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SHI family transcription factors regulate an interspecific barrier

Nature Plants volume 9, pages 1862–1873 (2023)Cite this article

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Abstract

Pre-zygotic interspecies incompatibility in angiosperms is an important mechanism to prevent unfavourable hybrids between species. Here we report our identification of STIGMATIC PRIVACY 2 (SPRI2), a transcription factor that has a zinc-finger domain and regulates interspecies barriers in Arabidopsis thaliana, via genome-wide association study. Knockout analysis of SPRI2/SRS7 and its paralogue SPRI2-like/SRS5 demonstrated their necessity in rejecting male pollen from other species within female pistils. Additionally, they govern mRNA transcription of xylan O-acetyltransferases (TBL45 and TBL40) related to cell wall modification, alongside SPRI1, a pivotal transmembrane protein for interspecific pollen rejection. SPRI2/SRS7 is localized as condensed structures in the nucleus formed via liquid–liquid phase separation (LLPS), and a prion-like sequence in its amino-terminal region was found to be responsible for the formation of the condensates. The LLPS-regulated SPRI2/SRS7 discovered in this study may contribute to the establishment of interspecific reproductive barriers through the transcriptional regulation of cell wall modification genes and SPRI1.

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Fig. 1: Identification of the SPRI2 gene via GWAS.
Fig. 2: SPRI2 positively regulates TBL40/TBL45 genes.
Fig. 3: Functional relationships between SPRI1 and SPRI2/SPRI2-like.
Fig. 4: LLPS-regulated nuclear localization of SPRI2.

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Data availability

The sequence data can be found at the Arabidopsis Information Resource database (https://www.arabidopsis.org/tools/bulk/sequences/index.jsp) or on the 1001 Genomes website (https://1001genomes.org/data/GMI-MPI/releases/v3.1/). The raw sequence data files have been deposited at the National Center for Biotechnology Information Sequence Read Archive under BioProject ID PRJNA874115.

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Acknowledgements

We thank M. Okamura, T. Manabe, Y. Yamamoto, M. Nara, M. Ishii, K. Mori and M. Saito for their technical assistance. This work was supported by JSPS KAKENHI grant nos. JP16H06464, JP16H06467, JP16H06380 and JP21H05030 to S. Takayama; JP15K14626, JP16H01467, JP18H02456, JP18H04776, JP22H05172, JP22H05174 and JP23K17987 to S.F.; JP22H05176 to T.I.; and JP21H05663 and JP23H04968 to N.Y. This work was also supported by the PRESTO programme (grant no. JPMJPR16Q8 to S.F.) of the Japan Science and Technology Agency and the Suntory Rising Stars Encouragement Program in Life Sciences (to S.F.).

Author information

Author notes

  1. Surachat Tangpranomkorn

    Present address: GRA&GREEN Inc., Nagoya, Japan

  2. These authors contributed equally: Sota Fujii, Eri Yamamoto.

Authors and Affiliations

  1. Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan

    Sota Fujii, Eri Yamamoto, Seitaro Ito, Surachat Tangpranomkorn, Yuka Kimura, Hiroki Miura, Yoshinobu Kato, Maki Niidome, Aya Yoshida & Seiji Takayama

  2. Suntory Rising Stars Encouragement Program in Life Sciences Fellow, Tokyo, Japan

    Sota Fujii

  3. Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan

    Nobutoshi Yamaguchi, Hiroko Shimosato-Asano, Yuko Wada & Toshiro Ito

  4. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Saitama, Japan

    Yoshinobu Kato

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Contributions

S.F. and S. Takayama conceived the study. S.F., Y. Kato, T.I. and S. Takayama supervised the study. S.F. and S. Takayama wrote the manuscript with input from all other authors. S.F., M.N., H.S.-A. and Y.W. conducted the GWAS. S.F. and E.Y. conducted the phylogenetic analysis. E.Y., H.M., M.N. and A.Y. generated the transgenic lines. E.Y., Y. Kimura, H.M. and M.N. analysed the phenotypes of the transgenic lines. Y. Kimura performed the histological analysis of the pistils. S.F. and E.Y. conducted the RNA-seq analysis. S.F. analysed the RNA-seq data. N.Y. conducted the ChIP experiment. S.F. analysed the ChIP-seq data. E.Y., S.I., S. Tangpranomkorn, Y. Kimura and A.Y. conducted the fluorescent protein localization experiment and analysed the data. S. Tangpranomkorn conducted the FRAP experiment and analysed the data.

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Correspondence to Sota Fujii or Seiji Takayama.

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Extended data

Extended Data Fig. 1 mRNA expression pattern of AT1G19790 (SPRI2/SRS7).

(a) Microarray expression analysis of candidate genes within the SPRI2/SRS7 locus in laser microdissected papilla cells. (b) Image obtained from the TAIR database, showing mRNA expression of AT1G19790 (SPRI2/SRS7) predominantly in the stigmatic papilla cells.

Extended Data Fig. 2 SHI family members.

