Male sterility enables hybrid crop breeding to increase yields and has been extensively studied. But thermo-sensitive female sterility, which is an ideal property that may enable full mechanization in hybrid rice breeding, has rarely been investigated due to the absence of such germplasm. Here we identify the spontaneous thermo-sensitive female sterility 1 (tfs1) mutation that confers complete sterility under regular/high temperature and partial fertility under low temperature as a point mutation in ARGONAUTE7 (AGO7). AGO7 associates with miR390 to form an RNA-Induced Silencing Complex (RISC), which triggers the biogenesis of small interfering RNAs (siRNAs) from TRANS-ACTING3 (TAS3) loci by recruiting SUPPRESSOR OF GENE SILENCING (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) to TAS3 transcripts. These siRNAs are known as tasiR-ARFs as they act in trans to repress auxin response factor genes. The mutant TFS1 (mTFS1) protein is compromised in its ability to load the miR390/miR390* duplex and eject miR390* during RISC formation. Furthermore, tasiR-ARF levels are reduced in tfs1 due to the deficiency in RDR6 but not SGS3 recruitment by mTFS1 RISC under regular/high temperature, while low temperature partially restores mTFS1 function in RDR6 recruitment and tasiR-ARF biogenesis. A miR390 mutant also exhibits female sterility, suggesting that female fertility is controlled by the miR390-AGO7 module. Notably, the tfs1 allele introduced into various elite rice cultivars endows thermo-sensitive female sterility. Moreover, field trials confirm the utility of tfs1 as a restorer line in fully mechanized hybrid rice breeding.
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All DNA sequencing and sRNA sequencing data from this study have been deposited in the National Center for Biotechnology Information (NCBI) with the accession number PRJNA827282. Source data for all graphs have been provided. All other data are available from the corresponding authors upon reasonable request.
Khush, G. S. Green revolution: the way forward. Nat. Rev. Genet. 2, 815 (2001).
Yuan, L. P. Hybrid rice technology for food security in the world. Crop Res. 18, 185–186 (2004).
Fei, Q., Xia, R. & Meyers, B. C. Phased, secondary, small interfering RNAs in posttranscriptional regulatory networks. Plant Cell 25, 2400–2415 (2013).
Chen, X. Small RNAs and their roles in plant development. Annu. Rev. Cell Dev. Biol. 25, 21 (2009).
Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. microRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005).
Axtell, M. J., Jan, C., Rajagopalan, R. & Bartel, D. P. A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565–577 (2006).
Montgomery, T. A. et al. Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133, 128–141 (2008).
Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L. & Poethig, R. S. SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18, 2368–2379 (2004).
Iwakawa, H. et al. Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants. Cell Rep. 35, 109300 (2021).
Sakurai, Y. et al. Cell-free reconstitution reveals the molecular mechanisms for the initiation of secondary siRNA biogenesis in plants. Proc. Natl. Acad. Sci. USA 118, 2102889118 (2021).
Adenot, X. et al. DRB4-dependent TAS3 trans-acting siRNAs control leaf morphology through AGO7. Curr. Biol. 16, 927–932 (2006).
Garcia, D., Collier, S. A., Byrne, M. E. & Martienssen, R. A. Specification of leaf polarity in Arabidopsis via the trans-Acting siRNA pathway. Curr. Biol. 16, 933–938 (2006).
Xu, L. et al. Genetic interaction between the AS1-AS2 and RDR6-SGS3-AGO7 pathways for leaf morphogenesis. Plant Cell Physiol. 47, 853–863 (2006).
Fahlgren, N. et al. Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. Curr. Biol. 16, 939–944 (2006).
Yoshikawa, M., Peragine, A., Park, M. Y. & Poethig, R. S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175 (2005).
Marin, E. et al. mir390, Arabidopsis TAS3 tasiRNAs, and their AUXIN RESPONSE FACTOR targets define an autoregulatory network quantitatively regulating lateral root growth. Plant Cell 22, 1104–1117 (2010).
