis. eLife 4, e9591 (2015).

[5] Fukudome, A. & Fukuhara, T. Plant dicer-like proteins: double-stranded RNA-cleaving enzymes for small RNA biogenesis. J. Plant. Res. 130, 33-44 (2017).

[6] Zhu, H. et al. Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1. Nat. Struct. Mol. Biol. 20, 1106 (2013).

[7] Kravchik, M. et al. Global and local perturbation of the tomato microRNA pathway by a trans-activated DICER-LIKE 1 mutant. J. Exp. Bot. 65, 725-739 (2014).

[8] Wu, Y. et al. DCL2-and RDR6-dependent transitive silencing of SMXL4 and SMXL5 in Arabidopsis dcl4 mutants causes defective phloem transport and carbohydrate over-accumulation. Plant J. 90, 1064-1078 (2017).

[9] Taochy, C. et al. A genetic screen for impaired systemic RNAi highlights the crucial role of DICER-LIKE 2. Plant Physiol. 175, 1424-1437 (2017).

[10] Kravchik, M., Damodharan, S., Stav, R. & Arazi, T. Generation and characterization of a tomato DCL3-silencing mutant. Plant Sci. 221, 81-89 (2014).

[11] Yifhar, T. et al. Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome. Plant Cell 24, 3575-3589 (2012).

[12] Song, X. et al. Roles of DCL4 and DCL3b in rice phased small RNA biogenesis. Plant J. 69, 462-474 (2012).

[13] Xie, Z. X., Allen, E., Wilken, A. & Carrington, J. C. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 102, 12984-12989 (2005).

[14] Fei, Q., Xia, R. & Meyers, B. C. Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell 25, 2400-2415 (2013).

[15] Deng, P., Muhammad, S., Cao, M. & Wu, L. Biogenesis and regulatory hierarchy of phased small interfering RNAs in plants. Plant. Biotechnol. J. 16, 965-975 (2018).

[16] Chen, H. et al. 22-nucleotide RNAs trigger secondary siRNA biogenesis in plants. Proc. Natl Acad. Sci. USA 107, 15269-15274 (2010).

[17] Xia, R. et al. MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA biogenesis in eudicots. Plant Cell 25, 1555-1572 (2013).

[18] Axtell, M. J., Jan, C., Rajagopalan, R. & Bartel, D. P. A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565-577 (2006).

[19] Bouche, N., Lauressergues, D., Gasciolli, V. & Vaucheret, H. An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 25, 3347-3356 (2006).

[20] Garcia-Ruiz, H. et al. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during turnip mosaic virus infection. Plant Cell 22, 481-496 (2010).

[21] Mlotshwa, S. et al. DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS ONE 3, e1755 (2008).

[22] Parent, J., Bouteiller, N., Elmayan, T. & Vaucheret, H. Respective contributions of Arabidopsis DCL2 and DCL4 to RNA silencing. Plant J. 81, 223-232 (2015).

[23] Qin, C. et al. Roles of Dicer-Like proteins 2 and 4 in intra- and intercellular antiviral silencing. Plant Physiol. 174, 1067-1081 (2017).

[24] Chen, W. et al. A genetic network for systemic RNA silencing in plants. Plant Physiol. 176, 2700-2719 (2018).

[25] Bergougnoux, V. The history of tomato: from domestication to biopharming. Biotechnol. Adv. 32, 170-189 (2014).

[26] Wang, T. et al. Cloning, identification, and expression analysis of a Dicer-Like gene family from Solanum lycopersicum. Biol. Plant. 60, 410-418 (2016).

[27] Xie, X. et al. CRISPR-GE: a convenient software toolkit for CRISPR-based genome editing. Mol. Plant 10, 1246-1249 (2017).

[28] Andrade, O., Latorre, B. A. & Escaffis, O. Tomato mosaic-virus associated with shoestring sympotem in chilean tomatoes. Plant Dis. 65, 761-762 (1981).

[29] Visser, M., Bester, R., Burger, J. T. & Maree, H. J., Next-generation sequencing for virus detection: covering all the bases. Virol. J. 13, 85 (2016).

[30] Kung, Y. et al. Genetic analyses of the FRNK motif function of turnip mosaic virus uncover multiple and potentially interactive pathways of cross-protection. Mol. Plant Microbe Interact. 27, 944-955 (2014).

[31] Andika, I. B., Jamal, A., Kondo, H. & Suzuki, N. SAGA complex mediates the transcriptional up-regulation of antiviral RNA silencing. Proc. Natl Acad. Sci. USA 114, E3499-E3506 (2017).

[32] Malpica-Lopez, N. et al. Revisiting the roles of tobamovirus replicase complex proteins in viral replication and silencing suppression. Mol. Plant Microbe Interact. 31, 125-144 (2018).

[33] Zhu, B. et al. RNA sequencing and functional analysis implicate the regulatory role of long non-coding RNAs in tomato fruit ripening. J. Exp. Bot. 66, 4483-4495 (2015).

[34] Cao, M. et al. Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis. Proc. Natl Acad. Sci. USA 111, 14613-14618 (2014).

[35] Vazquez, F. & Hohn, T. Biogenesis and biological activity of secondary siRNAs in plants. Scientifica 2013, 783253 (2013).

[36] Schmitz-Linneweber, C. & Small, I. Pentatricopeptide repeat proteins: a socket set for organelle gene expression. Trends Plant. Sci. 13, 663-670 (2008).

[37] Nagy, P. D. & Pogany, J. The dependence of viral RNA replication on co-opted host factors. Nat. Rev. Microbiol. 10, 137-149 (2011).

[38] Miguel, M., et al. Lipid involvement in viral infections: present and future perspectives for the design of antiviral strategies. in Lipid Metabolism, pp 291-322 (ed Rodrigo, V. B.) Ch. 13 (InTech, 2013).

[39] Li, F. et al. MicroRNA regulation of plant innate immune receptors. Proc. Natl Acad. Sci. USA 109, 1790-1795 (2012).

[40] Ishibashi, K. & Ishikawa, M. The resistance protein Tm-1 inhibits formation of a tomato mosaic virus replication protein-host membrane protein complex. J. Virol. 87, 7933-7939 (2013).

[41] Ishibashi, K. et al. Structural basis for the recognition-evasion arms race between Tomato mosaic virus and the resistance gene Tm-1. Proc. Natl Acad. Sci. USA 111, E3486-E3495 (2014).

[42] Deleris, A. et al. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313, 68-71 (2006).

[43] Andika, I. B. et al. Differential contributions of plant Dicer-like proteins to antiviral defences against potato virus X in leaves and roots. Plant J. 81, 781-793 (2015).

[44] Ma, X. et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274-1284 (2015).

[45] Van Eck, J., Kirk, D. D. & Walmsley, A. M. Tomato (Lycopersicum esculentum). Methods Mol. Biol. 343, 459-473 (2006).

[46] Whitham, S., McCormick, S. & Baker, B. The N gene of tobacco confers resistance to tobacco mosaic virus in transgenic tomato. Proc. Natl. Acad. Sci. USA 93, 8776-8781 (1996).

[47] Li, R. et al. Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum. Plant. Biotechnol. J. 16, 415-427 (2018).

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

[49] Larkin, M. A. et al. Clustal W and clustal X version 2.0. Bioinformatics 23, 2947-2948 (2007).

[50] He, Z. et al. Evolviewv2: an online visualization and management tool for customized and annotated phylogenetic trees. Nucleic Acids Res. 44, W236-W241 (2016).