(a) Phylogenetic analysis of the SHI family members. The protein sequences of the SHI family members were obtained from the databases for A. thaliana (those starting with AT), Brassica rapa (starting with Brara), Cleome violacea (starting with Clevi), Theobroma cacao (starting with Thecc), Solanum lycopersicom (starting with Solyc) and Oryza sativa (starting with LOC_Os). The node values indicate Bayesian posterior probabilities for topology support. The sequence of a protein with a zinc-finger domain (AT1G32730) was used as the outgroup. (b) Representative fluorescent images of aniline-blue-stained pistils of the SHI family mutants six hours after pollination with M. littorea pollen grains. Bars: 200 µm. (c) Bar graph showing the compatibility score of each SHI family mutant after pollination with M. littorea. Groups consisted of n = 7,3,3,10,5,6,4,3,6,10 pistil replicates. The p values by Dunnett’s two-sided test against Col-0 whenever significant difference was found were 8.30E-11 and 2.95E-05. Significant differences were indicated by *** (p < 0.005). The values indicate means of replicates and whiskers indicate standard deviations.

Extended Data Fig. 3 Functional difference of SPRI2_PRS and SPRI2_SRC alleles.

(a) Plot of the interspecific incompatibility phenotype of n = 36 strains carrying the SPRI2_PRS allele and n = 30 strains carrying the SPRI2_SRC allele. Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles. (b) Introduction of SPRI2_PRS and SPRI2_SRC into the spri2/spri2-like double mutant. Groups consisted of n = 5 biological replicates. Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles.

Extended Data Fig. 4 Summary of the genome-edited mutants of SPRI2/SRS7 and SPRI2-like/SRS5.

A diagram illustrating how frame shifts occur and lead to the generation of premature stop codons in mutants.

Extended Data Fig. 5 Pistil morphology of spri2/spri2-like and tbl40/tbl45 mutants.

(a) Bright field image from the top. Bars: 200 µm. Data are representative of three independent pistils. (b) Bright field image from the side. The sty1 mutant reported to exhibit severe pistil developmental defect was also observed as a comparison. Bars: 200 µm. Data are representative of three independent pistils. (c) Technovit thin sections of spri2/spri2-like and tbl40/tbl45 mutants to find any microscopic defects in these mutants. Bars: 100 µm for Col-0 and spri2/spri2-like; 200 µm for tbl40/tbl45. Data are representative of three independent pistils.

Extended Data Fig. 6 Association of the ChIP peaks with differentially expressed genes.

Boxplots with points overlayed showing the distributions of the distances from the nearest SPRI2-Venus ChIP peak for different gene categories. Groups consisted of n = 297, n = 299, and n = 37,231 genes, respectively. The p values by the two-tailed Student’s t-test after Bonferroni correction against the non-DEG group were 1.48E-06 and 5.32E-05. Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles.

Extended Data Fig. 7 Pollen penetration phenotypes of spri2/spri2-like and tbl40/tbl45 mutants.

(a) Bright field images (upper panels) stained with ruthenium red, and fluorescent images (lower panels) stained with calcofluor white, obtained from Technovit thin sections of papilla cells following pollination with M. littorea pollen. Data are representative of three independent pollination experiments. (b) Proportion of pollen tubes either stalled outside the papilla cell wall or penetrated the pistils. Groups consisted of n = 64, n = 102 and n = 80 pollen tubes, respectively. The p values by the two-sided Fisher’s exact test against Col-0 were 2.2E-16 and 6.5E-07. Significant differences are indicated by ***(p < 0.005).

Extended Data Fig. 8 Functional relationships between SPRI1 and SPRI2/SPRI2-like related to Fig. 3.

The box plots display the number of pollen tubes observed on pistils after pollination with seven species that were not rejected by the function of SPRI1. Groups consisted of n = 3 pollination replicates for the ♂Brassica cross and n = 5 for other crosses. The p value by Dunnett’s two-sided test against Col-0 when significant difference existed was 1.90E-02. The significant difference is indicated by * (p < 0.05). Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles.

Extended Data Fig. 9 Analysis of SPRI2 nuclear condensates related to Fig. 4.

(a) Violin plot showing the distribution of particle sizes with nuclear fluorescent signals, estimated from stigmatic cells of the lines stably expressing SPRI2-Venus. Groups consisted of n = 15 and n = 74 cells, respectively. The p value by the two-tailed Student’s t-test was 2.64E-05. The significant difference is indicated by *** (p < 0.005). Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles. (b) Fusion of SPRI2-Venus signals in protoplasts, presented as time-lapse images. Data are representative of five independent experiments. (c) Representative fluorescent images of 1,6-hexanediol treatment to protoplasts expressing SPRI2-Venus. (d) Box plots of Iinside / Itotal estimated from mesophyll protoplasts transiently expressing the wild-type SPRI2-Venus with (+) or without (–) 1,6-hexandiol. Groups consisted of n = 3 cells. Different lowercase letters above each plot indicate significant differences based on the Tukey multiple comparisons of means test with a 95% family-wise confidence level. Center lines: medians, box limits: the 25th and 75th percentiles, whiskers: extend 1.5 times the interquartile range from the 25th and 75th percentiles. (e) Representative fluorescent images of protoplast cells expressing SPRI2-CFP and SPRI2-like-Venus. Data are representative of four independent experiments.

Extended Data Fig. 10 Prion-like domains in SPRI2/SRS7 and SPRI2-like/SRS5.

Output of the prion domain prediction by the PLAAC program using the protein sequences of SPRI2/SRS7 and SPRI2-like/SRS5.

Supplementary information

Reporting Summary

Supplementary Data 1

Supplementary Tables 1–5.

Supplementary Video 1

FRAP of protoplast transiently expressing SPRI2–Venus.

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Fujii, S., Yamamoto, E., Ito, S. et al. SHI family transcription factors regulate an interspecific barrier. Nat. Plants 9, 1862–1873 (2023). https://doi.org/10.1038/s41477-023-01535-5

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