Yoon, E. K. et al. Auxin regulation of the microRNA390-dependent transacting small interfering RNA pathway in Arabidopsis lateral root development. Nucleic Acids Res. 38, 1382–1391 (2009).
Hobecker, K. V. et al. The microRNA390/TAS3 pathway mediates symbiotic nodulation and lateral root growth. Plant Physiol. 174, 2469–2486 (2017).
Xia, R., Xu, J. & Meyers, B. C. The emergence, evolution, and diversification of the miR390-TAS3-ARF pathway in land plants. Plant Cell 29, 1232–1247 (2017).
Hunter, C., Sun, H. & Poethig, R. S. The Arabidopsis heterochronic gene ZIPPY is an ARGONAUTE family member. Curr. Biol. 13, 1734–1739 (2003).
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).
Brooks, C., Nekrasov, V., Lipppman, Z. B. & van Eck, J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 166, 1292–1297 (2014).
Douglas, R. N. et al. ragged seedling2 encodes an ARGONAUTE7-like protein required for mediolateral expansion, but not dorsiventrality, of maize leaves. Plant Cell 22, 1441–1451 (2010).
Itoh, J.-I., Kitano, H., Matsuoka, M. & Nagato, Y. Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice. Plant Cell 12, 2161–2174 (2000).
Nagasaki, H. et al. The small interfering RNA production pathway is required for shoot meristem initiation in rice. Proc. Natl. Acad. Sci. USA 104, 14867–14871 (2007).
Li, S. et al. Natural variation in PTB1 regulates rice seed setting rate by controlling pollen tube growth. Nat. Commun. 4, 2793 (2013).
Zhang, T. et al. LATERAL FLORET 1 induced the three-florets spikelet in rice. Proc. Natl. Acad. Sci. USA 114, 9984–9989 (2017).
Abe, A. et al. Genome sequencing reveals agronomically important loci in rice using MutMap. Nat. Biotechnol. 30, 174 (2012).
Song, X. et al. Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development. Plant J. 71, 378–389 (2012).
Liu, B. et al. Oryza sativa Dicer-like4 reveals a key role for small interfering RNA silencing in plant development. Plant Cell 19, 2705–2718 (2007).
Yoshikawa, M. et al. 3′ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3. Proc. Natl. Acad. Sci. USA 110, 4117–4122 (2013).
Jouannet, V. et al. Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis. EMBO J. 31, 1704–1713 (2012).
Jiang, P. et al. 21-nt phasiRNAs direct target mRNA cleavage in rice male germ cells. Nat. Commun. 11, 5191 (2020).
Han, J. et al. A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming. Science 370, eabc9546 (2020).
Shi, C. Y. et al. The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation. Science 370, eabc9359 (2020).
Zhang, X. et al. The Arabidopsis thaliana F-box gene HAWAIIAN SKIRT is a new player in the microRNA pathway. PLoS One 12, e0189788 (2017).
Lang, P. L. M. et al. A role for the F-box protein HAWAIIAN SKIRT in plant microRNA function. Plant Physiol. 176, 730–741 (2018).
Yan, J. et al. Effective small RNA destruction by the expression of a short tandem target mimic in Arabidopsis. Plant Cell 24, 415–427 (2012).
Franco-Zorrilla, J. M. et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat. Genet. 39, 1033–1037 (2007).
Kuhn, C.-D. & Joshua-Tor, L. Eukaryotic Argonautes come into focus. Trends Biochem. Sci 38, 263–271 (2013).
Meister, G. Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459 (2013).
Parker, J. S. How to slice: snapshots of Argonaute in action. Silence 1, 3 (2010).
Höck, J. & Meister, G. The Argonaute protein family. Genome Biol 9, 210 (2008).
Hutvagner, G. & Simard, M. J. Argonaute proteins: key players in RNA silencing. Nat. Rev. Mol. Cell Biol. 9, 22–32 (2008).
Boland, A., Huntzinger, E., Schmidt, S., Izaurralde, E. & Weichenrieder, O. Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein. Proc. Natl. Acad. Sci. USA 108, 10466–10471 (2011).
Gan, H. H. & Gunsalus, K. C. Assembly and analysis of eukaryotic Argonaute–RNA complexes in microRNA-target recognition. Nucleic Acids Res. 43, 9613–9625 (2015).
Elkayam, E. et al. The structure of human argonaute-2 in complex with miR-20a. Cell 150, 100–110 (2012).
Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).
Miyoshi, T., Ito, K., Murakami, R. & Uchiumi, T. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute. Nat. Commun. 7, 11846 (2016).
Hu, Y. et al. Study of rice pollen grains by multispectral imaging microscopy. Microsc. Res. Tech. 68, 335–346 (2005).
Zeng, Y. X., Hu, C. Y., Lu, Y. G., Li, J. Q. & Liu, X. D. Diversity of abnormal embryo sacs in indica/japonica hybrids in rice demonstrated by confocal microscopy of ovaries. Plant Breeding 126, 574–580 (2007).
Fujii, S. & Toriyama, K. Molecular mapping of the fertility restorer gene for ms-CW-type cytoplasmic male sterility of rice. Theor. Appl. Genet. 111, 696–701 (2005).
Martin, F. W. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34, 125–128 (1959).
Patel, R. et al. Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel. PLoS One 7, e51153 (2012).
Cingolani, P. et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 6, 80–92 (2012).
Wang, J. et al. Tissue-specific ubiquitination by IPA1 INTERACTING PROTEIN1 modulates IPA1 protein levels to regulate plant architecture in rice. Plant Cell 29, 697–707 (2017).
Yang, X., Chen, L., He, J. & Yu, W. Knocking out of carotenoid catabolic genes in rice fails to boost carotenoid accumulation, but reveals a mutation in strigolactone biosynthesis. Plant Cell Rep. 36, 1533–1545 (2017).
Lyznik, L. A., Mitchell, J. C., Hirayama, L. & Hodges, T. K. Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Res. 21, 969–975 (1993).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet. J. 17, 10–12 (2011).
’t Hoen, P. A. C. et al. Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms. Nucleic Acids Res. 36, e141–e141 (2008).
Tang, C., Xie, Y. & Yan, W. AASRA: an anchor alignment-based small RNA annotation pipeline. Biol. Reprod. 105, 267–277 (2017).
Li, X. et al. Comparative small RNA analysis of pollen development in autotetraploid and diploid rice. Int. J. Mol. Sci. 17, 499 (2016).
Komoda, K., Naito, S. & Ishikawa, M. Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts. Proc. Natl Acad. Sci. USA 101, 1863–1867 (2004).
Komoda, K., Mawatari, N., Hagiwara-Komoda, Y., Naito, S. & Ishikawa, M. Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation. J. Virol. 81, 2584–2591 (2007).
Iki, T. et al. In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90. Mol. Cell 39, 282–291 (2010).
Iki, T., Ishikawa, M. & Yoshikawa, M. In vitro formation of plant RNA-induced silencing complexes using an extract of evacuolated tobacco protoplasts. Methods Mol. Biol. 1640, 39–53 (2017).
We thank Biogle GeneTech for providing a gene-editing vector and transgenic plants. This work was funded by the National Natural Science Foundation of China (NSFC31971924), Hong Kong Research Grant Council (GRF14122415), and JSPS KAKENHI (18H02380).
The authors declare no competing interests.
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Li, H., You, C., Yoshikawa, M. et al. A spontaneous thermo-sensitive female sterility mutation in rice enables fully mechanized hybrid breeding. Cell Res 32, 931–945 (2022). https://doi.org/10.1038/s41422-022-00711-0
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